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Medicine and Research Services

Baltimore Highlands, MD, United States
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Kaplan D.E.,Medicine and Research Services | Kaplan D.E.,University of Pennsylvania
Gastroenterology Clinics of North America | Year: 2015

Despite advances in therapy, hepatitis C virus infection remains a major global health issue with 3 to 4 million incident cases and 170 million prevalent chronic infections. Complex, partially understood, host-virus interactions determine whether an acute infection with hepatitis C resolves, as occurs in approximately 30% of cases, or generates a persistent hepatic infection, as occurs in the remainder. Once chronic infection is established, the velocity of hepatocyte injury and resultant fibrosis is significantly modulated by immunologic as well as environmental factors. Immunomodulation has been the backbone of antiviral therapy despite poor understanding of its mechanism of action. © 2015.

Adeyemo O.,University of Pennsylvania | Doi H.,University of Pennsylvania | Rajender Reddy K.,University of Pennsylvania | Kaplan D.E.,University of Pennsylvania | Kaplan D.E.,Medicine and Research Services
Journal of Viral Hepatitis | Year: 2013

Silymarin displays anti-inflammatory effects on T lymphocytes in vitro. The immunomodulatory properties of oral silymarin in vivo in humans with chronic hepatitis C have not previously been characterized. We hypothesized that silymarin would suppress T-cell proliferation and pro-inflammatory cytokine production of virus- and non-virus-specific T cells while increasing anti-inflammatory IL-10 production in vivo. Patients from one site of the SyNCH-HCV double-masked, placebo-controlled study of oral silymarin in prior interferon nonresponders with chronic hepatitis C provided blood samples at baseline and treatment week 20. Mononuclear cells were stimulated with recombinant HCV proteins and controls in 3H-thymidine proliferation assays, IFNγ ELISPOT and IL-10 ELISPOT. The frequency of CD4 +CD25hi and CD4+foxp3+ regulatory T cells, serum cytokine levels, serum IP-10 and lymphocyte interferon-stimulated gene expression were also quantified at baseline and week 20. Thirty-two patients were recruited (10; placebo, 11; 420 mg three times a day, 11; 700 mg three times a day). Serum ALT and HCV RNA titres did not change in any group. HCV-specific CD4+ T-cell proliferation and the frequency of IFNγ- and IL-10-producing T cells were not significantly changed in silymarin-treated subjects. However, C. albicans-induced T-cell IFNγ and phytohaemagglutinin-induced T-cell proliferation were suppressed by silymarin therapy. A trend towards augmentation of interferon-induced ISG15 expression was present in the high-dose silymarin group. While no effect on HCV-specific T cells was identified, these data confirm that high-dose oral silymarin exerts modest nonspecific immunomodulatory effects in vivo. The impact of this anti-inflammatory effect on long-term liver health in chronic hepatitis C merits future clinical investigation. © 2013 John Wiley & Sons Ltd.

Doi H.,University of Pennsylvania | Iyer T.K.,University of Pennsylvania | Carpenter E.,University of Pennsylvania | Li H.,University of Pennsylvania | And 5 more authors.
Hepatology | Year: 2012

Chronic hepatitis C virus (HCV) infection is a leading cause of cirrhosis and hepatocellular carcinoma (HCC). Both advanced solid tumors and HCV have previously been associated with memory B-cell dysfunction. In this study, we sought to dissect the effect of viral infection, cirrhosis, and liver cancer on memory B-cell frequency and function in the spectrum of HCV disease. Peripheral blood from healthy donors, HCV-infected patients with F1-F2 liver fibrosis, HCV-infected patients with cirrhosis, patients with HCV-related HCC, and non-HCV-infected cirrhotics were assessed for B-cell phenotype by flow cytometry. Isolated B cells were stimulated with anti-cluster of differentiation (CD)40 antibodies and Toll-like receptor (TLR)9 agonist for assessment of costimulation marker expression, cytokine production, immunoglobulin (Ig) production, and CD4 + T-cell allostimulatory capacity. CD27 + memory B cells and, more specifically, CD27 +IgM + B cells were markedly less frequent in cirrhotic patients independent of HCV infection. Circulating B cells in cirrhotics were hyporesponsive to CD40/TLR9 activation, as characterized by CD70 up-regulation, tumor necrosis factor beta secretion, IgG production, and T-cell allostimulation. Last, blockade of TLR4 and TLR9 signaling abrogated the activation of healthy donor B cells by cirrhotic plasma, suggesting a role for bacterial translocation in driving B-cell changes in cirrhosis. Conclusion: Profound abnormalities in B-cell phenotype and function occur in cirrhosis independent of HCV infection. These B-cell defects may explain, in part, the vaccine hyporesponsiveness and susceptibility to bacterial infection in this population. (HEPATOLOGY 2012) © 2011 American Association for the Study of Liver Diseases.

Xu Y.,University of Pennsylvania | Li H.,University of Pennsylvania | Gao R.L.,University of Pennsylvania | Adeyemo O.,University of Pennsylvania | And 3 more authors.
Clinical Immunology | Year: 2011

Glypican-3 is a promising target for immunotherapy for hepatocellular carcinoma, but limited data exist regarding its immunogenicity in patients with diverse HLA types, immunogenicity for CD4+ T-cells, and the impact of inhibitory co-stimulation on glypican-3-specific T-cells. Using a 15mer overlapping peptide library for glypican-3, PBMC from patients with HCC were assessed ex vivo and after short-term in vitro expansion for tumor antigen-specific T-cell responses with and without blockade of PD-1/PD-L1 and CTLA-4 signaling. Glypican-3-specific T-cells were undetectable ex vivo, but primarily IFNγ+TNFα+ CD4+ T-cells expanded with short-term in vitro stimulation in 10/19 (52%) patients. Glypican-3-specific CD8+ T-cells predominantly produced TNFα, but did not secrete IFNγ nor degranulate. CTLA-4 and PD-1 blockade minimally impacted the cytokine secretion and proliferation of glypican-3-specific T-cells. These data suggest that CD8+ T-cell-directed tumor vaccines in HCC may have limited potential for efficacy unless optimal co-stimulation conditions can be identified but CD4+-directed vaccines merit consideration. © 2011.

Doi H.,University of Pennsylvania | Tanoue S.,University of Pennsylvania | Kaplan D.E.,Medicine and Research Services | Kaplan D.E.,University of Pennsylvania
Clinical Immunology | Year: 2014

Hepatitis C cirrhosis is associated with a profound disappearance of memory B-cells. We sought to determine if this loss is associated with the expansion of the CD27-CD21- tissue-like memory B-cells with features of B-cell exhaustion. To this end, we quantified the frequency of CD27-CD21- B-cells in healthy, non-cirrhotic HCV-infected, and cirrhotic patients. We examined the expression of putative inhibitory receptors, the proliferative and immunoglobulin-secreting capacity of CD27/CD21-defined B-cell subsets upon B-cell receptor and/or CD40 stimulation. We found that CD27-CD21- B-cells are significantly increased in frequency relative to healthy donors in HCV-infected patients. CD27-CD21- B-cells were hypoproliferative relative to naïve and resting memory B-cells upon agonistic stimulation, but retained similar capacity for antibody secretion. Conclusion: CD27-CD21- tissue-like memory B-cells with exhausted proliferation circulate at increased frequency in cirrhotic and non-cirrhotic HCV-infected patients. This B-cell subset does not appear anergic, exhibiting immunoglobulin-secreting capacity on CD40 agonism indistinguishable from other CD27/CD21-defined B-cell subsets. © 2013.

Li Y.,University of Pennsylvania | Siegel D.L.,University of Pennsylvania | Scholler N.,University of Pennsylvania | Kaplan D.E.,Medicine and Research Services | Kaplan D.E.,University of Pennsylvania
BMC Biotechnology | Year: 2012

Background: Glypican-3 (GPC3) is a heparan-sulfate proteoglycan frequently expressed on the cell membrane of malignant hepatocytes in hepatocellular carcinoma. The capacity for screening potential antibodies in vitro using human hepatocellular lines is critical to ensure binding to this highly post-translationally modified glycophosphatidylinositiol-linked protein. We hypothesized that we could utilize a recently described paired display/secretory yeast library to isolate human-derived scFv against glypican-3 for potential diagnostic and/or therapeutic application.Results: Using two different biotinylated antigen targets, a synthesized 29mer fragment GPC3 550-558 and a truncated GPC3 368-548 fused with glutathione S-transferase (GST) we enriched the yeast display library to greater than 30% target-specific yeast with both positive selection and depletion of streptavidin- and GST-specific clones. After cloning of scFv cDNA from the enriched sub-library, scFv specificity was validated by ELISA for binding to recombinant protein from prokaryotic and eukaryotic sources and ultimately naturally presented human protein on the cell membrane of human hepatocellular cell lines. Specificity was confirmed using non-expressing cell lines and shRNA knockdown. Ultimately, five unique scFv with affinity EC 50 ranging from 5.0-110.9nM were identified.Conclusions: Using a paired display/secretory yeast library, five novel and unique scFvs for potential humoral or chimeric therapeutic development in human hepatocellular carcinoma were isolated and characterized. © 2012 Li et al; licensee BioMed Central Ltd.

Watkins R.R.,Cleveland Clinic | Watkins R.R.,Northeast Ohio Medical University | Bonomo R.A.,Medicine and Research Services | Bonomo R.A.,Case Western Reserve University
Infectious Disease Clinics of North America | Year: 2016

The rapid and ongoing spread of antibiotic resistance poses a serious threat to global public health. The indiscriminant use of antibiotics in agriculture and human medicine along with increasingly connected societies has fueled the distribution of antibiotic-resistant bacteria. These factors together have led to rising numbers of infections caused by multidrug-resistant and pan-resistant bacteria, with increases in morbidity and mortality. This article summarizes the trends in antibiotic resistance, discusses the impact of antibiotic resistance on society, and reviews the use of antibiotics in agriculture. Feasible ways to tackle antibiotic resistance to avert a post-antibiotic era are suggested. © 2016 Elsevier Inc.

Lee T.Y.,University of Maryland Baltimore County | Ezelle H.J.,University of Maryland Baltimore County | Venkataraman T.,University of Maryland Baltimore County | Lapidus R.G.,University of Maryland Baltimore County | And 3 more authors.
Journal of Interferon and Cytokine Research | Year: 2013

The endoribonuclease RNase-L is the terminal component of an interferon-regulated RNA decay pathway known as the 2′-5′- oligoadenylate (2-5A) system, whose established functions include antimicrobial and tumor suppressive activities. RNase-L activity requires binding of the small molecule 2-5A, leading to RNase-L dimerization and cleavage of single-stranded RNA. RNase-L expression is controlled post-transcriptionally by its 3′-untranslated region (3′ UTR), which exerts a strong negative effect on RNase-L levels. MicroRNAs (miRNAs) are a class of small noncoding RNAs that repress expression of target genes by binding to regions of complementarity often in the 3′ UTR. The miR-29 family acts as a tumor suppressor in several cancers, including acute and chronic myelogenous leukemia (CML), and has many oncogenic targets. We report that the miR-29 family represses RNase-L protein expression across several cell types. Using a luciferase reporter, we showed that miR-29 acts via 4 target sites within the RNASEL 3′ UTR. Mutation of all sites is required for abrogation of miR-29 repression. In light of the reported tumor suppressive role of miR-29 in K562 CML cells and miR-29 repression of RNase-L in these cells, we generated K562 cells with stable RNase-L knockdown and demonstrated that loss of RNase-L inhibits proliferation in vitro as well as tumor growth in a xenograft model. Our findings identify a previously unknown miRNA regulator of RNase-L expression and support a novel oncogenic role for RNase-L in CML and potentially other hematopoietic malignancies. © Copyright 2013, Mary Ann Liebert, Inc.

Potla R.,University of Maryland Baltimore County | Singh I.S.,University of Maryland Baltimore County | Singh I.S.,Medicine and Research Services | Atamas S.P.,Medicine and Research Services | And 3 more authors.
RNA | Year: 2015

Previous studies have revealed that clinically relevant changes in temperature modify clinically relevant gene expression profiles through transcriptional regulation. Temperature dependence of post-transcriptional regulation, specifically, through expression of miRNAs has been less studied. We comprehensively analyzed the effect of 24 h exposure to 32°C or 39.5°C on miRNA expression profile in primary cultured human small airway epithelial cells (hSAECs) and its impact on expression of a targeted protein, protein kinase C α (PKCα). Using microarray, and solution hybridization-based nCounter assays, with confirmation by quantitative RT-PCR, we found significant temperature-dependent changes in expression level of only five mature human miRNAs, representing only 1% of detected miRNAs. Four of these five miRNAs are the less abundant passenger (star) strands. They exhibited a similar pattern of increased expression at 32°C and reduced expression at 39.5°C relative to 37°C. As PKCα mRNA has multiple potential binding sites for three of these miRNAs, we analyzed PKCα protein expression in HEK 293T cells and hSAECs. PKCα protein levels were lowest at 32°C and highest at 39.5°C and specific miRNA inhibitors reduced these effects. Finally, we analyzed cell-cycle progression in hSAECs and found 32°C cells exhibited the greatest G1 to S transition, a process known to be inhibited by PKCα, and the effect was mitigated by specific miRNA inhibitors. These results demonstrate that exposure to clinically relevant hypothermia or hyperthermia modifies expression of a narrow subset of miRNAs and impacts expression of at least one signaling protein involved in multiple important cellular processes. © 2015 Potla et al.

Nagarsekar A.,University of Maryland Baltimore County | Tulapurkar M.E.,University of Maryland Baltimore County | Singh I.S.,University of Maryland Baltimore County | Singh I.S.,Medicine and Research Services | And 5 more authors.
American Journal of Respiratory Cell and Molecular Biology | Year: 2012

Hyperthermia has been shown to confer cytoprotection and to augment apoptosis in different experimental models. We analyzed the mechanisms of both effects in the same mouse lung epithelial (MLE) cell line (MLE15). Exposing MLE15 cells to heat shock (HS; 42°C, 2 h) or febrile-range hyperthermia (39.5°C) concurrent with activation of the death receptors, TNF receptor 1 or Fas, greatly accelerated apoptosis, which was detectable within 30 minutes and was associated with accelerated activation of caspase-2, -8, and -10, and the proapoptotic protein, Bcl2-interacting domain (Bid). Caspase-3 activation and cell death were partially blocked by inhibitors targeting all three initiator caspases. Cells expressing the IκB superrepessor were more susceptible than wild-type cells to TNFα-induced apoptosis at 37°C, but HS and febrile-range hyperthermia still increased apoptosis in these cells. Delaying HS for 3 hours after TNF-α treatment abrogated its proapoptotic effect in wild-type cells, but not in IκB superrepressor-expression cells, suggesting that TNF-α stimulates delayed resistance to the proapoptotic effects of HS through an NF-κB-dependent mechanism. Pre-exposure to 2-hour HS beginning 6 to 16 hours before TNF-α treatment or Fas activation reduced apoptosis in MLE15 cells. The antiapoptotic effects of HS pretreatment were reduced in TNF-α-treated embryonic fibroblasts from heatshock factor- 1 (HSF1)-deficient mice, but the proapoptotic effects of concurrent HS were preserved. Thus, depending on the temperature and timing relative to death receptor activation, hyperthermia can exert pro- and antiapoptotic effects through distinct mechanisms. Copyright © 2012 by the American Thoracic Society.

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