The Lymphatic Fluid and T-Cell Receptor Vβ expression in Peripheral Blood Mononuclear Cells (PBMCs) of patients with Wuchereria Bancrofti Infection

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

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.