A Multi-Institutional Description of Processes and Outcomes of Postbaccalaureate Research Education Programs in the Mid-Atlantic Region.

ABSTRACT: Outcome data from 6 National Institutes of Health-funded Postbaccalaureate Research Education Programs (PREPs) in the Mid-Atlantic region were combined to give a multi-institutional perspective on their scholars' characteristics and progress through biomedical research training. The institutions hosting these programs were Johns Hopkins University School of Medicine, the Medical University of South Carolina, the University of Maryland School of Medicine, the University of North Carolina at Chapel Hill, Virginia Commonwealth University, and Virginia Polytechnic Institute and State University. The authors summarize the institutional pathways, demographics, undergraduate institutions, and graduate institutions for a total of 384 PREP scholars who completed the programs by June 2021. A total of 228 (59.4%) of these PREP scholars identified as Black or African American, 116 (30.2%) as Hispanic or Latinx, and 269 (70.0%) as female. The authors found that 376 of 384 scholars (97.9%) who started PREP finished their program, 319 of 376 (84.8%) who finished PREP matriculated into PhD or MD/PhD programs, and 284 of 319 (89.0%) who matriculated have obtained their PhD or are successfully making progress toward their PhD.

Engineering the common cold to be a live-attenuated SARS-CoV-2 vaccine.

According to the American Centers for Disease Control and Prevention, people in all age groups catch two or more "colds" per year, at least half of which are caused by human rhinoviruses. Despite decades of effort, there are no vaccines or drugs against rhinovirus infections and even social distancing measures that were effective in reducing the spread of the pandemic coronavirus, SARS-CoV-2, did not reduce the rate of rhinovirus detection. Fortunately, most rhinovirus strains are naturally attenuated in that they are not associated with serious illness, hospitalization or mortality. Instead, rhinoviruses are one of the most frequent viruses found in nasal swabs of asymptomatic, healthy people. Since rhinovirus infections cannot be avoided, a rational approach would be to engineer them for the benefit of their human hosts. Rhinovirus infections naturally induce robust mucosal and serum immune responses to all virus-expressed proteins. Several replication-competent, human rhinovirus vaccine vectors able to express protective antigens for other pathogens have already been designed and tested in animal models. With this strategy, the inevitable common cold would be able to induce immunity not just to a specific rhinovirus serotype but to other more pathogenic respiratory viruses as well. This article reviews existing rhinovirus vaccine vector technology and describes the characteristics that make live-attenuated rhinoviruses attractive vaccine candidates for SARS-CoV-2 and other pathogenic respiratory viruses in the future.

Sex differences in the MB49 syngeneic, murine model of bladder cancer.

OBJECTIVE: The MB49 syngeneic, murine model of bladder cancer has been widely used for more than 35 years. In humans, bladder cancer is one third as prevalent in women as in men, with a trend toward lower prevalence in parous compared to nulliparous women. Our objective was to determine if the MB49 bladder cancer model reproduces the sex differences observed in humans, and to determine its sensitivity to testosterone and the pregnancy hormone, human chorionic gonadotropin (hCG). METHODS: Male and female C57BL/6 mice were implanted with MB49 murine bladder cancer cells, and observed for tumor growth. MB49 dose responses to hCG and dihydrotestosterone were determined in vitro. RESULTS: MB49 tumor growth was significantly greater in male mice than female mice. Pregnancy did not affect MB49 tumor growth in female mice. MB49 cells did not proliferate in response to hCG in vitro and the functional receptor for gonadotropins was absent. Dihydrotestosterone strongly stimulated growth of MB49 cells in vitro. CONCLUSIONS: The MB49 murine model of bladder cancer reproduced some aspects of the sex differences observed in humans. Our results suggest that testosterone may stimulate MB49 cell proliferation, which may explain the more rapid MB49 tumor growth observed in male mice.

Polymer-enhanced delivery increases adenoviral gene expression in an orthotopic model of bladder cancer.

Gene therapy has garnered significant attention as a therapeutic approach for bladder cancer but efficient delivery and gene expression remain major hurdles. The goal of this study was to determine if cationic polymers can enhance adenoviral gene expression in cells that are difficult to transduce in vitro and to subsequently investigate lead candidates for their capacity to increase adenoviral gene expression in an orthotopic in vivo model of bladder cancer. In vitro screening of linear polyamine-based and aminoglycoside-based polymer libraries identified several candidates that enhanced adenoviral reporter gene expression in vitro. The polyamine-based polymer NPGDE-1,4 Bis significantly enhanced adenoviral gene expression in the orthotopic model of bladder cancer but unfortunately further use of this polymer was limited by toxicity. In contrast, the aminoglycoside-based polymer paromomycin-BGDE, enhanced adenoviral gene expression within the bladder without adverse events. Our study demonstrates for the first time that cationic polymers can enhance adenoviral gene expression in an orthotopic model of bladder cancer, thereby providing the foundation for future studies to determine therapeutic benefits of polymer-adenovirus combination in bladder cancer gene therapy.

An orthotopic bladder cancer model for gene delivery studies.

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Histone deacetylase inhibitors restore cell surface expression of the coxsackie adenovirus receptor and enhance CMV promoter activity in castration-resistant prostate cancer cells.

Adenoviral gene therapy using the death receptor ligand TRAIL as the therapeutic transgene can be safely administered via intraprostatic injection but has not been evaluated for efficacy in patients. Here we investigated the efficacy of adenoviral TRAIL gene therapy in a model of castration resistant prostate cancer and found that intratumoral injections can significantly delay tumor growth but cannot eliminate established lesions. We hypothesized that an underlying cause is inefficient adenoviral delivery. Using the LNCaP progression model of prostate cancer we show that surface CAR expression decreases with increasing tumorigenicity and that castration resistant C4-2b cells were more difficult to transduce with adenovirus than castration sensitive LNCaP cells. Many genes, including CAR, are epigenetically silenced during transformation but a new class of chemotherapeutic agents, known as histone deacetylase inhibitors (HDACi), can reverse this process. We demonstrate that HDACi restore CAR expression and infectivity in C4-2b cells and enhance caspase activation in response to infection with a TRAIL adenovirus. We also show that in cells with high surface CAR expression, HDACi further enhance transgene expression from the CMV promoter. Thus HDACi have multiple beneficial effects, which may enhance not only viral but also non-viral gene therapy of castration resistant prostate cancer.

Adenoviral infectivity of exfoliated viable cells in urine: implications for the detection of bladder cancer.

BACKGROUND: Bladder cancer, the 5th most common malignancy in the USA, is often detected as a result of incidental findings or by presenting hematuria. Once diagnosed the disease is one of the costliest cancers to treat due to frequent, invasive and often lifelong follow-up procedures. Because cells are shed into urine, there has been an emerging effort to develop non-invasive tests for the detection of bladder cancer. Expression of survivin, a member of the inhibitor of apoptosis protein family, has been associated with bladder cancer. Therefore, the goal of this study was to determine the feasibility of transducing viable exfoliated cells obtained from urine with an adenoviral vector in which a reporter gene is under the control of the survivin promoter. METHODS: Exfoliated cells from urine were obtained from 36 human subjects (> 40 years old). An adenovirus in which GFP expression is under control of the survivin promoter (Ad.Surv.GFP) was generated. An adenovirus in which GFP is expressed from the CMV promoter served as a control. GFP expression was analyzed by fluorescent microscopy and quantified by flow cytometry. RESULTS: Short-term cultures from exfoliated cells in urine could be established in 16 of 31 samples. These cultures were successfully transduced with Ad.CMV.GFP. Analysis of GFP expression following transduction with Ad.Surv.GFP, indicated that the survivin promoter was preferentially active in UM-UC-3 bladder cancer cells compared to non-malignant UROtsa cells. Interestingly, baseline levels of GFP expression in cultures from exfoliated cells in urine exhibited higher baseline levels than UROtsa following transduction with Ad.Surv.GFP. CONCLUSIONS: We demonstrated the feasibility of establishing and analysing short-term cultures isolated from exfoliated cells in voided urine by means of adenoviral transduction, thereby forming the foundation for future studies to determine the specificity and sensitivity of a non-invasive test based on survivin promoter activity.

Salivary glands act as mucosal inductive sites via the formation of ectopic germinal centers after site-restricted MCMV infection.

We investigated the hypothesis that salivary gland inoculation stimulates formation of ectopic germinal centers (GCs), transforming the gland into a mucosal inductive site. Intraglandular infection of mice with murine cytomegalovirus (MCMV; control: UV-inactivated MCMV) induces salivary gland ectopic follicles comprising cognate interactions between CD4(+) and B220(+) lymphocytes, IgM(+) and isotype-switched IgG(+) and IgA(+) B cells, antigen presenting cells, and follicular dendritic cells. B cells coexpressed the GC markers GCT (57%) and GL7 (52%), and bound the lectin peanut agglutinin. Lymphoid follicles were characterized by a 2- to 3-fold increase in mRNA for CXCL13 (lymphoid neogenesis), syndecan-1 (plasma cells), Blimp-1 (plasma cell development/differentiation), and a 2- to 6-fold increase for activation-induced cytidine deaminase, PAX5, and the nonexcised rearranged DNA of an IgA class-switch event, supporting somatic hypermutation and class-switch recombination within the salivary follicles. Intraglandular inoculation also provided protection against a systemic MCMV challenge, as evidenced by decreased viral titers (10(5) plaque-forming units to undetectable), and restoration of normal salivary flow rates from a 6-fold decrease. Therefore, these features suggest that the salivary gland participates in oral mucosal immunity via generation of ectopic GCs, which function as ectopic mucosal inductive sites.

Polymer-enhanced adenoviral transduction of CAR-negative bladder cancer cells.

The application of adenoviral gene therapy for cancer is limited by immune clearance of the virus as well as poor transduction efficiency, since the protein used for viral entry (CAR) serves physiological functions in adhesion and is frequently decreased among cancer cells. Cationic polymers have been used to enhance adenoviral gene delivery, but novel polymers with low toxicity are needed to realize this approach. We recently identified polymers that were characterized by high transfection efficiency of plasmid DNA and a low toxicity profile. In this study we evaluated the novel cationic polymer EGDE-3,3' for its potential to increase adenoviral transduction of the CAR-negative bladder cancer cell line TCCSUP. The amount of adenovirus required to transduce 50-60% of the cells was reduced 100-fold when Ad.GFP was preincubated with the EGDE-3,3' polymer. Polyethyleneimine (pEI), a positively charged polymer currently used as a standard for enhancing adenoviral transduction, also increased infectivity, but transgene expression was consistently higher with EGDE-3,3'. In addition, EGDE-3,3'-supplemented transduction of an adenovirus expressing an apoptosis inducing transgene, Ad.GFP-TRAIL, significantly enhanced the amount of cell death. Thus, our results indicate that novel biocompatible polymers may be useful in improving the delivery of adenoviral gene therapy.

Oxidative stress sensitizes bladder cancer cells to TRAIL mediated apoptosis by down-regulating anti-apoptotic proteins.

PURPOSE: TRAIL, an endogenous protein involved in immunosurveillance and a novel drug in clinical trials, is of particular interest as cancer therapy because it can induce apoptosis in cancer cells but not in normal cells. Since some cancers develop resistance to TRAIL, safe and effective methods of TRAIL sensitization are of clinical interest. We explored how chemotherapy and oxidative stress affect TRAIL sensitivity and expression of proteins in the apoptotic pathway. MATERIALS AND METHODS: Sensitivity to TRAIL was assessed in viability assays. Apoptosis was measured by caspase-3/7 activity and/or nuclear condensation using Hoechst staining. Western blotting was used to determine cleavage, phosphorylation or alterations in protein expression. RESULTS: TRAIL decreased the viability of 5637 but not of J82 or T24 bladder carcinoma cells (ATCC(R)). Chemotherapy with doxorubicin or cisplatin (Ben Venue Laboratories, Bedford, Ohio) decreased the expression of the anti-apoptotic protein cFLIP(S) and increased caspase-8 cleavage, reversing TRAIL resistance in T24 cells. Specific targeting of cFLIP(S) by siRNA was insufficient for sensitization to TRAIL in T24 cells. However, chemotherapy mediated TRAIL sensitization was mimicked by low concentrations of H(2)O(2), which resulted in the phosphorylation of translation EF2 and decreased the expression of several short half-life, anti-apoptotic proteins, including FLIP(S), XIAP and survivin. CONCLUSIONS: Inducing oxidative stress by low H(2)O(2) concentrations may reverse TRAIL resistance. This warrants the further exploration of H(2)O(2) as an adjuvant intravesical treatment to lower the apoptotic threshold of bladder cancer cells.

Doxorubicin generates a proapoptotic phenotype by phosphorylation of elongation factor 2.

We have previously shown that doxorubicin sensitizes prostate cancer cells to tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL). Sensitization correlated with decreased expression of the antiapoptotic cellular FLICE-like inhibitor protein (cFLIP(S)). The decrease in cFLIP(S) could not be explained by transcriptional regulation or increased degradation, leading us to focus on translational mechanisms. In this study, we found that doxorubicin caused strong and sustained phosphorylation of elongation factor 2 (EF-2), which interferes with protein elongation. Phosphorylation of EF-2 appeared to occur in a kinase-independent manner. Treatment with hydrogen peroxide recapitulated the events observed after doxorubicin treatment. In addition, cells treated with hydrogen peroxide expressed less X-linked inhibitor of apoptosis protein (XIAP) and survivin which, like cFLIP(S), are short-half-life proteins with an antiapoptotic function while expression levels of DR5, caspases-8, -9, -3, and Bax are maintained. The doxorubicin-mediated decrease in cFLIP(S) and XIAP and the TRAIL-induced apoptosis were prevented by pretreatment with an iron chelator, indicating that expression of these proteins was affected by free radical generation upon interaction of iron with doxorubicin. In conclusion, our data suggest that free radicals can affect the phosphorylation of EF-2 resulting in a net loss of short-half-life proteins such as cFLIP(S) and XIAP, leaving a cell more vulnerable to apoptotic stimuli.

A focused salivary gland infection with attenuated MCMV: an animal model with prevention of pathology associated with systemic MCMV infection.

While the salivary gland has been recognized as an important effector site of the common mucosal immune system, a useful model for studying anti-viral salivary gland immune responses in vivo and for exploring the role of the salivary gland within the common mucosal system has been lacking. Murine cytomegalovirus (MCMV) is a beta-herpesvirus that displays a strong tropism for the salivary gland and produces significant morbidity in susceptible mice when introduced by intraperitoneal (i.p.) inoculation. This study tested the hypothesis that MCMV morbidity and pathology could be reduced by injecting the virus directly the submandibular salivary gland (intraglandular (i.g.)), using either in vivo derived MCMV or the less virulent, tissue-culture-derived MCMV (tcMCMV). Peak salivary gland viral titers were completely unaffected by infection route (i.p vs. i.g.) after inoculation with either MCMV or tcMCMV. However, i.g. tcMCMV inoculation reduced viremia in all systemic tissues tested compared to i.p. inoculation. Furthermore, systemic organ pathology observed in the liver and spleen after i.p. inoculation with either MCMV or tcMCMV was completely eliminated by i.g. inoculation with tcMCMV. Cellular infiltrates in the salivary glands, after i.p. or i.g. inoculation were composed of both B and T cells, indicating the potential for a local immune response to occur in the salivary gland. These results demonstrate that a focused MCMV infection of the salivary gland without systemic organ pathology is possible using i.g. delivery of tcMCMV.

Immunoglobulin gene implicated in murine herpes stromal keratitis is not associated with the human disease.

PURPOSE: Susceptibility to herpes stromal keratitis (HSK) is strongly influenced by genetic factors, as shown by multiple rodent models using human herpes simplex virus. A single gene, encoding the immunoglobulin G (IgG) 2a heavy chain protein, confers susceptibility or resistance through a mechanism involving molecular mimicry in one mouse model. However, other rodent studies have produced contradictory results. This study tested the hypothesis that the GM23 gene (the human IgG2a homolog) influences susceptibility to HSK in humans. METHODS: The study population consisted of all consenting patients diagnosed with HSK (25 whites, 2 African Americans) at the Medical University of South Carolina Storm Eye Institute Clinic in Charleston, SC, between August 2000 and June 2004. Healthy controls (23 white adults with no history of HSK) were recruited from the same local population. Genomic DNA from subjects was genotyped at the GM23 locus, which has been implicated as an HSK resistance gene in animal models, by polymerase chain reaction-restriction fragment length polymorphism (RFLP) analysis. RESULTS: No difference in GM23 genotype frequency was observed between patients with HSK and controls. CONCLUSION: Susceptibility to HSK in whites is not predicted by GM23 genotype.

Barriers to coliphage infection of commensal intestinal flora of laboratory mice.

BACKGROUND: Growth characteristics of coliphage viruses indicate that they are adapted to live with their Eschericia coli hosts in the intestinal tract. However, coliphage experimentally introduced by ingestion persist only transiently if at all in the gut of humans and other animals. This study attempted to identify the barriers to long term establishment of exogenous coliphage in the gastrointestinal (GI) tracts of laboratory mice. Intestinal contents were screened for the presence of coliphage and host bacteria, and strains of E. coli bacteria from different segments of the GI tract were tested for susceptibility to six common laboratory coliphages. RESULTS: Contrary to expectations, coliphage were not evident in the GI tracts of laboratory mice, although they were occasionally detected in feces. Commensal flora showed extreme variability within groups of mice despite identical handling and diet. Less than 20% of 48 mice tested carried E. coli in their gut, and of 22 commensal E. coli strains isolated and tested, 59% were completely resistant to infection by lambda, M13, P1, T4, T7, and PhiX174 coliphage. Lysogeny could not be demonstrated in the commensal strains as mitomycin C failed to induce detectable phage. Pre-existing immunity to phages was not evident as sera and fecal washes did not contain significant antibody titers to six laboratory phage types. CONCLUSION: Lack of sufficient susceptible host bacteria seems to be the most likely barrier to establishment of new coliphage infections in the mouse gut.

CD13/aminopeptidase N and murine cytomegalovirus infection.

CD13/aminopeptidase N is a membrane-bound metalloproteinase implicated in human cytomegalovirus (HCMV) infection and pathogenesis. Anti-CD13 antibodies can neutralize HCMV infectivity, and HCMV viremia after bone marrow transplantation induces anti-CD13 autoantibodies which correlate with development of chronic graft vs. host disease. We examined whether murine CD13/APN was similarly implicated in murine cytomegalovirus (MCMV) disease. MCMV infection did induce anti-CD13 antibodies in mice in a strain-specific manner. ICR and 129S mice developed high titers of anti-CD13 antibodies and anti-MCMV antibodies after MCMV infection, whereas CBA and CBAxC57BL/6 f1 hybrid mice produced antibodies against MCMV only. Unlike HCMV, no evidence was found for a correlation between host cell CD13/APN expression and infection, or for the presence of CD13/APN on MCMV particles, although APN inhibitors decreased MCMV plaque formation. Reproduction of CD13/APN autoantibody production in the murine system should make it possible to determine if these antibodies contribute to CMV pathogenesis.

Use of genetically engineered phage to deliver antimicrobial agents to bacteria: an alternative therapy for treatment of bacterial infections.

The emergence and increasing prevalence of multidrug-resistant bacterial pathogens emphasizes the need for new and innovative antimicrobial strategies. Lytic phages, which kill their host following amplification and release of progeny phage into the environment, may offer an alternative strategy for combating bacterial infections. In this study, however, we describe the use of a nonlytic phage to specifically target and deliver DNA encoding bactericidal proteins to bacteria. To test the concept of using phage as a lethal-agent delivery vehicle, we used the M13 phagemid system and the addiction toxins Gef and ChpBK. Phage delivery of lethal-agent phagemids reduced target bacterial numbers by several orders of magnitude in vitro and in a bacteremic mouse model of infection. Given the powerful genetic engineering tools available and the present knowledge in phage biology, this technology may have potential use in antimicrobial therapies and DNA vaccine development.

The role of infectious agents in the pathogenesis of systemic sclerosis.

Over the past few years, increasing evidence has accumulated to implicate infectious agents in the etiology of systemic sclerosis (SSc) and Raynaud phenomenon. Infection rates in patients with SSc compared with those in control populations do not provide clear support for any specific pathogen. However, increased antibody titers, a preponderance of specific strains in patients with SSc, and evidence of molecular mimicry inducing autoimmune responses suggest mechanisms by which infectious agents may contribute to the development and progression of SSc. Here we review studies examining the potential involvement of, cytomegalovirus, and parvovirus B19 in SSc pathogenesis.

Overcoming the phage replication threshold: a mathematical model with implications for phage therapy.

Prior observations of phage-host systems in vitro have led to the conclusion that susceptible host cell populations must reach a critical density before phage replication can occur. Such a replication threshold density would have broad implications for the therapeutic use of phage. In this report, we demonstrate experimentally that no such replication threshold exists and explain the previous data used to support the existence of the threshold in terms of a classical model of the kinetics of colloidal particle interactions in solution. This result leads us to conclude that the frequently used measure of multiplicity of infection (MOI), computed as the ratio of the number of phage to the number of cells, is generally inappropriate for situations in which cell concentrations are less than 10(7)/ml. In its place, we propose an alternative measure, MOI(actual), that takes into account the cell concentration and adsorption time. Properties of this function are elucidated that explain the demonstrated usefulness of MOI at high cell densities, as well as some unexpected consequences at low concentrations. In addition, the concept of MOI(actual) allows us to write simple formulas for computing practical quantities, such as the number of phage sufficient to infect 99.99% of host cells at arbitrary concentrations.