Zoology
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Item Antibody specificities of children living in a malaria endemic area to inhibitory and blocking epitopes on MSP-119 of Plasmodium falciparum(Elsevier, 2009) Omosun, Y. O.; Adoro, S.; Anumudu, C. I.; Odaibo, A. B.; Uthiapibull, C.; Holder, A. A.; Nwagwu, M.; Nwuba, R. I.Merozoite surface protein-119 (MSP-119) specific antibodies which include processing inhibitory, blocking and neutral antibodies have been identified in individuals exposed to Plasmodium falciparum. Here we intend to look at the effect of single and multiple amino acid substitutions of MSP-119 on the recognition by polyclonal antibodies from children living in Igbo-Ora, Nigeria. This would provide us with information on the possibility of eliciting mainly processing inhibitory antibodies with a recombinant MSP-119 vaccine. Blood was collected from children in the rainy season and binding of anti-MSP-119 antibodies to modified mutants of MSP-119 was analysed by ELISA. The MSP-119 mutant proteins with single substitutions at positions 22 (Leu→Arg), 43 (Glu→Leu) and 53 (Asn→Arg) and the MSP-119 mutant protein with multiple substitutions at positions 27 + 31 + 34 + 43 (Glu→Tyr, Leu→Arg, Tyr→Ser, Glu→Leu); which had inhibitory epitopes; had the highest recognition. Children recognised both sets of mutants with different age groups having different recognition levels. The percentage of malaria positive individuals (32–80%) with antibodies that bound to the mutants MSP-119 containing epitopes that recognise only processing inhibitory and not blocking antibodies, were significantly different from those with antibodies that did not bind to these mutants (21–28%). The amino acid substitutions that abolished the binding of blocking antibodies without affecting the binding of inhibitory antibodies are of particular interest in the design of MSP-119 based malaria vaccines. Although these MSP-119 mutants have not been found in natural population, their recognition by polyclonal antibodies from humans naturally infected with malaria is very promising for the future use of MSP-119 mutants in the design of a malaria vaccine.Item Genetic variants of Plasmodium falciparum infective Anopheles gambiae s.l. at a rural community in southwest Nigeria(Faulty of Science, University of Port Harcourt, Nigeria., 2009) Noutcha, M. A. E.; Ngoundou-Landji, J.; Anumudu, C. I.During studies on the epidemiology of malaria at a rural community, Igbo-Ora, Southwest Nigeria, genotyping of Plasmodium falciparum extracted from infective Anopheles gambiae s.l. was undertaken. Circumsporozoite (CSP) ELISA was used on crushes from head-thorax for DNA extraction and PCR amplification for the determination of P. falciparum genotypes on merozoite surface protein-I and 2 (MSP-I & 2). Of the 65 infective anophelines, P. falciparum genotypes were positively identified in 41. Mono-infections constituted 73.4% of all infections; the dominant mono-infections on MSP-1 and MSP-2 were MAD20 (18) and ICI (09) respectively; the rare RO33 (01) was recorded. Double infections were 20.20% (09) with both markers, while only one triple infection was observed on MSP-1. An anopheles was found with two double infections, one on each of the two blocks. Eight of the 12 multiple infections were on MSP-1, five on both MSP-1 and MSP-2. In addition to the multiplicity of proteins in these vectors, size polymorphism was observed in alleles, indicating vector/parasite interactions and environmental variations. These results were compared to those from human sera.Item Angiotensin II type 1 receptor A1166C gene polymorphism and essential hypertension in the Efiks of Calabar(Academic Journals, 2009) Kooffreh, M. E.; Anumudu, C. I.; Akpan, E. E.Hypertension is a multifactoral disorder because of the interaction of risk genes and environmental factors. The angiotensin II is a well known vasoconstrictor that exerts most of its influence through the angiotensin Il type 1 receptor The A 1166C polymorphism is a single base substitution of adenine for cytosine at position 1166 in the 3' untranslated region of the gene. There are conflicting reports on the association of the Al166C polymorphism with cardiovascular diseases such as prevalent hypertension, left ventricular hypertrophy, and pregnancy induced hypertension. These variations were attributed to ethnic differences in different populations. We investigated the association of the All66C polymorphism with hypertension in 100 individuals from the Efik tribe who were matched for gender and sample size. PCR-RFLP analysis was carried out to determine the allele frequency of the gene. The genotype frequencies were 48, 2 and 47, 3 for the AA, AC genotypes respectively in the patient and control groups. No individual with the CC genotype was observed in the study population. The frequency or the C allele was 0.03 and 0.02 in the control and the patient population. The genotype and allele frequency did not conform to the Hardy-Weinberg theory. Using regression analysis, age and diastolic were positive predictors for SBP, r=0.0.50; systolic was the only predictor for DBP in the patient group. Diastolic was the only predictor for SBP, r= 0.656 while age and systolic were positive predictors for DBP r= 0.718 in the control group. Gender, BMI, Al166C polymorphism and other independent varilables were not predictors for SBP and DBP in the population. P= 0.05, odds ratio 0.65, 95% CI (0.13 to 3.44). The Al166C polymorphism is not an independent risk factor for essential hypertension in the study population.Item Cellular responses to modified Plasmodium falciparum MSP 119 antigens in individuals previously exposed to natural malaria infection(Springer, 2009) Okafor, C. M. F.; Anumudu, C. I.; Omosun, Y. O.; Uthaipibull, C.; Ayede, I.; Awobode, H. O.; Odaibo, A. B.; Langhorne, J.; Holder, A. A.; Nwuba, R. I.; Troye-Blomberg, M.Background: MSP1 processing-inhibitory antibodies bind to epitopes on the 19 kDa C-terminal region of the Plasmodium falciparum merozoite surface protein 1 (MSP119), inhibiting erythrocyte invasion. Blocking antibodies also bind to this antigen but prevent inhibitory antibodies binding, allowing invasion to proceed. Recombinant MSP119 had been modified previously to allow inhibitory but not blocking antibodies to continue to bind. Immunization with these modified proteins, therefore, has the potential to induce more effective protective antibodies. However, it was unclear whether the modification of MSP119 would affect critical T-cell responses to epitopes in this antigen. Methods: The cellular responses to wild-type MSP119 and a panel of modified MSP119 antigens were measured using an in-vitro assay for two groups of individuals: the first were malaria-naïve and the second had been naturally exposed to Plasmodium falciparum infection. The cellular responses to the modified proteins were examined using cells from malaria-exposed infants and adults. Results: Interestingly, stimulation indices (SI) for responses induced by some of the modified proteins were at least two-fold higher than those elicited by the wild-type MSP119. A protein with four amino acid substitutions (Glu27→Tyr, Leu31→Arg, Tyr34→Ser and Glu43→Leu) had the highest stimulation index (SI up to 360) and induced large responses in 64% of the samples that had significant cellular responses to the modified proteins.Item Optimization of the T-cell proliferation assay in fascioliasis using a non-radioactive method, the Alamar Blue Assay(2009) Anumudu, C. I.; Molehin, A. J.; Nwuba, R. I.T-cell proliferation studies are traditionally carried out with radioactive reagents or fluorescent reagents that require measurement with advanced technology instrumentation. We attempted to calibrate the optimal conditions suitable for the use of a non-radioactive assay for the measurement of a T-cell proliferation assay in bovine fascioliasis, but applicable to the study of other infectious diseases in our developing 'country setting, Crude antigen extract was prepared from 15 adult Fasciola gigantica flukes: Cellular responses were detected by the proliferatjon of peripheral blood mononuclear cells (PBMC) in response to stimulation by serial dilutions of the crude antigen extract. The results showed that the antigen dilution 1:1,600 gave the highest PBMC proliferative response (Stimulation Index, S.I = 1.10± 0:2). Percentage reduced Alamar Blue was 27.3-71.6%. This suggests that the cell-mediated immune response in bovine immunity to Fasciola infection may be reliably measured in our setting with the Alamar Blue Assay.Item Entomological indices of Anopheles gambiae sensu lato at a rural community in south-west Nigeria(National Institute of Malaria Research, New Delhi on behalf of Indian Council of Medical Research, 2009) Noutcha, M. A. E.; Anumudu, C. I.Background & objectives: Investigations were conducted to obtain key entomological indices of Anopheles gambiae s.l. at Igbo-Ora, a rural community in south-west Nigeria. Methods: Mosquitoes were caught daily for a week from rooms where tenants had slept the previous night in each of the four months June, July (2001), and August, September (2002). Anopheles gambiae s.l. sibling species were PCR-identified, the blood meal origin was determined by direct ELISA, and the circumsporozoite antigen by sandwich ELISA. Mean weekly rates were calculated. Results: The mean human biting rates were 0.90 and 1.6 in 2001 and 2002 respectively. The mean weekly anthropophilic rates for An. gambiae s.l. were 82 and 86% in 2001 and 2002 respectively; they were high in An. gambiae s.s., An. arabiensis and non-identified species in the complex. The mean weekly circumsporozoite rates were 6.70% in 2001 and 6.30% in 2002. The mean weekly entomological inoculation rates (EIR) were 4.95 and 5.05 in 2001 and 2002 respectively; the seasonal (6-month) rates were high: 128.7 in 2001 and 131.3 in 2002, compared to data from other rural communities on the continent. Interpretation & conclusion: The implications of these findings on the role of An. gambiae s.l. in the holoendemicity of malaria at Igbo-Ora are discussed.Item Cytokine profiles and antibody responses to Plasmodium falciparum malaria infection in individuals living in Ibadan, southwest Nigeria(Faculty of Medicine, Makerere University, 2009) Iriemenam, N. C.; Okafor, C. M.; Balogun, H. A.; Ayede, I.; Omosun, Y.; Persson, J. O.; Hagstedt, M.; Anumudu, C. I.; Nwuba, R. I.; Troye-Blomberg, M.; Berzins, K.Background: The ability of the host immune system to efficiently clear Plasmodium falciparum parasites during a malaria infection depends on the type of immune response mounted by the host. Study design: In a cross-sectional study, we investigated the cellular-and antibody responses in individuals with P. falciparum infection, in an attempt to identify immunological signs indicative of the development of natural immunity against malaria in Ibadan, Nigeria. Levels of IL-10, IL-12(p70), IFN-γ, and IgM, IgG and IgG1-4 subclasses in the serum of 36 symptomatic children with microscopically confirmed malaria parasitaemia and 54 asymptomatic controls were analysed by ELISA. Results: IFN-γ and IL-10 were significantly higher in the symptomatic children (p=0.009, p=0.025 respectively) than in the asymptomatic controls but no differences were seen for IL-12(p70). Estimated higher ratios of IFN-γ/IL-10 and IFN-γ/IL-12 were also observed in the symptomatic children while the asymptomatic controls had higher IL-12/IL-10 ratio. The mean concentration levels of anti-P. falciparum IgG1, IgG2, IgG3 antibodies were statistically significantly higher in the individuals >5 years of age than <5 years while anti-P. falciparum IgG3 antibodies were notably low in <5 years category. Children <5 years had higher IgM antibodies than IgG and the expression of IgG subclasses increased with age. Conclusion: Taken together, malaria infection is on a delicate balance of pro- and anti-inflammatory cytokines. The higher levels of IFN-γ seen in the symptomatic children (<6months) may be instrumental in immune-protection against malaria by limiting parasite replication. The observed variations in immunoglobulin subclass levels were age-dependent and exposure-relatedItem Antibody specificities of children living in a malaria endemic area to inhibitory and blocking epitopes on MSP-119 of Plasmodium falciparum(Elsevier, 2009) Omosun, Y. O.; Adoro, S.; Anumudu, C. I.; Odaibo, A. B.; Uthiapibull, C.; Holder, A. A.; Nwagwu, M.; Nwuba, R. I.Merozoite surface protein-119 (MSP-119) specific antibodies which include processing inhibitory, blocking and neutral antibodies have been identified in individuals exposed to Plasmodium falciparum. Here we intend to look at the effect of single and multiple amino acid substitutions of MSP-119 on the recognition by polyclonal antibodies from children living in Igbo-Ora, Nigeria. This would provide us with information on the possibility of eliciting mainly processing inhibitory antibodies with a recombinant MSP-119 vaccine. Blood was collected from children in the rainy season and binding of anti-MSP-119 antibodies to modified mutants of MSP-119 was analysed by ELISA. The MSP-119 mutant proteins with single substitutions at positions 22 (Leu→Arg), 43 (Glu→Leu) and 53 (Asn→Arg) and the MSP-119 mutant protein with multiple substitutions at positions 27 + 31 + 34 + 43 (Glu→Tyr, Leu→Arg, Tyr→Ser, Glu→Leu); which had inhibitory epitopes; had the highest recognition. Children recognised both sets of mutants with different age groups having different recognition levels. The percentage of malaria positive individuals (32–80%) with antibodies that bound to the mutants MSP-119 containing epitopes that recognise only processing inhibitory and not blocking antibodies, were significantly different from those with antibodies that did not bind to these mutants (21–28%). The amino acid substitutions that abolished the binding of blocking antibodies without affecting the binding of inhibitory antibodies are of particular interest in the design of MSP-119 based malaria vaccines. Although these MSP-119 mutants have not been found in natural population, their recognition by polyclonal antibodies from humans naturally infected with malaria is very promising for the future use of MSP-119 mutants in the design of a malaria vaccine.Item Detection of p53 codon 249 mutation in Nigerian patients with hepatocellular carcinoma using a novel evaluation of cell-free DNA(Mexican Association Hepatology, Medica Sur Clinic and Foundation, 2008) Igetei, R.; Otegbayo, J. A.; Ndububa, D. A.; Lesi, O. A.; Anumudu, C. I.; Hainaut, P.; Gorrnally, E.Objectives: This case-control study was done to determine the association and prevalence of p53 codon 249 mutation using cell-free DNA in the plasma of patients with hepatocellular carcinoma (HCC) in South-Western Nigeria. Method: Eighty-five adults with HCC and seventy-seven age and gender matched controls without evidence of liver disease or malignancy involving any part of the body, were recruited. Plasma DNA was analyzed for p53 codon 249 by restriction fragment length polymorphism. Patient evaluation was done by means questionnaire interview, clinical examination, laboratory and radiological tests. The prevalence of the p53 codon 249 mutation was expressed as a percentage amplifiable DNA samples analyzed from HCC patients while that of controls was expressed in the same way. Fisher’s exact test or the student t-test where appropriate were used to assess statistical significance of prevalence between both groups as well as comparison of some characteristics in the HCC cases between those who had codon 249 mutation and those who did not. Associations between the various parameters assessed were determined by odds ratio and significant difference was specified at p < 0.05. Results: p53 codon 249 mutation was present in 6 (7.6%) of the 79 samples from the HCC patients with amplifiable plasma DNA while none (i.e. 0%) of the 73 samples with amplifiable plasma DNA from the controls had this mutation. This prevalence is significantly higher among HCC patients than controls (0.029). The mutation was also found to be significantly associated with HCC (odds ratio = 2.00; 95% C I: 1.70 – 2.35). Conclusion: The prevalence of the p53 codon 249 mutation from plasma DNA of hepatocellular carcinoma patients is significantly higher than among controls in South-Western Nigeria and the presence of this mutation is significantly associated with HCC in this region.Item Epidemiology of malaria in children living at Igbo-Ora, South Western Nigeria(Faculty of Science Obafemi Awolowo University, Ile-Ife, Nigeria, 2008) Nwuba, R. I.; Omosun, Y. O.; Anumudu, C. I.; Adoro, S.; Odaibo, A. B.; Nwagwu, M.Malaria transmission is seasonal with higher transmission occurring in the rainy season. The burden of malaria falls mainly on children and causes anaemia and fever. Children of school going age are affected and this leads to absence from school. Blood samples were collected from children aged 10 days to 15 years in dry and rainy seasons. Parasite densities were determined by microscopy. Malaria prevalence was higher in the rainy season than in the dry season. In the dry season, 42.4% of the children studied were positive for P. falciparum. While at the end of the rainy season 48.4% of the children were malaria positive. The parasite prevalence was not significantly different between males and females. Parasite densities varied from 18 to 52174 parasites per 111 of blood. The most abundant group ranged from 1-100 (59%). There was a significant correlation between parasite density and age with the mean parasite density decreasing with age group. The study shows that malaria is more prevalent in the rainy season, and children in rural areas have high prevalence of asymptomatic parasitemia which might lead to symptomatic malaria. The results show that malaria immunity increases with age in both seasons.
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