Ashby B. Bodine

Genetic selection for aflatoxin B1 resistance influences chicken T-cell and thymocyte proliferation

Studies were conducted with two lines of chickens that were selected for high and low plasma protein concentrations in response to aflatoxin B1 (AFB1) exposure. The experiments were designed to determine genetic differences in the responses of T cells and thymocytes to the toxin. Chicks were orally administered AFB1 at a rate of 0, 100, or 500 micrograms/kg body weight up to 21 days of age. At 4 weeks of age, concanavalin A (Con A, 2.5 micrograms/mL) stimulated T-cell proliferation was similar for untreated chicks from the low line (LL) and the high line (HL). However, AFB1 reduced the responses of T cells with HL cells being more sensitive. In a second experiment, immature chickens were bled and peripheral blood lymphocytes were cultured with Con A and either 0, 3.125, 6.25, 12.5, or 25 micrograms/mL AFB1. T cells from LL had greater responses to Con A than those from HL, and LL T-cells were also more resistant to in vitro AFB1 exposure. Furthermore, thymocyte proliferation was greater for LL chicks; but when thymocytes were cultured with 25 micrograms/mL AFB1, 3H-thymidine incorporation was similarly reduced in both lines. Cell cycle analysis indicated that there were more LL thymocytes in S phase, and the percentages for both lines decreased with AFB1 treatment. Although there were no differences between the lines for percent G2/M cells, AFB1 treatment increased the percentages of thymocytes in G2/M. These studies showed that selection for plasma protein response also changed T-cell and thymocyte proliferative activity.

Dexamethasone-induced apoptosis in immune cells from peripheral circulation and lymphomyeloid tissues of juvenile clearnose skates, Raja eglanteria

Juvenile clearnose skates (Raja eglanteria) were injected intramuscularly with dexamethasone-21-phosphate at 50, 75, and 100mg/kg body weight. After 24h, skates were sacrificed and lymphomyeloid tissues (thymus, spleen, Leydig organ, and epigonal organ) were removed and whole blood was sampled. Tissues were used fresh for imprints or prepared for histology by solvent fixation or freezing in liquid nitrogen. Apoptosis in fixed tissues was assessed by transmission electron microscopy. Frozen sections and cytospin preparations of peripheral blood leukocytes (PBL) were evaluated by the TUNEL reaction to detect DNA strand breaks. Dexamethasone treatment increased apoptotic activity in all lymphomyeloid tissues as well as in PBL. These studies demonstrate that immune cells of elasmobranchs have the capacity for glucocorticoid-driven apoptosis, and that programmed cell death as a mechanism to regulate immune cell production appears to have been conserved during vertebrate evolution.

CLINICAL ASSISTED REPRODUCTION: Seminal Plasma and IVF Potential

Purpose: Many components of seminal plasma play a role in sperm motility by serving as energy sources. Human seminal plasma contains over 30 proteins, including forward motility proteins, antifertility proteins, and coagulation/liquefaction proteins. This study was designed to determine any correlation between motility or fertilization rates and concentrations of fructose, lactic acid, citric acid, carnitine, and protein in human seminal plasma.Methods: Fertilization rates were determined by in vitro methods. Fructose, lactic acid, citric acid, and carnitine concentrations were ascertained using high performance liquid chromatography. Protein concentration was determined by Bradford assay.Results: Protein concentrations were significantly different as a function of sperm motility levels. Other constituents of human seminal plasma showed an overall correlation, though not significant. No constituent exhibited significant differences as a function of fertility levels.Conclusions: Protein concentration was significantly lower for samples with high motility. No significant differences between fertility levels and constituents measured were found.

In vitro metabolism of the pro-carcinogen aflatoxin B1 by liver preparations of the calf, nurse shark and clearnose skate

1. Liver postmitochondrial supernatant preparations of calf, clearnose skate, and nurse shark were able to metabolize the fungal toxin aflatoxin B1 to various metabolites. 2. Calf liver produced aflatoxin M1 and Q1 as the major chloroform soluble metabolites, with small amounts of aflatoxicol formed during incubation. 3. Liver preparations of the elasmobranchs, however, produced aflatoxicol as the major chloroform soluble metabolite with no other metabolite being detected. 4. The water soluble metabolite profiles for the three species were also quite different with the tris diol adduct being produced to a much greater extent in calf liver preparations. 5. Aflatoxicol production by the elasmobranch liver homogenates was reversible with the skate reconverting a large amount (30%) of aflatoxicol to AFB1. The nurse shark, however, appeared to convert a portion of aflatoxicol to an unknown metabolite more polar than AFB1. 6. Calf liver DNA bound approximately 3 x more 3H-AFB1 than shark liver DNA.

Epigonal Conditioned Media from Bonnethead Shark, Sphyrna tiburo, Induces Apoptosis in a T-Cell Leukemia Cell Line, Jurkat E6-1

Representatives of Subclass Elasmobranchii are cartilaginous fish whose members include sharks, skates, and rays. Because of their unique phylogenetic position of being the most primitive group of vertebrates to possess all the components necessary for an adaptive immune system, the immune regulatory compounds they possess may represent the earliest evolutionary forms of novel compounds with the potential for innovative therapeutic applications. Conditioned medium, generated from short term culture of cells from the epigonal organ of bonnethead sharks (Sphyrna tiburo), has been shown to have potent reproducible cytotoxic activity against a variety of human tumor cell lines in vitro. Existing data suggest that epigonal conditioned medium (ECM) exerts this cytotoxic activity through induction of apoptosis in target cells. This manuscript describes apoptosis induction in a representative tumor cell line, Jurkat E6-1, in response to treatment with ECM at concentrations of 1 and 2 mg/mL. Data indicate that ECM exposure initiates the mitochondrial pathway of apoptosis through activation of caspase enzymes. Future purification of ECM components may result in the isolation of an immune-regulatory compound with potential therapeutic benefit for treatment of human cancer.