KSHV Susceptibility and Transmission Within Tonsillar Specimens

Despite nearly three decades of research, not much is known regarding the early stages of development for KSHV lymphoproliferative disorders, hindering our ability to develop prophylactic measures or effective treatments. This dissertation will focus on the host and viral factors influencing the magnitude and dynamics of KSHV infection in the human tonsil to pave the way for future interventions directed at limiting person-to-person transmission of KSHV. To understand the contribution of host factors to KSHV susceptibility in B lymphocytes, we generated a library of 40 tonsillar specimens. Our results indicate that the immunological composition of tonsillar lymphocytes varies across our donor samples, and KSHV possess a diverse B lymphocyte tropism. Furthermore, the highly specific targeting of plasma cells is not due to CD138 (Syndecan-1) being used as an attachment factor, and heparan sulfates, in general, do not play an important role in KSHV infection of B cells. Finally, the donor-dependent immunological factors and immune status of individual samples influences the overall susceptibility, as well as specific targeting of B cell lineages. To understand the significance of viral gH/gL glycoprotein interaction with some of the key EphA family of receptors in the process of KSHV entry into B lymphocytes, we demonstrate that the expression of EphA2, EphA4, and EphA7 are donor and subset-specific and gH/gL-EphA interactions are important for KSHV-WT infection of primary tonsil lymphocytes. These interactions are critical for establishing KSHV-WT infection of plasma cells and germinal center cells, and KSHV exploits alternative mechanism of entry in absence of gH. Finally, we develop a model for cell-to-cell transmission of KSHV within human tonsils by examining KSHV spread within and between primary cell types derived from tonsil specimens, located at the proximity of the crypt lumen, which are in direct contact with external pathogens present in saliva. We show that a variety of primary tonsillar cells are susceptible to KSHV infection and can differentially transmit the infection into B cells. We demonstrate that KSHV spread between and within tonsil cell types is directed, suggesting, this tropism may be influenced by differential virion composition arising from each cell type

Molecular Virology of KSHV in the Lymphocyte Compartment—Insights From Patient Samples and \u3cem\u3eDe Novo\u3c/em\u3e Infection Models

The incidence of Kaposi’s sarcoma-associated herpesvirus (KSHV)-associated Kaposi Sarcoma has declined precipitously in the present era of effective HIV treatment. However, KSHV-associated lymphoproliferative disorders although rare, have not seen a similar decline. Lymphoma is now a leading cause of death in people living with HIV (PLWH), indicating that the immune reconstitution provided by antiretroviral therapy is not sufficient to fully correct the lymphomagenic immune dysregulation perpetrated by HIV infection. As such, novel insights into the mechanisms of KSHV-mediated pathogenesis in the immune compartment are urgently needed in order to develop novel therapeutics aimed at prevention and treatment of KSHV-associated lymphoproliferations. In this review, we will discuss our current understanding of KSHV molecular virology in the lymphocyte compartment, concentrating on studies which explore mechanisms unique to infection in B lymphocytes

Analysis of KSHV B Lymphocyte Lineage Tropism in Human Tonsil Reveals Efficient Infection of CD138+ Plasma Cells

Despite 25 years of research, the basic virology of Kaposi Sarcoma Herpesviruses (KSHV) in B lymphocytes remains poorly understood. This study seeks to fill critical gaps in our understanding by characterizing the B lymphocyte lineage-specific tropism of KSHV. Here, we use lymphocytes derived from 40 human tonsil specimens to determine the B lymphocyte lineages targeted by KSHV early during de novo infection in our ex vivo model system. We characterize the immunological diversity of our tonsil specimens and determine that overall susceptibility of tonsil lymphocytes to KSHV infection varies substantially between donors. We demonstrate that a variety of B lymphocyte subtypes are susceptible to KSHV infection and identify CD138+ plasma cells as a highly targeted cell type for de novo KSHV infection. We determine that infection of tonsil B cell lineages is primarily latent with few lineages contributing to lytic replication. We explore the use of CD138 and heparin sulfate proteoglycans as attachment factors for the infection of B lymphocytes and conclude that they do not play a substantial role. Finally, we determine that the host T cell microenvironment influences the course of de novo infection in B lymphocytes. These results improve our understanding of KSHV transmission and the biology of early KSHV infection in a naïve human host, and lay a foundation for further characterization of KSHV molecular virology in B lymphocyte lineages

Suppression of DC-SIGN and gH Reveals Complex, Subset-Specific Mechanisms for KSHV Entry in Primary B Lymphocytes

Kaposi sarcoma-associated herpesvirus (KSHV) is the causative agent of multiple cancers in immunocompromised patients including two lymphoproliferative disorders associated with KSHV infection of B lymphocytes. Despite many years of research into the pathogenesis of KSHV associated diseases, basic questions related to KSHV molecular virology remain unresolved. One such unresolved question is the cellular receptors and viral glycoproteins needed for KSHV entry into primary B lymphocytes. In this study, we assess the contributions of KSHV glycoprotein H (gH) and the cellular receptor DC-SIGN to KSHV infection in tonsil-derived B lymphocytes. Our results show that (1) neither KSHV-gH nor DC-SIGN are essential for entry into any B cell subset, (2) DC-SIGN does play a role in KSHV entry into tonsil-derived B cells, but in all B cell subtypes alternative entry mechanisms exist, (3) KSHV-gH can participate in KSHV entry into centrocytes via a DC-SIGN independent entry mechanism, and (4) in the absence of KSHV-gH, DC-SIGN is required for KSHV entry into centrocytes. Our results provide a first glimpse into the complexity of KSHV entry in the lymphocyte compartment and highlight that multiple subset-dependent entry mechanisms are employed by KSHV which depend upon multiple cellular receptors and multiple KSHV glycoproteins

Association of matrix metalloproteinase-1, 9, 12 and tissue inhibitor of metalloproteinase-1 gene polymorphisms in malay male essential hypertensive subject

Genetic polymorphisms are the modified sequences of the DNA and they serve as molecular biomarkers for the detection of the individual at risk of developing the disease. Essential hypertension (EH) are majority of hypertensive cases and diagnosed where there is no clear evidence of medical condition predisposing to the high BP. There have been variety of the genetic studies in relation to hypertension and some of them showed association with occurrence of hypertension. Family of the matrix metalloproteinases (MMP) belong to the large family of the zinc-dependent endopeptidases that are involved in many physiological disorders ranging from cancer to cardiovascular disorders. Matrix metalloproteinases are implicated in degradation of the extracellular matrix (ECM) which is fundamental in many aspects, both physiologically and pathologically. These include: normal functioning of the cells from development to growth and proliferation, as well as pathological conditions such as cardiac remodeling and cancer development. Matrix metalloproteinases play important role in hypertensive vascular stiffness, remodeling and dysfunction. They may be involved in the excessive degradation of ECM components, vascular smooth muscle cells migration and proliferation and intima layer invasion by monocytes. Besides,ECM remodeling is largely determined by the balance of MMPs with respect to tissue inhibitor of metalloproteinases (TIMP). Several studies have been reported the imbalanced MMP:TIMP-1 ratio in hypertensive subjects, indicating the depressed systematic degradation of collagenase in etiology of hypertension. The main objective of this study was to determine the candidate gene polymorphisms involved in ECM metabolism among Malaysian male subject with EH. Since, there have been variety of genetic association studies of MMPs and TIMPs conducted on different populations,but no study was done on Malaysian populations and in relation to hypertension. A total of 133 newly diagnosed EH subjects and 129 unrelated healthy individuals were requited under this study. The genomic DNA of these individuals were extracted from buffy coat and the plasma was separated for biochemical analysis. The genotyping of the polymorphisms were done by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) method. The PCR product and the restricted fragment product were run on agarose gel electrophoresis. All the statistical analysis were done by using Statistical Package for the Social Sciences (SPSS) version no. 21.0. The demographic characteristic of the subjects such as age, body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), low density lipoprotein (LDL), triglyceride (TG) and cholesterol (Chol) were shown to be differentially significant (p 0.05) in case subjects when compared to the controls, high density lipoprotein (HDL) did not show any significance. The genotype and allelic distribution of TIMP-1 372 T/C polymorphism was highly significant in hypertensive subjects as compared to the controls (p 0.05). Whilst, SNPs in position -1607 (1G/2G) in the MMP-1 gene, position -1562 (C/T) and 279 (R/Q) of the MMP-9 gene as well as site -82 (A/G) in the MMP-12 gene did not differ significantly (p 0.05) when compared to the controls. However, the data showed that the SNP in TIMP- 1 gene at site 372 (T/C) was associated with EH in Malay male hypertensive subjects. Hence, the allele and genotype of TIMP-1 polymorphisms may be considered as a possible genetic biomarker and a risk factor for EH

Analysis of KSHV B lymphocyte lineage tropism in human tonsil reveals efficient infection of CD138+ plasma cells.

Despite 25 years of research, the basic virology of Kaposi Sarcoma Herpesviruses (KSHV) in B lymphocytes remains poorly understood. This study seeks to fill critical gaps in our understanding by characterizing the B lymphocyte lineage-specific tropism of KSHV. Here, we use lymphocytes derived from 40 human tonsil specimens to determine the B lymphocyte lineages targeted by KSHV early during de novo infection in our ex vivo model system. We characterize the immunological diversity of our tonsil specimens and determine that overall susceptibility of tonsil lymphocytes to KSHV infection varies substantially between donors. We demonstrate that a variety of B lymphocyte subtypes are susceptible to KSHV infection and identify CD138+ plasma cells as a highly targeted cell type for de novo KSHV infection. We determine that infection of tonsil B cell lineages is primarily latent with few lineages contributing to lytic replication. We explore the use of CD138 and heparin sulfate proteoglycans as attachment factors for the infection of B lymphocytes and conclude that they do not play a substantial role. Finally, we determine that the host T cell microenvironment influences the course of de novo infection in B lymphocytes. These results improve our understanding of KSHV transmission and the biology of early KSHV infection in a naïve human host, and lay a foundation for further characterization of KSHV molecular virology in B lymphocyte lineages