Joshua Nyagol

MYC translocation-negative classical Burkitt lymphoma cases: an alternative pathogenetic mechanism involving miRNA deregulation

The molecular feature of Burkitt lymphoma (BL) is the translocation that places c‐Myc under the control of immunoglobulin gene regulatory elements. However, there is accumulating evidence that some cases may lack an identifiable MYC translocation. In addition, during the EUROFISH project, aiming at the standardization of FISH procedures in lymphoma diagnosis, we found that five cases out of 35 classic endemic BLs were negative for MYC translocations by using a split‐signal as well as a dual‐fusion probe. Here we investigated the expression pattern of miRNAs predicted to target c‐Myc, in BL cases, to clarify whether alternative pathogenetic mechanisms may be responsible for lymphomagenesis in cases lacking the MYC translocation. miRNAs are a class of small RNAs that are able to regulate gene expression at the post‐transcriptional level. Several studies have reported their involvement in cancer and their association with fragile sites in the genome. They have also been shown to control cell growth, differentiation, and apoptosis, suggesting that these molecules could act as tumour suppressors or oncogenes. Our results demonstrated a modulation of specific miRNAs. In particular, down‐regulation of hsa‐let‐7c was observed in BL cases, compared to normal controls. More interestingly, hsa‐mir‐34b was found to be down‐regulated only in BL cases that were negative for MYC translocation, suggesting that this event might be responsible for c‐Myc deregulation in such cases. This hypothesis was further confirmed by our in vitro experiments, which demonstrated that increasing doses of synthetic hsa‐mir‐34b were able to modulate c‐Myc expression. These results indicate for the first time that hsa‐mir‐34b may influence c‐Myc expression in Burkitt lymphoma as the more common aberrant control exercised by the immunoglobulin enhancer locus. Copyright

The different epidemiologic subtypes of Burkitt lymphoma share a homogenous micro RNA profile distinct from diffuse large B-cell lymphoma

Sporadic Burkitt lymphoma (sBL) can be delineated from diffuse large B-cell lymphoma (DLBCL) by a very homogeneous mRNA expression signature. However, it remained unclear whether all three BL variants—sBL, endemic BL (eBL) and human immunodeficiency virus-associated BL (HIV-BL)—represent a uniform biological entity despite their differences in geographical occurrence, association with immunodeficiency and/or incidence of Epstein–Barr virus (EBV) infection. To address this issue, we generated micro RNA (miRNA) profiles from 18 eBL, 31 sBL and 15 HIV-BL cases. In addition, we analyzed the miRNA expression of 86 DLBCL to determine whether miRNA profiles recapitulate the molecular differences between BL and DLBCL evidenced by mRNA profiling. A signature of 38 miRNAs containing MYC regulated and nuclear factor-kB pathway-associated miRNAs was obtained that differentiated BL from DLBCL. The miRNA profiles of sBL and eBL displayed only six differentially expressed miRNAs, whereas HIV and EBV infection had no impact on the miRNA profile of BL. In conclusion, miRNA profiling confirms that BL and DLBCL represent distinct lymphoma categories and demonstrates that the three BL variants are representatives of the same biological entity with only marginal miRNA expression differences between eBL and sBL.

B-cell differentiation in EBV-positive Burkitt lymphoma is impaired at posttranscriptional level by miRNA-altered expression.

Endemic, sporadic and HIV‐associated Burkitt lymphoma (BL) all have a B‐cell phenotype and a MYC translocation, but a variable association with the Epstein‐Barr virus (EBV). However, there is still no satisfactory explanation of how EBV participates in the pathogenesis of BL. A recent investigation suggested that EBV‐positive and EBV‐negative BL have different cells of origin. In particular, according to immunoglobulin gene mutation analysis, EBV‐negative BLs may originate from early centroblasts, whereas EBV‐positive BLs seem to arise from postgerminal center B cells or memory B cells. The appearance of a germinal center phenotype in EBV‐positive cells might thus derive from a block in B‐cell differentiation. The exit from the germinal center involves a complex series of events, which require the activation of BLIMP‐1, and the consequent downregulation of several target genes. Here, we investigated the expression of specific miRNAs predicted to be involved in B‐cell differentiation and found that hsa‐miR‐127 is differentially expressed between EBV‐positive and EBV‐negative BLs. In particular, it was strongly upregulated only in EBV‐positive BL samples, whereas EBV‐negative cases showed levels of expression similar to normal controls, including microdissected germinal centers (GC) cells. In addition, we found evidence that hsa‐miR‐127 is involved in B‐cell differentiation process through posttranscriptional regulation of BLIMP1 and XBP1. The overexpression of this miRNA may thus represent a key event in the lymphomagenesis of EBV positive BL, by blocking the B‐cell differentiation process.

The effects of HIV-1 Tat protein on cell cycle during cervical carcinogenesis

The role of HPV in the carcinogenesis of intraepithelial and invasive anogenital lesions is currently well established. E6 and E7 oncoproteins of high risk HPV genotypes are known to inactivate p53 and pRb pathways. Several studies have described an increased prevalence and recurrence of both cervical HPV infection and invasive cervical cancer among HIV-1 positive women compared to HIV-1 negative cases. For these reasons, cervical cancer is considered an AIDS-defining neoplasm. Unlike other AIDS-associated neoplasms, the occurrence of cervical cancer is independent of immune suppression. HIV-1 infection in patients with high grade precancerous lesions and cervical cancers results in a therapy refractory and more aggressive disease phenotype, which is not yet well understood at the molecular level. An upregulation of HPV E6 and E7 gene expressions by HIV-1 proteins such as Tat has been documented by some authors. However, the role of HIV-1 in cervical carcinomas is still unclear. It is already known that HIV-1 Tat protein is able to influence cell cycle progression. Altogether, these facts led us to investigate the effects of Tat on the expression of cell cycle regulator genes. After transfection of HeLa cells with Tat, we analyzed the expression of cell cycle regulators from these cells by IHC and Real-time PCR. A significant reduction in the expression of cell cycle inhibitors of transcription and an increase in the levels of proliferation markers were observed. These results suggest that HIV-1 may enhance cervical carcinogenesis by promoting cell cycle progression. We also found that this HIV-1 Tat-induced cell proliferation was not dependent on the E2F family of transcription factors, and therefore postulate that Sp factors may be involved.

HIV-1 Tat mimetic of VEGF correlates with increased microvessels density in AIDS-related diffuse large B-cell and Burkitt lymphomas

Angiogenic switch marks the beginning of tumor’s strategy to acquire independent blood supply. In some subtypes of non-Hodgkin’s lymphomas, higher local vascular endothelial growth factor (VEGF) expression correlates with increased microvessel density. However, this local VEGF expression is higher only in tumors with elevated expression of the receptors of the growth factor, suggesting an autocrine growth-promoting feedback loop. Several studies have indicated that VEGF receptors are also targeted by Tat protein from the HIV-1-infected cells. Given the similarity of the basic region of Tat to the angiogenic factors (basic fibroblast growth factor, VEGF), Tat mimics these proteins and binds to their receptors. We evaluated the role of HIV-1 Tat in regulating the level of VEGF expression and microvessel density in the AIDS-related diffuse large B-cell (DLBCL) and Burkitt lymphomas (BL). By luciferase assay, we showed that VEGF promoter activity was downregulated in vitro in cells transfected with Tat. Reduced VEGF protein expression in primary HIV-1 positive BL and DLBCL, compared to the negative cases, supported the findings of promoter downregulation from the cell lines. Microvascular density assessed by CD34 expression was, however, higher in HIV-1 positive than in HIV-1 negative tumors. These results suggest that Tat has a wider angiogenic role, besides the regulation of VEGF expression. Thus, targeting Tat protein itself and stabilizing transient silencing of VEGF expression or use of monoclonal antibodies against their receptors in the AIDS-associated tumors will open a window for future explorable pathways in the management of angiogenic phenotypes in the AIDS-associated non-Hodgkin’s lymphomas.

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

BackgroundSquamous 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.ObjectiveThe present study aimed to investigate if immunohistochemical expressions of EGFR, pEGFR and p16, could predict infection with high risk HPV in HIV-related SCCC.MethodsFFPE 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.ResultsOut 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.ConclusionThis study points to an association of high risk HPV with over expressions of p16INK4A and EGFR proteins in AIDS-associated SCCC.

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

Background 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. Methods 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. Results 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. Conclusion 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.

Cytokine Expression and Hypertension Comorbidity in HIV/AIDS Patients at Kenyatta National Hospital HIV Care Centre, Nairobi, Kenya

Chronic inflammation in HIV disease is sustained by immune reconstitution that results from use of combination therapy by patients and manifested by increased expression of inflammatory cytokines. Chronic inflammation in HIV disease is associated with endothelial dysfunction of the cardio vasculature giving rise to reduced elasticity resulting in high blood pressure. This state is marked by increased IL-6 cytokine expression as manifested in patients with type 2 diabetes and hypertension. This study sought to establish cytokine expression in HIV seropositive patients with hypertension comorbidity and correlate with HIV/AIDS progression. We hypothesized that chronic inflammation and presence of hypertension comorbidity presents higher levels of expression of selected interleukins namely IL-6, IL-8 and IL-17A. This study demonstrated that the IL-6 cytokine expression have a positive correlation with the release of IL-8 cytokine as patients with higher IL-6 cytokine values were more likely to have higher IL-8 cytokine levels. This shows that a synergistic effect from both cytokines may accelerate the development of atherosclerosis on blood vessels. Therefore routine measurement of these cytokines may increase prediction of risk of development of hypertension in patients on HIV care.

Risk indicators and effects of hypertension on HIV/AIDS disease progression among patients seen at Kenyatta hospital HIV care center.

ABSTRACT There is paucity of data on prevalence of hypertension and its effects on HIV/AIDS progression among patients at Kenyatta National Hospital (KNH), Kenya. This was a cross sectional study conducted between January and May 2015 at the KNH HIV Care Centre. Ethical approval was obtained from institutional ethics review board. HIV positive adult patients were recruited sequentially, and written informed consent obtained from each participant. Systematic sampling was used to select participants who were screened for blood pressure, body mass index (BMI) and lifestyle characteristics. Data on clinical parameters were extracted from patient records. A total of 297 participants (89 males and 208 females) were enrolled in the study. The participants were socially diverse in cultural beliefs, religious practices and lifestyles. Their ages ranged from 30 to 57 years, and the average age of males (M = 44.56, SD = 6.05) was higher than females (M = 42.29, SD = 6.16), p < .01. The prevalence of hypertension was found to be 23.2%. The relation between CD4 counts and creatinine was statistically significant, p < .01, as was the association between CD4 counts and BMI, p < .01. Hypertension is a highly prevalent co-morbidity in HIV patients. The risk factors include prolonged use of ART as well as increased body mass index. The effects of hypertension on HIV progression include low CD4+ T cell counts which complicate the underlying immunosuppression.