Synthesis, properties and thermal studies of oxorhenium(V) complexes with 3-hydrazino-5,6-diphenyl-1,2,4-triazine, benzimidazolethione and 2-hydrazinobenzimidazole. Mixed ligand complexes, pyrolytical products and biological activity.

A series of biologically active complexes of oxorhenium(V), were prepared by using the organic ligands 3-hydrazino-5,6-diphenyl-1,2,4-triazine (HL1), benzimidazolethione (H2L2) and 2-hydrazinobenzimidazole (H2L3). The mixed ligand complexes of oxorhenium(V) with the previous ligands and one of the following ligands: NH4SCN, 1,10-phenanthroline (1,10-phen), 8-hydroxyquinoline (8-OHquin) or glycine (Gly), were isolated. All the binary and mixed ligand complexes have monomeric structures and exist in the octahedral configuration. Thermal studies on these complexes showed the possibility of structural transformation from mononuclear into binuclear ones. The structures of all complexes and the corresponding thermal products were elucidated by elemental analyses, IR, electronic absorption and 1H NMR spectra, magnetic moments, conductance and TG-DSC measurements. The antifungal activities of the metal complexes towards Alternaria alternata and Aspergilus niger were tested and showed comparable behaviour with some well known antibiotics.

Relationship between circulating thyroid hormones and cell-mediated immunity in immature male chickens.

The objective of this experiment was to examine the relationship between circulating triiodothyronine (T3), thyroxine (T4) and cell-mediated immunity in immature male chickens. Three week old Single Comb White Leghorn male chicks were used as the experimental animals. In order to produce a wide range of circulating thyroid hormone concentrations, birds were divided into groups and received one of nine treatments including surgical thyroidectomy; 0.1% propylthiouracil (PTU) in the feed; 1 ppm T3 and 10 ppm T4 in the feed. The graft versus host (GvH) response and response to phytohemagglutinin (PHA) were measured at 6 weeks of age. Total and differential white blood cells were counted. Birds were bled, weighed, sacrificed, and the lymphoid organs were removed and weighed. Concentrations of T3 and T4 were measured in the birds from all treatment groups. There were positive correlations between thyroid hormones, mainly T3, and weights of thymus and spleen. T4 but not T3 was positively correlated with number of circulating lymphocytes. There were no significant correlations between circulating T3 and T4 and either PHA or GvH responses. It was concluded that physiological levels of thyroid hormones are needed to maintain normal weights of thymus and spleen and a normal level of circulating lymphocytes. Furthermore, we conclude that lower than physiological levels might be sufficient to maintain normal cell-mediated immunity. Finally, in order to enhance cell-mediated immune responses using thyroid hormones, different doses than were used might be required.

Relationship between circulating thyroid hormones and humoral immunity in immature male chickens.

The objective of this experiment was to examine the relationship between levels of circulating T3, T4, and humoral immunity in immature male chickens. Three week old Single Comb White Leghorn male chicks were used as the experimental animals. In order to produce a wide range of circulating thyroid hormone concentrations, birds were divided into groups and received one of nine treatments including surgical thyroidectomy; 0.1% propylthiouracil (PTU) in the feed; 1 ppm T3 and 10 ppm T4 in the feed. Antibody production against sheep red blood cells (SRBC) (thymus-dependent antigen) and Brucella abortus (BA) (thymus-independent antigen) was tested at 6 weeks of age. Concentrations of T3 and T4 were measured in birds from each treatment group at 7 and 11 weeks of age. At 11 weeks of age, birds were weighed, sacrificed and lymphoid organs removed and weighed. There were positive correlations between circulating thyroid hormones and weights of bursa of Fabricius and spleens. There were no significant correlations between circulating thyroid hormones and antibody production. It was concluded that physiological levels of thyroid hormones are needed to maintain normal weights of bursa and spleen. Furthermore, we conclude that lower than physiological levels might be sufficient for normal antibody production. Finally, stimulation of antibody production using thyroid hormones may require different doses than what were utilized in this study.

Chicken growth hormone, triiodothyronine and thyrotropin releasing hormone modulation of the levels of chicken natural cell-mediated cytotoxicity.

Newly hatched White Leghorn male chicks received dietary supplements of either Triiodothyronine (T3) or Thyrotropin Releasing Hormone (TRH) until 8 weeks of age. Chicken growth hormone (cGH) (10 micrograms/kg BW) was injected into different chicks twice daily for 1 week starting at 7 weeks of age. Separate groups received both T3 and cGH. Natural cell-mediated cytotoxic (NCMC) activity against different target cells was tested. It was found that cytotoxic activity in cells involved in NCMC against P815 mouse mastocytoma was stimulated by cGH alone or in combination with T3 (1 ppm). These findings indicate that cGH and T3 stimulate NCMC activity; and that the cells responsible for this activity may be Natural Killer (NK) cells.

Characterization by scanning and transmission electron microscopy of avian peripheral blood mononuclear cells exhibiting natural killer-like (NK) activity.

Peripheral blood mononuclear cells (MN) were isolated from 10-week-old Single Comb White Leghorn female chickens (Cornell K strain) and incubated with either LSCC-RP9 or P815 tumor cells at a ratio of 200:1. Samples were filtered through a 2 micron nucleopore filter at 15 min, 30 min, 1 hr, 2 h, 3 h, and 4 h following incubation to isolate conjugates for scanning electron microscopy (SEM). For transmission electron microscopy (TEM), MN were incubated with LSCC-RP9 tumor cells at a ratio of 200:1 for 1 h. Conjugates in the suspension were fixed, stained, and embedded for thin sectioning. Samples were viewed to determine morphological characteristics of cells having NK-like ability and tumor cell destruction process. The MN possessing NK-like activity appear to be small lymphocytes (5-6 microns in diameter) with a small cytoplasm to nuclear ratio and many surface projections. In addition, it was shown that conjugate formation occurred in both tumor cell lines and the lymphocyte surface projections appear to play a role in the destruction process. Tumor cell destruction did occur with both cell lines, however, the LSCC-RP9 tumors cells were destroyed more rapidly.

Changes in the profiles of circulating white blood cells, corticosterone, T3 and T4 during the initiation of humoral immunity in immature male chickens.

Eight-week-old male White Leghorn chickens were injected intravenously with either 0.1 ml of Brucella abortus (BA) or saline. Blood samples were collected before injection, 1 hr, 3 hr, 6 hr, 12 hr, 24 hr, and then daily for the following 6 days after the injection. Total white blood cells (WBC), differential counts and corticosterone (CS), T3 and T4 levels were measured in all blood samples. These data represented the results obtained from the primary response. To obtain data for the secondary response, the same birds were injected 4 weeks following the first challenge and the same procedure was followed. During primary and secondary responses, circulating lymphocytes and monocytes decreased significantly 3 hr following BA injection and then increased steadily and reached a peak approximately 5 days post-BA injection, however, circulating heterophils increased significantly 6-12 hr and returned to normal levels 2 days post-BA injection. Serum corticosterone increased significantly 3 hr following BA injection. Serum T3 decreased significantly 3 to 12 hr then increased significantly 2 days following BA injection. There were no significant changes in any of the measurements following saline injection. Correlations between circulating hormones and white blood cells were calculated and are reported. These results indicate that an early step in the initiation of humoral immunity is probably an increase in serum CS. This increase in CS is followed by a decrease in circulating lymphocyte and monocyte numbers. These decreases may be the result of lymphocyte trapping in secondary lymphoid organs. This series of events may occur to modulate and activate the immune response.

Initiation of humoral immunity. I. The role of cytokines and hormones in the initiation of humoral immunity using T-independent and T-dependent antigens.

The early events during the initiation of immune responses following the injection of T-independent (lipopolysaccharide, LPS) and T-dependent (bovine serum albumin, BSA) antigens were studied in immature male chickens. Specifically, the role of cytokines and hormones in the initiation of humoral immunity against these antigens was investigated. Both interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) increased significantly post-LPS but not post-BSA injection. While interleukin-2 (IL-2) significantly decreased post-LPS injection, IL-2 significantly increased post-BSA injection. Furthermore, corticosterone levels significantly increased and tri-iodothyronine (T(3)) levels significantly decreased post-LPS but not post-BSA injection. In this study, the results indicate that although LPS and BSA can induce a humoral antibody response in chickens, they activate different cytokines and neuroendocrine network systems.

Initiation of humoral immunity. II. The effects of T-independent and T-dependent antigens on the distribution of lymphocyte populations.

The effect of injecting T-independent (lipopolysaccharide, LPS) and T-dependent (bovine serum albumin, BSA) antigens on the redistribution of lymphocyte populations in immature male chickens was investigated. In the blood, percentages of total T-cells (CD3+), T-helper cells (CD4+), and T-cytotoxic/suppressor cells (CD8+) significantly decreased post-LPS injection (PLI) but not post-BSA injection (PBI), while percentages of monocytes/thrombocytes (K1+) significantly increased PLI. Interleukin-1 receptor expression on blood lymphocytes increased significantly PLI and PBI. In the spleen, the percentages of total T-cells (CD3+) increased significantly PLI and PBI, macrophage (K1+) percentages increased significantly PLI, while B-cell percentages decreased significantly PLI. These results indicate that following antigen injection, there is a redistribution of peripheral blood lymphocytes (specifically T-lymphocytes) to secondary lymphoid organs and the kinetics and magnitude of the changes can differ according to the type of antigen used.

Effect of genetic selection and MHC haplotypes on lymphocyte proliferation and interleukin-2 like activity in chicken lines selected for high and low antibody production against sheep red blood cells.

Chickens from third generation matings of lines of chickens selected for high (HA) and low (LA) antibody production to sheep red blood cells (SRBC) and typed for MHC genotypes B13/13, B13/21, and B21/21 were used in this study. Chickens from both lines carried all the three genotypes B13/13, B13/21, and B21/21. To study T- and B-lymphocytes mitogenic activity, 12-week-old female chickens were injected intravenously with 0.2 ml of 9% SRBC and spleens were collected at 0, 6 h, and 6 day post-antigen injection (pAg). Isolated lymphocytes were incubated with either Concanavalin-A (Con-A) for T-cell activity, or Pokeweed mitogen (PWM) for B-cell activity and thymidine 3H uptakes were measured. To study the Interleukin-2 (IL-2)-like activity in the same lines and genotypes, splenic lymphocytes from 12-week-old chickens were passed through nylon wool columns to enrich the T-cell population. After a 24 h incubation with Con-A, the conditioned media (CM) were collected. The CM were tested for IL-2 like activity by determining whether they altered the proliferation of Con-A stimulated T cells. This proliferation effect was then compared to that of a reference conditioned media (RCM) prepared from K-strain birds and that were used as the standard for the assay. There was no significant difference (p > 0.05) in IL-2 like activity between HA and LA lines, however, the LA was significantly higher than HA (p < 0.05) in T- and B-cell mitogenic activity. The genotype B13/13 had significantly higher (p < 0.05) IL-2 like activity than the B21/21. The genotype B13/13 was also significantly higher (p < 0.05) in T- and B-cell mitogenic activity than the B21/21. At 0 h, pAg T- and B-mitogenic activity was significantly higher (p < 0.05) than 6 h. In summary, our results indicate that although the birds were selected for high antibody production to SRBC, their lymphocyte mitogenic activity was lower than those selected for low antibody production. Hence, humoral and cell-mediated immune responses appear to be under different genetic controls, and that selection for greater humoral response may be at the expense of cellular responses. Our results also suggest differences in IL-2 like activity production between chickens carrying different MHC B-haplotypes, and that genetic control of such activity is possibly linked to the MHC genes.

The role of neuroendocrine immune interactions in the initiation of humoral immunity in chickens.

The presence of neuroendocrine immune interaction in mammalian species has been studied extensively and has been established. However, such an interaction is not as well established in avian species. Furthermore, the role of such an interaction in the initiation of humoral immunity is not well understood. Therefore, the present studies were conducted to determine mechanisms involved in the initiation of humoral immunity in chickens. Cornell K-strain White Leghorn immature male chickens were used for all the experiments. Changes in hormonal and leukocyte profiles after antigen stimulation were studied. The ability of different leukocytes to produce ACTH was also investigated. It was concluded that the first step in the initiation of humoral immunity after antigen exposure is the release of interleukin-1 by macrophages, which in turn stimulates the production of CRF by hypothalamus and/or leukocytes. It is important to mention that CRF production could also be a direct effect of antigen stimulation. The CRF will then stimulate ACTH production by anterior pituitary and/or leukocytes. In addition, CRF will directly enhance lymphocyte activities in the spleen. Corticosteroid production will be stimulated by ACTH and will cause redistribution of lymphocytes from circulation to secondary lymphoid organs such as the spleen for antigen processing and eventual production of antibodies against the invading antigens. Finally, both ACTH and corticosteroids will later act in a negative feedback manner to regulate and control the process of antibody production by inhibiting lymphocyte activities and/or reducing the responsiveness to different stimuli.

Synthesis of novel heterocycles through reaction of indolin-2-one derivatives with active methylene and amino reagents.

Several new spiro compounds were synthesized via one-pot ternary condensation of isatin, malononitrile and each of thiobarbituric acid, barbituric acid, 3-methyl-pyrazolin-5-one, 1-phenyl-3-methyl-pyrazolin-5-one, acetylacetone, benzoylacetone, ethyl acetoacetate, phenacyl cyanide or ethyl-cyanoacetate dimer. Structures and reaction mechanism were reported and supported via a second synthetic route.

Bursectomy and its influence on circulating corticosterone, triiodothyronine, and thyroxine in immature male chickens.

This investigation was carried out to determine the effect of bursectomy on circulating corticosterone, triiodothyronine (T3), and thyroxine (T4) in immature male chickens. One-day-old Single Comb White Leghorn male chicks were surgically bursectomized ( BSX ). Shamoperated chicks were used as control. Blood samples were collected from different BSX and sham birds at 2, 3, and 4 weeks of age. Serum concentrations of corticosterone, T3 and T4 were measured using radioimmunoassays. Serum concentrations of corticosterone and T3 were higher in BSX than sham-operated birds at all ages. Concentrations of T4, however, were similar in both groups. It was concluded that the bursa, directly or indirectly, affected hormone secretion from the thyroid and adrenal glands.

Effect of caponization on cell-mediated immunity of immature cockerels.

The effect of gonadectomy was determined on cell-mediated immune response of immature male chickens. Single Comb White Leghorn males were castrated at 3 weeks of age. Response to phytohemagglutinin (PHA) injection as well as graft vs. host (GvH) response were measured at 5, 10, and 15 weeks of age to evaluate cell-mediated immunity. The PHA and GvH responses were inhibited in castrated males. The results suggest that physiological levels of testosterone are needed to stimulate cell-mediated immune response in immature male chickens.

Effect of exogenous corticosterone on chicken embryonic development.

Embryonic corticosterone (CS) levels were altered by dipping 3-day-old fertile Leghorn eggs in 0, .01, .05, and 1% CS dissolved in absolute ethanol. Mortality was determined by candling from Day 4 through hatch. Body weights and serum CS were measured daily from Day 14 until hatch. Embryos of eggs dipped in 1% CS in ethanol solution had reduced circulating CS. Embryo weights were reduced and mortality increased as concentration of hormone increased. A CS peak was observed just before hatch for control, .01 and .05% groups, but not for the .1% group. All embryos in the .1% treatment group died before hatch. The importance of embryonic CS for normal development and hatching was indicated.

Effect of thyrotropin-releasing hormone, triiodothyronine, and chicken growth hormone on plasma concentrations of thyroxine, triiodothyronine, growth hormone, and growth of lymphoid organs and leukocyte populations in immature male chickens.

One-day-old White Leghorn male chicks were fed different levels of Thyrotropin Releasing Hormone (TRH) (1 and 5 ppm) or Triiodothyronine (T3) (.1 and 1 ppm) for an 8-wk period. In a second experiment, chicken growth hormone (cGH) (10 micrograms/kg of BW) was injected (iv) into different birds daily for 7 days starting at 7 wk of age. Different groups of birds received both T3 (.1 and 1 ppm) and cGH. Serum concentrations of thyroxine (T4), T3, and growth hormone (GH), lymphoid organ weights, total circulating white blood cells (WBC), and differential counts were measured following hormone treatments. It was found that T3, cGH, or a combination of both significantly lowered serum T4 concentrations. Triiodothyronine supplementation at 1 ppm, alone or with cGH significantly increased serum T3 concentrations. Chicken GH with T3 (.1 ppm) significantly increased serum GH concentrations. Thyrotropin releasing hormone supplementation did not affect serum concentrations of either T4, T3, or cGH. Relative bursa weights were greater in chicks that received T3 (1 ppm) or TRH (1 or 5 ppm) but not cGH. Relative spleen weights were enhanced in response to cGH alone or with T3 (1 ppm) but not TRH. Total WBC count was significantly increased in response to T3 (1 ppm). Supplementation of T3 (.1 or 1 ppm), TRH (1 ppm), and the combination of cGH and T3 (1 ppm) significantly increased the percentage of lymphocyte cell population. These results demonstrate the impact of feeding hormones on T3, T4, and cGH concentrations in the serum and suggest the involvement of the above hormones in the growth of lymphoid organs as well as the production of lymphocytes.

Maturation of the diurnal rhythm of corticosterone in female domestic fowl.

The purpose of this study was to examine the maturation of the avian corticosterone diurnal rhythm in relation to other indices of adrenocortical maturation. Female chickens were studied at 1, 5, 11, 17, 23, and 55 weeks of age. Blood samples were drawn at 4-hr intervals during a 24-hr (14 hr light: 10 hr dark) photoperiod. The diurnal rhythm in corticosterone concentration was not apparent until 11 weeks of age. At this age, unlike at 17 weeks of age, the maximal corticosterone concentration occurred during the light period. This indicates that although a rhythm is present at 11 weeks, a phase shift apparently occurs before the adult-like diurnal rhythm is attained. Injection of adrenocorticotropin (ACTH) produced an increase in the circulating corticosterone concentration in chickens at all ages. These results indicate 1) a phase shift must occur between the time of the initial emergence of a diurnal corticosterone rhythm and the attainment of the characteristic adult-like diurnal rhythm, and 2) the avian adrenal cortex is capable of responding to an ACTH stimulus with an increase in circulating corticosterone before the central nervous system rhythmically controls the pattern of circulating ACTH or corticosterone.

The effects of adrenocorticotropic hormone and heat stress on the distribution of lymphocyte populations in immature male chickens.

This study investigated the effects of heat stress and adrenocorticotropic hormone (ACTH) on the distribution of lymphocyte populations. Two experiments were conducted; each used 12-wk-old Cornell K-strain male chickens. In Experiment 1, birds were exposed to a temperature of 35 C, 6 h/d for 5 d and control birds were maintained at 24 C. In Experiment 2, birds received daily i.m. injections of 50 IU/kg body weight of ACTH for 5 d and control birds received .9% saline. On Day 6, blood and spleens were collected and lymphocytes were isolated. Indirect immunofluorescent labeling of lymphocytes were performed to detect B lymphocytes, CD4+ cells, and CD8+ cells. Neither ACTH injection nor heat stress affected percentages of B lymphocytes in the blood or the spleen. The CD4+ cells decreased significantly in the blood of ACTH-injected and heat-stressed birds and significantly increased in the spleens of heat-stressed birds. The CD4+ cells in the spleens of ACTH-injected birds did not differ from controls. The CD8+ cells significantly declined in the blood following both ACTH injection and heat stress and significantly increased in the spleen of the ACTH-injected birds but did not differ from controls in the heat-stressed birds. These results indicate that stress factors lead to redistribution of different lymphocyte populations.

Effect of thyroidectomy of immature male chickens on circulating thyroid hormones and on response to thyroid-stimulating hormone and chronic cold exposure.

Previous studies in the author's laboratory established that thyroidectomized (Tx) immature male chickens had significant levels of circulating thyroid hormones, and it was proposed that extrathyroidal tissue might be present. Three experiments were conducted to further investigate this possibility. In all experiments, thyroid glands were removed surgically at 3 wk of age. In the first experiment, birds were kept until 20 wk of age. It was found that only triidothyronine levels were reduced significantly in the Tx birds. In the second experiment, Tx as well as sham-operated control groups received a single iv injection of bovine thyroid-stimulating hormone (TSH) to determine if extrathyroidal tissue in Tx birds would respond to exogenous TSH. It was found that circulating thyroxine (T4) concentrations in sham-operated control birds, but not Tx birds, were increased following TSH injection. In the third experiment, Tx and sham-operated birds were exposed chronically to cold (7 C), and only circulating T4 was found to be elevated in both groups. It was concluded that extrathyroidal tissue in Tx birds does not respond to TSH.

Effect of induced molting in laying hens on production and immune parameters.

A total of 600 commercial strain (DeKalb) Single Comb White Leghorn hens, 80 wk of age, were used in this study to determine the effects of different induced molting programs on production and immune parameters. The hens were randomly divided into four treatment groups (three experimental and one control) of 150 hens each. The hens in the first treatment group were fed a layer ration containing 20,000 ppm of zinc for 5 d, and received a reduced photoperiod of 8 h/d for 5 d (Zn group). In the second group, feed was withdrawn for 10 d, the photoperiod was reduced to 8 h/d and oyster shell and water were provided for ad libitum consumption. At Day 11, hens consumed corn and oyster shell ad libitum until Day 30 and at Day 31, hens were returned to a full feed layer ration and received 16 h of light/d [California treatment (CAL group)]. In the third treatment, light was reduced to 8 h/d, and oyster shell was provided for ad libitum consumption until Day 60. Feed and water were removed for the first 2 d and on Days 4, 6, and 8. On Days 3, 5, 7, and 9, hens were fed 45 g of feed per hen. On Day 10 until Day 60, hens were fed 90 g/hen and at Day 61, hens were returned to the layer ration ad libitum and received 16 h of light/d [on-again, off-again program (ON-OFF group)]. The last group served as controls (CONT). Body weight, egg production, egg size, internal egg quality, shell weight, and mortality were determined. Total circulating leukocytes, differential leukocyte counts, and antibody production were also measured. The results demonstrated that induced molting significantly increased egg production from 64% to 77 to 83%, Haugh units from 80.4 to 85.9 to 87.3, and shell weight from 5.3 g to 6.3 to 6.4 g when compared to CONT. The body weight of the molted hens decreased significantly to 84.8, 74.5, and 88% of the initial body weight for Zn, CAL, and ON-OFF groups, respectively. The total circulating leukocytes was significantly lower in molted hens than in CONT hens. Differential leukocyte counts were affected by all induced molting programs and the heterophil to lymphocyte ratio was significantly increased, reaching 0.61, whereas that of CONT was only 0.20. Antibody production was largely unaffected by any of the induced molting programs.

Effect of photoperiod on body weight and reproductive performance of ringneck pheasants.

This study was conducted to determine the effects of exposing ringneck pheasants to different photoperiods on their body weights and reproductive performance. Ringneck pheasants were obtained from commercial sources for the first experiment and from the Pennsylvania Game Commission for the second. Birds were exposed to different periods of nonstimulatory light (8 hr of constant light and 16 hr of darkness daily) before they received 16 hr of light and 8 hr of darkness daily (stimulatory light). Data on body weight, feed consumption, semen volume, fertility, and egg production were obtained. The results indicated that ringneck pheasants grow faster when exposed to a period of nonstimulatory light. It was concluded that, if pheasant hens are to be raised under confinement and receive stimulatory light at 27 weeks of age, they should be exposed prior to that time to at least 6 weeks of nonstimulatory light.