Dharma R. Thapa

Differential regulation of beta-defensin expression in human skin by microbial stimuli

In response to infection, epithelia mount an innate immune response that includes the production of antimicrobial peptides. However, the pathways that connect infection and inflammation with the induction of antimicrobial peptides in epithelia are not understood. We analyzed the molecular links between infection and the expression of three antimicrobial peptides of the β-defensin family, human β-defensin (hBD)-1, hBD-2, and hBD-3 in the human epidermis. After exposure to microbe-derived molecules, both monocytes and lymphocytes stimulated the epidermal expression of hBD-1, hBD-2, and hBD-3. The induced expression of hBD-3 was mediated by transactivation of the epidermal growth factor receptor. The mechanisms of induction of hBD-1 and hBD-3 were distinct from each other and from the IL-1-dependent induction of hBD-2 expression. Thus during inflammation, epidermal expression of β-defensins is mediated by at least three different mechanisms.

Urinary hepcidin in congenital chronic anemias.

Hepcidin, a regulator for iron homeostasis, is induced by inflammation and iron burden and suppressed by anemia and hypoxia. This study was conducted to determine the hepcidin levels in patients with congenital chronic anemias.

Injury-induced innate immune response in human skin mediated by transactivation of the epidermal growth factor receptor

We found that sterile wounding of human skin induced epidermal expression of the antimicrobial (poly)peptides human beta-defensin-3, neutrophil gelatinase-associated lipocalin, and secretory leukocyte protease inhibitor through activation of the epidermal growth factor receptor. After skin wounding, the receptor was activated by heparin-binding epidermal growth factor that was released by a metalloprotease-dependent mechanism. Activation of the epidermal growth factor receptor generated antimicrobial concentrations of human beta-defensin-3 and increased the activity of organotypic epidermal cultures against Staphylococcus aureus. These data demonstrate that sterile wounding initiates an innate immune response that increases resistance to overt infection and microbial colonization.

CD40 Ligand (CD154) Incorporated into HIV Virions Induces Activation-Induced Cytidine Deaminase (AID) Expression in Human B Lymphocytes

Most AIDS-associated non-Hodgkins lymphoma (AIDS-NHL) arises from errors in immunoglobulin heavy-chain gene (IgH) class switch recombination (CSR) or somatic hypermutation (SHM), events that occur in germinal center (GC) B cells and require the activity of activation induced cytidine deaminase (AID). Several oncogenic viruses (EBV, HCV, HPV) can induce AID gene (AID) expression, and elevated AID expression is seen in circulating lymphocytes prior to AIDS-NHL diagnosis. Here, we report that HIV produced in peripheral blood mononuclear cells (PBMC) induced AID expression in normal human B cells. Since HIV produced in PBMC contains host cell CD40 ligand (CD40L) incorporated into the viral membrane, and CD40L is known to induce AID expression in human B cells, the role of virion-associated CD40L in HIV-induced AID expression was examined. Only viruses expressing functional CD40L were seen to induce AID expression; CD40L-negative HIV did not induce AID expression. The induction of AID expression by CD40L+ HIV was abrogated by addition of blocking anti-CD40L antibody. AID protein was detected in B cells exposed to CD40L+ HIV using intracellular multicolor flow cytometry, with most AID producing B cells expressing the CD71 activation marker on their surface. Therefore, HIV virions that express CD40L induce AID expression in B cells, and this induction appears to be due to a direct interaction between CD40L on these viruses and CD40 on B cells. These findings are consistent with a role for HIV in the direct stimulation of B cells, potentially leading to the accumulation of molecular lesions that have the potential to contribute to the development of NHL.

Decreased clearance of Pseudomonas aeruginosa from airways of mice deficient in lysozyme M

Lysozyme is a ubiquitous and abundant, cationic, antimicrobial polypeptide of leukocytes and epithelia, but its biological function in host defense is largely unexplored. To ascertain the role of lysozyme during bacterial infection of murine airways, we exposed the airways of lysozyme M‐deficient (lys M−/−) mice to the pulmonary pathogen Pseudomonas aeruginosa and examined the host’s response to infection. Despite partial compensation as a result of the appearance of lysozyme P in the infected airways of lys M−/− mice, these lys M−/− mice showed decreased clearance of P. aeruginosa compared with their lys M+/− or lys M+/+ littermates. Lysozyme contributes to optimal clearance of P. aeruginosa from the murine airways.

Overexpression of microRNAs from the miR-17-92 paralog clusters in AIDS-related non-Hodgkin's lymphomas.

Background Individuals infected by HIV are at an increased risk for developing non-Hodgkins lymphomas (AIDS-NHL). In the highly active antiretroviral therapy (HAART) era, there has been a significant decline in the incidence of AIDS-associated primary central nervous system lymphoma (PCNSL). However, only a modest decrease in incidence has been reported for other AIDS-NHL subtypes. Thus, AIDS-NHLs remain a significant cause of morbidity and mortality in HIV infected individuals. Recently, much attention has been directed toward the role of miRNAs in cancer, including NHL. Several miRNAs, including those encoded by the miR-17-92 polycistron, have been shown to play significant roles in B cell tumorigenesis. However, the role of miRNAs in NHL in the setting of HIV infection has not been defined. Methodology/Principal Findings We used quantitative realtime PCR to assess the expression of miRNAs from three different paralog clusters, miR-17-92, miR-106a-363, and miR-106b-25 in 24 cases of AIDS-NHLs representing four tumor types, Burkitts lymphoma (BL, n = 6), diffuse large B-cell lymphoma (DLBCL, n = 8), primary central nervous system lymphoma (PCNSL, n = 5), and primary effusion lymphoma (PEL, n = 5). We also used microarray analysis to identify a differentiation specific miRNA signature of naïve, germinal center, and memory B cell subsets from tonsils (n = 4). miRNAs from the miR-17-92 paralog clusters were upregulated by B cells, specifically during the GC differentiation stage. We also found overexpression of these miRNA clusters in all four AIDS-NHL subtypes. Finally, we also show that select miRNAs from these clusters (miR-17, miR-106a, and miR-106b) inhibited p21 in AIDS-BL and DLBCL cases, thus providing a mechanistic role for these miRNAs in AIDS-NHL pathogenesis. Conclusion Dysregulation of miR-17-92 paralog clusters is a common feature of AIDS-associated NHLs.

B-cell activation induced microRNA-21 is elevated in circulating B cells preceding the diagnosis of AIDS-related non-Hodgkin lymphomas.

We show that microRNA-21 is significantly elevated in peripheral B cells of HIV-infected individuals who go on to develop AIDS-related non-Hodgkin lymphoma (n = 13, <3 years prior to diagnosis) when compared with HIV-negative (n = 18) or HIV-positive controls (n = 21) (P < 0.01). Moreover, miR-21 is overexpressed in activated B cells and can be induced by interleukin 4 alone, or with CD40 or immunoglobulin M co-stimulation, and lipopolysaccharides, suggesting that miR-21 may help maintain B-cell hyperactivation, contributing to lymphomagenesis.

Serum microRNAs in HIV-infected individuals as pre-diagnosis biomarkers for AIDS-NHL.

Objective:To determine if changes in levels of serum microRNAs (miRNAs) were seen preceding the diagnosis of AIDS-related non-Hodgkin lymphoma (AIDS-NHL). Design:Serum miRNA levels were compared in 3 subject groups from the Multicenter AIDS Cohort Study: HIV-negative men (n = 43), HIV-positive men who did not develop NHL (n = 45), and HIV-positive men before AIDS-NHL diagnosis (n = 62, median time before diagnosis, 8.8 months). Methods:A total of 175 serum-enriched miRNAs were initially screened to identify differentially expressed miRNAs among these groups and the results validated by quantitative polymerase chain reaction. Receiver-operating characteristic analysis was then performed to assess biomarker utility. Results:Higher levels of miR-21 and miR-122, and a lower level of miR-223, were able to discriminate HIV-infected from the HIV-uninfected groups, suggesting that these miRNAs are biomarkers for HIV infection but are not AIDS-NHL specific. Among the HIV-infected groups, a higher level of miR-222 was able to discriminate diffuse large B-cell lymphoma (DLBCL) and primary central nervous system lymphoma (PCNSL) subjects from HIV-infected subjects who did not develop NHL, with area under the receiver-operating characteristic curve of 0.777 and 0.792, respectively. At miR-222 cutoff values of 0.105 for DLBCL and 0.109 for PCNSL, the sensitivity and specificity were 75% and 77%, and 80% and 82%, respectively. Conclusions:Altered serum levels of miR-21, miR-122, and miR-223 are seen in HIV-infected individuals. Higher serum level of miR-222 has clear potential as a serum biomarker for earlier detection of DLBCL and PCNSL among HIV-infected individuals.

MicroRNA-related polymorphisms and non-Hodgkin lymphoma susceptibility in the Multicenter AIDS Cohort Study.

BACKGROUND MicroRNAs, small non-coding RNAs involved in gene regulation, are implicated in lymphomagenesis. We evaluated whether genetic variations in microRNA coding regions, binding sites, or biogenesis genes (collectively referred to as miRNA-SNPs) were associated with risk of AIDS-associated non-Hodgkin lymphoma (AIDS-NHL), and serum levels of four lymphoma-related microRNAs. METHODS Twenty-five miRNA-SNPs were genotyped in 180 AIDS-NHL cases and 529 HIV-infected matched controls from the Multicenter AIDS Cohort Study (MACS), and real-time polymerase chain reaction was used to quantify serum microRNA levels. Adjusted odds ratios (ORs) estimated using conditional logistic regression evaluated associations between miRNA-SNPs and AIDS-NHL risk. A semi-Bayes shrinkage approach was employed to reduce likelihood of false-positive associations. Adjusted mean ratios (MR) calculated using linear regression assessed associations between miRNA-SNPs and serum microRNA levels. RESULTS DDX20 rs197412, a non-synonymous miRNA biogenesis gene SNP, was associated with AIDS-NHL risk (OR=1.34 per minor allele; 95% CI: 1.02-1.75), and higher miRNA-222 serum levels nearing statistical significance (MR=1.21 per minor allele; 95% CI: 0.98-1.49). MiRNA-196a2 rs11614913 was associated with decreased central nervous system (CNS) AIDS-NHL (CT vs. CC OR=0.52; 95% CI: 0.27-0.99). The minor allele of HIF1A rs2057482, which creates a miRNA-196a2 binding site, was associated with systemic AIDS-NHL risk (OR=1.73 per minor allele; 95% CI: 1.12-2.67), and decreased CNS AIDS-NHL risk (OR=0.49 per minor allele; 95% CI: 0.25-0.94). CONCLUSIONS This study suggests that a few miRNA-SNPs are associated with AIDS-NHL risk and may modulate miRNA expression. These results support a role for miRNA in AIDS-NHL and may highlight pathways to be targeted for risk stratification or therapeutics.

Abstract 1240: A flexible antibody-based strategy targeting CD71 for the treatment of AIDS-related non-Hodgkin's lymphoma.

AIDS related non-Hodgkin9s lymphoma (AIDS-NHL) remains a significant clinical problem in the era of effective multiple-agent highly active anti-retroviral therapy (HAART). While an overall decrease in the incidence of AIDS-NHL has been seen in the HAART era, the risk for AIDS-NHL remains elevated, and not all AIDS-NHL subtypes have decreased in incidence, with the incidence of Burkitt9s lymphoma (BL) remaining unchanged. In fact, AIDS-NHL is now the most common AIDS-related cancer in developed countries, where NHL accounts for 23-30% of all AIDS-related deaths. We have previously developed an antibody-avidin fusion protein (ch128.1Av) specific for the human transferrin receptor 1 (TfR1/CD71), a receptor that is overexpressed on cancer cells due to their high demand for iron. This fusion protein exhibits an increased in vitro cytotoxicity against malignant hematopoietic cells compared to the parental antibody without avidin (ch128.1). This cytotoxicity is due to the ability of ch128.1Av to decrease cell surface TfR1, triggering its intracellular sequestration and degradation, with the subsequent induction of lethal iron deprivation. However, both ch128.1Av and ch128.1 confer protection in murine models of human disseminated multiple myeloma. ch128.1Av is also a universal delivery system and its cytotoxicity can be further enhanced by its conjugation with biotinylated drugs making it a unique molecule capable of a two-pronged attack against malignant cells through drug delivery and direct cytotoxic activity through iron starvation. The main goal of this study is to evaluate the potential use of ch128.1Av alone and as a delivery vehicle as a possible AIDS-NHL therapy. We found that, when used alone, ch128.1Av exhibits significant cytotoxic activity against two representative AIDS-NHL cell lines: 2F7 (AIDS-Burkitt9s NHL) and RRBL (AIDS-diffuse large B-cell lymphoma). Importantly, the level of cytotoxicity was similar to that observed using the highly sensitive human B lymphoblastoid cell line IM-9. In addition, the cytotoxicity dramatically increases when this fusion protein is conjugated to biotinylated saporin 6 (b-SO6), a plant ribosome inactivating protein that blocks protein synthesis, resulting in an immunotoxin. We also found that non-activated (resting) B cells isolated from the peripheral blood of healthy individuals are resistant to ch128.1Av, but sensitive to ch128.1Av complexed to b-SO6, which can be explained by the different mechanisms of action of the fusion protein alone (iron starvation) and that of the immunotoxin (protein synthesis inhibition). We have previously reported ch128.1Av alone, or complexed to b-SO6, are not toxic to normal human hematopoietic stem cells, which can be explained by the lack of TfR1 expression on such cells. Our results suggest the potential use of anti-TfR1 antibody-mediated approaches as therapeutic interventions against AIDS-NHL. Citation Format: Tracy R. Daniels-Wells, Lai Sum Leoh, Daniel Widney, Dharma R. Thapa, Jose Leon Merino, Larry Magpantay, Otoniel Martinez-Maza, Manuel L. Penichet. A flexible antibody-based strategy targeting CD71 for the treatment of AIDS-related non-Hodgkin9s lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1240. doi:10.1158/1538-7445.AM2013-1240