Hypoglycemic and Toxic Effect of Morus mesozygia Leaf Extract on the Liver and Kidneys of Alloxan-Induced Hyperglycemic Wistar Rats.

PURPOSE: We investigated the hypoglycemic and toxic effect of Morus mesozygia leaf extract on the liver and kidneys of alloxan-induced hyperglycemic wistar rats. METHOD: Phytochemical analysis was done. Diabetes was induced by the use of alloxan monohydrate in six groups of rats, i.e., 200 mg/kg, 400 mg/kg, 800 mg/kg, glibenclamide, normal saline, and normal control group. Blood glucose was measured at the time of inoculation, then at 1, 2, 3, and 4 hours after. After 14 days, rats were killed under anesthesia; blood collected for measurement of total protein, albumin, TAGs, cholesterol, AST, ALT, urea, and creatinine; and whole tissue of liver and kidneys used for histological studies. RESULTS: The extract possessed antidiabetic effects between 400 mg/kg and 800 mg/kg doses, which we attributed to the presence of flavonoids, tannins, terpenoids, and amino acids. There was a drop in total protein and albumin with no statistical significance (P ≥ 0.05). The changes in levels of ALT, TAGs, cholesterol, AST, creatinine, and urea were not statistically different from the standard diabetic drug. The extract was protective against histological damage as there were no significant lesions suggestive of toxicities in the liver and kidneys at doses below 800 mg/kg. CONCLUSION: We established credible evidence that Morus mesozygia leaf extract has hypoglycemic effects between 400 mg/kg and 800 mg/kg and that it is safe on the liver and kidneys of wistar rats at doses less than 800 mg/kg.

Targeting wild-type Erythrocyte receptors for Plasmodium falciparum and vivax Merozoites by Zinc Finger Nucleases In- silico: Towards a Genetic Vaccine against Malaria.

BACKGROUND: Malaria causes immense human morbidity and mortality globally. The plasmodium species vivax and falciparum cause over 75 % clinical malaria cases. Until now, gene-based strategies against malaria have only been applied to plasmodium species and their mosquito-vector. Merozoites of these two respective plasmodium species target and invade red blood cells (RBCs) by using the duffy antigen receptor for chemokines (DARC), and Sialic Acid (SLC4A1) residues of the O-linked glycans of Glycophorin A. RBCs of naturally selected duffy-negative blacks are resistant to P.vivax tropism. We hypothesized that artificial aberration of the host-pathway by target mutagenesis of either RBC -receptors, may abolish or reduce susceptibility of the host to malaria. As a first step towards the experimental actualization of these concepts, we aimed to identify zinc finger arrays (ZFAs) for constructing ZFNs that target genes of either wild-type host-RBC- receptors. METHODS: In-Silico Gene & Genome Informatics RESULTS: Using the genomic contextual nucleotide-sequences of homo-sapiens darc and glycophorin-a, and the ZFN-consortia software- CoDA-ZiFiT-ZFA and CoDA-ZiFiT-ZFN: we identified 163 and over 1,000 single zinc finger arrays (sZFAs) that bind sequences within the genes for the two respective RBC-receptors. Second, 2 and 18 paired zinc finger arrays (pZFAs) that are precursors for zinc finger nucleases (ZFNs) capable of cleaving the genes for darc and glycophorin-a were respectively assembled. Third, a mega-BLAST evaluation of the genome-wide cleavage specificity of this set of ZFNs was done, revealing alternate homologous nucleotide targets in the human genome other than darc or glycophorin A. CONCLUSIONS: ZFNs engineered with these ZFA-precursors--with further optimization to enhance their specificity to only darc and glycophorin-a, could be used in constructing an experimental gene-based-malaria vaccine. Alternatively, meganucleases and transcription activator-like (TAL) nucleases that target conserved stretches of darc and glycophorin-a DNA may serve the purpose of abrogating invasion of RBCs by falciparam and vivax plasmodia species.

EBV, HHV8 and HIV in B cell non Hodgkin lymphoma in Kampala, Uganda.

BACKGROUND: B cell non Hodgkin lymphomas account for the majority of lymphomas in Uganda. The commonest is endemic Burkitt lymphoma, followed by diffuse large-B-cell lymphoma (DLBCL). There has been an increase in incidence of malignant lymphoma since the onset of the HIV/AIDS pandemic. However, the possible linkages of HHV8 and EBV to the condition of impaired immunity present in AIDS are still not yet very clearly understood. OBJECTIVES: 1. To describe the prevalence of Epstein-Barr virus, Human Herpes virus 8 and Human Immunodeficiency Virus-1 in B cell non Hodgkin lymphoma biopsy specimens in Kampala, Uganda.2. To describe the histopathology of non Hodgkin lymphoma by HIV serology test result in Kampala, Uganda METHOD: Tumour biopsies specimens from 119 patients with B cell non Hodgkin lymphoma were classified according to the WHO classification. Immunohistochemistry was used for detection of HHV8 and in situ hybridization with Epstein Barr virus encoded RNA (EBER) for EBV. Real time and nested PCR were used for the detection of HIV.The patients from whom the 1991-2000 NHL biopsies had been taken did not have HIV serology results therefore 145 patients biopsies where serology results were available were used to describe the association of HIV with non Hodgkin lymphoma type during 2008-2009. RESULTS: In this study, the majority (92%) of the Burkitt lymphomas and only 34.8% of the diffuse large B cell lymphomas were EBV positive. None of the precursor B lymphoblastic lymphomas or the mantle cell lymphomas showed EBV integration in the lymphoma cells.None of the Burkitt lymphoma biopsies had HIV by PCR. Of the 121 non Hodgkin B cell lymphoma patients with HIV test results, 19% had HIV. However, only 1(0.04%) case of Burkitt lymphoma had HIV. All the tumours were HHV8 negative. CONCLUSIONS: The majority of the Burkitt lymphomas and two fifths of the diffuse large B cell lymphomas had EBV. All the tumours were HHV8 negative. Generally, the relationship of NHL and HIV was weaker than what has been reported from the developed countries. We discuss the role of these viruses in lymphomagenesis in light of current knowledge.

An up-date on the prevalence of sickle cell trait in Eastern and Western Uganda.

BACKGROUND: The first survey on sickle cell disease (SCD) done in Uganda in 1949, reported the district of Bundibugyo in Western Uganda to have the highest sickle cell trait (SCT) prevalence (45%). This is believed to be the highest in the whole world. According to the same survey, the prevalence of SCT in the districts of Mbale and Sironko in the East was 20-28%, whilst the districts of Mbarara and Ntungamo in the West had 1-5%. No follow-up surveys have been conducted over the past 60 years. SCA accounts for approximately 16.2% of all pediatric deaths in Uganda. The pattern of SCT inheritance, however, predicts likely changes in the prevalence and distribution of the SCT. The objective of the study therefore was to establish the current prevalence of the SCT in Uganda. METHODS: This study was a cross sectional survey which was carried out in the districts of Mbale and Sironko in the Eastern, Mbarara/Ntungamo and Bundibugyo in Western Uganda. The participants were children (6 months-5 yrs). Blood was collected from each subject and analyzed for hemoglobin S using cellulose acetate Hb electrophoresis. RESULTS: The established prevalence of the SCT (As) in Eastern Uganda was 17.5% compared to 13.4% and 3% in Bundibugyo and Mbarara/Ntungamo respectively. 1.7% of the children in Eastern Uganda tested positive for haemoglobin ss relative to 3% in Bundibugyo, giving gene frequencies of 0.105 and 0.097 for the recessive gene respectively. No ss was detected in Mbarara/Ntungamo. CONCLUSIONS: A shift in the prevalence of the SCT and ss in Uganda is notable and may be explained by several biological and social factors. This study offers some evidence for the possible outcome of intermarriages in reducing the incidence of the SCT.

Immunohistochemical and other prognostic factors in B cell non Hodgkin lymphoma patients, Kampala, Uganda.

BACKGROUND: Non Hodgkin lymphomas are the most common lymphomas in Uganda. Recent studies from developed countries have shown differences in survival for the different immunophenotypes. Such studies are lacking in Africa where diagnosis is largely dependent on morphology alone. We report immunohistochemical and other prognostic factors in B cell non Hodgkin lymphoma patients in Kampala, Uganda. METHODS: Non Hodgkin lymphoma tissue blocks from the archives of the Department of Pathology, Makerere University College of Health Sciences, Kampala, Uganda, from 1991-2000, were sub typed using haematoxylin and eosin, Giemsa as well as immunohistochemistry. Using tissue micro array, 119 biopsies were subjected to: CD3, CD5, CD10, CD20, CD23, CD30, CD38, CD79a, CD138, Bcl-6, Bcl-2, IRTA-1, MUM1/IRF4, Bcl-1/cyclin D1, TdT, ALKc, and Ki-67/Mib1. Case notes were retrieved for: disease stage, chemotherapy courses received and retrospective follow up was done for survival. RESULTS: Non Hodgkin B cell lymphomas comprised of Burkitt lymphoma [BL] (95/119) diffuse large B cell lymphoma (19/119), mantle cell lymphoma (4/119) and precursor B lymphoblastic lymphoma (1/119). For Burkitt lymphoma, good prognosis was associated with receiving chemotherapy, female gender and CD30 positivity. Only receiving chemotherapy remained significant after Cox regression analysis. Diffuse large B cell lymphomas with activated germinal centre B cell (GCB) pattern (CD10+/-, BCL-6+/-, MUM+/-, CD138+/-) had better survival (98.4 months; 95% CI 89.5 -107.3) than the others (57.3 months; 95% CI 35.5 - 79.0) p = 0.027 (log rank test). CONCLUSIONS: Activated GCB diffuse large B cell lymphoma had a better prognosis than the others. For Burkitt lymphoma, not receiving chemotherapy carried a poor prognosis. Availability of chemotherapy in this resource limited setting is critical for survival of lymphoma patients.

In silico evidence for the species-specific conservation of mosquito retroposons: implications as a molecular biomarker.

BACKGROUND: Mosquitoes are the transmissive vectors for several infectious pathogens that affect man. However, the control of mosquitoes through insecticide and pesticide spraying has proved difficult in the past. We hypothesized that, by virtue of their reported vertical inheritance among mosquitoes, group II introns - a class of small coding ribonucleic acids (scRNAs) - may form a potential species-specific biomarker. Structurally, introns are a six-moiety complex. Depending on the function of the protein encoded within the IV moiety, the highly mobile class of group II introns or retroposons is sub-divided into two: Restriction Endonuclease (REase)-like and Apurinic aPyramydinic Endonuclease (APE)-like. REase-like retroposons are thought to be the ancestors of APE retroposons. Our aim in this study was to find evidence for the highly species-specific conservation of the APE subclass of mosquito retroposons. METHODS AND RESULTS: In silico targeted sequence alignments were conducted across a 1,779-organism genome database (1,518 bacterial, 59 archeal, 201 eukaryotic, and the human), using three mosquito retroposon sequence tags (RST) as BLASTN queries [AJ970181 and AJ90201 of Culex pipien origin and AJ970301 of Anoplese sinensis origin]. At a calibration of E = 10, A & D = 100, default filtration and a homology cut-off of >95% identity, no hits were found on any of the 1,518 bacterial genomes. Eleven (100%) and 15 (100%) hits obtained on the 201-eukaryote genome database were homologs (>95% score) of C.pipien quinquefasciatus JHB retroposons, but none of An. sinensis. Twenty and 221 low score (30-43% identity) spurious hits were found at flanking ends of genes and contigs in the human genome with the C.pipien and An. sinensis RSTs respectively. Functional and positional inference revealed these to be possible relatives of human genomic spliceosomes. We advance two models for the application of mosquito RST: as precursors for developing molecular biomarkers for mosquitoes, and as RST-specific monoclonal antibody (MAb)-DDT immunoconjugates to enhance targeted toxicity. CONCLUSION: We offer evidence to support the species-specific conservation of mosquito retroposons among lower taxa. Our findings suggest that retroposons may therefore constitute a unique biomarker for mosquito species that may be exploited in molecular entomology. Mosquito RST-specific MAbs may possibly permit synthesis of DDT immunoconjugates that could be used to achieve species-tailored toxicity.

Sickling and solubility tests and the peripheral blood film method for screening for sickle cell disease. [corrected].

OBJECTIVE: To determine the cost benefit of screening for sickle-cell disease among infants at district health centres in Uganda using sickling, solubility tests and the peripheral blood film method. METHODS: Pilot screening services were established at district health centres. Cost benefit analysis (CBA) was performed in four scenarios: A1 - where there are no sickle-cell screening services at district health centres and all children are referred either to Mulago tertiary referral hospital or A2 - a regional hospital for haemoglobin (Hb) electrophoresis; B1 - when there are screening services at district health centres, only positive samples are taken either to Mulago Hospital or B2 - the regional hospital for confirmation using haemoglobin electrophoresis. Calculations were done in Uganda shillings (USh). RESULTS: Initial operational costs were high for all scenarios but variably reduced in the subsequent years. Scenarios A1 and A2 were very sensitive compared with B1 and B2. Scenario A1 had the highest screening costs in the subsequent years, costing over 62 000 USh per test in both eastern and western Uganda. Scenario B2 was sensitive and cheaper when using the sickling test, but was expensive and insensitive when using the solubility test and more insensitive though cheaper when using the peripheral blood film method. CONCLUSIONS AND RECOMMENDATION: Screening children in Mulago hospital using haemoglobin electrophoresis (A1) was very expensive although it was sensitive. Screening the children at four health centres using the sickling method and confirming positive samples at a regional hospital (B2) was both cheap and sensitive and is therefore recommended.

Identification of restriction endonuclease with potential ability to cleave the HSV-2 genome: inherent potential for biosynthetic versus live recombinant microbicides.

BACKGROUND: Herpes Simplex virus types 1 and 2 are enveloped viruses with a linear dsDNA genome of approximately 120-200 kb. Genital infection with HSV-2 has been denoted as a major risk factor for acquisition and transmission of HIV-1. Developing biomedical strategies for HSV-2 prevention is thus a central strategy in reducing global HIV-1 prevalence. This paper details the protocol for the isolation of restriction endunucleases (REases) with potent activity against the HSV-2 genome and models two biomedical interventions for preventing HSV-2. METHODS AND RESULTS: Using the whole genome of HSV-2, 289 REases and the bioinformatics software Webcutter2; we searched for potential recognition sites by way of genome wide palindromics. REase application in HSV-2 biomedical therapy was modeled concomitantly. Of the 289 enzymes analyzed; 77(26.6%) had potential to cleave the HSV-2 genome in > 100 but < 400 sites; 69(23.9%) in > 400 but < 700 sites; and the 9(3.1%) enzymes: BmyI, Bsp1286I, Bst2UI, BstNI, BstOI, EcoRII, HgaI, MvaI, and SduI cleaved in more than 700 sites. But for the 4: PacI, PmeI, SmiI, SwaI that had no sign of activity on HSV-2 genomic DNA, all 130(45%) other enzymes cleaved < 100 times. In silico palindromics has a PPV of 99.5% for in situ REase activity (2) Two models detailing how the REase EcoRII may be applied in developing interventions against HSV-2 are presented: a nanoparticle for microbicide development and a "recombinant lactobacillus" expressing cell wall anchored receptor (truncated nectin-1) for HSV-2 plus EcoRII. CONCLUSION: Viral genome slicing by way of these bacterially- derived R-M enzymatic peptides may have therapeutic potential in HSV-2 infection; a cofactor for HIV-1 acquisition and transmission.

B-cell non-Hodgkin lymphomas in Uganda: an immunohistochemical appraisal on tissue microarray.

The most common non-Hodgkin lymphomas in Uganda are neoplasms of B-cell derivation. The field of B-cell lymphoma immunophenotype has rapidly progressed because of the increasing availability of markers applicable to routine sections. Although the latter have allowed the identification of distinctive lymphoma entities in the developed countries, such approach has not yet been used in Uganda. One hundred twenty-nine formalin-fixed, paraffin-embedded tissue samples from the Department of Pathology of Makerere University were used for tissue micro-array (TMA) construction. Four-micrometer-thick sections were cut from TMAs and stained with hematoxylin and eosin and Giemsa. They were also used for immunohistochemistry and in situ hybridization. According to morphology and immunohistochemistry, lymphoid neoplasms were classified as Burkitt's lymphoma (BL) (95 cases), diffuse large B-cell lymphoma (19 cases), mantle cell lymphoma (4 cases), and B-cell lymphoblastic lymphoma (1 case). In BL, a homogeneous phenotype (CD10(+), Bcl-6(+), Bcl-2(-), MUM1/IRF4-, and Ki-67 approximately 100%) and a stable Epstein-Barr virus integration were found. A distinctive and unusual feature was the frequent plasma cellular differentiation, along with the positivity for CD30 and CD138 (recorded in 35 and 43 cases, respectively). According to our findings, most non-Hodgkin B-cell tumors in Uganda are endemic BLs followed by diffuse large B-cell lymphomas. The rest consist of rare but clinically important entities such as mantle cell lymphoma and B-cell lymphoblastic lymphoma. The availability of TMAs and immunohistochemistry has enabled us to precisely categorize tumors that have so far been diagnosed in Uganda as "high-grade/aggressive" lymphomas on the basis of cell morphology alone.

Harnessing pharmacogenomics to tackle resistance to the "nucleoside reverse trancripatse inhibitor" backbone of highly active antiretroviral therapy in resource limited settings.

BACKGROUND: The sustainable use of HAART within the sub-Saharan and other developing world settings faces the emerging challenge of drug resistance. Nucleoside reverse transcriptase inhibitors (NRTI) form the backbone of HAART and preserving their "antiviral efficacy" is thus critical to sustainable HAART use. METHODS: A systematic review of the "mechanisms of evolution" of resistance to NRTI at the HIV genome level, and the phenotypic manifestations on drug pharmacokinetics was done. CONCLUSION: This paper provides an evidence based account of how the knowledge of pharmacogenomics may be exploited to tackle NRTI resistance within limited resource.

Monitoring the severity of iodine deficiency disorders in Uganda.

BACKGROUND: Iodine deficiency disorders (IDD) cover a variety of pathological conditions including goitre, mental retardation and perinatal mortality in millions of individuals globally. IDD was initially identified as a problem in 1970 and was confirmed in 1991. In 1993, the Uganda government introduced a policy of Universal Salt Iodization (USI) requiring all household salt to be iodized. After 5 years this study evaluates the USI programme. OBJECTIVES: To determine goitre prevalence rate, establish the proportion of household consuming iodized salt and determine the levels of iodine intake in the sample districts. METHODS: A sample of 2880 school children aged 6-12 years from 72 Primary schools in 6 districts of Uganda was studied in October 1999. Goitre was established by palpation, salt iodine was analysed by thiosulphate titration, while urinary iodine was analyzed using ICCIDD recommended method F in which iodine is detected colorimetrically at 410 nm. RESULTS: The over all total goitre rate was 60.2% down from 74.3 in 1991 and visible goitre was 30% down from 39.2% in 1991. The proportion of households taking adequately iodized salt was 63.8% and the median urinary iodine was 310 microg/L. Whereas 36% of 95 urine samples analysed in 1991 had urinary iodine below 50 microg/L, only 5% of the 293 urine samples studied in 1999 had the same urine levels. This represents a considerable improvement in iodine intake, which is confirmed by the fact that 63.8% of the study households consume adequately iodized salt. If maintained and evenly spread, this will enable Uganda to control IDD. CONCLUSION: USI has improved iodine intake in Uganda. However, iodine malnutrition is still a severe public health problem because some communities in this study such as in Kisoro still have low iodine consumption, while others such as Luwero now have iodine excess. The latter is likely to predispose to hyperthyroidism. RECOMMENDATION: The national set standard of household salt iodine of 100 ppm be revised. Locally produced salt be iodized, and a national iodine monitoring programme be instituted to ensure evenly spread consumption of adequately iodized salt by all communities in the country.