Kaposi Sarcoma-Associated Herpesvirus Glycoprotein H Is Indispensable for Infection of Epithelial, Endothelial, and Fibroblast Cell Types

Kaposi sarcoma-associated herpesvirus (KSHV) is an emerging pathogen and is the causative infectious agent of Kaposi sarcoma and two malignancies of B cell origin. To date, there is no licensed KSHV vaccine. Development of an effective vaccine against KSHV continues to be limited by a poor understanding of how the virus initiates acute primary infection in vivo in diverse human cell types. The role of glycoprotein H (gH) in herpesvirus entry mechanisms remains largely unresolved. To characterize the requirement for KSHV gH in the viral life cycle and in determination of cell tropism, we generated and characterized a mutant KSHV in which expression of gH was abrogated. Using a bacterial artificial chromosome containing a complete recombinant KSHV genome and recombinant DNA technology, we inserted stop codons into the gH coding region. We used electron microscopy to reveal that the gH-null mutant virus assembled and exited from cells normally, compared to wild-type virus. Using purified virions, we assessed infectivity of the gH-null mutant in diverse mammalian cell types in vitro. Unlike wild-type virus or a gH-containing revertant, the gH-null mutant was unable to infect any of the epithelial, endothelial, or fibroblast cell types tested. However, its ability to infect B cells was equivocal and remains to be investigated in vivo due to generally poor infectivity in vitro. Together, these results suggest that gH is critical for KSHV infection of highly permissive cell types, including epithelial, endothelial, and fibroblast cells. IMPORTANCE All homologues of herpesvirus gH studied to date have been implicated in playing an essential role in viral infection of diverse permissive cell types. However, the role of gH in the mechanism of KSHV infection remains largely unresolved. In this study, we generated a gH-null mutant KSHV and provided evidence that deficiency of gH expression did not affect viral particle assembly or egress. Using the gH-null mutant, we showed that gH was indispensable for KSHV infection of epithelial, endothelial, and fibroblast cells in vitro. This suggests that gH is an important target for the development of a KSHV prophylactic vaccine to prevent initial viral infection

Kaposi Sarcoma-associated Herpesvirus Glycoprotein H is Indispensable for Infection of Epithelial, Endothelial, and Fibroblast Cell Types

Kaposi sarcoma-associated herpesvirus (KSHV) is an emerging pathogen and is the causative infectious agent of Kaposi sarcoma and two malignancies of B cell origin. To date, there is no licensed KSHV vaccine. Development of an effective vaccine against KSHV continues to be limited by a poor understanding of how the virus initiates acute primary infection in vivo in diverse human cell types. The role of glycoprotein H (gH) in herpesvirus entry mechanisms remains largely unresolved. To characterize the requirement for KSHV gH in the viral life cycle and in determination of cell tropism, we generated and characterized a mutant KSHV in which expression of gH was abrogated. Using a bacterial artificial chromosome containing a complete recombinant KSHV genome and recombinant DNA technology, we inserted stop codons into the gH coding region. We used electron microscopy to reveal that the gH-null mutant virus assembled and exited from cells normally, compared to wild-type virus. Using purified virions, we assessed infectivity of the gH-null mutant in diverse mammalian cell types in vitro Unlike wild-type virus or a gH-containing revertant, the gH-null mutant was unable to infect any of the epithelial, endothelial, or fibroblast cell types tested. However, its ability to infect B cells was equivocal, and remains to be investigated in vivo due to generally poor infectivity in vitro Together, these results suggest that gH is critical for KSHV infection of highly permissive cell types including epithelial, endothelial, and fibroblasts. MPORTANCE: All homologues of herpesvirus gH studied to date have been implicated in playing an essential role in viral infection of diverse permissive cell types. However, the role of gH in the mechanism of KSHV infection remains largely unresolved. In this study, we generated a gH-null mutant KSHV and provided evidence that deficiency of gH expression did not affect viral particle assembly or egress. Using the gH-null mutant, we showed that gH was indispensable for KSHV infection of epithelial, endothelial, and fibroblast cells in vitro. This suggests that gH is an important target for the development of a KSHV prophylactic vaccine to prevent initial viral infection

Correlation of EGFR, pEGFR and p16INK4 expressions and high risk HPV infection in HIV/AIDS-related squamous cell carcinoma of conjunctiva

Background: Squamous cell carcinoma of conjunctiva has increased tenfold in the era of HIV/AIDS. The disease pattern has also changed in Africa, affecting young persons, with peak age-specific incidence of 30-39 years, similar to that of Kaposi sarcoma, a well known HIV/AIDS defining neoplasm. In addition, the disease has assumed more aggressive clinical course. The contributing role of exposure to high risk HPV in the development of SCCC is still emerging. Objective. The present study aimed to investigate if immunohistochemical expressions of EGFR, pEGFR and p16, could predict infection with high risk HPV in HIV-related SCCC. Methods. FFPE tissue blocks of fifty-eight cases diagnosed on hematoxylin and eosin with SCCC between 2005-2011, and subsequently confirmed from medical records to be HIV positive at the department of human pathology, UoN/KNH, were used for the study. Immunohistochemistry was performed to assess the expressions of p16INK4A, EGFR and pEGFR. This was followed with semi-nested PCR based detection and sequencing of HPV genotypes. The sequences were compared with the GenBank database, and data analyzed for significant statistical correlations using SPSS 16.0. Ethical approval to conduct the study was obtained from KNH-ERC. Results: Out of the fifty-eight cases of SCCC analyzed, twenty-nine (50%) had well differentiated (grade 1), twenty one (36.2%) moderately differentiated (grade 2) while eight (13.8%) had poorly differentiated (grade 3) tumours. Immunohistochemistry assay was done in all the fifty eight studied cases, of which thirty nine cases (67.2%) were positive for p16INK4A staining, forty eight cases (82.8%) for EGFR and fifty one cases (87.9%) showed positivity for p-EGFR. HPV DNA was detected in 4 out of 40 SCCC cases (10%) in which PCR was performed, with HPV16 being the only HPV sub-type detected. Significant statistical association was found between HPV detection and p16INK4 (p=0.000, at 99% C.I) and EGFR (p=0.028, at 95% C.I) expressions, but not pEGFR. In addition, the expressions of these biomarkers did not show any significant association with tumor grades. Conclusion: This study points to an association of high risk HPV with over expressions of p16INK4A and EGFR proteins in AIDS-associated SCCC. © 2014 Mwololo et al.; licensee BioMed Central Ltd

Prevalence of Drug Resistance Mycobacterium Tuberculosis among Patients Seen in Coast Provincial General Hospital, Mombasa, Kenya

<div><p>Background</p><p>Although prevention and control of spread of multi-drug resistant tuberculosis strains is a global challenge, there is paucity of data on the prevalence of DR-TB in patients diagnosed with TB in referral hospitals in Kenya. The present study assessed patients’ characteristics and prevalence of drug resistant TB in sputa smear positive TB patients presenting to Coast Provincial General Hospital (CPGH) in Mombasa, Kenya.</p><p>Methods</p><p>Drug resistance was evaluated in 258 randomly selected sputa smear TB positive cases between the periods of November 2011 to February 2012 at the CPGH-Mombasa. Basic demographic data was obtained using administered questionnaires, and clinical history extracted from the files. For laboratory analyses, 2mls of sputum was obtained, decontaminated and subjected to mycobacteria DNA analyses. Detection of first line drug resistance genes was done using MDRTDR plus kit. This was followed with random selection of 83 cases for second line drug resistance genes testing using Genotype MDRTBsl probe assay kit (HAINS Lifesciences, GmbH, Germany), in which ethambutol mutation probes were included. The data was then analyzed using SPSS statistical package version 19.0.</p><p>Results</p><p>Male to female ratio was 1:2. Age range was 9 to 75 years, with median of 30 years. New treatment cases constituted 253(98%), among which seven turned out to be PTB negative, and further grouped as 4 (1.6%) PTB negative and 3(1.1%) NTM. 237(91.7%) new cases were fully susceptible to INH and RIF. The remaining, 8 (3.1%) and 1(0.4%) had mono- resistance to INH and RIF, respectively. All the retreatment cases were fully susceptible to the first line drugs. HIV positivity was found in 48 (18.6%) cases, of which 46(17.8%) were co-infected with TB. Of these, 44 (17.1%) showed full susceptibility to TB drugs, while 2 (0.8%) were INH resistant. For the second line drugs, one case each showed mono resistance to both and FQ. Also, one case each showed drug cross poly resistance to both ETH and FQ, with second line injectable antibiotics. However, no significant statistical correlation was established between TB and resistance to the second line drugs p = 0.855.</p><p>Conclusion</p><p>The findings of this study showed the existence of resistance to both first and second line anti-tubercular drugs, but no MDR-TB and XDR-TB was detected among patients attending TB clinic at CPGH using molecular techniques.</p></div

Second line drug susceptibility testing stratified by age, study type and HIV status.

<p>Second line drug susceptibility testing stratified by age, study type and HIV status.</p

Gene-expression analysis identifies novel RBL2/p130 target genes in endemic Burkitt lymphoma cell lines and primary tumors.

16Burkitt lymphoma (BL) is a B-cell tumor whose characteristic gene aberration is the translocation t(8;14), which determines c-myc overexpression. Several genetic and epigenetic alterations other than c-myc overexpression have also been described in BL. It has been demonstrated that the RBL2/p130 gene, a member of the retinoblastoma family (pRbs), is mutated in BL cell lines and primary tumors. The aim of this study was to investigate the biologic effect of RBL2/p130 in BL cells and its possible role in lymphomagenesis. Therefore, we reintroduced a functional RBL2/p130 in BL cell lines where this gene was mutated. Our results demonstrated that RBL2/p130-transfected cells regain growth control. This suggests that RBL2/p130 may control the expression of several genes, which may be important for cell growth and viability. Gene-expression analysis revealed a modulation of several genes, including CGRRF1, RGS1, BTG1, TIA1, and PCDHA2, upon RBL2/p130 reintroduction. We then monitored their expression in primary tumors of endemic BL as well, demonstrating that their expression resembled those of the BL cell lines. In conclusion, these data suggest that, as RBL2/p130 modulates the expression of target genes, which are important for cell growth and viability, its inactivation may be relevant for the occurrence of BL.reservedmixedDE FALCO G; LEUCCI E; LENZE D; PICCALUGA PP; CLAUDIO PP; ONNIS A; CERINO G; NYAGOL J; MWANDA W; C. BELLAN; HUMMEL M; PILERI S; TOSI P; STEIN H; GIORDANO A; LEONCINI LDE FALCO, G; Leucci, E; Lenze, D; Piccaluga, Pp; Claudio, Pp; Onnis, A; Cerino, G; Nyagol, J; Mwanda, W; Bellan, C.; Hummel, M; Pileri, S; Tosi, P; Stein, H; Giordano, A; Leoncini,

Additional file 1: of Effects of HIV-1 infection on malaria parasitemia in milo sub-location, western Kenya

Table S1. Expressions age, sex, malaria parasitaemia and density against HIV status

Immune landscape in Burkitt lymphoma reveals M2-macrophage polarization and correlation between PD-L1 expression and non-canonical EBV latency program

Background: The Tumor Microenviroment (TME) is a complex milieu that is increasingly recognized as a key factor in multiple stages of disease progression and responses to therapy as well as escape from immune surveillance. However, the precise contribution of specific immune effector and immune suppressor components of the TME in Burkitt lymphoma (BL) remains poorly understood. Methods: In this paper, we applied the computational algorithm CIBERSORT to Gene Expression Profiling (GEP) datasets of 40 BL samples to draw a map of immune and stromal components of TME. Furthermore, by multiple immunohistochemistry (IHC) and multispectral immunofluorescence (IF), we investigated the TME of additional series of 40 BL cases to evaluate the role of the Programmed Death-1 and Programmed Death Ligand-1 (PD-1/PD-L1) immune checkpoint axis. Results: Our results indicate that M2 polarized macrophages are the most prominent TME component in BL. In addition, we investigated the correlation between PD-L1 and latent membrane protein-2A (LMP2A) expression on tumour cells, highlighting a subgroup of BL cases characterized by a non-canonical latency program of EBV with an activated PD-L1 pathway. Conclusion: In conclusion, our study analysed the TME in BL and identified a tolerogenic immune signature highlighting new potential therapeutic targets

Tumor microenvironment of Burkitt lymphoma: different immune signatures with different clinical behavior

: Burkitt lymphoma (BL) is characterized by tumor microenvironment (TME) in which macrophages represent the main component, determining a distinct histological appearance known as "starry sky" pattern. However, in some instances, BL may exhibit a granulomatous reaction that has been previously linked to a favorable prognosis and spontaneous regression. The aim of our study was to deeply characterize the immune landscape of 7 cases of EBV + BL with granulomatous reaction compared to 8 cases of EBV + BL and 8 EBV- BL, both with typical "starry sky" pattern, by Gene expression profiling performed on the NanoString nCounter platform. Subsequently, the data were validated by multiplex and combined immunostaining. Based on unsupervised clustering of differentially expressed genes, BL samples formed 3 distinct clusters differentially enriched in BL with a diffuse granulomatous reaction (cluster 1), EBV+ BL with typical starry sky pattern (Cluster 2), EBV - BL with typical "starry sky" (cluster 3). We observed variations in the immune response signature among BL with granulomatous reaction and BL with typical "starry sky", both EBV + and EBV -. The TME signature in BL with diffuse granulomatous reaction showed a proinflammatory response, while BLs with "starry sky" were characterized by up-regulation of M2- polarization and pro-tumor response. Moreover, the analysis of additional signatures revealed an up-regulation of Dark zone-signature and epigenetic-signature in BL with typical "starry sky". Tumor associated macrophages (TAM) and epigenetic regulators may be promising targets for additional therapies in BL lymphoma opening novel immunotherapeutic strategies