Effect of Concanavalin A (ConA) on
lymphocytes of thalassemia Patients
Neeraj Kumar
4th Semester, Zoology, Patna Science College, Patna
Kanjaksha Ghosh
Director - Research, Surat Raktadan Kendra & Research Centre, Surat
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Effect of concanavalin A on lymphocytes of thalasemmia patients, Study Guides, Projects, Research of Cell Biology
Culture of lymphocytes of major thalasemmia patients
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Effect of Concanavalin A (ConA) on
lymphocytes of thalassemia Patients
Neeraj Kumar
4th Semester, Zoology, Patna Science College, Patna
Kanjaksha Ghosh
Director - Research, Surat Raktadan Kendra & Research Centre, Surat
References
Table of Contents
ABSTRACT
1 INTRODUCTION
2 OBJECTIVE
3 REVIEW
4 MATERIALS AND METHODS
5 RESULTS
6 DISCUSSION
ACKNOWLEDGEMENT
REFERENCES
2 E ect of Concanavalin A (ConA) on lymphocytes of thalassemia Patients
carbohydrate which is normally termed carbohydrate recognition domain (CRD) or carbohydrate binding domain (CBD). The property of lectin to bind carbohydrate at specific sites has distributed the lectins in all living organisms from virus to vertebrates. In the microbial world they are called by other names such as hemaglutinins, adhesion, and toxins. In animals, lectins present serve much different biological function such as cell adhesion, pathogen recognition, and glycoprotein synthesis. In leguminosae family of plant kingdom lectins are present abundantly. Lectins in plants found in tissues including seeds, roots, and fruits. And the lesser amount of lectins also also present in the bark, flower, and ovaries, and nectar but the seeds contains about upto 10-15% of the total protein content. Plants lectins can be easily extracted because of property of solubility. Concanavalin A is a plant lectin
extracted from the jack beans (Canavalia ensiformis) has been extensively as a probe to study cell surface interaction and cell division. T cells respond to the concanavalin A (Con A ) only in the presence of Ia-positive accessory cells ( AC). There are two steps for the chain of events leading to T cell proliferation (a) Con A induces the production of interleukin 2 (IL2), a growth factor for activated T cells; it is now known that IL2 is produced by T cells of the helper (Lyt- 2-) phenotype; and (b) Con A induces reactivity to IL2 in resting T cells which then respond to IL2 with proliferation. β-Thalassemia is a blood genetic inherited disease from parents to
offspring. B-thalassemia is can be broadly divided into three categories.
[1]
[2]
(a) β-Thalassemia trait- the individual having one defective gene and one normal gene, individual don’t require transfusion but may have experience mild anemia.
(b) β-Thalassemia intermedia– the individual having both defective gene but have some β- globin production and need intermittent transfusion.
(c) β-Thalassemia major (Cooley’s anemia)- the individual have both gene defective and there is no β-globin production and the individual need lifelong transfusion of blood(RBC) and may have short lifespan[3]
β-Thalassemia major patient need blood transfusion within the second birthday to prevent anaemia and physical consequences. The standard of transfusion should maintain hemoglobin level 10gm/dl. The combination of regular blood transfusion and chelation therapy improved overall survival of major patients.[4] Thalassaemia patients have also option of bone marrow or stem cell transplantation but it is out of reach for most of the patients because of money problem hence they have only option of blood transfusion for treatment of thalassaemia. The thalassaemia patients have several complications due to multiple transfusion of blood the patient’s body start making or development of alloantibody against the donor RBC this is called alloimmunization. Alloimmunization further complicates in the problem of getting compatible blood, increased incidence of additional alloantibody and autoantibody (antibody against self RBC antigen). Alloimmunization rate of 5-30% has been found in thalassaemia patients and for prevention of the alloimmunization extended phenotype matched, lecodepleted red cell transfusion is recommended. The number of major thalassaemia patients in India is so high and data of alloimmunization is not through. [5]
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Looking to alloimmunization of the β-Thalassemia patient and their property of producing alloantibodies due to multitransfusion we chose these patients to check for antibody secretion using culture techniques. By culturing its supernatant can be tested for its secretion. The lectin ConA enhances the transformation of B-lymphocytes so it was used for culturing the buffy coat obtained from patients.
2 OBJECTIVE
To evaluate transformation of lymphocyte as well as to evaluate anti red cell antibody secretion from these lymphocytes in culture.
3 REVIEW
Lectins are binding proteins that can bind to carbohydrate moieties of complex macromolrecules which acts as an antigen on the surface of various cells including red cells. Lectins are of non- immune origins proteinaceous in nature and can activate / agglutinate different cells in a very specific manner. It is capable of specific binding to carbohydrates by reversal binding and doesn’t change covalent structure of glycosyl ligands. Lectins can be detected by red cell agglutination or precipitation of glycoconjucates and agglutination can be increased by treating proteolytic enzymes and neuraminidase [6] to better expose these antigens on cells.
Lectins can be easily detected by agglutination assays because lectins interact with carbohydrate or glycoproteins are linked to cell membrane and forms various reversible linkages by binding their sites that interact with cells.The 3-D structure helps to find lectins specivity to carbohydrate and it shows conserved amino acid profile.lectins have a wide range of effects on cells such as agglutination, mitogenic stimulation ,redistribution of cell surface components, modifying the activity of membrane enzymes, inhibition of bacterial and fungal growth, cell aggregation, toxicity, immunomodulation.Because of carbohydrate binding sites it diagonase many disease like malaria. [7]
Table 1 Types of lectins and differentiation between them
POKEWEED MITOGEN (PWM)
PHYTOHEMAGGLUTINAN (PHA) CONCANAVALIN A (ConA)
- Pokeweed, the lectin was first isolated from Phytolacca Americana for transformation of human lymphocytes in vitro. 1. PHA, the lectin extracted from the red kidney beans (Phaseolus vulgaris) contents potents, cell agglutination and mitogenic activities against T lymphocytes. 1. ConA, the lectin first isolated from jack beans (Canavalia ensiformis), the lectin concanavalinA has been used extensively as a probe to studycell surface interactions and cell division.
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Due to the tetramer structure, conA forms clusters of carbohydrate binding receptors by crosslinking and lead to change the membrane or cytoplasmic structures;
after binding to the cell surface of carbohydrates,ConA can interact with with the cell membrane via portions of the molecular surface that alone it not bind to the cell;this secondry interaction may provide the necessary surface alternations to activate other cellular systems for stimulation. [12]
B lymphocyte also called the B cells, its letter derives its designation of its site of maturation at bursa fabricus in birds; bone marrow is the major site for development of B cells in mammalian species including human and mice. Mature B cells are different from the other lymphocytes and the other cells by their display of membrane bound immunoglobulin. [13] From the hematopoietic stem cell (HSC) the development of B cell initiates. After maturation B cells can be divided on the basis on their location, cell surface phenotype, Ag specificity, and activation routes.
Transitional B Cells- This B cell considered to the first stage of the development of B cell, in the bone marrow.
Follicular B cells (FoB) – It is formed in the bone marrow and move to the secondary lymphoid organs and the circulation, constitute the largest subpopulation of B cells. They are also considered as resting cells and called as naïve cells.
Marginal zone B Cells (MZB) –Marzinal B cells present in the marginal zones of spleen. It corresponds to the interphase between the circulation and splenic lymphoid tissue.
B1 B Cells - B1 or B Cells are the first B Cells that form in the in the fetal liver. They are further divides in two parts as B1a and B1b. B1a express the glycoprotein of membrane CD but absent in B1a. [14]
T lymphocytes also called T cells; its letter derives its designation of its maturation in the thymus. When the T Cells bind to the antigen it is called the T-cell receptor. T-cells receptors only recognize MHC molecules. Types of T cells on the basis of membrane glycoproteins, there are two namely T helper (T H) and T cytotoxic (Tc) cells but recently a third is found T regulatory (T reg) cells.
T helper cells – The T cells displays CD4 membrane gycoprotien on thier surface as T helper Cells. TH Cells differentiate into effector cells and it helps in the activation of B Cells but sometimes TH Cells differentiate into memory cells instead of the effector cells.
T cytotoxic cells- The T cells display CD8 membrane glycoprotein on their surface as T cytotoxic cells.
T regulatory cells – T cells which have both the membrane glycoproteins on their surface i.e. (CD4 and CD8). [13]
6 E ect of Concanavalin A (ConA) on lymphocytes of thalassemia Patients
We can use Con A to stimulate B cells ( memory B cells )and from the stimulated B cells supernatant culture can be used to test the immunoglobulin specificity secreted by this cells. CON A has a dose response curve [15] and at optimum dose mainly B cells may be stimulated. Now a day’s various specific B cell growth factors are used as a cocktail for stimulating B lymphocytes.
4 MATERIALS AND METHODS
Isolation of lymphocytes from peripheral blood
Blood Samples
The fresh 10 blood samples were collected from Beta- thalassemia major patients coming for transfusion at Atilakshmi Center after taking informed consent from the patient or their parents if they were below 18 years of age. The samples were collected in 2ml BD Vacutainer® Heparin Tubes from the blood bank. Peripheral blood smears were made of every sample and stained using Giemsa staining technique for morphological examination. In addition total blood counts were done in a semi automated (Nihon Kohden) haematology analyser. This gave us total leukocyte count in the patient’s blood and its differential.
Isolation of lymphocytes from peripheral blood (Ficoll Method)
Isolation of lymphocytes from whole blood was carried out using Ficoll Histopaque. Whole blood was fractionated into different components by differential centrifugation and selective removal. Aqueous density gradient media was used with centrifugation to separate and isolate different blood cell types. The separation medium, HISTOPAQUE-1077, is an aqueous solution of a high molecular weight polysaccharide and sodium diatrizoate, an iodinated non-ionic compound, adjusted to a density of 1.077 ± 0.001. Differential migration of cells during centrifugation results in the formation of layers containing different cell types. The bottom layer contains erythrocytes, which have been aggregated by Ficoll-400 and therefore sediment completely in the Ficoll-Paque density gradient media layer. The layer immediately above the erythrocyte layer contains mostly granulocytes, which at the osmotic pressure of the Ficoll- Paque media solution; attain a density high enough to migrate through the Ficoll Paque media layer. At the interface between the plasma and the Ficoll-Paque layer, mononuclear cells are found together with other slowly sedimenting particles (e.g., platelets) with low density. [16]
Blood was allowed to stand at room temperature (~30 minutes) before processing. 2.5ml of Histopaque-1077 was aliquoted into sterile centrifuge tubes. Carefully 5 ml blood was layered in tube. The tube was centrifuged at 1800 rpm at 4°C for 30 minutes. Following the centrifugation, the lymphocytes appeared in the middle layer as a buffy coat between the yellowish plasma layer on top and red blood cell layer at the bottom. Carefully the yellow-
8 E ect of Concanavalin A (ConA) on lymphocytes of thalassemia Patients
were used to capture red blood cell specific IgG antibodies from patient plasma. Plasma was
added to the red blood cell coated test wells and the wells were incubated at 37 C. Following incubation unbound residual immunoglobulins were rinsed from the wells and a suspension of anti-IgG coated indicator red cell was added. Centrifugation brings the red cells in contact with antibodies bound to the immobilised reagent red cells.
0
Flow cytometry
The immunofluorescence experiments were performed using cultured cells and conjugate which was FITC labelled anti-human IG globulin. The cells were also checked for CD 34 and CD45 markers along with 7 – AAD. [18]
5 RESULTS
Blood smear staining
The figure given below depicts the smear morphology of the cells obtained from whole blood.
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Ficoll-Paque layer, mononuclear cells are found together with other slowly sedimenting particles (e.g., platelets) with low density. (Figure 2 & 3)
Figure 2 – Buffy coat containing lymphocytes
Figure 3 - Lymphocytes aggregate together when spun at high
12 E ect of Concanavalin A (ConA) on lymphocytes of thalassemia Patients
speeds.
Cell count using hemocytometer
To determine the total number of cells isolated, counting was performed using a hemocytometer. Figure 4 describes how the cells looked under the neubauer chamber and table 2 indicates the number of cells found in the four coner chambers.
Figure 4 Cells under Neubauer Chamber (20X)
Table 2 Total number of cells in each chamber in haemocytometer
14 E ect of Concanavalin A (ConA) on lymphocytes of thalassemia Patients
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S5 S
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Figure 6: Smear of cultured cells
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Figure 5 Cells in lymphocyte culture under transformation
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Figure 6 and 7 demonstrates the colour development after incubation at 37 C. The more colour development was obtained in test tubes having high protein content.
0
Figure 6 Tube 1 is water blank and tube 2 is reagent blank. Tube 3, 6 and 7 depict serum sample while the tubes
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18 E ect of Concanavalin A (ConA) on lymphocytes of thalassemia Patients
4, 5, 8 and 9 depicts supernatant of cultured cells.
This depicts the colour difference obtained after incubation. Left tube is reagent control and right tube is serum sample.
Figure 7.
Protein electrophoresis