Abdel Hakim Saad

Blood Testosterone Level: A Season-Dependent Factor Regulating Immune Reactivity in Lizards

An attempt to study the interaction between testosterone (Ts) and the immune system of the lizard Chalcides ocellatus led to three major findings: 1) Endogenous serum Ts levels in both males and females peak in spring and are minimal during summer; 2) Injection of Ts in either male or female lizards induces significant depletion of lymphoid elements, reduction in serum antibody titers to rat erythrocytes and increase in skin allograft survival; 3) A distinct inverse correlation between endogenous serum Ts levels and lizard immunocompetence is observed from March to September. The data obtained strongly suggested that concentration of circulating Ts is a season-related factor that is critical in defining the immune profile of lizards.

Mixed Leukocyte Reaction, Graft-Versus-Host Reaction, and Skin Allograft Rejection in the Lizard, Chalcides ocellatus

In optimal seasonal conditions, splenocytes derived from adult lizards, Chalcides ocellatus, responded to vigorous proliferation in two- and one-way mixed leukocyte reaction cultures (MLRC). Data based on a large number of reciprocal MLRC provided evidence for the presence of strong and diverse lymphocyte activating determinants. These findings were fully confirmed in studies of systemic graft-versus-host reaction as intraperitoneal injection of splenocytes into newborn allogeneic recipients consistently induced splenomegaly, retarded growth and mortality. In favourable ambient conditions, adult lizards were also able to reject skin allografts in a subacute manner (mean survival time +/- S.E. = 28.8 +/- 0.88). The results clearly indicate that the lizard, Chalcides ocellatus, is endowed with an advanced type of cell-mediated immunity, and possesses strong and polymorphic histocompatibility antigens. Cellular alloreactivity in MLRC and towards skin grafts was, however, abrogated in winter and significantly diminished during spring through mid-summer as compared to mid-summer till autumn. The present study, thus, suggests that immunological defects attributed to reptiles might be more apparent than real, and essentially ascribable to the fact that the immune capacity of these ectothermic vertebrates is profoundly modulated by environmental conditions.

Effect of hydrocortisone on immune system of the lizard, Chalcides ocellatus. I. Response of lymphoid tissues and cells to in vivo and in vitro hydrocortisone.

A single dose of 1 mg/g body weight of hydrocortisone acetate (HC) administered intraperitoneally to adult lizards, Chalcides ocellatus induced rapidly a reduction of about 85% of thymic lymphocytes. Histological evidence indicated that cortical, as well as, medullary thymocytes are sensitive to HC exposure. Around 40-50% of lymphocytes in peripheral blood (PB) and spleen were depleted at 3-7 days post-HC injection; such depletion durated about 4 weeks for PB but was rather temporary in spleen. Increase in number of bone marrow (BM) lymphocytes was negligible and transient and could by no way account for the dramatic cell losses in the different lymphoid tissues. The findings thus suggested that HC-mediated lymphocyte depletion in lizards is not attributable to redistribution between the different lymphoid compartments but rather to destruction. In direct conformation, lymphocytes were readily lysed in vitro by 10(-3)M HC, thymocytes being more vulnerable greater than PB greater than spleen greater than BM lymphocytes.

Concanavalin A responsiveness and interleukin 2 production in the snake Spalerosophis diadema

Thymocytes and splenocytes (SC) of adult snakes, Spalerosophis diadema, responded to concanavalin A (Con A) in vitro by strong proliferation during the spring and autumn seasons. Con A-mediated mitogenesis was, however, abrogated in summer and winter. Conditioned medium (CM) collected from snake SC cultures stimulated with Con A in spring or autumn could enhance the Con A summer and winter responses and support the proliferation of splenic lymphoblasts. Gel filtration of native CM on Sephadex G-100 revealed the presence of two biologically active peaks of molecular weight 39-42 and 15 KD. However, only one peak of activity corresponding to molecular weight (m.w.) of 14-15 KD was observed when CM was subjected to analysis by sodium dodecyl sulphate gel electrophoresis (SDS-PAGE). The active molecular forms exhibited isoelectric points of 5.5-5.8 and 6.4-6.6. The findings suggest that Con A activation of snake lymphocytes in optimal seasonal conditions is associated with the secretion of a lymphokine analogous to the interleukin 2 (IL 2) of endothermic vertebrates.

Characterization of Biomphalaria alexandrina-derived lectins recognizing a fucosyllactose-related determinant on schistosomes.

Two novel lectins that bind selectively to a schistosome-associated fucosyllactose-related determinant have been characterized and purified from the hemolymph of Biomphalaria alexandrina, the snail vector of Schistosoma mansoni. Both lectins were purified by affinity chromatography on a column of equimolar mixture of D- and L-glucose coupled to epoxy-activated Sepharose 6B and sequential elution by D-glucose (designated BaSI) and L-fucose (designated BaSII). Assessment of the structural characteristics, by one- and two-dimensional polyacrylamide gel electrophoresis, indicated that BaSI and BaSII were structurally distinct, and exist in their native forms as multimers of non-covalently associated subunits, that were of different sizes in BaSI and of equal size in BaSII. Removal of N-linked glycans by Endo-beta-N-acetylglucosaminidase F resolved the heterodisperse pattern of BaSI subunits into two spots of 13.2 kDa (pI 7.2) and 10.1 kDa (pI 5.8), and collapsed the acidic charge microheterogeneity of the BaSII subunit into a single spot that corresponded in terms of molecular weight and pI to the basic 13.2-kDa subunit of BaSI. In miracidial binding and inhibition assays with different sugars, both lectins exhibited selectivity towards a fucosyllactose sequence, but BaSII had a higher binding preference to fucose moieties. BaSII-Sepharose 4B affinity chromatography and analysis on two-dimensional gels indicated that multiple copies of the fucosyllactose-related determinant were expressed by heterogeneous, acidic glycoproteins in the miracidial stage of S. mansoni.

Sexual dimorphism on the immune responses of the snake, Psammophis sibilans.

Recent trends in research have documented the influence of sex factors on the immune response of higher vertebrates. The present study is the first to indicate the existence of sex-dependent differences in the immune potentiality in reptiles. In optimal seasonal conditions, kinetics of the primary anti-rat erythrocytes (RRBC) antibody response were compared in individual male and female snakes, Psammophis sibilans. Antigen specific rosette forming cells (RFC) were enumerated in the spleen after in vivo primary immunization with 0.5 ml of 10% RRBC. A greater RFC activity as well as highest antibody titers were noted in females as compared to males. In addition, the peak of primary antibody response to RRBC in males was somewhat delayed in comparison with females. A similar pattern of sex-dependent differences was also observed regarding mitogen responsiveness to Con A, PHA and LPS with female lymphocytes being always the highest responders. In conclusion, female snakes demonstrated an elevated response in almost all comparative tests. Thereafter, at least under physiological conditions, our observations suggest a possible influence of sex hormones as immunoenhancers/immunosuppressors in reptiles.

Effect of hydrocortisone on immune system of the lizard, Chalcides ocellatus. III: Effect on cellular and humoral immune responses

Administration of a single injection of 1.0 mg/g body weight hydrocortisone acetate (HC) to adult lizards, Chalcides ocellatus in summer elicited a high and lasting rise in serum corticosterone and cortisol, peak levels being, however, in the physiologic ranges of 10 micrograms % (2 X 10(-7) M) and 40 micrograms % (1 X 10(-6) M), respectively. Elevation of serum corticosteroid (CS) concentrations by exogenous HC impaired the proliferative capacity of spleen cells in mixed leukocyte reaction (MLR) and significantly delayed skin allograft rejection. In vivo HC also abrogated the primary in vivo and in vitro immune responses of lizards to rat erythrocytes (RRBC). Reactivity to allogeneic cells in MLR, and antibody production against RRBC were, however, recovered when serum CS levels resumed normal, basal values i.e. at three weeks post-HC injection. These data indicate that rise in circulating CS induces severe immunosuppression in lizards.

Effect of hydrocortisone on immune system of the lizard, Chalcidesocellatus II. Differential action on T and B lymphocytes

Lymphocytes of thymus, spleen, peripheral blood (PB) and bone marrow (BM) collected from adult lizards, Chalcides ocellatus were cultured for 24 hr in the presence of 10(-3)M hydrocortisone acetate (HC) in order to assess the effect of in vitro HC on lizard T and B cell viability. The results indicated that HC induced stepwise, time-dependent mortality of the majority of thymocytes carrying T cell specific antigen(s) (TSA), 30-50% of T cells of spleen, PB and BM, and of a proportion of splenic B lymphocytes. Administration of 1 mg/g body weight HC to adult Ch. ocellatus lead to depletion of all TSA+ thymocytes. In contrast, T lymphocytes in the peripheral lymphoid compartments revealed both sensitivity and resistance to HC; similarly, B lymphocytes constituted susceptible and resistant subpopulations.

Sex-associated differences in the mitogenic responsiveness of snake blood lymphocytes

The blastogenic response of male and female snake (Psammophis sibilans) peripheral blood lymphocytes to concanavalin A, phytohaemagglutinin, and E. coli lipopolysaccharide was tested for putative sex-associated differences. Data obtained suggest that normal female blood lymphocytes respond relatively vigorous in comparison to male cells or cells taken from gravid female snakes. The data are discussed in terms of possible correlations with the normal status of the animal.

Characterization of diverse hemolymph lectins in the cotton caterpillar Spodoptera littoralis

Abstract 1. 1. Hemolymph lectins (agglutinins) of the cotton caterpillar Spodoptera littoralis were analyzed by agglutination, cross-absorption and carbohydrate-hemagglutination inhibition using several vertebrate erythrocytes. 2. 2. Lectins were found to interact, with all tested erythrocytes, by binding to carbohydrate moieties but showing no definite specificity. 3. 3. Disulphide bonds were probably absent as 2-ME treatment was ineffective. 4. 4. By cross-absorption studies, we have proposed that the hemolymph contains multiple lectins.