Department of Biochemistry, Cell and Molecular Biology
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Item Mapping The Kinetics And Diversity Of Adaptive Immune Responses In Children Over The Course Of An Acute Plasmodium Falciparum Infection(University Of Ghana, 2021-07) Nyakoe, N.K.Naturally acquired immunity to malaria is Plasmodium species strain- and variant- specific. It is non-sterile and short-lived therefore requiring repeated exposure. These protective immune responses are directed against pre-erythrocytic parasites, blood-stage merozoites, or Plasmodium antigens on the surface of infected red blood cells and can be humoral or cellular. The importance of the various immune response components during malaria episodes in exposed populations remains unknown, as conclusive interpretation of data from previous studies has been limited by sampling design, disease definition, patient selection and approaches in natural infections. Long-term malaria protection in humans is likely to be determined by the cumulative effect of multiple low-level immune responses to various antigens, with each person having a unique "protective signature" based on their genetic background. Differences in transmission intensity observed in malaria-endemic areas significantly influence the acquisition of natural immunity to malaria. Antibody and cytokine responses, which contribute to malaria protective immunity, are exposure-dependent; hence, are significantly influenced by transmission intensity. Cytokine levels change rapidly upon exposure to malaria and often dictate the extent of clinical disease, whereas antibodies increase gradually and are long-lasting, and act to control and eliminate parasites. A variety of molecular mechanisms are involved in regulating these immune responses; however, it is still unclear what combination of immune features is essential for protection. Characterization of these host immune responses is important in understanding the bidirectional host-parasite interactions which will aid in effective vaccine development and eventual malaria control and elimination. This study employed a systems immunological approach to profile changes in the cellular, humoral, and molecular components of the immune system during acute P. falciparum infection and after treatment to identify immune signatures associated with varying malaria transmission intensity. Methods: Samples were collected from children of ages 5-14 years in two regions in Ghana with distinct malaria transmission intensities: Accra (low transmission) and Kintampo (high transmission). Whole blood samples were collected and a portion separated to obtain plasma from the participants when they present at the hospital with confirmed P. falciparum infection (Day 0), and convalescent phases (Day 7 and Day 21). Levels of 25 plasma cytokines were determined using a multiplex Luminex human magnetic 25-plex bead array kit. A custom-made protein microarray was used to detect IgG responses to 190 antigenic targets for blood-stage, pre-erythrocytic, and gametocyte stage parasite proteins. Antibodies and cytokine levels from the 2 sites were compared during infection and convalescent phases. Additionally, whole-blood transcriptomes were profiled by RNA-sequencing and differential gene expression compared during infection and convalescent in the study participants. Correlation and machine-learning classifier approaches were used to model immune responses to identify predictive responses correlating with distinct transmission intensities. Gene ontology modules associated with changes in immune responses in the two transmission areas were identified using gene set enrichment analysis. Results: Acute P. falciparum infection was associated with pro-inflammatory cytokines IL-6, IFN-γ, IFN-α, MIG, MCP-1, and IP-10, as well as an immunomodulatory profile mediated by IL-10, IL-2R and IL-1RA. In children from low transmission areas, they were significantly higher than in children in the high transmission areas, whereas, IFN-α, IL-6, IL-1RA, and MCP-1 were significantly associated with parasitemia. In addition, analysis of the correlation network revealed a distinctive signature between individuals from the low and high transmission areas. Of interest, is a subset of children from the high transmission area with detectable parasitemia at day 21 (D21) after treatment, with a unique cytokine signature dominated by IL-10 and IL-1RA. This was associated with asymptomatic parasitemia. Of the 190 Plasmodium antigens tested, 118 had antibody reactivity in more than 80% of the children in our study cohorts. Though overall breadth and magnitude of antibody responses was similar, the composition of these responses was different between individuals from high and low transmission areas. Hierarchical clustering revealed IgG response clusters to antigens associated with different malaria endemicity, with 48/118 antigens including EBA140, ETRAMP5, GLURP, MSP1 and MSP6 associated to low transmission area and 30/118 antigens associated to high transmission area, including Rh5.1, EBA181, VAR2CSA, ACS5, HSP40, MSP2, MSP3, MSP4, and MSP7. Machine-learning and feature selection approaches further predicted 17 antibody signatures (MSP4, MSP2 3D7, MSP1-19 2A, Etramp 5 Ag1 His var 3, MSP1-19 2B, Etramp 5 Ag1 His var 2, MSP2 [15-46b], MSP2 [5-36A], ACS5 Ag 3, PfMSP1_19, MSP3 FVO, Var2CSA, SE36/SERA 5 (T), HSP40 Ag 3, GLURP R2, Rh4.2_2030, and MSP2 CH150/9) that distinguish individuals from these two regions. Multivariable linear regression models predicted age and transmission intensity as factors that highly affect antibody reactivity to most antigens in our panel. Gene expression levels differ with transmission intensity More genes were differentially expressed during infection (D0) compared to convalesces (D7 and D21). Comparison of the DEGs between the two sites show that more genes were highly expressed in low transmission area compared to the high transmission area at D0. In the high transmission area, the DEGs enriched in gene ontology (GO) modules related to B cell and surface signature, T cell development and activation, platelet activation-actin binding, enriched cell cycle, and regulation of transcription and transcription factor modules, were up-regulated. In the low transmission area, there was DEGs in modules associated with cell cycle and transcription, immune activation- generic cluster, E2F1 targets, plasma cell surface signature, immunoglobulins, enriched monocytes, enriched NK cells, platelet activation, enriched neutrophils, enriched activated dendritic cells, NK cell surface signature, chemokine and inflammatory molecules in myeloid cells, Golgi membrane and TBA modules, were up-regulated. Cellular deconvolution revealed an increased proportion of neutrophils cell type in both high and low transmission areas, with decreased CD4 naïve, CD8, B-cells and T-follicular cells, during infection compared to convalescence in both high and low transmission. Our data identified transcription patterns that are distinguish by malaria transmission intensity, where most genes highly expressed in the high transmission area are those involved in adaptive immune response. While genes highly expressed in the low transmission area are those involve in the innate immune response. These data provide insight into molecular and cellular immune response kinetics in natural infection. Conclusion: The findings show that cytokine responses during active malaria infection varies significantly between individuals with differing levels of prior exposure, with individuals from low transmission settings having higher levels of pro-inflammatory cytokines. However, these differences are transient and do not persist during recovery. Whereas antibodies levels remained relatively stable across the timepoints for most antigens in our panel. We show that models trained to capture distinct antibody response patterns predicted 17 antibody responses that are key in distinguishing between individuals from different intensities. Given the important role that exposure plays in the acquisition of immunity these could be useful antigens as possible targets of protective immunity and provides clues for potential vaccine candidates that can be prioritized in evaluation. Transcriptomic analysis of whole blood over the course of infection revealed molecular signatures that can provide insights into the protective immune responses against P. falciparum infection. We identified genes whose expression patterns can help differentiate changes during acute P. falciparum infection and convalesces in varying transmission intensity. Taken together, this study predicts humoral and cellular signatures associated with the acquisition of naturally acquired immunity that can fairly distinguish individuals from two distinct transmission areas based on their immune profileItem Tolerance Of Plasmodium Falciparum To Artemetherlumefantrine In The Gambia(University Of Ghana, 2020-12) Mbye, H.Antimalarial drug resistance contributes significantly to obstacles in reducing the global burden of malaria especially in sub-Saharan Africa (sSA) where the disease is most prevalent. Resistance to artemisinin-based combination therapies (ACTs), the only recommended frontline drugs for the treatment of uncomplicated malaria is now widespread in South East Asia (SEA). However, ACTs remain efficacious in sSA though in vivo delayed parasite clearance and in vitro reduced susceptibility to both components of the drug has been reported. Resistance to ACTs is therefore anticipated especially with its sustained use in endemic regions and the recent report of the emergence of de novo Pfk13 mutation that is now spreading in Rwanda. In The Gambia where artemether-lumefantrine (AL) is the first-line drug used for over 10 years, a steady increase in parasite tolerance to lumefantrine (LUM) was observed over a period of 4 years which strongly correlated with reported directional selection on a cysteine desulfarase gene (Pfnfs1). These findings are concerning and require continuous drug surveillance to track spontaneous development of AL resistant parasites and determine pathways to resistance development. This study therefore sought to investigate the prevalence and mechanisms of parasite tolerance to AL in The Gambia. A novel ex vivo drug susceptibility assay suitable to simultaneously assess parasite responses to both drugs used in AL was developed and used to assess drug susceptibility profiles of circulating parasites in western Gambia. This assay was then used to confirm identified potent compounds from the Medicines for Malaria Venture pathogen box effective against the erythrocytic stages of the parasite for future development into new antimalarial drugs. The prevalence of known drug resistance markers was assessed and novel markers that could be associated with drug resistance identified using both regression analysis and GWAS approach. Finally, CRISPR-Cas9 genome editing was used to functionally validate Pfnfs1 for its involvement in LUM tolerance using gene editing approaches. Antimalarial drug resistance contributes significantly to obstacles in reducing the global burden of malaria especially in sub-Saharan Africa (sSA) where the disease is most prevalent. Resistance to artemisinin-based combination therapies (ACTs), the only recommended frontline drugs for the treatment of uncomplicated malaria is now widespread in South East Asia (SEA). However, ACTs remain efficacious in sSA though in vivo delayed parasite clearance and in vitro reduced susceptibility to both components of the drug has been reported. Resistance to ACTs is therefore anticipated especially with its sustained use in endemic regions and the recent report of the emergence of de novo Pfk13 mutation that is now spreading in Rwanda. In The Gambia where artemether-lumefantrine (AL) is the first-line drug used for over 10 years, a steady increase in parasite tolerance to lumefantrine (LUM) was observed over a period of 4 years which strongly correlated with reported directional selection on a cysteine desulfarase gene (Pfnfs1). These findings are concerning and require continuous drug surveillance to track spontaneous development of AL resistant parasites and determine pathways to resistance development. This study therefore sought to investigate the prevalence and mechanisms of parasite tolerance to AL in The Gambia. A novel ex vivo drug susceptibility assay suitable to simultaneously assess parasite responses to both drugs used in AL was developed and used to assess drug susceptibility profiles of circulating parasites in western Gambia. This assay was then used to confirm identified potent compounds from the Medicines for Malaria Venture pathogen box effective against the erythrocytic stages of the parasite for future development into new antimalarial drugs. The prevalence of known drug resistance markers was assessed and novel markers that could be associated with drug resistance identified using both regression analysis and GWAS approach. Finally, CRISPR-Cas9 genome editing was used to functionally validate Pfnfs1 for its involvement in LUM tolerance using gene editing approaches. Keywords: Malaria, antimalarial drug resistance, ex vivo drug assays, high throughput screening genotyping, functional analysis, association studiesItem Development Of DNA-Based Assays For Molecular Surveillance And Point-Of-Care Diagnosis Of Non-Falciparum Malaria(University Of Ghana, 2022-09) Ansah, F.Background In recent years, the clinical significance of Plasmodium malariae and Plasmodium ovale is increasingly gaining public health attention as the global transmission of falciparum malaria is decreasing. However, the most readily available and cost-effective malaria diagnostic tools, which include microscopy and antigen-based rapid diagnostic tests, lack adequate sensitivity and specificity for accurate surveillance and point-of-care diagnosis of these non-falciparum species. This challenge poses a major setback to global efforts aimed towards malaria control and elimination. In this study, DNA-based assays with improved sensitivity and specificity were developed for species-specific detection of P. malariae and P. ovale. Methods SYBR Green-based real-time quantitative polymerase chain reaction (qPCR) assays were developed for the detection of P. malariae and P. ovale using a new set of primers called cooperative primers. The cooperative primer-based qPCR assays were used in a cross-sectional study to determine the prevalence rates of P. malariae and P. ovale using field samples obtained from three malaria transmission settings in Ghana. Following this, the associations between P. malariae and P. ovale infections and haematological indices were assessed. For point-of-care diagnosis of P. malariae and P. ovale associated malaria, in-house real-time loop-mediated isothermal amplification (RT-LAMP) assays were developed and the diagnostic performance of the RT-LAMP assays were compared to the cooperative primer-based qPCR assays. In addition, label-free DNA-based electrochemical biosensors were developed for ultrasensitive detection of P. malariae and P. ovale. Results Both the P. malariae and the P. ovale cooperative primer-based qPCR assays had a detection limit of approximately 1.0 parasite/μL, which is at least 10-fold lower than the corresponding conventional primer-based qPCR assays. Using the cooperative primer-based qPCR assays in a cross-sectional study, the prevalence rates of P. malariae and P. ovale infections among the combined study population were 13.3% and 4.8%, respectively. Notably, study participants harbouring mixed infections of P. falciparum with either P. malariae or P. ovale had the greatest risk of developing anaemia. The diagnostic sensitivity and specificity of the in-house RT-LAMP assays were in the range of 86.2% - 97.5% when compared to the cooperative primer-based qPCR assays. Remarkably, both the P. malariae and the P. ovale DNA-based biosensors showed a sensitivity of 100% using purified genomic DNA samples. However, the specificities of the biosensors were 100% and 66.7% for P. malariae and P. ovale, respectively. Conclusion In summary, the results demonstrate that the DNA-based assays described in this study have adequate sensitivity and specificity for the detection of P. malariae and P. ovale in clinical isolates. This study highlights the importance of including detection tools with lower detection limits in the routine surveillance and point-of-care diagnosis of non-falciparum species. The availability of reliable and cost-effective species-specific detection tools for P. malariae and P. ovale will be necessary for comprehensively assessing the effectiveness of malaria interventions and control measures aimed towards global malaria elimination.Item Understanding The Mechanisms Of Anti-Inflammatory Activities Of Cryptolepine(University Of Ghana, 2020-10) Abdulrahman, A.R.Although inflammation coordinates host immunity against infectious pathogens and alarmins, its dysregulation results in severe pathological conditions. Dysregulated inflammatory responses come about as a result of many factors including hyperactivity of pro-inflammatory signaling pathways. Hyperactivity of the Toll like receptor (TLR) - nuclear factor kappa B (NF-κB) signaling pathway is reported to be associated with many inflammatory diseases, which makes the pathway a good therapeutic target. Although cryptolepine, an alkaloid obtained from Cryptolepis sanguinolenta, has been demonstrated to possess anti-inflammatory properties in vivo, its mechanisms of action are not fully understood. This study sought to determine the anti-inflammatory effects of cryptolepine and its underlying mechanisms of action in a murine macrophage cell line (RAW Blue cells) stably transfected with secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of a promoter inducible by NF-κB transcription factor. The cytotoxic effect of cryptolepine on the cells was determined by 3-(4, 5Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. The activity of the TLR1/TLR2-NF-κB signaling pathway was induced with Pam3CSK4 (100 ng/mL) in the absence or presence of cryptolepine (0.5 - 1 μM) for 24 hours. Afterwards, culture supernatants were harvested and the activity of the pathway measured by assessing the levels of SEAP using Quanti-Blue assay. In addition, the effect of cryptolepine on the transcript levels of Tnf-α, Il-6, Il-1β, Il-23, Ccl2, Cxcl2, Ikbkb, Nfkb1, Rela, Tlr2 and Tlr1 were assessed by RT-qPCR and the levels of TNF-α, IL-6, IL-1β, IL-23, MIP-2α and MCP-1 determined using multiplex ELISA technique. Compared to untreated cells, cryptolepine-treated cells showed a dose-dependent decline in viability with 1 μM being the maximum non-toxic concentration of cryptolepine. Cryptolepine (0.5 – 1 μM) also dose-dependently inhibited the TLR1/TLR2-NF-κB signaling pathway activity. Additionally, the transcript levels of Tnf-α, Il-6, Il-1β, Il-23, Mip-2α, Mcp1,Tlr1, Tlr2, Rela, Ikkβ and Nf-κb1 were all attenuated by cryptolepine. Cryptolepine also suppressed the protein levels of TNF-α, IL-6, IL-23, MCP-1 but not IL-1β and MIP-2α. In summary, the results from this study demonstrated that cryptolepine inhibited the activity of the TLR1/TLR2 signaling pathway, attenuated the transcript levels of key signaling molecules and suppressed the levels of key pro-inflammatory cytokines and chemokines, which suggest that cryptolepine may be a good anti-inflammatory therapeutic agent.Item Construction Of Potent Immunogenic Epitopes Of The Haemagglutinins Of The Seasonal Influenza A Viruses(University Of Ghana, 2021-01) Kotey, E.N.Seasonal influenza viruses are renowned for recurring annual epidemics worldwide. The Influenza A subtypes H1 and H3 are the most dominant and prevalent in recent outbreaks in humans. As with other infectious diseases, vaccines are an important public health tool. However, influenza viruses continue to evolve evading pre-existing or transient vaccine-induced immunity in addition to antigenic pressures associated with antiviral drugs. For this reason, current seasonal influenza vaccines require annual review. Vaccine (immunogen) design, efficacy, and effectiveness presents a formidable seasonal influenza management issue. Passive immunotherapy has been proposed to offer tremendous protection when appropriately used in the management of influenza, either as a substitute or to complement vaccines. A well-designed immunogen that elicits a strong antibody response towards the conserved domains of the surface Haemagglutinin (HA) protein would be critical to avert virus evolution. A detailed analysis of the highly conserved regions spanning the fusion peptide, cleavage site, and the two heptad repeats for the HA gene in over 1000 and 21,000 H1 and H3 strains, respectively was therefore conducted. Chimeric haemagglutinins (cHAs) of these conserved regions were constructed by alignment with consensus sequences generated from exotic HAs (H5 and H9 for H1-based cHAs; H7 for H3-based cHA). These cHAs were successfully expressed in Drosophila S2 cell lines. Mice were then immunized with these cHAs to determine protection against lethal doses in virus challenges against H1 and H3 seasonal viruses. Serum from seroconverted mice applied in challenge experiments indicated the presence of anti-HA specific antibodies with broadly cross-reactive potential against H5 and H7 viruses for H1 and H3-based cHAs, respectively. This study offers an alternative approach whereby multi-subtype or pan-group immunogens could be utilized for the design and generation of cross-reactive antibodies of potential therapeutic value for influenza in humansItem The Genetics Of Congenital Non-Syndromic Hearing Impairment In Ghana(University of Ghana, 2020-07) Adadey, S.M.Abstract Background: The partial or total inability of an individual to hear sound is known as hearing impairment (HI). Globally, over 466 million people are living with HI, with the majority of cases from developing countries. Although over 123 genes have been associated with HI, only one gene (GJB2) has been studied in Ghana. This thesis therefore sought to investigate variants in GJB2, GJB4, GJB6, and GJC3 genes that are associated with HI in Ghana as well as to investigate other non-genetic causes of HI among Ghanaian children. Method: Hearing-impaired students from 11 schools for the deaf and Adamorobe, a village in Ghana, were enrolled and categorized as familial or non-familial. Control participants who did not have any known family history of HI were also enrolled. DNA was obtained from the blood samples collected from all the participants in 81 families from the schools for the deaf, 8 families from Adamorobe, and 166 non-familial cases. From the DNA samples, the regions that code for GJB2, GJC3, and GJB4 proteins were polymerase chain reaction (PCR) amplified using specific primers, Sanger sequenced and analyzed. The large genomic deletion of the GJB6 gene (GJB6-D3S1830) was investigated using multiplex PCR and confirmed with Sanger sequencing. A rapid diagnostic test was designed for GJB2-p.Arg143Trp (rs80338948) using restriction fragment length polymorphism (RFLP) and NciI restriction enzyme. The test was optimized and validated using Sanger sequencing. Bioinformatic tools and online databases were employed in predicting the clinical significance/pathogenicity of the identified variants in the connexin genes. In silico protein modeling techniques were used to model the protein structure for a likely pathogenic GJB4-p.Asn119Thr (rs190460237) mutant and wild-type proteins. The ligand-binding properties of the modeled proteins were studied. Results: The hearing-impaired participants enrolled on the study had severe to profound HI with the majority (68.3%) of them having prelingual HI. Nearly all the prelingual HI cases seemed congenital based on their parent’s reports, however, 54% of the hearing-impaired students received the first comprehensive hearing test when they had grown past the age of language development, thus between the ages of 6-11years. Cerebrospinal meningitis (CSM) was found to be the most frequent environmental cause of HI. The genetic analyses revealed that GJB2-Arg143Trp accounted for HI in 25.9% of the families studied, and 7.9% of isolated cases. A carrier frequency of 1.4% was estimated from randomly selected hearing controls in Ghana. Seven out of eight families from Adamorobe tested positive for the GJB2-Arg143Trp founder mutation. We were the first to report the presence of a GJB2-Trp44Ter variant in a hearing-impaired family in Ghana. To facilitate rapid screening of the variant within the population, a rapid GJB2- p.Arg143Trp-Nci-RFLP test was developed and found to be 100% sensitive, with no false positive or false negative observed. The test is highly specific for any variant within the recognition site of the restriction enzymes; however, it cannot differentiate between these variants. When screening other genes associated with HI, we identified one GJC3 variant that may not associate with HI. Also identified were seven GJB4 variants, of which 5 were predicted to be as either benign or synonymous, and the remaining 2 were predicted likely pathogenetic. One of the two variants may not be associated with HI because the variant’s homozygous form was observed in both patients and controls. We modelled the protein structure and function of the other likely pathogenic GJB4 variant (p.Asn119Thr) and found subtle but important alterations in the structure and binding characteristics of the mutated protein compared to the wildtype. Conclusion: We have obtained many important results through these studies. We have confirmed meningitis as the major cause of environmental HI in Ghana. Variations within the GJB2 gene account for the most HI cases of genetic origin in Ghana and hence we have identified the need to include GJB2 gene investigations in the national newborn hearing screening (NHS) program. The GJB2- p.Arg143Trp-Nci-RFLP test will therefore be instrumental in this capacity. Finally, based on data presented in this thesis, GJB4, GJB6, and GJC3 gene variants were not likely associated with HI in the Ghanaian population.Item The Detection and Molecular Characterisation of Bacterial Symbionts of Anopheles Gambiae S.L.(University of Ghana, 2003-09) Brown, C.A.The aim of the present study was to identify and characterise bacterial symbionts in An. gambiae s.l. mosquitoes, and ultimately select one that occurs in all its life stases. Mosquito larvae and pupae samples were collected from six locations in the Greater Accra region of Ghana and some reared to adults in the laboratory. Wild adult An. gambiae mosquitoes from Navrongo and Dodowa (Ghana) and Jaribuni (Kilifi District, Kenya) and laboratory colonies of adult An. gambiae mosquitoes from Kilimanjaro (Tanzania), Suakoko (Liberia) and Kisumu (Kenya) and An. lU'abknsu (Wqcningen SU'ain) were alw studied.Item Chemokine Mediators of Cerebral Malaria: A Comparative Study of the Role of Rantes in Murine and Human Cerebral Malaria(University of Ghana, 2004-09) Sarfo, B.Y.Although the involvement of cytokines and adhesion molecules in malaria-induced brain inflammation has been established, the role of chemokines and chemokine receptors remain unclear. This unexplained component of cerebral malaria (CM) pathology may be responsible for the heterogeneity in the observed features of CM and the difficulty in its characterization. RANTES (regulated on activation normal T cell expressed and secreted), a chemokine involved in the generation of inflammatory infiltrates plays a special role in the modulation of inflammation. Trafficking of inflammatory T helper 1 (THL) cells into the brain is mediated partly by RANTES interactions with c-c chemokine receptor 5 (CCR5) receptor. The main hypothesis of this study are:(a) RANTES and corresponding receptors mediate malaria induced brain immunopathogenesis (b) Blocking RANTES which is upregulated during malaria infection will abrogate or minimize the outcome of the disease. Studies were directed at i) Characterizing and analyzing the expression of RANTES and corresponding receptors CCR1, CCR3 and CCR5 in a mouse model of CM (SW mice/P.yoelii 17X) using eDNA microarray. RT-PCR and Western Blot analyses ii) Evaluating effect of P.yoelii 17X infection on mouse brain by electron microscopy and immunohistological analyses iii) Evaluating expression of RANTES and receptors in cerebellum, cerebrum, brain stem and hippocampus of post-mortem CM and non-malaria (NM) tissue samples using RT-PCR and Western Blot analyses iv) Comparing and contrasting levels of RANTES in plasma of rodent malaria model and malaria-positive human subjects using ELISA v) Determine the functional role of RANTES by anti-RANTES antibody blocking experiment using P.yeolii 17X infected mice. Transcriptional analyses results indicate significant upregulation of chemokines; macrophage inflammatory protein-2a(MIP-2a.), monocyte chemotactic protein-1 (MCP-1) and RANTES chemokine receptors; CCR1, CCR3, and CCR5, adhesion molecules; platelet endothelial cell adhesion molecule-1 (PECAM-1), intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule-l (VCAM-l) and cytokines; intereferon-gamma (INF-y), tumour necrosis factor-alpha (TNF-a) and interleukin-12 (IL-12) at peak parasitemia during P.yoelii 17X infection. Western Blot analysis revealed upregulation of RANTES protein in P.yoelii 17X infected mouse brain. Ultrastructural analysis showed that P.yoelii 17X infection induces perivascular oedema in cerebellum in mouse brain at peak parasitemia. lmmunohistological analysis demonstrates high immunoreactivity of glial fibrillary acidic protein (GFAP) ill P.yoelii l7X infected mouse brain. RANTES, CCR3 and CCR5 but not CCR1 mRNA are significantly upregulated in the cerebellum and cerebrum (P < 0.0001) in CM than NM samples. There were no changes in the expression of CCR1, CCR3 and CCR5 mRNA in brain stem and hippocampus of CM and NM. RANTES mRNA expression in cerebellum and cerebrum is highly significant(p < 0.0001) compared with the brain stem(p = 0.0018) and hippocampus (p=0.0027) in CM group. CCR5 and RANTES proteins were significantly upregulated in cerebellum (P < 0.0013 for CCR5, P < 0.0001 for RANTES) and cerebrum (P < 0.0124 for CCR5, P < 0.0001 for RANTES) but not brain stem and hippocampus of CM than in NM. Western Blot analysis could not detest CCR3 protein RANTES was significantly upregulated in plasma of murine malaria model and malaria positive subjects compared with controls. RANTES concentration in plasma correlated with P. falciparum infection. At day of sacrifice. level of parasitemia (4.2x 10^6/ml ±O.2) in mock antibody treated mice was higher (P < 0.05) than in mice in which RANTES was blocked with anti-RANTES antibody(1.2 x 10^6/ml ± 0.2). Anti-RANTES antibody treated mice survived longer (14 days) than mock antibody treated mice (10 days). This is the first temporal study of murine malaria associated RANTES and receptors CCR1, CCR3 and CCR5 expression. P.yoelii 17X murine malaria model is useful in characterizing differentially expressed genes associated with human clinical malaria. There is an association of RANTES expression in malaria-induced brain immunopathogenesis and endothelial lesions in infected mice. Cerebellum and cerebrum in humans were the focal points for increased malaria-induced RANTES and CCR5 expression. Active sequestration of infected red blood cells (IRBCs) and platelets in addition to leukocytes in these regions of the brain could exacerbate CM immnopathology. Blocking of RANTES decreased parasitemia and mortality associated with P.yoelii 17X infection.Item Molecular Epidemiology of Noroviruses in Ghanaian Children(University of Ghana, 2020-07) Lartey, B.N.L.Background: The human noroviruses are a highly diverse group of diarrheagenic RNA viruses which are globally distributed, and a cause of acute gastroenteritis in all age groups with the elderly and young children usually experiencing severe clinical outcomes. To date, at least 40 different genotypes of noroviruses belonging to two major genogroups have been observed in humans. These different genotypes have been suggested to be associated with different transmission patterns and disease severity in humans. Also, host genetic factors including the presence of ABO antigens and mutations in the fucosyltransferase (FUT2 and FUT3) genes affect the susceptibility of individuals to infection with these diverse norovirus genotypes. This has raised questions on whether the prevalence, as well as the percentage of circulating Histo-Blood Group Antigen (HBGA) mutations within a population, would influence the prevalence of specific norovirus genotypes as a function of their ability to infect certain HBGA types. Moreover, the continuous changes observed in the genetic diversity of the noroviruses highlight the need for sustained surveillance to provide a full overview of norovirus epidemiology for future vaccine policy decisions. The overall aim of this thesis was to get a better understanding of norovirus infection dynamics, strain diversity, evolutionary dynamics, and the host genetic factors associated with the risk of norovirus infection in the Ghanaian pediatric population. Methods: A chronologically comprehensive 10-year study was conducted with diarrheic stool samples collected during active surveillance for diarrhoea in Ghanaian children between January 2008 and December 2017. A total of 1,337 stool specimens were obtained and subjected to RT-PCR and partial nucleotide sequencing for the typing of the polymerase and capsid genes of the norovirus genome. Phylodynamic and evolutionary relationships were performed using MEGA 6.0 and BEAST software, respectively, to analyze sequences that overlapped at open reading frame (ORF) 1 and ORF2 regions. The entire coding region of the FUT2 gene was also amplified from saliva samples collected from a cohort of the study population and genotyped using RLFP. Results: Overall positivity for norovirus infection among the Ghanaian pediatric population was 36.2%. Infection was most commonly (82.7%) observed in children aged between 6-24 months, suggesting that 0-6 months would be the most appropriate age range for effective norovirus vaccination, as early prevention is critical. Results from this thesis showed broad norovirus genotype diversity characterized by the circulation of both GII.4, and non-GII.4 strains. The evolution of these norovirus strains was usually a result of both intra- and intergenic recombination that occurred within the capsid and polymerase genes. A total of 25 capsid/RdRP combinations were detected with GII.4[P4] (25.9%); GII.4[P16] (9.2%); GII.3[P21] (6.3%); GII.4[P31] (4.6%) and GII.6P[7] (4.0%) being the most common norovirus strains. Children infected with non-GII.4 norovirus strains recorded equally severe clinical illness comparable to that caused by GII.4 norovirus strains. Data from this thesis also suggest that most norovirus strains circulated at low prevalence within the population before their recognition as epidemic and pandemic strains associated with increased norovirus outbreaks. The study results further indicate that neither secretor status nor genotype difference affects the susceptibility of an individual to norovirus infection in Ghana. More than half (62.0%; 8/13) of symptomatic patients were found to be carriers of the G428A mutation for inactivation of the FUT2 enzyme. Comparing norovirus genotype GII.4 with non-GII.4 genotypes, we observed that GII.4 norovirus strains infected more secretor-positive children who possessed heterozygous allele of the FUT2 gene than non-GII.4 strains (60.0% vs 40.0%, p=0.035) whereas non-GII.4 norovirus strains had a preference for secretor-positive children with the homozygous allele of the gene (62.5% vs 37.5%, p<0.05) in the study population. Conclusions: In summary, the study confirmed the significant role that noroviruses play in the cause of acute gastroenteritis among Ghanaian children and further contributes to our understanding of the epidemiology and evolution of the virus which hopefully can lead to better preventive measures for norovirus disease as well as baseline data for vaccine policy decisions. Since the epidemiology of norovirus changes rapidly, the establishment of systematic surveillance within sentinel sites across the country would enhance the monitoring of circulating norovirus strains and allow us to have a continuous understanding of the current state of norovirus infection within our settings.Item Transcriptional Regulatory Patterns of Activation-Induced Cytidine Deaminase (AID) Expression in the Context of Plasmodium Falciparum Infection(University of Ghana, 2019-07) Ayivor-Djanie, R.Activation-Induced cytidine Deaminase (AID) initiates two important immunoglobulin gene modification processes in B lymphocytes; somatic hypermutation (SHM) and Class Switch Recommendation (CSR). Despite this important function, the aberrant expression of AID is associated with a number of cancers. Although the regulation of AID is strict and complex, there is evidence that Plasmodium falciparum infections can lead to a deregulated expression of the enzyme. It is not known how infection with the malaria parasite mediate this deregulated expression and activity. The aim of this work was to investigate the transcriptional regulatory patterns of AID and transcriptional factors (TFs) associated with AID expression. First, the transcriptional regulatory patterns of AID expression in P. falciparum-exposed geminal center Bcells was investigated in tonsillar mononuclear cells (MNCs) obtained from six (6) children from a malaria holoendemic region (Ghana) and one (1) child from a malaria free region (New Mexico, USA). Total MNCs were sorted into four distinct sub-populations; naive B lymphocytes (NB), germinal center B lymphocytes (GC), memory B lymphocytes (MB) and plasmablasts (PB). The expression patterns of AID and its alternatively spliced variants, as well as the AID related TFs, Pax5, HoxC4, Bcl6, Bach2, Irf8, Prdm-1, XBP-1, miR181b and miR155 were investigated in the B-cell sub-populations by qRT-PCR. The Ghanaian tonsils had GC B-cell frequencies ranging from 14% to 51%, and GC B cells accounted for only 7% of the total MNCs in the New Mexican tonsil. While the expression of positive AID TFs Pax5, HoxC4, Bcl6, Bach2 and Irf8 were significantly upregulated (p < 0.0001), negative regulatory TFs of AID, Prdm-1 and XBP-1 were significantly downregulated (p < 0.0001) in GC B cells compared to PBs. Together with the fulllength AID mRNA transcript (AIDFL), AID-ΔE4, AID-ΔE4, AID-ΔE4a, and AID-ivs3 were upregulated (p < 0.0001) in GC B cells compared to NB, MB and PBs. The expression of miR181b was down-regulated in all the B-cell subsets, but miR155 was significantly (p < 0.0001) downregulated in both MB and GC B lymphocytes. The expression patterns of AID and related TFs were investigated in children with asymptomatic P. falciparum infection. In asymptomatic infected children, significantly (p = 0.00197) higher levels of AID transcripts were observed compared to uninfected children, independent of the EBV infection status. The expression of Bcl6, Pax5 Prdm- 1 and Irf4 were also higher in children with asymptomatic infection compared to uninfected children, but the expression of XBP-1 and Irf8 did not vary significantly (p > 0.05). Lastly, the expression patterns of AID and related TFs were assayed in total white blood cells (WBCs) isolated from whole blood of children between ages 0 -3 years with either severe malaria anemia (SMA), uncomplicated malaria (Non-SMA) or febrile but aparasitemic (AP). Elevated AID transcripts levels were observed in children with SMA, which was not associated with Hb levels (Pearson’s R, p value: AP = -0.36, 0.35, non-SMA = 0.26, 0.16 and SMA = 0.09, 0.62), fever, or body temperature (Pearson’s coefficient, R, p: AP = 0.29, 0.36, Non-SMA = 0.20, 0.24 and SMA = 0.16, 0.38). The levels of AID transcripts correlated with parasite loads in children with SMA (Pearson’s R = 0.41, p = 0.018), but not with titers of IgG (Pearson’s coefficient, R, p: Non-SMA = 0.1946, 0.6022 and SMA = -0.0414, 0.1903) and IgM (Pearson’s coefficient, R, p: Non-SMA = -0.1241, 0.6022 and SMA = 0.26, 0.1903) to EBV viral capsid antigen (VCA). While Irf8 and XBP-1 levels were higher in P. falciparum infected children, irrespective of the SMA status (p < 0.01), Pax5 transcript levels were higher (p < 0.01) in SMA children than non-SMA children. The expression levels of Prdm-1 and Irf4 were not different (p > 0.05) among SMA, non-SMA and Aparasitemic children. The expression of miR181b was lowest in SMA children, and miR155 levels were not different in all three groups. All four splice variants of AID were significantly higher in SMA children than non-SMA and aparasitemic children. In conclusion, enhanced AID transcription in GC B lymphocytes from Ghanaian tonsils was associated with elevated levels of Pax5, HoxC4, Bcl6, Bach2 and Irf8, as well as the down-regulation of miR155 and the expression of AID alternative splice variants. P. falciparum infection is associated with increased levels of AID and Pax5 transcripts in both asymptomatic and SMA children. This study is the first to document the effect of asymptomatic and acute P. falciparum infection on the transcriptional regulatory patterns of AID.
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