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

Doktorarbeit / Dissertation 2006 232 Seiten

Medizin - Biomedizinische Technik

Leseprobe

TABLE OF CONTENTS

ABSTRACT

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

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

APPENDIX 1

REFERENCES

LIST OF PUBLICATIONS

VITAE

ABSTRACT

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. As a result, a public health dimension with a focus on affected populations, now supplements the earlier, predominantly patient-oriented clinical approach to LF.

The Indian government launched a nationwide LF control programme with the components of transmission interruption by annual mass-drug therapy, using diethylcarbamazine with/without albendazole, and the alleviation of disability and suffering among affected people.

In pursuit of understanding such a disease to which a social stigma is attached, the chronic pathology 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. Hence, an attempt was made in this thesis to identify parasite specific antigens and antibodies in lymphatic fluid, which accumulates in CP conditions. At the same time, the corresponding serum from the same patient who was suffering from chronic pathology of filariasis, was also taken for analysis. 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 also made to examine T cell by TCR V analysis using RT PCR and antigen stimulated PBMC’s. Therefore, 24 V gene families in the given repertoire was the choice for experimental analysis. Along with all CP cases for these studies in both the objectives, appropriate controls were also included in this study as EN, who are normal healthy individuals and MF, who are carriers of this disease. At the onset, it was thought that bacterial infections could predominate the sequence of infections in CP. As such, an attempt was also made to study the effect of lymphatic fluid on the bacterial growth in vitro, and the presence of antibodies against bacteria that cause secondary infections, was assessed.

Lymphatic fluid and corresponding serum contains parasite specific antigens and antibodies when analyzed by SDS-PAGE and Western blot. At the sametime, LF promoted the growth of beta -hemolytic streptococci bacteria which usually predominates the secondary infections. Specific T Cell Receptors which get overrepresented in the CP subjects when the PBMCs of these subjects come in contact with crude antigen of the parasite BmA but MF’s, do not show any overrepresentation for BmA under similar conditions.

ACKNOWLEDGEMENT

I thank Prof. R.B. Narayanan, my mentor, for providing me with the opportunity to work on filarial chronic pathology for immunopathological studies. I thank Prof. P. Kaliraj, Dr. V. Murugan and Dr. K. Sankaran from the Centre for Biotechnology for providing me with the facilities available in their laboratories and their encouragement. I thank my Doctoral Committee Members Dr. V. Kumaraswami and Dr. V.D. Ramanathan, Deputy Directors from TRC, Chennai, for their valuable suggestions and sparing their valuable time to critically read the methods, results and discussion of my thesis.

I am grateful to Prof. K.V. Alalasundaram Head, Plastic Surgery Dept., Govt. Roypettah Hospital, Chennai, for providing me with lymphoedema cases. I also thank Dr. Dhanaraj of Municipal Corp., Chennai, for mobilizing Microfilaraemics. I thank Dr. S.V. Chiplunkar, Head, Immunology Division ACTREC, TMH, Mumbai for training me in T-cell Receptor studies. I thank Dr. M. Chandy, Head, Hematology Dept., CMC, Vellore, for permitting me to work in his lab. I also thank Dr. Rama Raja Ram, HOD, Biochemistry Dept., CLRI, Chennai, for allowing me to carry out proliferation studies. I thank Dr. R. Kirubagaran, Scientist-E NIOT, Govt. of India, and Dr. B. Nagarjan HOD, Microbiology Dept., Cancer Institute, Chennai, for perusing my thesis and their suggestions. I thank Dr. R. Ravanan, Reader Dept of Statistics Presidency College, Chennai, for his statistical analyses. I express my gratitude to LTMT, Mumbai for awarding SRF for 2 years. I thank my father Dr. M. Bhaskara Rao, for his moral encouragement and financial support for the last six years. I thank all my friends for their sincere support.

MAGAPU SOLOMON SUDHAKAR

LIST OF TABLES

1.1 State Wise Distribution of Filarial Endemic Regions in India
1.2 Classification of Filarial Lymphoedema

2.1 TCR V Primers

3.1 Demographic details of study population for Lymphatic Fluid Analysis
3.2 Triglycerides profile and Protein Concentration in the Pooled Lymphatic fluid and Serum of CP
3.3 Parasite antigens in the lymphatic fluid and serum of CP patients identified using Anti-BmA and Anti-ES antibodies
3.4 Parasite antigens identified by Western blot using Lymphatic fluid and Serum of CP patients as source of antibodies and S. digitata as antigen
3.5 Parasite antigens in the Serum Immune complexes of the filarial patients identified using Anti-BmA and Anti-ES
3.6 Effect of Lymphatic Fluid on the Growth of Beta Hemolytic Streptococci
3.7 Demographic details of study population for T Cell Receptors Repertoire
3.8 ANOVA for Significant Difference between Genes for Each Stimulant in EN
3.9 ANOVA for Significant Difference between Stimulants for Each TCRBV 1-24 Genes in EN
3.10 Comparative Analysis and Statistical Significance of Gene Expressions in EN
3.11 ANOVA for Significant Difference between TCRBV 1-24 Genes for Each Stimulants in CP
3.12 ANOVA for Significant Difference between Stimulants for Each TCRBV 1-24 Genes in CP
3.13 Comparative Analysis and Statistical Significance of Gene Expressions in CP
3.14 ANOVA for Significant Difference between Genes for Each Stimulant for MF
3.15 ANOVA for Significant Difference between Stimulants for Each TCRBV 1-24 Genes for MF
3.16 Comparative Analysis and Statistical Significance of Gene Expressions in MF

LIST OF FIGURES

1.1 Life Cycle of Wuchereria bancrofti
1.2 Areas endemic for lymphatic filariasis
1.3 Four major stages of Lymphoedema
1.4 Pneumatic Compression Treatment for Filarial CP
1.5 Molecular structure of the TCR
1.6 Some members of the Ig-gene superfamily of surface glycoproteins
1.7 Organization of human germline and rearranged TCR genes
1.8 Class I MHC Molecular Structure
1.9 Class II MHC Molecular Structure
1.10 T-cell activation

2.1 Immunization and bleeding schedule for polyclonal antibodies production for ES antigens similar schedule was also adopted for BmA antigen (crude extract)

3.1 SDS-PAGE of lymphatic fluid (A) and serum (B) on 15% gel
3.2 Western blot analysis of pooled Lymphatic fluid and Serum of CP patients
3.3 Western blot analysis of Immune complexes of EN, MF and CP probed with anti-ES
3.4 Western Blot of Immune Complexes from EN, MF and CP Probed with anti-BmA antiserum
3.5 Total IgG against S. aureus, alpha and beta-hemolytic Streptococci in the lymphatic fluid and serum of CP patients measured by ELISA. Horizontal bar shows the average value
3.6 A Profile of Total RNA obtained from PBMC’s (5×106 ) of Endemic Normal. 5 μg/ml of RNA was taken for cDNA synthesis
3.6 B Profile of cDNA obtained from Total RNA (5 μg/ml) of PBMC’s of EN 1 and 2 Samples converted by SuperScript™ First-Strand Synthesis System for RT-PCR kit and run in the 2% agarose gel
3.7 Beta-Actin of 202 bp amplified by the cDNA and constructed by SuperScriptTM First-Strand Synthesis System for RT-PCR kit
3.8 Statistically Significant Stimulants in EN and TCRBV1-24 Profile Percentage Expression
3.9 TCRBV1-24 Profile for various cDNA of Endemic Normal (EN)
3.10a Statistically Significant Stimulant (PHA) CP and TCRBV1-24 Profile Percentage Expression
3.10b Statistically Significant Stimulant (BmA) CP and TCRBV1-24 Profile Percentage Expression
3.11 TCRBV1-24 Profile for various cDNA of Chronic Pathology Patient
3.12 Statistically Significant Stimulants in MF and TCRBV1-24 Profile Percentage Expression
3.13 TCRBV1-24 Profile of Microfilaraemic Patient

LIST OF ABBREVIATIONS

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CHAPTER 1
INTRODUCTION

Lymphatic filariasis caused by Wuchereria bancrofti and Brugia malayi is a major debilitating disease in the developing countries, particularly in the tropics. It is currently estimated that around 1.1 billion people are at risk of being infected, with 120 million people already infected in 73 endemic countries (World Health Organization, unpublished data). India accounts for 41% of global burden of lymphatic filariasis. According to a report from National Filariasis Control Programme (NFCP), India has a total of 33 million people who are at risk of infection and there are approximately 21 million people with symptomatic filariasis (7.8 million lymphoedema / elephantiasis cases, 12.9 million hydrocele cases) and 27 million microfilaria carriers. (ICMR Bulletin: Vol. 32, No. 5 and 6 May-June 2002: Prospects of Elimination of Lymphatic Filariasis in India.). In Tamil Nadu there are 40 districts of endemic regions and only 20 of these had MDA programme implemented but on the other hand there are 25864 Lymphoedema cases, 8794 Hydrocele cases, 3 core people are living in 20 Filaria endemic districts, Tamil Nadu loses 112 million man days a year, Rs. 335 cores is the annual economic loss (Health Policy Report by Government of Tamil Nadu 2004).

The Global Programme for the Elimination of Lymphatic Filariasis (GPELF) was recognized in early 2000. This was followed by the World Health Assembly Resolution 50.29 (WHA 50.29) in 1997 for a mission on the member states of the World Health Organization (WHO) to eliminate the disease as a public health problem. Exhaustive research in lymphatic filariasis over the last decade has proved the efficacy of new drug combinations (Ismail et al 1998) and led to the identification of simple diagnostic tools (Weil et al 1997) like the ICT (Immunochromatographic Card Test) procedure which is a simple, rapid test for detecting adult worm antigen in human blood and permits the rapid evaluation of prevalence, facilitates mapping of disease distribution and assessment of levels of transmission in populations under mass drug administration. The studies in human lymphatic filariasis has also enhanced the knowledge of disease pathogenesis (Amaral et al 1994 and Mand et al 2003) and demonstrated that those with existing disability could have symptoms of lymphoedema and elephantiasis, which can be alleviated by community or home-based care (Dreyer et al 2002).

The development of a new strategy - time limited (at least 5 years) - annual co-administration of two drugs and the creation of GPELF and the Global Alliance for the Elimination of Lymphatic Filariasis (GAELF) build on successful lymphatic filariasis elimination programmes in several countries (Molyneux et al 2000, Molyneux and Zagaria 2002 and Ottesen et al 1997). The number of people treated annually was observed to be rising from 2.9 million in 12 countries in 2000 to 28.89 million in 2001 and an estimated 60 million in 34 countries in 2002 itself. This trend indicates the total commitment toward global eradication of Lymphatic filariasis by the member states of WHO.

The range of clinical manifestations in bancroftian filariasis includes elephantiasis, hydrocele formation, recurrent febrile episodes associated with lymphangitis and lymphadenitis. The syndrome of tropical pulmonary eosinophilia is an asymptomatic condition in which individuals manifest persistent microfilaremia. The principal pathological changes in chronic lymphatic filariasis result from dysfunction of or inflammatory damage to the lymphatics. Adult worms live in the afferent lymphatics or sinuses of the lymph nodes and induce local changes that result in dilatation of the lymphatics and thickening of the vessel walls. Histologically, there is infiltration with plasma cells, eosinophils, and macrophages in and around the infected vessels. There is endothelial and connective tissue proliferation with tortuosity of the lymphatics.

The clinical manifestations of chronic lymphatic dysfunction in filariasis are fairly well characterized, though the pathogenic mechanisms responsible for this disease, particularly lymphoedema, remain unclear (Ottesen 1987 and 1992). Very little information is available on the parasite antigens present in the lymphatic fluid, although earlier reports of parasite antigens were reported in urine and hydrocele fluid of chronic pathology patients (Ashok et al 1985). On the other hand Circulating immune complexes (CIC) capable of immunomodulating the immune responses (Barnett 1986) occurring in most of the parasitic diseases, are formed in the circulation or in tissues, as a result of interaction between exogenous and filaraemics. This can be demonstrated using polyclonal antibodies raised against adult S. digitata worms (Dissanayake et al 1981 and 1982). The polyclonal antibodies are reactive with the antigens derived CIC and can bind to adult worms, but not to microfilariae. Therefore, CICs were also analyzed in CP serum to identify parasite specific antigens.

There are, however, strong correlations between the host immune response and the spectrum of clinical infection, which imply that induction and type of anti-filarial immunity may determine the outcome of infection (Ottesen 1992) and thus the clinical presentation.

So the current study has been undertaken to examine the antigenic profile in the serum and lymphatic fluid of lymphoedema patients and to assess the contribution of lymphatic fluid and opportunistic infections causing bacteria, in the disease pathogenesis and to study the immune complexes involved in antigen antibodies interaction of patients with chronic pathology.

An effective immune response to a parasite antigen depends on the interaction between APCs and the MHC-Ag-TCR complex, along with the expression of co-stimulatory molecules (secondary signals) like CD40, CD80, and CD86 etc. The affinity and specificity of the TCR binding to the MHC, is of primary concern as weak or inappropriate TCR binding results in hyporesponsiveness. Studies in various diseased conditions have shown that there is a biased T cell repertoire selection that was responsible for the outcome of the disease.

T cell repertoire bias in human lymphatic filariasis has been shown to be antigen specific in a Brazilian population (Freedman 1998). However, there have been no other studies with respect to the APC activation with the biased T cells and their responses towards crude extracts filarial antigens in Indian populations, although such studies were earlier reported in Indonesian populations (Sartano 1997). Consequently, this present study was undertaken to examine the T-Cell receptors of beta variables 24 families in the presence of Brugia malayi adult crude antigen in Endemic Normal individuals and compare the same with Chronic pathology and Microfilaremics.

1.1 OVERVIEW OF THE THESIS

As mentioned above, Lymphatic filariasis is an up-and-coming disease in many areas of the tropics, where vector habitations have stretched because of large-scale water projects and declining sanitation associated with uncontrolled urbanization (Harb et al 1993; Albuquerque et al 1995; Dhanda et al 1996). In many countries, where filariasis has been mapped systematically for the first time, its geographic distribution is much more extensive than previously believed (Gyapong et al 2002 and Beau et al 2004). Lymphoedema of the limb is a physically deforming and socially stigmatizing consequence of filarial infection. Although the factors responsible for the instigation and development of filarial lymphoedema to its most severe form, elephantiasis, have been debated, recurrent episodes of bacterial acute dermatolymphangioadenitis (ADLA) play a major role (Dreyer et al 1999a; Olszewski et al 1997; Shenoy et al 1998). Illustrated by painful swelling of the limb, ADLA go together with fever and chills lasting for several days, sometimes with nausea and vomiting (Dreyer et al 1999b; Ramaiah et al 1997; Shenoy et al 1998). As lymphoedema develops, the frequencies of ADLA episodes normally amplify (Pani et al 1995; Pani and Srividya 1995). Skin changes of chronic lymphoedema comprise thickening, nodular lesions, and pigmentary changes (Burri et al 1996; Olszewski et al 1993). Histopathologic studies have found evidence of inflammatory infiltrate in lymphoedematous tissue (Burri et al 1996; Olszewski et al 1993).

Globally, lymphoedema following infection with the filarial parasite, Wuchereria bancrofti, is more common in women than in men (Michael et al 1996; Lammie et al 1993; Gyapong et al 1994). Reasons for this incongruity are unclear, but may be related to differences in the “favored” anatomic location of the adult filarial worm between men and women (Noroes et al 1996a; 1996b) and biologic factors, particularly pregnancy, that further stress the lymphatic system in women. Thus, in many filariasis-endemic areas, lymphoedema is primarily a disease of women. Both the functional limitations caused by chronic lymphoedema and the short-range mutilation that go together with episodes of ADLA compromise the capability of women to perform household chores and to participate in income-generating activities outside the home. This results in domestic and economic difficulties for their families and communities (Ramaiah et al 1998; Babu et al 2002; Bandyopadhyay et al 1996; Gyapong et al 1996a, 1996b and Coreil et al 1998). In 1998, the Global Program to Eliminate Lymphatic Filariasis embraced lymphoedema management as an elemental component of its strategy to eliminate lymphatic filariasis (Seim et al 1999). Supported by the evidence of the bacterial etiology of ADLA, current World Health Organization (WHO) recommendations for management of lymphoedema, stress basic skin care and hygiene, using soap, water, and antiseptics, as well as rising of the leg, exercise, and proper footwear (WHO Learner’s guide The Organization 2003). Application of these measures improves skin condition, reduces the frequency of ADLA attacks, and reverses or arrests the progression of lymphoedema, and thus improves quality of life (Coreil et al 1998; Dreyer et al 2002; McPherson et al 2003; Suma et al 2002).

In spite of a high parasite load or worm burden in patients with lymphoedema it was observed that, antifilarial responses, especially cell mediated immune responses are high, compared to microfilaremic persons (Freedman 1998; Ottesen 1984; Grenfell 1991; Ottesen 1992; Lammie 1993 and Maizels 1995).

The parasite antigen stimulation of T cells might be allied with increased local inflammatory responses that promote lymphatic damage, ultimately eliciting a cascade of responses that lead to pathology. As reported earlier, the advancement of chronic, immune-mediated inflammation is reliant upon the penetration of circulating PBMC’s across the endothelial cell lining of postcapillary venules into affected tissues. Filarial lymphoedema and elephantiasis most frequently go along with comprehensive tissue inflammatory component in which the dominant local lesion is a CD8+ T cell pericapillary/perivenular infiltrate and Ag-4(VLA-4)/VCAM-1 pathway has been implicated (Freedman et al 1996; Freedman et al 1995). A subclinical CD4+ infiltrate was also present in the majority of clinical asymptomatic individuals with filariasis. Characterization of the antigenic specificity and of the functionality of these infiltrating T cell populations could serve to augment understanding of disease pathogenesis.

Finally, it is important to understand the mechanisms underlying pathogenesis, since the consequences of Lymphatic Filariasis initiated damage to the lymphatics may persist long after infection has been eliminated. It is unknown to what degree this lymphatic damage is reversible, and it is clear that such damaged lymphatics predispose individuals, by some undefined mechanism, to prolonged periods of increasingly damaging lymphoedema and recurrent bacterial infections. In this regard, understanding the mechanism of Lymphoedema formation can result in a better Lymphoedema management, based on inexpensive and practical elements of self-care at home, which can improve the quality of life. In pursuit of this, the current study was initiated to identify and characterize the filarial parasite specific antigens in lymphatic fluid and serum of chronic pathology patients and to look at the molecular mechanism of interaction of T-cells with Brugia malayi adult antigen (BmA) crude extract in vitro and compare the same with non-parasite antigen like Purified Protein Derivative (PPD) which was also acting as mitogen to T-cells therefore employed as positive control.

1.2 OBJECTIVES

- To identify and characterize parasite antigens in the lymphatic fluid of CP patients.
- To study the effect of lymphatic fluid on the growth of micro organisms that causes secondary infections in filarial patients.
- T-cell repertoire following interaction of PBMC’s with Brugia malayi adult worm crude extract antigen (BmA) in Endemic Normal and filarial patients.

The outcome of the above studies has been summarized as follows

The main focus of the study was to comprehend the intricate mechanisms leading to parasite related molecules attributing to the formation of lymphoedema in CP patients.

Therefore, the first section of the thesis deals with the collection of lymphatic fluid and serum from various hospitals where lymphoedema treatment and management was being done on chronic pathology patients and how this fluid was processed and analyzed on SDS-PAGE gels. Western Blot was carried out using rabbit anti-BmA and mouse anti-ES polyclonal antibodies and also the identification of antigens in immune complexes by running native gel for Setaria digitata (SD) and then performing the Western blot. These had shown that 47 kDa and 32 kDa appears to be common immunodominant epitopes of the filarial antigens recognized in both the lymphatic fluid and serum when probed with rabbit anti-BmA and mouse anti-ES antisera. On the other hand, the antigens that were common as immunodominant epitopes which were recognized in the immune complexes of the serum of filarial patients when probed with antisera of rabbit anti-BmA was 17 kDa, whilst 68 kDa was exhibited with mouse anti-ES.

The second part of the thesis deals with secondary infections in CP patients, where bacteria predominant in ADLA of CP patients was cultured from pure strains and was processed through sonication procedures and was used in ELISA as a source of antigen to detect antibodies in the lymphatic fluid and serum of CP patients. In parallel to this study, the predominant bacteria were also cultured for assessment of the biological property of the lymphatic fluid as a potential growth medium for these bacteria. These studies have shown that antibodies against these bacteria were absent in the lymphatic fluid and serum of CP patients. However the lymphatic fluid promoted the growth of the beta hemolytic Streptococci.

The third part of the thesis deals T-Cell Receptor (TCR) analysis using PBMCs from CP patients as an in vitro model system using crude extract of adult filarial parasite antigen of Brugia malayi and non-parasite antigen PPD, which is a mitogen. These studies were extended to healthy individuals from endemic region (EN) as controls and carriers of the Lymphatic filariasis known as Microfilaremics (MF). The findings of these studies in the given limited range of samples had shown that parasite e antigen BmA was a potential stimulant where there was overrepresentation of TCRVß genes in CP patients when compared to EN, but MF failed to show any overrepresentation of TCRVß genes. However this group of MF individuals showed substantial representation with non-parasite antigen PPD.

1.3 REVIEW OF LITERATURE

1.3.1 Filarial Parasites

Filariasis is a disease of wide spectrum, caused by infection with the nematode parasites (roundworms) that inhabit the lymphatics and subcutaneous tissues. Eight main species infect humans. Three of these are responsible for most of the morbidity. Wuchereria bancrofti and Brugia malayi cause lymphatic filariasis, and Onchocerca volvulus causes onchocerciasis (river blindness). The other five species are Loa loa, Mansonella perstans, M. streptocerca, M. ozzardi, and Brugia timori. (The last species also causes lymphatic filariasis), which are transmitted by mosquitoes.

1.3.2 Life Cycle

The discovery of filarial parasite life cycle by Patrick Manson in 1877 is regarded as one of the most significant discoveries in tropical medicine. Infective larvae (L-3) are transmitted by infected, blood sucking arthropods during a blood meal. The larvae migrate to the appropriate site of the host's body, where they develop into microfilariae-producing adults. The adults of lymphatic filariasis reside in lymphatic vessels and lymph nodes (Figure 1.1). The female worms produce microfilariae which circulate in the blood during night time. The periodicity of Wuchereria and Brugia species is dependent primarily on the daily activities of the host and not on the alterations of day and night. Thus if the human host reverses its routine sleep- and- wake cycle, the periodicity of the microfilariae is also reversed. Studies of Wuchereria and Brugia species suggest their periodicity, is due to the differences in the oxygen tension between the arterial and venous blood in the lungs (Burren 1972; Hawking and Gammage 1968). When the microfilariae are absent from the peripheral circulation they accumulate primarily in the

illustration not visible in this excerpt

Figure 1.1 Life Cycle of Wuchereria bancrofti

Different species of the following genera of mosquitoes are vectors of W. bancrofti filariasis depending on geographical distribution. Among them are: Culex (C. annulirostris, C. bitaeniorhynchus, C. quinquefasciatus, and C. pipiens); Anopheles (A. arabinensis, A. bancrofti, A. farauti, A. funestus, A. gambiae, A. koliensis, A. melas, A. merus, A. punctulatus and A. wellcomei); Aedes (A. aegypti, A. aquasalis, A. bellator, A. cooki, A. darlingi, A. kochi, A. polynesiensis, A. pseudoscutellaris, A. rotumae, A. scapularis, and A. vigilax); Mansonia (M. pseudotitillans, M. uniformis); Coquillettidia (C. juxtamansonia). During a blood meal, an infected mosquito introduces third-stage filarial larvae onto the skin of the human host, where they penetrate into the bite wound[illustration not visible in this excerpt]. They develop in adults that commonly reside in the lymphatics[illustration not visible in this excerpt]. The female worms measure 80 to 100 mm in length and 0.24 to 0.30 mm in diameter, while the males measure about 40 mm by .1 mm. Adults produce microfilariae measuring 244 to 296 m by 7.5 to 10 m, which are sheathed and have nocturnal periodicity, except the South Pacific microfilariae which have the absence of marked periodicity. The microfilariae migrate into lymph and blood channels moving actively through lymph and blood[illustration not visible in this excerpt]. A mosquito ingests the microfilariae during a blood meal[illustration not visible in this excerpt]. After ingestion, the microfilariae lose their sheaths and some of them work their way through the wall of the proventriculus and cardiac portion of the mosquito's midgut and reach the thoracic muscles[illustration not visible in this excerpt]. There the microfilariae develop into first-stage larvae[illustration not visible in this excerpt] and subsequently into third-stage infective larvae[illustration not visible in this excerpt]. The third-stage infective larvae migrate through the hemocoel to the mosquito's proboscis[illustration not visible in this excerpt] and can infect another human when the mosquito takes a blood meal[illustration not visible in this excerpt].

Adopted from CDC Website:http//www.dpd.cdc.gov/dpdx

arterioles of the lungs. It was suggested that, if the difference in the arterial-venous oxygen concentrations is <50mm Hg, microfilariae will accumulate in the lungs (Hawking and Clark 1967; Kazura, Spark et al 1984). The microfilariae are taken up by the mosquitoes during blood meal and they develop into the infective larval stage (L-3) in the arthropod host. This development of microfilariae to L-3 in the mosquito requires 7-14 days. During a subsequent blood meal by the mosquito, the larvae, infect the vertebrate host where it migrate to the appropriate site of the host's body, and develop into adults. The prepatent period begins, when the L-3 enters the definitive host and ends when microfilaraie become detectable in the blood is around 60-90 days (Eberhard and Lammie 1991).

1.3.3 Geographic Distribution of Filarial Parasites

Wuchereria bancrofti which is one of the causative agents for human lymphatic filariasis is encountered in tropical areas worldwide; where as Brugia malayi is limited to Asia; and Brugia timori is restricted to some islands of Indonesia (Figure 1.2). In fact Asian sub-continent Lymphatic filariasis distribution map has been created for India, based on the historical data available in 2000 (Sabesan et al 2000). As per the Annual Report on “National Programme to Eliminate Lymphatic Filariasis in India”, 2004. The following Table 1.1 shows the geographic distributions of endemic areas in India.

1.3.4 Periodicity

Some species of microfilariae circulate in peripheral blood at all hours of the day and night, while others are present only during certain periods. The fluctuation in numbers of microfilariae present in peripheral

illustration not visible in this excerpt

Figure 1.2 Areas endemic for lymphatic filariasis

Table 1.1 State Wise Distribution of Filarial Endemic Regions in India

illustration not visible in this excerpt

* indicates the number which are districts of endemic regions covered and uncovered under Mass Drug Administration Programme of DEC. In Tamil Nadu 20 districts and West Bengal 7 districts are not covered under MDA implementation. Data as available for the Year 2004.

blood during a 24 hour period is referred to as periodicity. Species that are found in the blood during night-time hours but are absent at other times, are designated nocturnally periodic (e.g. Wuchereria bancrofti, Brugia malayi); those that are present only during certain daytime hours are designated diurnally periodic (e.g. Loa loa).Microfilariae that are normally present in the blood at all hours but whose density increases significantly during either the night or the day are referred to as subperiodic. Microfilariae that circulate in the blood throughout a 24-hour period without significant changes in their numbers are referred to as nonperiodic or aperiodic (e.g. Mansonella spp). The periodicity of a given species or geographical variant is especially useful in determining the best time of day to collect blood samples for examination. To determine microfilarial periodicity in an individual, it is necessary to examine measured quantities of peripheral blood collected at consecutive intervals of 2 or 4 hours over a period of 24-30 hours.

1.3.5 Vectors

There exist many mosquito species, which act as vectors for the three types of filarial nematodes. The most important vectors of W. bancrofti are Culex quinquefasciatus, Anopheles gambiae, Anopheles funestus, Aedes polynesiensis, Aedes scapularis, Aedes pseudoscutellaris etc. For B. malayi, Anopheles barbirostris, Anopheles sinensis, Anopheles donaldi, several species of Aedes and Mansonia and for B. timori infection, Anopheles species serve as vectors.

1.3.6 Clinical Groups in Filariasis

Lymphatic filariasis is characterized by a variety of clinical manifestations which enables the classification of people residing in an endemic region into different groups.

1.3.6.1 Asymptomatic amicrofilaremics (Endemic Normals)

Although the entire population of an endemic area is exposed to the parasite antigens, large groups of healthy people exhibit no signs of infection, either parasitological or clinical and are classified as endemic normal (EN) (Ramzy and Gad et al 1991).

1.3.6.2 Asymptomatic microfilaremics (MF)

This form is described by loads of microfilariae (mf) in blood and adult worms in lymph nodules of infected individuals, causing lymph node and duct dilation leading to lymph retention in the area drained by the affected nodes and vessels.

1.3.6.3 Chronic Pathology

Adenolymphangitis (ADL), an important manifestation that occurs due to the reaction of parasite products, released either by the adult worm or mf (Ottesen 1984) , or due to secondary infections. These agents constantly invade the affected lymphatics, generating trauma and acute inflammation of the skin, lymph nodes and lymphatic vessels. This causes lymphadenitis, lymphangitis, lymphoedema, chyluria, hydrocele leading to a chronic obstructive form of disease called elephantiasis. This is initially reversible, but later becomes permanent and irreversible. Elephantiasis is characterized by solid non-pitting edema (permanent swelling), fibrosis and hyperplasia (excess growth) of the affected organ, viz. foot, legs, vulva, and male genitalia. Rarely, the female breasts are affected.

Adenolymphangitis (ADL) and Acute Filarial Lymphangitis (AFL) are the acute manifestations in lymphatic filariasis. ADL is characterized by recurrent attacks of fever associated with inflammation of the lymph node or lymph vessels, which is recurrently seen in brugian filariasis rather than in bancroftian filariasis (Partono 1987). The lymphatic system of the male genitalia is repeatedly affected leading to funiculitis, epididymitis or orchitis, or a combination of these (Pani et al 1995). The limb, breast or male genitalia are implicated in acute attacks of ADL.

1.3.6.4 Non-filarial elephantiasis

Non-filarial elephantiasis may be produced by deposits of inorganic materials in the lymph nodes of the lower extremities. Its prevalence is higher in males, indicating sex differences in occupationally linked trauma to the feet and cumulative effect of long-term exposure to volcanic soils (Kloos et al 1991, 1992). So keeping in mind the similar manifestations observed in both the cases it becomes imperative to identify a specific diagnostic test that differentiates elephantiasis of filarial and non-filarial etiology.

1.3.6.5 Tropical pulmonary eosinophilia (TPE)

This is an interstitial lung disease characterized by cough, dyspnoea and nocturnal wheezing (resulting from hyper immune response to filarial antigens). These individuals have high levels of eosinophils and parasite antigen specific IgE (Ottesen and Nutman 1992). Studies of Paxton et al (1993) show that these individuals show high levels of total serum IgE and filarial specific IgG.

1.3.7 Filarial Genome Project

The complete genome sequencing for filarial parasite is not yet available. However the sequences of key antigens and the gene involved in immune evasion are currently available (Maizels et al 2001). The identification of the prophylactic candidates and drug targets to counter Human Lymphatic Filariasis began as a part of Filarial Genome Project launched by WHO in the year 1994. Genes of diagnostic importance like SXP-1 (Rao et al 2000), prophylactic candidates like Thieredoxin (Kunchitapautham et al 2003 etc have been identified which are currently being worked on. Novel methods like Phage Display-Based Subtractive Screening were performed to identify vaccine candidates from B. malayi (Gnanasekar et al 2004) and their efficacy in terms of protective immune response is currently being assessed.

1.3.8 Vaccine for Filariasis

1.3.8.1 Need for the vaccine

Till date no vaccines are available for humans against filariasis. There is an urgent need for it due to the increasing infection rate particularly in developing countries. The situation is complicated by the non-availability of the drugs which can ensure complete cure from the parasite. Further difficulty lies on the various stages of the parasite where there occurs various kind of immune response in the host that makes hard to define the correlates of protective immune response, which forms the basis for vaccine construction. This implies that multiple vaccines will be needed, if each stage of a parasite’s life cycle will need to be targeted.

A promising vaccine candidate against these parasites of complex life cycle patterns should be endowed with the following properties: a) to elicit a high titer of protective antibodies and preferably of IgG1 and IgG3 subclass (Garruad et al 2003a and 2003b) should promote T cell activation, especially the T cytotoxic cells. A high antibody titer against an immunized antigen necessities a prolonged circulation of antigen in circulation and enhanced immunogenicity. This can be attained only by the usage of Adjuvants.

1.3.8.2 Currently available vaccines

Experimental studies comparing the protective efficacy with ALT-2 DNA vaccines and recombinant protein vaccines implicated a greater protection with recombinant protein compared to DNA which may be attributed to a low antibody response with DNA vaccines. Recently (Thirugnanam et al 2007) reported a higher protective efficacy of recombinant BmALT-2 protein (75%) compared to DNA vaccination (57%) and prime boost vaccine strategy in Jird experimental models. The immune response analysis in this regard revealed that immunization with recombinant Bm ALT-2 protein generates a strong TH-2 response with higher levels of IL-5, IL-4 and IgG1 in comparison with DNA immunization which generates TH-1 responses and the prime boost strategy which results in both Th-1 and Th-2 responses with lesser levels of Th-2 responses which is required for protection.

A zinc containing metalloprotease, 175 kDa collagenase, purified from adult female Setaria cervi showed strong cross-reactivity with sera from putatively immune (PI) individuals (unpublished observation) and induced cytotoxicity to B. malayi L3 larvae and microfilariae by ADCC mechanism (Srivastava et al 2004). Vaccination with this protease resulted into a mean protection level of 75.86% and produced high level of protease neutralizing antibodies (Pokharel et al 2006). Vaccination with irradiated larvae provided long-term protection against the third larval stage but not against subsequent life cycle stages (Babayan et al 2006).

1.3.8.3 DNA vaccines

Keeping in view the complex life cycle of Filarial parasite and its disease dynamics, it was perceived that Human Lymphatic Filariasis may be better encountered through DNA vaccination in terms of achieving protective immunity against this deadly disease. Li et al had evaluated the DNA of Brugia malayi paramyosin protein (BM5) as a DNA vaccine (Li et al 1999). A comparison of protective immune responses induced by Bm-alt-2 DNA, recombinant Bm-ALT-2 protein and prime-boost vaccine regimens in a jird model was conducted by Thirugnanam et al (2007). They suggested that Bm-ALT-2 protein vaccination regimen may be slightly better than prime-boost vaccine regimen and the DNA vaccine (Thirugnanam et al 2007).

A trial was made with the DNA of chitinase gene from Onchocerca volvulus which was shown to confer resistance against post-infective L3 larvae (Harrison et al 1999), immunized mice with DNA plasmids expressing the O. volvulus antigens, Ov-TMY-1 (tropomyosin) and OvB20 (a nematode specific gene product) and showed that the DNA immunization has good potential for induction of humoral responses against nematode infections (Harrison and Bianco 2000).

1.3.8.4 Potential vaccine candidates from B. malayi

It has been shown that the major transcript present in mosquito- borne infective larvae, Bm-ALT, is a credible vaccine candidate for use against lymphatic filariasis, while a second abundantly-expressed gene, Bm- VAL-1, is similar to a likely vaccine antigen being developed against hookworm parasites. A few other interesting vaccine candidate identified from B. malayi cDNA library include Abundant Larval Transcript (ALT-1 and 2) and Vespid or Venom Allergin Homologue (VAH) (Maizels et al 2001) etc. Recent studies also indicate that the Secreted Larval Acidic Protiens (SLAPS) (Wu et al 2004) are members of the ALT gene family. These two related but unique genes, expressed strongly in the larval stages are considered to be the potential candidates for vaccination against filarial infection (Sabarinathan et al 2004 and Murray et al 2001). The high level of sequence similarity between the ALT sequences from B.malayi and W. bancrofti suggests a good immunological cross-protectivity (Garraud et al 2005).

1.3.8.5 Adjuvants

Adjuvants are a group structurally heterogenous compounds that augment or transform the immunogenicity of poorly immunogenic vaccine proteins or peptides (Vogel 1995). Adjuvants, presently licenced for human use include alum, squaline oil/water emulsion (mf 59), influenza viriosomes and some cytokines like IFN- and IL-2 Besides these, there are a number of other adjuvanats that are currently under investigation like, DNA motifs, monophosphoryl lipid A, Cholera toxin (CT) E. coli heat labile toxin (LT) , immunostimulating complexes (ISCOMs) etc). Unmethylated CpG dinucleotide motifs present in bacterial DNA (uncommon in mammalian DNA) are powerful stimulators of immune responses in mammalian hosts. CpG-ODN induces a Th1-biased immune response, including direct stimulation of APC through a toll-like receptor (TLR) 9 (Hemmi et al 2000). Thus, CpG-ODN acts as a potent adjuvant, with the result that some studies have employed CpG-ODN for more successful immunization in animal models and in non-human primates (Stacey et al 1999; Brazolot et al 1998; Davis et al 2000) and even in very young mice than an HbsAg-expressing DNA vaccine (Brazolot et al 1998). CpG motifs with DSP30 can favour the production of IgG1 and IgG3 and not IgG2 and IgG4, IgG1 and foremost IgG3, represent antibody subclasses that mediate protection against viral and certain parasite pathogens (Garraud et al 2003a and 2003b). Alum (aluminium based mineral salt) stimulates strong Th-2 type responses and recently it has been observed that it up regulates costimulatory signals on monocytes and results in IL-4 production (Ulanova et al 2001). Abraham et al have shown that when mice were immunized with the five individual recombinant antigens (Ov7, Ov64, OvB8, Ov9M, and Ov73k) statistically significant reductions in parasite survival were induced in mice immunized with Ov7, OvB8, or Ov64, when administered in alum but not when injected in Freund's complete adjuvant (FCA) (Abraham et al 2001). The aqueous suspensions of trehalose 6-6' dimycolate (TDM) has been successfully used as an effective immunomodulator in experimental studies on filariasis by Sharma and Upadhyay (1993).

1.3.8.6 Animal models in lymphatic filariasis

Meriones unguiculatus, commonly known as jirds or gerbils have been used extensively as animal models to study animal and human parasites infections. Gerbils have been successfully used as a source of different developmental stages of filarial parasites, like B.malayi which is a natural parasite of humans and B.pahangi, a parasite infecting cats and dogs (Junhom et al 2006; Ash and Riley 1970; Lok and Abraham 1992). Nevertheless, humans are the only definitive hosts for lymphatic filariasis caused by W. bancrofti (accounting for 90% of the cases) and no animal models are available to study this parasite development.

1.3.9 Diagnosis of lymphatic filariasis

1.3.9.1 Parasitological diagnosis

This requires night blood specimens which is a major drawback, due to failure of the population to cooperate. Other methods like counting chamber membrane filtration, Knotts concentration and DEC challenging day test (Denham 1995) are less sensitive in identifying low numbers of mf or mf sequestered in inaccessible sites (Wamae 1994). Membrane filtration technique is time consuming and requires large volumes of blood for examination.

1.3.9.2 Lymphatic imaging

Ultrasonography is a non-invasive, cheap, rapid and a sophisticated test with no side effects but unsuitable for field studies (Doldi et al 1992). Using this method live W. bancrofti adult worms were directly localized along with structures of peculiar alleviatory movements called “filarial dance” and isolated from the scrotal lymphatics of asymptomatic microfilaraemic men (Amaral et al 1994).

Lymphoscintigraphy is safe and non-invasive technique, but is costly and inappropriate for field evaluations. It examines the peripheral lymphatic system, including truncal and nodal abnormalities, in endemic populations with occult and overt lymphatic filariasis (Fredman et al 1994).

1.3.9.3 Lymphangiography

Though this technique recognizes alterations in lymphatic anatomy of filarial patients (Sen et al 1969), it is laborious, impracticable for mass screening, time consuming, invasive and uses oil based contrast material for imaging (Miller et al 1987), which can induce local morbidity and aggravate the pathology.

1.3.9.4 DNA based diagnosis

Nucleic acid probe based assays are always sensitive and specific for the corresponding parasite DNA. DNA probe sensitivity relies on the presence of highly repeated sequences, generally non-coding, which evolve more rapidly than the rest of the genome, thus making them potential targets for the genus and species-specific identification. Species-specific DNA probes developed for Brugia malayi, Wuchereria bancrofti, Onchocerca volvulus and Loa loa (Nutman et al 1994) enable the detection of parasite DNA in whole blood and vector (Chandrasekhar 1997). Highly repeated sequence is generally used to make the most sensitive DNA probes in Onchocerca volvulus (Zimmerman et al 1994).

1.3.9.5 Immunodiagnosis

These methods, which rely on the detection of circulating antigen or antibody in the serum, are based on heterologous or homologous, either crude or fractionated filarial worm antigens, recombinant antigens or monoclonal antibodies.

1.3.9.5.1 Antibody detection assay

W. bancrofti mf antigens have been used to develop latex agglutination, indirect haemagglutination and ELISA for the diagnosis of filariasis (Kaliraj et al 1981; Dasgupta et al 1984). Antibodies against both crude and recombinant antigens are evaluated for the diagnosis of filariasis. BmSxp is the most commonly used recombinant antigen for the diagnosis of filariasis by ELISA. This antigen is either used alone or in combination with other recombinant antigens like BmR1 WbSXP-1, WbVAH (Lammie et al 2004; Rao et al 2000; Baskar 2004) these tests prove to be cheaper and faster, however the specificity ranges from 85-95%.

1.3.9.5.2 IgG4 antibody based assays

IgG4 based assays using either crude or recombinant antigens are used to diagnose individuals with active infection. Amicrofilaraemia, with clinical filariasis undetectable by routine parasitological methods, are detected by these assays. Using soluble B. malayi antigen, anti-filarial IgG4-ELISA can detect 4-6 times more positive cases (microfilaraemic) than mf detection by night blood smear (Rahmah et al 1994). So, active filarial infections can be identified by demonstrating IgG4 antibodies in the patients’ sera. In this regard, anti-Wb-SXP-1 IgG4 ELISA was developed and it was found to be 100% sensitive for patients with patent W. bancrofti infection (Rao et al 2000).

1.3.9.5.3 Homologous antigen based antibody assays

These antigens can be obtained either by fractionation of the crude worm extracts or by recombinant DNA technology. Several fractionated homologous antigens from W. bancrofti and B. malayi are preferred over the heterologous antigens, since they exhibit diminished cross reactivity to different nematode species.

In this regard it is note worthy that purified surface antigens of the bovine filarial parasite S. digitata were sensitive and specific in the detection of antibodies in filarial sera and showed least cross reactivity with sera of other parasitic infections (Theodore and Kaliraj 1990). Though several homologous antigens have been characterized and proved to be of diagnostic value, they remain unsuitable for field applications, due to scarcity of parasitic material, lack of animal models for W. bancrofti and tedious fractionation procedures.

A rapid-format, simple and quantitative immuno filtration test was developed in our laboratory which detects total IgG antibodies to recombinant antigen Wb-SXP-1. This test system employs colloidal gold-protein A, reagent for antibody capture and was evaluated with serum samples from patients with filarial infection and with various control samples. The sensitivity was found to be 91.8% with brugian and 86% with bancroftian microfilaraemic subjects. Specificity was found to be 94.4% for brugian and 79.1% for bancroftian microfilaraemic subjects. Another attribute of this method is its non reactivity to most of other parasitic disease like malarial infections and its ability to detect mf positive subjects from both brugian and bancroftian filariasis.

1.3.9.5.4 Heterologous antigen based antibody assays

Heterologous antigens include soluble whole worm antigens or mf antigen extracts, excretory-secretary (ES) antigens and circulating immune complex antigens (CIC). The non-availability of homologous antigens has led to the identification and purification of heterologous filarial antigens that show specificity to human filariasis.

1.3.9.5.5 Excretory Secretory (ES) antigen based antibody assays

Due to non-availability of sufficient parasite material from

W. bancrofti, a heterologous ES antigen from B. malayi has been used to diagnose bancroftian filariasis. MF individuals had high levels of antibodies to the mf ES antigen suggesting that microfilaremic state is associated with a stronger antibody response to mf ES. The two recombinant proteins of diagnostic importance, Bm 12 and Bm 14 identified from mf ES (Kumari et al 1994) are highly species specific.

1.3.9.5.6 Circulating immune complexes (CIC)

CICs in the sera of filarial patients can be confirmed using polyclonal antibodies raised against adult S. digitata worms (Dissanayake et al 1982, 1983 and 1984). These polyclonal antibodies are reactive with the antigens derived from CIC and adult worms, but not to microfilarial antigens.

1.3.9.5.7 Monoclonal antibodies

Hybridoma derived mAb is useful for diagnosis. MAb Gib13 raised against O. gibsoni can detect circulating antigen in the sera or urine samples of W. bancrofti-infected persons in an immuno-radiometric assay (Forsyth et al 1985). Another mAb, E34 raised against W. bancrofti mf ES antigens were able to detect filarial antigen associated with active infection (Reddy et al 1989). MAb raised against a major 200 kDa circulating antigen was directed against phosphoryl choline (PC) epitopes of W. bancrofti. Though this PC determinant is not filarial specific, its abundance in PC-bearing filarial antigen in circulation, makes it a useful target for immuno diagnosis (Lal et al 1987). MAb-Og4C3 of O. gibsoni for the detection of circulating antigen in human sera is highly specific for W. bancrofti infections and can detect circulating ES antigens from W. bancrofti adult worms. Further it does not cross react with sera of patients infected with B. malayi, B. timori, O. volvulus or Loa loa (More and Copemann 1990). In our laboratory, this mAb assay was evaluated and standardized to suit field conditions (Lalitha et al 1998). ICT diagnostics, Balgowlah, New South Wales, Australia had developed a rapid filarial antigen card test based on AD12.1 mAb (Weil et al 1997).

1.3.9.5.8 Antigen detection assays

Since most of the antibody detection systems cannot differentiate between current and past infection, parasite antigen detection in blood and other body fluid has been the focus of research for the past 20 years (Dissanayake et al 1982; Reddy et al 1984; Hamilton et al 1984).

Antigens from Setaria digitata (Theodore and Kaliraj 1990), Dipetalonema vitae (Baschong et al 1982), O. gibsoni (Forsyth et al 1981) have been studied for their use in diagnosis. The WbE34 monoclonal antibody raised against W. bancrofti microfilarial ES antigen is useful in detecting the filarial antigen in W. bancrofti and B. malayi infected sera (Reddy et al 1989). Filarial antigen detection system “Seva-Filachek” based on B. malayi mf ES antigens is useful in detecting occult filarial infections (Harinath et al 1996).

The AMRAD-ICT Filariasis Test (ICT-Fil), a new, rapid-format card test for the detection of bancroftian antigenaemia in human blood has been developed by Ramzy et al (2000). A Wb-SXP-1 antigen capture ELISA which can detect both brugian and bancroftian filariasis was recently developed using antibodies against the antigen was developed in our lab and the reactivity was found to be 83.33% and 88.33% for brugian and bancroftian filariasis respectively.

1.3.10 Lymph (Formation, Absorption and Flow)

The present theory of the formation of lymph was proposed by Starling in 1896. He hypothesized that blood capillary endothelium is a semi permeable membrane i.e. permeable to water and crystalloids and impermeable to plasma proteins. Hydrodynamic pressure induces ultra filtration in the arterial limb of the capillaries. This gradually gets further reduced in the venous limb and becomes lesser than the colloidal osmotic pressure of the plasma protein. Since very little plasma protein diffuses out, the pericapillary tissues are practically free of them, so the colloidal osmotic pressure acts as a force of absorption in the venous limb of the capillary. Thus filtration ceases and the protein free interstitial fluid is absorbed back. The fluid along with the corpuscular elements are pulled out and transported via the lymphatics, which finally empty into the veins. Under normal conditions, force of filtration and reabsorption are equal, but if the former is elevated (e.g. venous congestion) or the latter is decreased (hypoproteinemia), it results in a pathological collection of fluid in the interstitial space and increased lymph flow.

According to Casley Smith, the mechanism of lymphatic absorption is as follows. The capillary holes held apart by the microfibrils open up and allow the interstitial water and dissolved molecules to enter the lymph capillaries. The slowly diffusing protein macromolecules are also allowed to enter. At rest, there is little flow of lymph, at least in the peripheries. A little movement compresses the lymph capillaries to push up the fluid. Simultaneously, the openings (which act as flap valves) are shut by any increase in pressure. Thus massage and movement push up the fluid and relaxation of pressure allows more fluid to enter (Drinker and Yoffrey 1941). In this regard it was also observed that pressure also pushes out some of the fluid and crystalloids back into the tissue spaces, to decrease the colloidal pressure there, as compared to the intra lymphatic colloidal pressure (Casley Smith 1976a, 1976b). Rusznayak et al (1967) stated that some water and colloids were reabsorbed by the vascular compartment from the lymph vessel to make lymphatic protein content higher than the tissue protein content. Massage and movement thus promote the vascular absorption of fluid and allow larger particles to diffuse. The smooth muscle lining the lymphatics has an automatic tone and undergoes spasm in acute inflammation. The loss of this tone may be one cause of edema in paralysis.

1.3.10.1 Lymphoedema

Lymphoedema has been defined as swelling of the soft tissues caused by abnormal quantity of lymph. This definition can be further expanded to include the after effects of this accumulation i.e., proliferation of connective tissue, a variable degree of cell infiltration, pigmentation and fibrotic thickening of the dilated lymphatics. In long standing cases, these result in a firm enlarged limb with hardened skin and papillomatosis, justifying the name ‘ELEPHANTIASIS’ in severe cases.

Lymphoedema is a common clinical problem in many parts of our country (Ahuja 1976). The magnitude of this problem can be seen from the fact that nearly 200 million (20% of the population) live in the endemic zone and more than 19 million; actually suffer from the disease (Priya Ritu 1994). Repeated attacks of fever, lymphangitis and lymphadenitis are accompanied by progressive edema of the limbs. This is widely regarded as the most significant discovery in tropical medicine, with implications that went far beyond helminthology into such diverse areas as malaria and the arboviruses.

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Details

Seiten
232
Jahr
2006
ISBN (eBook)
9783656083665
ISBN (Buch)
9783656083764
Dateigröße
6.9 MB
Sprache
Englisch
Katalognummer
v183781
Institution / Hochschule
Anna Universität
Note
TEXTBOOK-PUBLICATION
Schlagworte
antigenic analysis lymphatic fluid t-cell receptor repertoire gene expression peripheral blood mononuclear cells pbmcs patients with chronic pathology harbouring wuchereria bancrofti infection

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