Detection of cell-mediated immunity to sheep erythrocytes by the capillary migration inhibition technique in the lizard, Calotes versicolor

Utilizing the in vitro capillary migration inhibition (MI) technique, the cell-mediated immune response to sheep erythrocytes (SRBC) has been studied in the lizard, Calotes versicolor. The viability of spleen cells in culture was above 85%, irrespective of the presence or absence of antigen. Both plaque-forming cell (PFC) and MI responses were found to be antigen-specific. Heat-labile serum factors did not seem to have a role in the MI phenomenon. Both cellular and sonicated membrane preparations of SRBC induced a similar pattern and degree of MI of sensitized spleen cells. Migration of spleen cells was observed within 1 h of the initiation of cultures. The maximum difference between control and experimental cultures occurred by 12 h of incubation. There was an insignificant escape from inhibition after 24 h of culture. Administration of 6×10(8) SRBC via the intramuscular route favoured both PFC and MI responses. Although the PFC generation was favoured, only a low level of MI was induced by the intraperitoneal and intracardiac injections. MI and PFC responses are inversely related to the amount of antigen injected. Administration of 10(4) SRBC resulted in a high degree of MI without the production of PFC. On the other hand, 6×10(8) SRBC produced an abundant PFC response with a lesser degree of MI. Incorporation of SRBC into Freund's complete adjuvant resulted in the production of MI response with a concurrent reduction in the number of PFC. Formalized SRBC generated a good MI response without the induction of PFC. Sonicated SRBC induced both PFC and MI responses. MI was shown to be mediated by sensitized lymphoid cells. As few as 5% sensitized spleen cells were enough to bring about significant MI of unsensitized spleen cells. Thus, MI has been shown to be an in vitro manifestation of CMI to SRBC in the lizard

The immune response of the teleost, Tilapia mossambica to soluble and cellular antigens

The immune response of Tilapia mossambica to bovine serum albumin (BSA) and sheep red blood cells (SRBC) was characterized in detail in terms of the appearance of hemolysin plaque-forming cells and circulating antibodies at 30°C. Plaque-forming cells (PFC) were detected in the spleen, head-kidney and thymus of immunized fish and the maximum number was observed in these organs on the fifth day after immunization with SRBC. Peak circulating antibody response occurred on day 8 for SRBC and on day 11 for BSA. Following the second injection of the same antigen, a specific anamnestic response was observed with increased production of PFC and serum antibody. No cross reactivity was found when anti-SRBC antibody was tested with rat erythrocytes. Tests with 2-mercaptoethanol showed that all of the agglutinating antibody produced after both the first and second injection was mercaptoethanol sensitive. Analysis of histological and smear preparations revealed that there were consistent cellular changes occurring in the spleen as well as the head-kidney due to immunization

Immunoglobulin classes in the garden lizard, Calotes versicolor

Two classes of immunoglobulins have been purified from lizard serum using a combination of ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200 or on Sepharose 6B. Lizard IgM is 2-ME sensitive and has an intact molecular weight similar to human IgM. On SDS-PAGE, reduced IgM dissociates into heavy and light chains of molecular weight 70,000 and 23,500 daltons respectively. Lizards also possess a 2-ME resistant, low molecular weight immunoglobulin designated as IgY similar to avian and amphibian IgY. IgY dissociates on SDS-PAGE to yield 59,500 dalton heavy and 26,000 dalton light chains. Antisera raised in rabbits to each of the two Ig classes could be made class-specific by cross-absorption, thus indicating that IgM and IgY represent distinct isotypes

Effect of splenectomy on the immune response in the lizard, Calotes versicolor

After removal of the spleen, the EidechseCalotes versicolor can no longer respond to Immunizierung with sheep erythrocytes. If the spleen is removed, but only in part, is still a perfectly normal reaction exists

The kinetics of rosette-forming cell response against sharp erythrocytes in the lizard

The immune response of the lizard Calotes versicolor to sheep erythrocytes (SRBC) has been studied in terms of the appearance of rosette-forming cells (RFC) and plaque-forming cells (PFC). Two distinct RFC peaks were observed in the spleen, one within three days and the other, between six and ten days after immunization with 0.1 ml of 2.5%, 0.25% or 0.025% SRBC. Lower doses of antigen induced greater and quicker first RFC peaks, whereas higher doses stimulated increasing numbers of PFC as well as quicker appearance of second RFC peak. The early increase in RFC response is suggested to be due to "helper" activity and the second peak to the proliferation of precursors of antibody producing cells. Formalinised SRBC induced the formation of the first RFC peak but not the second, thus supporting the above suggestion. The interesting possibilities with regard to the dissociation of various cellular events during the development of antibody response in the lizard immune system are discussed

Lymphoid differentiation and organization of the spleen in the lizard, Calotes versicolor

In Calotes versicolor, splenic primordium appears at stage 30 as a protuberance of mesenchymal cells from the dorsal mesentery. Lymphopoiesis is initiated at stage 40, followed by an increase in the lymphoid population of the rudiment during the successive stages of development. In the adult, splenic pulp is poorly demarcated into white pulp and red pulp. The former is in the form of closely arranged lymphoid follicles and the latter is highly restricted to narrow strands of blood spaces. A comparative study on the lymphoid organization of the spleen of a few other species of reptiles was also carried out

Morphology of lymphoid organs in a cichlid teleost, Tilapia mossambica (Peters)

In Tilapia mossambica organized lymphoid tissues are present in the thymus, head-kidney and spleen, whereas they are lacking in pericardial tissue, liver, mesonephros, intestine and rectum. No lymphoid tissue was observed in the chondrocranium and cartilaginous viscerocranium of young adults. The thymus in Tilapia is encapsulated by thin strands of collagen fibers and consists of outer, middle and inner zones. While middle and inner zones are comparable to the thymic cortex and medulla of higher vertebrates, the homology of the outer zone is not clear. At the anterior end of the thymus, a loose aggregation of lymphocytes without a definite boundary has been observed. The head-kidney is characterized by the presence of lymphoid follicles, a subcapsular sinus, a hilus-like area and lymphatic vessels. The spleen is grossly divisible into white pulp and red pulp; the white pulp contains only a reticular area without definite lymphoid centers and the latter contains predominantly erythrocytes. Morphological changes in the lymphoid organs associated with immune response have been discussed

Analysis of the development of the lizard, Calotes versicolor. I. A series of normal stages in the embryonic development

It has been shown that the external parameters of eggs of the garden lizard, Calotes versicolor, are not suitable for assessing the exact developmental stages of embryos. In order to make use of this lizard's embryos for experimental work, a series of developmental stages has been characterized, using various morphological features