121 Records out of 22207 Records

Combination biomarkers for diagnosis of latent tuberculosis infection

Author: Kibanya, Michael M

Awarding University: University of Nairobi, Kenya

Level : MSc

Year: 2012

Holding Libraries: University of Nairobi Jomo Kenyatta Memorial Library ;

Subject Terms: Medical immunity/Cytokines/Tuberculosis/Immunology ;

Abstract:

Pulmonary tuberculosis is a worldwide public health problem affecting about 10 million individuals globally, causing 1.6 million deaths annually, despite availability of inexpensive effective ant TB therapy. Sub Saharan part of Africa is the'most affected .especially as a result of HIV/AIDS pandemic as evidenced by high HIV prevalence among TB patients. Kenya has been ranked as a high burden TB country, with TB as a major cause of morbidity and mortality especially in the most productive age group of 15-44 years. Some socio economic factors like poor housing in peri urban slums contribute to disease transmission. The etiologic agent, Mycobacterium tuberculosis, is transmitted through droplets of respiratory secretions from infected persons to other individuals. About 90% of individuals exposed to the bacilli contain the infection for decades and therefore become latently infected. Latently infected persons are at risk of reactivation resulting to clinical disease. Studies on mRNA gene expression of IL-402 and Rab- 33A in latently infected individuals clinical TB patients and healthy controls have previously shown differential expression of these genes. This suggests their involvement in an additional adaptive immune mechanism to TB. The aim of this study was to investigate a combination of these protective immune markers in latently infected persons for possible utilization in TB control programs. Objectives ? To quantify changes in fold mRNA gene expression of small GTPase Rab33A and IL- 4 delta 2 as diagnostic markers of latent tuberculosis infection. ? To compare changes in fold mRNA gene expression of small GTPase Rab33A with IL- 4 delta 2 in diagnosis of latent tuberculosis infection. ? To establish the predictor of diagnosis of latent tuberculosis using changes in fold mRNA gene expression of Rab 33A and IL-402. ? To establish the association of exposure to development of latent tuberculosis infection. Materials and Methods Using a cross sectional study design,64 sputum smear positive patients consecutively diagnosed of T. B. at Mbagathi District Hospital TB clinic ,39 of their household contacts and 30 healthy controls from University of Nairobi level 2 medical students were recruited into the study. A structured questionnaire was used to collect socio demograghic, exposure and clinical data: Four milliliters of venous blood samples were also taken aseptically using vacutainers at recruitment. Total RNA was separated from the whole blood samples using QIAmp RNA blood minikit (Qiagen), and then quantified using NanoDrop1000 UV. Primers and probes were designed using published sequences and purchased from Bioneer. Assays were done using Agpath 10 RT-PCR kit and Power SYBR Green RT- peR master mix (ABI). Relative quantification method was used to determine change in gene expression of Rab 33A, IL-4, and IL-402 using Ct values. Data entry was done using Excel software and then analyzed using STAT A 11. Results The study found significant up regulation of interleukin 4 (IL-4) mRNA gene expression in tuberculosis patients as compared to their close contacts and controls with median rank 77.97 (p <0.001). Interleukin 4delta 2 (IL-402.) mRNA gene expression was significantly higher in contacts as compared to tuberculosis patients and controls with median rank 101.18, (p <0.001). Rab 33A protein mRNA gene expression was higher in contacts than tuberculosis patients and controls although the difference was not sig~ificant. (p 0.127) Exposure characteristics had no significant association with increased IL-402.mRNA gene expression in contacts. Conclusion : Elevated production of interleukin 4 delta2 (IL-402) can be used .as a pointer of robust immunity against TB and as index of suspicion of latent TB infection. It is not sufficient alone as a diagnostic tool for latent TB.

Polyepitope design optimization system : a web based application prototype

Author: Maingi, Julius Kairithia

Awarding University: University of Nairobi, Kenya

Level : MSc

Year: 2012

Holding Libraries: University of Nairobi Jomo Kenyatta Memorial Library ;

Subject Terms: Immunology ; Bioinformatics ; Antigens ; Vaccines ;

Abstract:

Immunogenic epitopes in a polyepitope construct may induce immunity against multiple antigenic targets only if epitopes are correctly processed and presented. Applications that combine multiple supertypes and integrate variables that measure the quality of polyepitopes can be used to make better polyepitopes yet there are no publicly accessible tools. In this research project, the researcher developed a web-based polyepitope optimization prototype. The prototype runs on 3-tier application architecture: MYSQL was used for the database tier, PHP for the application tier that runs on Apache HTTP server and the presentation layer was implemented on a web browser. The researcher collected epitopes data and information from online databases; the Immune Epitope Database (IEDB), HIV molecular immunological database and National Center for Biotechnology Information (NCBI).The epitopes were analyzed using Microsoft Excel 2003 and uploaded onto a local database. Polyepitopes were generated, optimized and tested on an online web server NetChop 3.1 which was used to validate one of the polyepitope quality measurement variables- proteasome cleavage predictions. The results of data analysis indicate that the immune system recognize epitopes in clusters and the main clusters are 9- mers(9 amino acid long). 15mers,2Omers and 25mers. The polyepitope optimization results indicate that polyepitope optimization algorithms that integrate proteasome cleavage prediction, transporter associated with antigen processing (TAP) binding prediction and Major Histocompatibility Complex (MHC) binding predictions can be used to develop better polyepitopes which can be used to make better vaccines within a shorter time and at a lower cost. Key words: polyepitope, epitopes, optimization, mer, TAP, proteasome, MHC, prototype.

Comparison of immune with sputum smears status in Tuberculosis patients co-infected with Human Immunodeficiency virus

Author: Wapang'ana, Job Kisuya

Awarding University: Moi University, Kenya

Level : MSc

Year: 2010

Holding Libraries: Moi University Margaret Thatcher Library ;

Subject Terms: Tuberculosis/HIV infection/Diagnostic tests/Immunology ;

Abstract:

To compare the immune profiles with sputum smear status in pulmonary tuberculosis patients co-infected with human immunodeficiency virus. Background The interaction between mycobacterium tuberculosis and HIV results in more rapid progression of tuberculosis (TB) and HIV -induced immunosuppresion. Diagnosis of TB in these patients is more difficult due to its atypical presentation. There are contradicting results from studies investigating TB diagnostic methods and immune profiles. The study aimed at comparing the diagnostic methods (Direct AFB smear, Concentration smear and Immunofluorescence microscopy) with immune profiles in adult pulmonary tuberculosis patients co-infected with HIV. Study Area: AMPATH site, MTRH Study Design: It was a Cross sectional study. Methods: Blood and sputum samples from 73 eligible subjects were analyzed in the laboratory. The blood samples were analyzed by FACScalibur flow cytometer and coulter counter to determine immunophenotype and complete blood counts of the patients. Smear status was determined by direct microscopy and immunofluorescence microscopy. The laboratory results were then coded and analyzed using SPSS version 12.01 for statistical analysis. Standard statistical procedures including frequency tables, crosstabulation and measures of central tendency (Mean (sd) and Median (IQR) were employed. Kappa statistic was' used to determine the agreement between tests. A pvalue less than 0.05 was considered statistically significant. Results The mean age was 36.22 years (Sd 9.45). The median CD4 count was 94 cells/ul (IQR 25 cellsl Ill, 194 cells /ul). There was a substantial agreement between direct AFB microscopy smears and immunofluroscence AFB smears tests (kappa = 0.707, 95% CI [0.513-0.760]). The sensitivity of direct AFB smear test was 0.68 (95% CI [0.0561-0.713]) and its specificity was 0.979 (95% CI [0.917-0.996]). The positive predictive value (PPV) and the negative predictive value (NPV) were 0.944(95% CI [0.780-0.990]) and 0.855 (95% CI [0.801-0.869]), respectively. The immune cell markers medians of females were higher compared to males except CD 19. With regard to blood cells, males had higher median RBC, HGB, HCT and monocyte while the females had higher median in WBC, PLT, lymphocyte and eosinophils. There was a significant difference in the median of CD 19 between AFB positive smears and AFB negative smears among suspected TB co-infected patient (p=0.006). Significance of Findings There was a decline in levels of CD4 and that the levels of CD8 counts remained unchanged (within the reference ranges) among these patients. There no was no significant differences among the two CD markers in relationship to the smear status of the patient. It is possible that the B cells might have a role in the immunity against tuberculosis, though this could not be conclusive proved in the study. This was due to the significant difference in the relation of B cells and the smear status of the patients. Furthermore sensitivity of AFB direct microscopy was lower in TB patient coinfected with HIV.

Immunomodulatory effects of brucella abortus s19 on cytokine production that promote spontaneous abortion in swiss white mice

Author: Wamonje, Francis Onono

Awarding University: Jomo Kenyatta University of Agriculture and Technology, Kenya

Level : MSC

Year: 2010

Holding Libraries: Jomo Kenyatta University of Agriculture and Technology Library ;

Subject Terms: Abortion/Brucellosis/Brucella abortus/Women/Pregnancy/Immunology/ ;

Abstract:

Brucellosis is the most common zoonotic disease in the world. It has been implicated in spontaneous abortions in animals and man. Spontaneous abortion is the loss of the uterine product prior to the viability of the foetus. There is a progressive shift towards Th2 (antibody) profile in pregnancy with a concurrent diminishing role for Thl (cellular) immune response in successful pregnancy. A Thl profile is considered unfavourable to pregnancy. The immune mechanisms that lead to spontaneous abortion in Brucella infection are not known. Brucella lives intracellularly and elicits a Thl response. By use of a murine model and the S 19 vaccine strain of Brucella abortus, this study created these, conditions to examine if this is the mechanism by which Brucella causes spontaneous abortions. Serum samples were collected and analyzed by Cytometric Bead Array. Five cytokines were targeted; IL-2, IFN-y and TNF-a represented the Th 1 cytokines while IL-4 and IL-5 represented the Th2 cytokines. IL-2 was analyzed but its levels fell below detectable threshold. Data was analyzed using the Paired T- test and p< 0.05 was considered significant. IFN-y and TNF-a infected (test) uninfected (control) and groups had no significant differences in the mean levels of cytokine levels. IL-4 had significant differences between control and test groups in all three trimesters of pregnancy (t=13, P= 0.036, 0.0071 and 0.0277). IL-5 levels had significant difference between the control and test groups in the second trimester of pregnancy (t =14, P= 0.0075) These results show that the maternal immune system is skewed towards a Th2 response during pregnancy even when infected with intracellular bacteria like Brucella that would normally elicit a Th 1 response. The implication of this study is that while the mechanisms by which Brucella causes spontaneous abortion remains unknown, it is clear that Brucella elicits a strong hummoral response from mice during pregnancy and this immunomodulatory action is mediated through IL-4 suppressing IFN-y, hence protecting the foetus from the harmful effects of a cellular response. Therefore, Th2 cytokine levels can be used as a marker for successful pregnancy in Brucella infection.

Prevalence of Helicobacter pylori infection , the virulence genotypes and host immunological responses in disease outcomes among Kenyan dyspectic patients

Author: Kimanga, Andrew Nyerere

Awarding University: Jomo Kenyatta University of Agriculture and Technology, Kenya

Level : PhD

Year: 2010

Holding Libraries: Jomo Kenyatta University of Agriculture and Technology Library ;

Subject Terms: Helicobacter pylori/Immunology /Deoxyribonucleic acid--DNA/Molecular biology/Peptic ulcer/Dyspepsia USE Indigestion/Genetics/Gastro-esophageal reflux disease/Bacteria/Water/Indigestion/ ;

Abstract:

The current study was carried out to determine the genotypic characteristics and host immunologic responses of H pylori among patients presenting with dyspepsia at Aga Khan Hospital in Nairobi and to investigate the occurrence of H pylori DNA in samples from rivers, boreholes and wells around Nairobi. All the H pylori investigated in this study were largely sensitive to clarithromycin amoxicillin with occasional resistance to metronidazole. It was found that the precedence of H pylori tended to elevate the risk of peptic ulcer disease, implying that H pylori has a critical role to play in the development of gastric pathologies. In this study, peptic ulcer disease was more associated with carriage of cagA and vacA genotype s 1 a. The 16S rRNA gene sequences of one sample (here in designated as strain 2) had a mutation ofT370 to A. The 16S rRNA genes from all samples were identical along their entire length, with the, exception of the cytosine insertion in strain 2, and differed from the rest of the H. pylori 16S sequences, a strong indication that they are both derivatives of the same strain. Mutations found in the 16S rRNA genes of clinical isolates of H. pylori have been shown to confer tetracycline resistance. A profile of proinflammatory cytokines in relation to gastric pathologies revealed that provocative allele T of IL-l ~ -511 and IL-l RN*212 were more frequent in complex pathologies. This finding augments their pathological roles in the study population. Carriers of the IL-l B-511 allele and IL-l RN*2/2 have been reported to be at increased risk of hypochlorhydria and gastric cancer. IL-l genetic polymorphisms also influences H. pylori-related gastric mucosal IL-l beta levels and are related to gastric inflammation and atrophy, factors thought to be important in gastric carcinogenesis. The study found that the expression of IL-17 A is augmented in IL-17F in Kenyan H. pylori positive dyspepsia patients compared with Germany patients with gastro-esophageal reflux disease (GERD). Excess IL-17F signal induced the increased production of IL-17 A and both were up-regulated in H pylori infection. IL-17 A and IL-17F are potentially critical regulators of H pylori induced gastric inflammation. The aetipathological role of H pylori in Kenyan (African) subjects is not different from that observed in the west and elsewhere in the world.The role of the host immunology in disease progress may be different from that observed in the rest of the world. The detection of H pylori DNA in the Nairobi River basin waters confirms the fears that this important commodity may be a vehicle for these potentially pathogenic bacteria. The presence and abundance of fecal indicator bacteria is not predictive of either H pylori and may not be a telling sign of the H pylori in water bodies. All the H pylori investigated in this study were largely sensitive to clarithromycin amoxicillin and metronidazole. H pylori tended to elevate the risk of peptic ulcer disease, implying that H pylori has a critical role to play in the development of gastric pathologies. In this study, peptic ulcer disease was more associated with carriage of cagA and vacA -S I a.

The effect of pregnancy on immune responses in HIV infection : a comparison of immunological indicators in pregnant and non-pregnant HIV infected women

Author: Musyoki, Stanslaus Kiilu

Awarding University: Moi University, Kenya

Level : MSc

Year: 2010

Holding Libraries: Moi University Margaret Thatcher Library ;

Subject Terms: HIV infection ; Pregnancy ; Women ; Immunology ;

Abstract:

Background: The distribution and function of immune cells vary in different clinical tares. Not onlv is the immune resnonse chancing as a consequence of pregnancy but also the immune response is changing as a result oflllV infection. my causes destruction of CD4 cells, through activation of CDS cells; a phenomenon associated with increased destruction of CD4 cells and therefore a fall in CD4 count in HfV infection. On the contrary, pregnancy inhibits production and activation of CDS cells. To date, the effect of pregnancy on the immune responses that may affect lllV disease progression has not been well studied and remains unclear. Objective: To assess the influence of pregnancy on immune responses in my infection by comparing the immunological indicators in pregnant and non-pregnant women with lllV infection. Study design: Cross-sectional and comparative. Study site: AMPATH clinics and MCR clinics at MTRH. Study subjects: mv positive adult women not on antiretroviral therapy. Study methods: Blood samples from 72 eligible subjects were collected. Total White blood cell (WBC) count, lymphocyte count, lymphocyte subsets (CD19, CD56/CD16, CD3), T lymphocyte subsets (CD4, ens and their ratio), and activated CDS T cells (CD3S and lILA-DR percent counts) were performed using flow cytometer. The counts were compared across the 3 trimesters of pregnancy in the pregnant women and between pregnant and non-pregnant illY positive women. Statistical package for social sciences (SPSS) version 12.0 was used for descriptive analysis focusing on frequency, correlation, and cross-tabulation of the study data. Results: No statistically significant differences were found in the distribution of age, marital status, and income between the two significant groups. Pregnant myinfected women had lower activated CDS cell (HLA and CD38) count and higher total WBC, CD4 counts and CD4/CD8 ratio than non-pregnant Hl'V infected women. Total WBC and CD4 counts were noted to increase with advancing pregnancy. Activated CD8 cell percent counts were noted to decrease with advancing pregnancy. Low activated CD8-r percent counts were associated with higher CD4+ cell counts and vice versa. Conclusion: By measuring the immunological indicators in pregnancy by flow cytometer, a technical innovation, it is possible to show that pregnancy does not seem to accelerate HfV disease progression; but on the contrary, it suppresses activation ofT cells expressing CD8 molecules. Thus, reduced rate of depletion of CD4 cells in pregnant HfV positive women. Recommendations: Further studies to confirm the findings of this study.

Immunological effects of Solanum incanum and Carica papaya extracts in swiss mice infected with Schistosoma mansoni

Author: Mose, John Mokua

Awarding University: Jomo Kenyatta University of Agriculture and Technology, Kenya

Level : MSc

Year: 2010

Holding Libraries: Jomo Kenyatta University of Agriculture and Technology Library ;

Subject Terms: Solanum incanum ; Schistosomiasis ; Schistosoma mansoni ; Carica papaya ; Medicinal plants ; Immunology ;

Abstract:

Schistosomiasis is a chronic parasitic disease in tropical and subtropical regions and is associated with a variety of clinical syndromes that may lead to severe morbidity. Despite the existence of the highly effective antischistosome drug praziquantel (PZQ), schistosomiasis is spreading into new areas, and although it is the cornerstone of current control programs, PZQ chemotherapy does have limitations. In particular, mass treatment does not prevent reinfection. Furthermore, there is increasing concern about the development of parasite resistance to PZQ in addition to its high cost, hence a need for an alternative drug. Additionally, the drug has two major administration drawbacks, first being the high dose needed, and its well documented bitter and disgusting taste. Many plant species have been used worldwide in traditional medicine for the treatment of human helminthes but few have been screened for activity against adult Schistosoma sp. This study therefore sought to determine the effectiveness of Solanum incanum and Carica papaya extracts as possible novel antischistosomal agents. For each of the plant species, both aqueous [Solanum incanum aqueous (SIA) and Carica papaya aqueous (CPA)] and methanol [Solanum incanum methanol (S1M) and Carica papaya methanol (CPM) extracts were used. A total of 114 mice were used in the study out of which 72 were in the experimental groups; 18 in the positive control group and 24 in the infected control group. Mice in groups of six were individually infected with 250 Schistososma mansoni cercariae. Four weeks post infection, they were orally treated with 300mg/kg of Solanum incanum and Carica papaya extracts two days apart and at week six, all animals were perfused to evaluate the efficacy of Solanum incanum and Carica papaya in treatment of the infection. The following were also performed: Sampling for blood, spleen and lymph node cells; ELISA and gross pathology. The results obtained showed a 16.7 % maturation of penetrant cercariae. Indeed both the extracts had immunological effects in swiss mice infected with Schistosoma manson. but however, the Solanum incanum extracts had the greatest effect on worm reduction, worm recovery and IgO specific immunological responses compared to Carica papaya. The Solanum incanum aqueous group recorded the highest worm reduction of 46.3% compared to control infected animals where a percentage worm recovery of 53.7% was observed. The Solanum incanum stimulated high IgO levels signifying a high protective immunity and it was significantly different from the Infected control group (p<O.05). The Solanum incanum extracts comparably showed similar IL-5, IFN-'Y responses and gross pathology to PZQ than Carica papaya.

Natural monocytic acquisition of haemozoin and rantes polymorphisms : association with malarial disease outcomes and functional changes in children from Western Kenya

Author: Were Tom

Awarding University: Kenyatta University, Kenya

Level : PhD

Year: 2009

Holding Libraries: Kenyatta University Moi Library ;

Subject Terms: Malaria/Medical immunity/Immunology/Babies/Children and youth/Plasmodium falciparum/Siaya District ;

Abstract:

Plasmodium falciparum is an important cause of morbidity and mortality in children residing in holoendemic transmission areas largely from severe malarial anaemia (SMA). Although overproduction of inflammatory-derived cytokines are implicated in the immunopathogenesis of severe malaria, the chemokine regulated on activation, normal T-cell expressed and secreted (RANTES, CCL5) is largely unexplored in the context of malaria. Additionally, pro-inflammatory cytokines, such as tumour necrosis factor (TNF)-?, interleukin (IL)-1 and interferon (IFN)- promote increased RANTES production, while anti-inflammatory cytokines (e.g., IL-4, IL-10 and IL-13) down-regulate RANTES biosynthesis. However, the effect of these cytokines on RANTES production in children with malaria anaemia (MA) is poorly understood. Although genetic variation in RANTES is associated with inflammatory, autoimmune and infectious diseases, the role of single nucleotide polymorphisms (SNPs) in conditioning disease outcomes and RANTES production in malaria remains unexplored. These studies investigated the relationship between circulating RANTES with MA severity, thrombocytopaenia, suppression of erythropoiesis, and naturallyacquired haemozoin (pfHz) in monocytes. The functional influence of promoter (403G/A, -4120A/T, and -415]C/T) and intronic (+307A/G) SNPs on malaria outcomes was also examined. These hospital-based cross-sectional studies were performed in infants and young children (age <36mos.) enrolled at Siaya District Hospital, western Kenya. RANTES levels in circulation and from peripheral blood mononuclear cell (PBMC) cultures were measured by a 25-plex cytokine assay and enzyme-linked immunosorbent assay, respectively. Genotyping of -4120A/T, - 4151C/T and +307A/G polymorphisms was performed using Taqman? 5'-allelic discrimination assay, while -403G/A was genotyped by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) method. Results presented here show that suppression of circulating RANTES is associated with increasing severity of MA (p=0.002), decreased erythropoiesis (p=0.049), and malaria-induced thrombocytopaenia (p=0.036). Additional results demonstrate that suppression of circulating RANTES is associated with increasing levels of pigment-containing monocytes (PCM) (p=0.035). In vitro experiments indicate that monocytic acquisition of pfHz is associated with suppression of RANTES from PBMC under both baseline (p=0.001) and stimulated conditions (p=0.072). Although high levels of pfHz in monocytes caused reduced production of IFN- (p=0.003) and IL-10 (p=0.010), multivariate modelling revealed that only PCM (p=0.048, =-0.171) and IL-10 (p<0.0001, =-0.476) were independently associated with RANTES. Subsequent experiments in cultured PBMC from children with naturally-acquired Hz revealed that blockade of endogenous IL-10 caused significant increases in RANTES production (p=0.028). Haplotypic constructs of RANTES (+307/-403/-4120/-4151) revealed higher frequencies of SMA (Hb<5.0g/dL) in AGTC carriers (75.0% vs. 21.5%, p=0.010) and lower prevalence of thrombocytopaenia in AATC carriers (15.4% vs. 84.6%, p=0.036). Multivariate logistic regression analyses illustrated increased susceptibility to SMA (Hb<5.0g/dL) in AGTC carriers (OR, 13.4; 95% CI, 1.3-133.6; p=0.027) and protection from thrombocytopaenia in AATC carriers (OR, 0.2; 95% CI, 0.1-1.0; p=0.05). Moreover, circulating RANTES levels were lower in AGTC (p=0.049), and higher in AATC carriers (p=0.043). Taken together, these results demonstrate that thrombocytopaenia and natural monocytic acquisition of pfHz are a source of reduced RANTES that may contribute to suppression of erythropoiesis in children with MA. Additional results presented here demonstrate that genetic variation in RANTES is important for mediating SMA and thrombocytopaenia during P. falciparum infection. These findings have the potential to contribute to better understanding of SMA pathogenesis, and lead to b

Cytokine responses to plasmodium falciparum malaria peptides in Kenyan adults living in a sporadic malaria transmission area

Author: Kisia Lily

Awarding University: Moi University, Kenya

Level : MMED

Year: 2009

Holding Libraries: Moi University Margaret Thatcher Library ;

Subject Terms: Malaria/Plasmodium falciparum/Cytokines/Immunology/Adults/Kipsamoite, Nandi North District ;

Abstract:

Studies have shown that cytokine responses are important in protection from Pjalciparum malaria infection and disease. However, convincing T-cell correlates of protection have not been demonstrated in naturally exposed populations. Recent studies have shown that highland malaria transmission can be characterized as seasonal or sporadic, a situation that could lead to differences in the host immune response to malaria in highland populations. This study was undertaken to describe cytokine responses to various Pfalciparum malaria peptides in a sporadic malaria transmission area of Western Kenya by use of the cytometric bead assay. The results were compared with those obtained from standard ELISA especially in measuring the levels of IFN-y. To determine cytokine responses to P. Jalciparum malaria peptides in Kenyan adults living in a sporadic malaria transmission area by using the cytometric bead assay. The study was conducted on 29 randomly selected healthy adults living in Kipsamoite village in Nandi North District of Western Kenya during the month of August 2007 after a prolonged period of low malaria transmission. PBMC's were isolated from whole blood by density gradient centrifugation then challenged with malaria peptides/mitogen for l20hrs under C02 (5%) and humid conditions. Supernatants were collected and stored at -80?C. The Cytometric bead assay was used to determine the levels ofIL-2, IL-6, IL-8, IL-10, IL-12, IFN-y, TNFa and RANTES while standard ELISA was used to determine the levels of IFN-y in the supernatants. Descriptive statistics were used to determine the geometric means, ranges and frequencies of cytokines produced to the malaria peptides tested. Spearman's rank correlation was used to determine the correlations in the levels of IFN-y to various malaria peptides between standard ELISA and the cytometric bead assay. Informed consent was sought from the participants and they were free to withdraw at any time. Ethical approval to conduct the study was obtained from Moi University Institutional Review Committee, KEMRI Ethical Review Committee and University of Minnesota Institutional Review Board. The data from this study showed high IL-8 (36-947pg/ml) and moderate IL-6 (0- l7.11pg/ml) and RANTES (0.3-9.03pg/ml) levels in response to CSP-22, CSP-35, LSA-l T3, LSA-l T4, LSA-l 94, TRAP-TR539, TRAP-TP6 and AMA-l (n=29) to P. Jalciparum malaria peptides. TNF-a, IFN-y, IL-12, IL-lO and IL-2 levels were <l.5pg/ml. The frequencies of positive cytokine responses were between 17-56% for IL-8, IL-6, IFN-y and RANTES and <4% for IL-12, IL-10, IL-2 and TNF-a to the same peptides. On comparing the cytometric bead assay to standard ELISA, the data indicated that there were poor correlations (rho<0.5, p>O.l) between the two assays in measuring the levels ofIFN-y to all peptides tested except to AMA-1P where the correlation was weak but significant (rho<0.46, p>0.02) (n=25). This study concluded that IL-8 responses to TRAP-TP6, LSA-1T3, LSA-l 94 and LSA-l T4, IL-6 responses to LSA-l T3 and RANTES responses to LSA-l T4 were higher and more frequent than cytokine responses to other peptides. Though at low levels, IFN-y responses were frequent. It was also concluded that standard ELISA and the cytometric bead assay are not comparable in measuring the levels ofIFN-y to malaria peptides tested.

Evaluation of immune responses of a recombinant Theileria parva 2 antigen fused with a cell penetrating peptide from human immunodeficiency virus-1

Author: Tinega, Alex Nyaribo

Awarding University: Kenyatta University, Kenya

Level : MSc

Year: 2008

Holding Libraries: International Livestock Research Institute Library ;

Subject Terms: Theileria parva/Immunology/East Coast Fever ;

Abstract:

East Coast fever (ECF) is a tick-borne protozoal disease of cattle confined to Eastern, Central and Southern Africa. It is caused by an intracellular apicomplexan parasite, Theileria parva which is transmitted by the brown ear tick Rhipicephalus appendiculatus. The disease threatens the livelihoods of many African livestock farmers and it constitutes a major constraint to dairy industries. Control of ECF is mainly dependent on the use of acaricides, drugs and a live vaccine. However increasing prevalence of acaricide resistance in tick populations coupled with the high cost and cold chain requirements of the live vaccine has necessitated search for alternative control measures. Efforts are being focused on the development of a cheap and easy-to-deliver vaccine based on components of the parasite. Immunity to T. parva is associated with major histocompatibility complex class I (MHC-I) restricted CD8+ cytotoxic T lymphocytes (CTL) that kill lymphocytes infected with the schizont stage of the parasite. A number of T. parva vaccine candidate antigens have been identified that are the targets of CTL from immune cattle. Vaccination of cattle with recombinant DNA and viral vectors expressing these antigens have been shown to induce protective CD8+ CTL responses in a proportion of cattle. However an effective delivery strategy that consistently induces protective CTL responses remains to be identified. This study was aimed at determining whether fusing a cell penetrating peptide (CPP) from human immunodeficiency virus-1 trans-acting activator of transcription (HI V -1; TAT) to CTL target antigens from T. parva vaccine candidate antigen (Tp2) enhances the stimulation of bovine CD8+ CTL responses in vitro and induction of murine CD8+ CTL responses in vivo. Bovine in vitro experiments were carried out to assess the stimulation of CTL responses and a spectrum of MHC-I biochemical inhibitors were used to investigate the MHC-I processing and presentation pathway utilised. An experiment was conducted in thirty mice to assess the ability of CPP-antigen fusion proteins to stimulate CTL responses in vivo. Western blot and IFN-y ELISpot assays were used to evaluate antibody and CD8+ CTL responses respectively. Analysis of variance was used to compare the differences in immune responses between the experimental groups. The study has shown that the induction of bovine T. parva 2 (Tp2) specific CD8+ T cell responses in vitro can be achieved by antigen delivery to bovine antigen presenting cells (APC) by trans-acting activator of transcription- cell penetrating peptide (TAT -CPP). Interestingly, immunisation of mice with Tp2 protein induced CD8+ T cell IFN-y responses that were greater in magnitude compared to Tp2-CPP while immunisation with Tp2-CPP fusion protein induced greater antigen-specific antibody responses compared to Tp2-protein. The study has further shown that Tp2 CPP enhanced the induction of CD8+ T cell responses in vitro but failed to enhance significant CD8+ T cell responses in vivo. This area will require further investigation to explain these unexpected results. This study has contributed to the understanding and development of vaccination strategies for the delivery of CTL targeted T. parva antigens.