Lymphatic filariasis (LF) is a disease that is currently the target of a major global initiative for elimination.During the past decade, both the treatment and the control strategies for LF have undergone major paradigm shifts–due to rapid increase in knowledge and understanding of LF that is derived directly from a series of commendable progress made by scientific and medical research communities.In pursuit of understanding LF a disease to which a social stigma is attached, the chronic pathology (CP) of LF was taken as a model to understand the immunopathological mechanisms that lead to acute lymphangitis and lymphadenitis,in filarial infections, harboring Wuchereria bancrofti.It is a well established fact that elephantiasis is a consequence of immune-reactivity to adult worm antigens. Therefore,it was thought that T-cells infiltrating the lesions in chronic-pathology disease could augment for elevated inflammation seen in CP.An attempt was made to examine T cells by T Cell Receptor Vß analysis using parasite (BmA) and non parasite (PPD) antigen stimulated PBMC’s for RT PCR and agarose gel electrophoresis followed by Integrated Density Values(IDV),analysis which were further validated by Statistical analysis using One-Way ANOVA followed by Tukeys-HSD Test.Therefore,primers designed for specific 24 Vß gene families in the given repertoire were the choice for experimental analysis by PCR. Five CP cases and 5 Endemic Normals who were normal healthy individuals’ considered as controls,and also 2 Microfilaraemics, who are carriers of this disease were inducted into the current study. Specific T-Cell Receptors-TCRVβ1,TCRVβ2,TCRVβ7,TCRVβ14,TCRVβ20, TCRVβ24 which got overrepresented in the CP subjects when the PBMCs of these subjects came in contact with crude antigen of the parasite BmA in an In-Vitro culture system, but MF’s, do not show any overrepresentation for BmA under similar conditions. Similarly TCRVβ5.2 and TCRVβ11 are genes which were preferentially expressed out of TCRBV1-24 gene repertoire in EN upon stimulation with BmA. PPD seems to be a potential stimulator for MF’s. TCRBV21 of CP subjects was expressed high upon PPD stimulation along with EN in which batteries of receptors are expressed which does not carry any statistical significance.PHA stimulated and an unstimulated culture of PBMCs does not show any statistical significant difference in overrepresentation of TCRVβ1-24 genes with respect to EN and MF but PHA did showed significance with CP.
Table of Contents
1 INTRODUCTION
1.1 OVERVIEW OF THE THESIS
1.2 OBJECTIVES
1.3 REVIEW OF LITERATURE
1.3.1 Filarial Parasites
1.3.2 Life Cycle
1.3.3 Geographic Distribution of Filarial Parasites
1.3.4 Periodicity
1.3.5 Vectors
1.3.6 Clinical Groups in Filariasis
1.3.6.1 Asymptomatic amicrofilaremics (Endemic Normals)
1.3.6.2 Asymptomatic microfilaremics (MF)
1.3.6.3 Chronic Pathology
1.3.6.4 Non-filarial elephantiasis
1.3.6.5 Tropical pulmonary eosinophilia (TPE)
1.3.7 Filarial Genome Project
1.3.8 Vaccine for Filariasis
1.3.8.1 Need for the vaccine
1.3.8.2 Currently available vaccines
1.3.8.3 DNA vaccines
1.3.8.4 Potential vaccine candidates from B. malayi
1.3.8.5 Adjuvants
1.3.8.6 Animal models in lymphatic filariasis
1.3.9 Diagnosis of lymphatic filariasis
1.3.9.1 Parasitological diagnosis
1.3.9.2 Lymphatic imaging
1.3.9.3 Lymphangiography
1.3.9.4 DNA based diagnosis
1.3.9.5 Immunodiagnosis
1.3.10 Lymph (Formation, Absorption and Flow)
1.3.10.1 Lymphoedema
1.3.10.2 Lymphoedema Mechanism of Formation
1.3.10.3 Lymphoedema in conditions other than Filariasis
1.3.10.4 Stages of lymphoedema
1.3.10.5 Treatment of lymphoedema
1.3.10.6 Treatment of Filariasis
1.3.10.7 Surgical treatment
1.3.11 Immunity to Filariasis
1.3.11.1 Role of lymphocytes, antigen presenting cells and other cells in Filariasis
1.3.11.2 Role of Cytokines in Filariasis
1.3.11.3 Receptor signaling in Filariasis
1.3.11.4 Antibody Responses in Filarial infections
1.3.11.5 Filarial antigens
1.3.12 A Prelude to T-Cell Receptors (TCR’S)
1.3.12.1 Gene Organization TCR
1.3.12.2 TCR diversity
1.3.12.3 Association of TCR with CD3 complex
1.3.12.4 TCR and Trimolecular Complex
1.3.12.5 T-Cell activation
1.3.13 MHC recognition in filariasis
1.3.14 T-Cell Receptor Studies in Disease pathogenesis
2 MATERIALS AND METHODS
2.1 LYMPHATIC FLUID ANALYSIS
2.1.1 Study Population
2.1.2 Sample Collection
2.1.2.1 Protein Estimation
2.1.3 Preparation of Parasite Antigens
2.1.3.1 Preparation of Brugia malayi (BmA) and Setaria digitata (SD) total crude extracts
2.1.3.2 Excretory Secretory Antigens
2.1.4 Production of Antisera
2.1.4.1 Animals Used
2.1.4.2 Mice
2.1.4.3 Rabbit
2.1.4.4 Determination of titers of anti-BmA and anti-ES antibodies using ELISA
2.1.5 Identification, Characterization of Antigens and Antibodies in Lymphatic fluid and Serum from Patients with Chronic Pathology
2.1.5.1 Antisera
2.1.5.2 SDS-Polyacrylamide Gel Electrophoresis
2.1.5.3 Western blot analysis of lymphatic fluid and serum of CP patients
2.1.5.4 Isolation of Circulating Immune Complex (CIC) from CP patients
2.1.6 Secondary Bacterial Infections in Chronic Pathology Patients
2.1.6.1 Bacterial strains used in this study
2.1.6.2 Culture Media
2.1.6.3 ELISA
2.1.6.4 Biological Effects of Lymphatic fluid
2.2 T-CELL BETA VARIABLE RECEPTORS Of 1-24 GROUPS IN PATIENTS WITH W. BANCROFTI INFECTION
2.2.1 Donors
2.2.2 Antigens and Mitogens
2.2.3 T-Cell Receptor Beta Variable Primers
2.2.4 Beta Actin Primers as Positive Controls
2.2.5 Cellular Responses to Filarial Antigens Brugia malayi antigen (BmA-Crude Extract) and Non-Filarial Antigen Purified Protein Derivative (PPD)
2.2.5.1 Lymphocyte Responses
2.2.6 Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
2.2.6.1 Extraction of RNA
2.2.6.2 Reverse transcription reaction
2.2.6.3 PCR Amplifications of cDNAs
2.3 STATISTICAL ANALYSIS
3 RESULTS
3.1 LYMPHATIC FLUID ANALYSIS
3.1.1 Study Population
3.1.2 Sample Collection
3.1.3 Production of Antisera
3.1.3.1 Determination of the titers of rabbit polyclonal anti-BmA antibodies and mice polyclonal anti-ES antibodies
3.1.4 Identification, Characterization of Antigens and Antibodies in Lymphatic fluid and Serum from Chronic Pathology Patients
3.1.4.1 SDS-Polyacrylamide gel electrophoresis
3.1.4.2 Western blot analysis of Lymphatic Fluid and Serum of CP Patients
3.1.4.3 Parasite antigens identified in the Immune Complexes (I/C) from serum of filarial patients
3.1.5 Secondary Bacterial Infections in Chronic Pathology Patients
3.1.5.1 Reactivity of the lymphatic fluid and serum with Staphylococcus aureus and, - hemolytic Streptococci
3.1.5.2 ELISA
3.2 T-CELL BETA VARIABLE RECEPTORS OF 1-24 GROUPS, IN PATIENTS WITH W. BANCROFTI INFECTION
3.2.1 Donors
3.2.2 Cellular Responses
3.2.2.1 Lymphocyte Cultures for T-Cell Receptor Repertoire Expressions
4 DISCUSSION
4.1 WESTERN BLOT ANALYSIS OF LYMPHATIC FLUID AND SERUM OF CP PATIENTS AND BACTERIOLOGICAL STUDIES
4.2 TCR BETA VARIABLE GENE
4.3 CONCLUSIONS
4.4 FUTURE STUDIES
Objectives & Themes
This thesis aims to investigate the immunopathological mechanisms of lymphatic filariasis (LF) caused by Wuchereria bancrofti, with a focus on chronic pathology. The primary objective is to characterize parasite-specific antigens and antibodies within lymphatic fluid and serum of affected patients, while also analyzing the role of secondary bacterial infections and T-cell receptor (TCR) repertoire biases in disease pathogenesis.
- Identification of parasite-specific antigens and antibodies in lymphatic fluid and serum.
- Evaluation of the growth-promoting effects of lymphatic fluid on bacteria associated with secondary infections.
- Analysis of T-cell receptor (TCR) beta variable gene expression in response to Brugia malayi antigens.
- Comparison of immune responses across different clinical groups (Endemic Normals, Microfilaremics, and Chronic Pathology patients).
Excerpt from the Book
1.3.11.1.7 Tregs in Filariasis
The physiology of a suppressive T-cell population called the T regulatory cells or Tregs deserve a special mention in the immunobiology of Filarial infections due to the ability of the parasite antigens to suppress the host T-cell responses. Recent studies on immunogenetics in patients with onchocerciasis and LF have supported a dichotomy along the ‘Th1+Th2 vs. Treg’ divide, rather than favouring a Th1/ Th2 pendulum.
The role of these T-cell population that operate through TGF- and IL-10 in the suppression of parasite specific immune responses leading to parasite survival has been hypothesized by Hoerauf et.al (2002) and Maizels et al (2003). Subsequently Treg activity in human helminth infections has been reported by Hoerauf et al (2002) where they demonstrated the expression of regulatory cytokines (IL-10, TGF- and CTLA-4) in onchocerciasis patients. In another study by Steel and Nutman (2003) it was observed that the CTLA-4 expressed on T cells from lymphatic filariasis patients can suppress IL-5 production. In a different study on a related helminth parasite Schsitosoma mansoni it was observed that CD4+CD25+ T cells and IL-10 inhibit Th1 development (Mc Knee and Pearce 2004) and egg-induced pathology (Hesse et al 2004). The role of the T reg population in Filarial antigen induced hyporesponsivness can be better appreciated in the work of Taylor et.al 2005, where the removal of Treg cell activity resulted in to the reversal of hyporesponsiveness leading to the filarial parasite clearance in vivo. A recent report by Taylor et al has suggested that during filarial infection CTLA-4 coinhibition and CD4+ CD25+ Treg cells form complementary components of immune regulation that inhibit protective immunity in vivo (Matthew D. Taylor et al (2007). Enhanced levels of Foxp3 positive natural Tregs have been shown in Filariasis. Live parasite stimulation but not the antigens significantly up-regulated the expression of Foxp3 in infected individuals, indicating heightened Treg activity. Foxp3 expression was also shown to be higher at baseline in infected individuals, indicating the presence of chronic parasite-stimulated regulatory networks occurring in vivo (Subash et al 2006).
Summary of Chapters
1 INTRODUCTION: This chapter provides an overview of the global health burden of lymphatic filariasis, summarizes the current understanding of the disease, and outlines the thesis objectives.
2 MATERIALS AND METHODS: This section details the protocols used for sample collection from patients, preparation of parasite antigens, and the experimental methods including Western blotting, ELISA, and RT-PCR for T-cell receptor analysis.
3 RESULTS: This chapter presents the data regarding antigen identification in lymphatic fluid and serum, the role of lymphatic fluid in bacterial growth, and the analysis of T-cell receptor beta variable gene expression in different patient groups.
4 DISCUSSION: This final section interprets the findings, linking the observed antigenic profiles and TCR repertoire biases to the broader context of disease pathogenesis and host immune modulation.
Keywords
Lymphatic filariasis, Wuchereria bancrofti, Brugia malayi, Chronic pathology, Lymphatic fluid, T-cell receptors, TCR repertoire, Immunodiagnosis, Secondary bacterial infections, Immune complexes, Immunopathology, Antigenic profile, Host immune response, RT-PCR, Western blot.
Frequently Asked Questions
What is the core focus of this research?
The research focuses on understanding the immunopathological mechanisms of chronic lymphatic filariasis by examining the antigenic profile in lymphatic fluid and analyzing T-cell receptor repertoire bias.
What are the primary themes investigated?
The work covers parasite antigen identification, the impact of lymphatic fluid on secondary bacterial growth, and T-cell immune responses in different clinical groups.
What is the main objective of the thesis?
To identify parasite-specific antigens in lymphatic fluid and serum, and to examine the molecular mechanism of T-cell interaction with Brugia malayi antigens.
Which scientific methods are employed?
The methodology includes SDS-PAGE, Western blot analysis, ELISA for antibody detection, and RT-PCR for analyzing T-cell receptor (TCR) beta variable gene expression.
What is the focus of the study's main body?
The study examines the antigenic profile of serum and lymphatic fluid, evaluates bacterial growth in the presence of lymphatic fluid, and investigates biased T-cell repertoire selection in infected individuals.
Which keywords characterize this work?
Key terms include lymphatic filariasis, Wuchereria bancrofti, Brugia malayi, chronic pathology, TCR repertoire, and immune modulation.
How does lymphatic fluid affect bacterial growth?
The study found that lymphatic fluid specifically promotes the growth of beta-hemolytic streptococci, which often causes secondary bacterial infections in patients with chronic filariasis.
What role does the TCR repertoire play in the study?
The research analyzes whether there is a biased selection of T-cell receptor families when stimulated with Brugia malayi antigens, which provides insights into the immune regulation and potential anergy in filarial patients.
- Arbeit zitieren
- Dr. Magapu Solomon Sudhakar (Autor:in), 2006, The Lymphatic Fluid and T-Cell Receptor Vβ expression in Peripheral Blood Mononuclear Cells (PBMCs) of patients with Wuchereria Bancrofti Infection, München, GRIN Verlag, https://www.hausarbeiten.de/document/183781
