Evidence of HIV exposure and transient seroreactivity in archived HIV-negative severe hemophiliac sera

BACKGROUND: Approximately 25% of hemophiliacs that were frequently exposed to blood clotting factor concentrates (CFCs) contaminated with human immunodeficiency virus (HIV) are presently HIV seronegative. In this study, we sought to determine if some of these individuals were at any time transiently HIV seropositive. In the early to mid-1980s the majority of severe hemophilia patients were exposed to CFCs contaminated with HIV. Although many of these hemophiliacs became HIV-positive, a small percentage did not become infected. To determine if some of these individuals successfully resisted viral infection, we attempted to document the presence of transient HIV reactive antibodies in archived plasma samples (1980–1992) from currently HIV-negative severe hemophiliacs who had a high probability of repeated exposure to HIV contaminated CFC. Archived plasma samples were retrospectively tested using an FDA approved HIV-1Ab HIV-1/HIV-2 (rDNA) enzyme immunoassay (EIA) and a HIV-1 Western blot assay (Wb), neither of which were commercially available until the late 1980s, which was after many of these samples had been drawn. RESULTS: We found that during the high risk years of exposure to HIV contaminated CFC (1980–1987), low levels of plasma antibodies reactive with HIV proteins were detectable in 87% (13/15) of the haemophiliacs tested. None of these individuals are presently positive for HIV proviral DNA as assessed by polymerase chain reaction (PCR). CONCLUSION: Our data suggest that some severe hemophiliacs with heavy exposure to infectious HIV contaminated CFC had only transient low-level humoral immune responses reactive with HIV antigens yet remained HIV-negative and apparently uninfected. Our data supports the possibility of HIV exposure without sustained infection and the existence of HIV-natural resistance in some individuals

Enhancing Grant-Writing Expertise in BUILD Institutions: Building Infrastructure Leading to Diversity

Background The lack of race/ethnic and gender diversity in grants funded by the National Institutes of Health (NIH) is a persistent challenge related to career advancement and the quality and relevance of health research. We describe pilot programs at nine institutions supported by the NIH-sponsored Building Infrastructure Leading to Diversity (BUILD) program aimed at increasing diversity in biomedical research. Methods We collected data from the 2016–2017 Higher Education Research Institute survey of faculty and NIH progress reports for the first four years of the program (2015–2018). We then conducted descriptive analyses of data from the nine BUILD institutions that had collected data and evaluated which activities were associated with research productivity. We used Poisson regression and rate ratios of the numbers of BUILD pilots funded, students included, abstracts, presentations, publications, and submitted and funded grant proposals. Results Teaching workshops were associated with more abstracts (RR 4.04, 95% CI 2.21–8.09). Workshops on grant writing were associated with more publications (RR 2.64, 95% CI 1.64–4.34) and marginally with marginally more presentations. Incentives to develop courses were associated with more abstracts published (RR 4.33, 95% CI 2.56–7.75). Workshops on research skills and other incentives were not associated with any positive effects. Conclusions Pilot interventions show promise in supporting diversity in NIH-level research. Longitudinal modeling that considers time lags in career development in moving from project development to grants submissions can provide more direction for future diversity pilot interventions

Rapidly Self-Renewing Human Multipotent Marrow Stromal Cells (hMSC) Express Sialyl Lewis X and Actively Adhere to Arterial Endothelium in a Chick Embryo Model System

There have been conflicting observations regarding the receptors utilized by human multipotent mesenchymal bone marrow stromal cells (hMSC) to adhere to endothelial cells (EC). To address the discrepancies, we performed experiments with cells prepared with a standardized, low-density protocol preserving a sub-population of small cells that are rapidly self-renewing.Sialyl Lewis X (SLeX) and α4 integrin expression were determined by flow cytometry. Fucosyltransferase expression was determined by quantitative realtime RT-PCR. Cell adhesion assays were carried out with a panel of endothelial cells from arteries, veins and the microvasculature in vitro. In vivo experiments were performed to determine single cell interactions in the chick embryo chorioallantoic membrane (CAM). The CAM is a well-characterized respiratory organ allowing for time-lapse image acquisition of large numbers of cells treated with blocking antibodies against adhesion molecules expressed on hMSC.hMSC expressed α4 integrin, SLeX and fucosyltransferase 4 and adhered to human EC from arteries, veins and the microvasculature under static conditions in vitro. In vivo, hMSC rolled on and adhered to arterioles in the chick embryo CAM, whereas control melanoma cells embolized. Inhibition of α4 integrin and/or SLeX with blocking antibodies reduced rolling and adhesion in arterioles and increased embolism of hMSC.The results demonstrated that rapidly self-renewing hMSC were retained in the CAM because they rolled on and adhered to respiratory arteriolar EC in an α4 integrin- and SLeX-dependent manner. It is therefore important to select cells based on their cell adhesion receptor profile as well as size depending on the intended target of the cell and the injection route

Rapidly Self-Renewing Human Multipotent Marrow Stromal Cells (hMSC) Express Sialyl Lewis X and Actively Adhere to Arterial Endothelium in a Chick Embryo Model System

There have been conflicting observations regarding the receptors utilized by human multipotent mesenchymal bone marrow stromal cells (hMSC) to adhere to endothelial cells (EC). To address the discrepancies, we performed experiments with cells prepared with a standardized, low-density protocol preserving a sub-population of small cells that are rapidly self-renewing.Sialyl Lewis X (SLeX) and α4 integrin expression were determined by flow cytometry. Fucosyltransferase expression was determined by quantitative realtime RT-PCR. Cell adhesion assays were carried out with a panel of endothelial cells from arteries, veins and the microvasculature in vitro. In vivo experiments were performed to determine single cell interactions in the chick embryo chorioallantoic membrane (CAM). The CAM is a well-characterized respiratory organ allowing for time-lapse image acquisition of large numbers of cells treated with blocking antibodies against adhesion molecules expressed on hMSC.hMSC expressed α4 integrin, SLeX and fucosyltransferase 4 and adhered to human EC from arteries, veins and the microvasculature under static conditions in vitro. In vivo, hMSC rolled on and adhered to arterioles in the chick embryo CAM, whereas control melanoma cells embolized. Inhibition of α4 integrin and/or SLeX with blocking antibodies reduced rolling and adhesion in arterioles and increased embolism of hMSC.The results demonstrated that rapidly self-renewing hMSC were retained in the CAM because they rolled on and adhered to respiratory arteriolar EC in an α4 integrin- and SLeX-dependent manner. It is therefore important to select cells based on their cell adhesion receptor profile as well as size depending on the intended target of the cell and the injection route

A Novel Peptide Derived from Human Apolipoprotein E Is an Inhibitor of Tumor Growth and Ocular Angiogenesis

Angiogenesis is a hallmark of tumor development and metastasis and now a validated target for cancer treatment. We previously reported that a novel dimer peptide (apoEdp) derived from the receptor binding region of human apolipoprotein E (apoE) inhibits virus-induced angiogenesis. However, its role in tumor anti-angiogenesis is unknown. This study demonstrates that apoEdp has anti-angiogenic property in vivo through reduction of tumor growth in a mouse model and ocular angiogenesis in a rabbit eye model. Our in vitro studies show that apoEdp inhibits human umbilical vein endothelial cell proliferation, migration, invasion and capillary tube formation. We document that apoEdp inhibits vascular endothelial growth factor-induced Flk-1 activation as well as downstream signaling pathways that involve c-Src, Akt, eNOS, FAK, and ERK1/2. These in vitro data suggest potential sites of the apoE dipeptide inhibition that could occur in vivo

Lytic HSV-1 infection induces the multifunctional transcription factor Early Growth Response-1 (EGR-1) in rabbit corneal cells

<p>Abstract</p> <p>Background</p> <p>Herpes simplex virus type-1 (HSV-1) infections can cause a number of diseases ranging from simple cold sores to dangerous keratitis and lethal encephalitis. The interaction between virus and host cells, critical for viral replication, is being extensively investigated by many laboratories. In this study, we tested the hypothesis that HSV-1 lytic infection triggers the expression of important multi-functional transcription factor Egr1. The mechanisms of induction are mediated, at least in part, by signaling pathways such as NFκB and CREB.</p> <p>Methods</p> <p>SIRC, VERO, and 293HEK cell lines were infected with HSV-1, and the Egr-1 transcript and protein were detected by RT-PCR and Western blot, respectively. The localization and expression profile of Egr-1 were investigated further by immunofluorescence microscopy analyses. The recruitment of transcription factors to the Egr-1 promoter during infection was studied by chromatin immunoprecipitation (ChIP). Various inhibitors and dominant-negative mutant were used to assess the mechanisms of Egr-1 induction and their effects were addressed by immunofluorescence microscopy.</p> <p>Results</p> <p>Western blot analyses showed that Egr-1 was absent in uninfected cells; however, the protein was detected 24-72 hours post treatment, and the response was directly proportional to the titer of the virus used for infection. Using recombinant HSV-1 expressing EGFP, Egr-1 was detected only in the infected cells. ChIP assays demonstrated that NFкB and cAMP response element binding protein (CREB) were recruited to the Egr-1 promoter upon infection. Additional studies showed that inhibitors of NFкB and dominant-negative CREB repressed the Egr-1 induction by HSV-1 infection.</p> <p>Conclusion</p> <p>Collectively, these results demonstrate that Egr-1 is expressed rapidly upon HSV-1 infection and that this novel induction could be due to the NFкB/CREB-mediated transactivation. Egr-1 induction might play a key role in the viral gene expression, replication, inflammation, and the disease progression.</p

Design, Synthesis, and Biological Evaluation of Novel Pyridine-Bridged Analogues of Combretastatin-A4 as Anticancer Agents.

A series of novel pyridine-bridged analogues of combretastatin-A4 (CA-4) were designed and synthesized. As expected, the 4-atom linker configuration retained little cytotoxicities in the compounds 2e, 3e, 3g, and 4i. Activities of the analogues with 3-atom linker varied widely depending on the phenyl ring substitutions, and the 3-atom linker containing nitrogen represents the more favorable linker structure. Among them, three analogues (4h, 4s, and 4t) potently inhibited cell survival and growth, arrested cell cycle, and blocked angiogenesis and vasculature formation in vivo in ways comparable to CA-4. The superposition of 4h and 4s in the colchicine-binding pocket of tubulin shows the binding posture of CA-4, 4h, and 4s are similar, as confirmed by the competitive binding assay where the ability of the ligands to replace tubulin-bound colchicine was measured. The binding data are consistent with the observed biological activities in antiproliferation and suppression of angiogenesis but are not predictive of their antitubulin polymerization activities

Metformin Inhibits Migration and Invasion by Suppressing ROS Production and COX2 Expression in MDA-MB-231 Breast Cancer Cells

Background: Several mechanisms of action have been proposed to explain the apparent antineoplastic functions of metformin, many of which are observed at high concentrations that may not be reflective of achievable tissue concentrations. We propose that metformin at low concentrations functions to inhibit ROS production and inflammatory signaling in breast cancer, thereby reducing metastasis. Methods: Using the highly invasive MDA-MB-231 breast carcinoma model, we ascertained the impact of metformin on cell viability by DNA content analysis and fluorescent dye exclusion. Migration and invasion assays were performed using a modified Boyden chamber assay and metastasis was ascertained using the chorioallantoic membrane (CAM) assay. PGE2 production was measured by Enzyme-Linked Immunosorbent Assay (ELISA). COX2 and ICAM1 levels were determined by flow cytometry immunoassay. Results: Metformin acutely decreased cell viability and caused G2 cell cycle arrest only at high concentrations (10 mM). At 100 &#181;M, however, metformin reduced ICAM1 and COX2 expression, as well as reduced PGE2 production and endogenous mitochondrial ROS production while failing to significantly impact cell viability. Consequently, metformin inhibited migration, invasion in vitro and PGE2-dependent metastasis in CAM assays. Conclusion: At pharmacologically achievable concentrations, metformin does not drastically impact cell viability, but inhibits inflammatory signaling and metastatic progression in breast cancer cells

Obesity-Associated Dysregulation of Calpastatin and MMP-15 in Adipose-Derived Stromal Cells Results in Their Enhanced Invasion

Adipose tissue maintains a subpopulation of cells, referred to as adipose-derived stromal/stem cells (ASCs), which have been associated with increased breast cancer tumorigenesis and metastasis. For ASCs to affect breast cancer cells, it is necessary to delineate how they mobilize and home to cancer cells, which requires mobilization and invasion through extracellular matrix barriers. In this study, ASCs were separated into four different categories based on the donor\u27s obesity status and depot site of origin. ASCs isolated from the subcutaneous abdominal adipose tissue of obese patients (Ob +Ab+) demonstrated increased invasion through Matrigel as well as a chick chorioallantoic membrane, a type I collagen-rich extracellular matrix barrier. Detailed mRNA and protein analyses revealed that calpain-4, calpastatin, and MMP-15 were associated with increased invasion, and the silencing of each protease or protease inhibitor confirmed their role in ASC invasion. Thus, the data indicate that both the donor\u27s obesity status and depot site of origin distinguishes the properties of subcutaneous-derived ASCs with respect to enhanced invasion and this is associated with the dysregulation of calpain-4, calpastatin, and MMP-15

Distribution of hMSC to arteries/arterioles, veins and capillaries/end arterioles in the CAM.

<p><b>A.</b> Distribution of hMSC compared to lymphocytes and effects of pre-treatment with anti-SLeX and/or anti-α4 integrin (n = 5). <b>B</b>. Distribution in arteries of hMSC from 5 preparations from 5 different donors of marrow repeated 5 times.</p