Equine chorionic gonadotropin improves the efficacy of a timed artificial insemination protocol in buffalo during the nonbreeding season.

Two experiments were conducted to evaluate the effects of equine chorionic gonadotropin (eCG) treatment on ovarian follicular response, luteal function, and pregnancy in buffaloes subjected to a timed artificial insemination (TAI) protocol during the nonbreeding season. In experiment 1, 59 buffalo cows were randomly assigned to two groups (with and without eCG). On the first day of the synchronization protocol (Day 0), cows received an intravaginal progesterone (P4) device plus 2.0 mg estradiol benzoate im. On Day 9, the P4 device was removed, all cows were given 0.150 mg PGF(2α) im, and half were given 400 IU eCG im. On Day 11, all cows were given 10 µg of buserelin acetate im (GnRH). Transrectal ultrasonography of the ovaries was performed on Days 0 and 9 to determine the presence and diameter of the largest follicle; between Days 11 and 14 (12 hours apart), to evaluate the dominant follicle diameter and the interval from device removal to ovulation; and on Days 16, 20, and 24 to measure CL diameter. Blood samples were collected on Days 16, 20, and 24 to measure serum P4. In experiment 2, 256 buffaloes were assigned to the same treatments described in experiment 1, and TAI was performed 16 hours after GnRH treatment. Pregnancy diagnosis was performed by ultrasonography 30 days after TAI. Treatment with eCG increased the maximum diameter of dominant follicles (P = 0.09), ovulation rate (P = 0.05), CL diameter (P = 0.03), and P4 concentrations (P = 0.01) 4 days after TAI, and pregnancy per AI (52.7%, 68/129 vs. 39.4%, 50/127; P = 0.03). Therefore, eCG improved ovarian follicular response, luteal function during the subsequent diestrus, and fertility for buffalo subjected to a TAI synchronization protocol during the nonbreeding season.

Patterns of intracellular cytokines in CD4 and CD8 T cells from patients with mycobacterial infections

Using a short-term bulk culture protocol designed for an intracellular-staining method based on a flow cytometry approach to the frequencies of cytokine-producing cells from tuberculosis and leprosy patients, we found distinct patterns of T cell subset expression. The method also reveals the profile of peak cytokine production and can provide simultaneous information about the phenotype of cytokine-producing cells, providing a reliable assay for monitoring the immunity of these patients. The immune response of Mycobacterium leprae and purified protein derivative (PPD) in vitro to a panel of mycobacteria-infected patients from an endemic area was assessed in primary mononuclear cell cultures. The kinetics and source of the cytokine pattern were measured at the single-cell level. IFN-gamma-, TNF-alpha-, IL-4- and IL-10-secreting T cells were intracytoplasmic evaluated in an attempt to identify M. leprae- and PPD-specific cells directly from the peripheral blood. The analysis by this approach indicated that TNF-alpha was the first (8 h) to be produced, followed by IFN-gamma (16 h), IL-10 (20 h) and IL-4 (24 h), and double-staining experiments confirmed that CD4+ were a greater source of TNF-alpha than of CD8+ T cells (P < 0.05). Both T cell subsets secreted similar amounts of IFN-gamma. We conclude that the protocol permits rapid evaluation of cytokine production by different T cell populations. The method can also be used to define immune status in non-infected and contact individuals.

Patterns of intracellular cytokines in CD4 and CD8 T cells from patients with mycobacterial infections.

Using a short-term bulk culture protocol designed for an intracellular-staining method based on a flow cytometry approach to the frequencies of cytokine-producing cells from tuberculosis and leprosy patients, we found distinct patterns of T cell subset expression. The method also reveals the profile of peak cytokine production and can provide simultaneous information about the phenotype of cytokine-producing cells, providing a reliable assay for monitoring the immunity of these patients. The immune response of Mycobacterium leprae and purified protein derivative (PPD) in vitro to a panel of mycobacteria-infected patients from an endemic area was assessed in primary mononuclear cell cultures. The kinetics and source of the cytokine pattern were measured at the single-cell level. IFN-gamma-, TNF-alpha-, IL-4- and IL-10-secreting T cells were intracytoplasmic evaluated in an attempt to identify M. leprae- and PPD-specific cells directly from the peripheral blood. The analysis by this approach indicated that TNF-alpha was the first (8 h) to be produced, followed by IFN-gamma (16 h), IL-10 (20 h) and IL-4 (24 h), and double-staining experiments confirmed that CD4+ were a greater source of TNF-alpha than of CD8+ T cells (P < 0.05). Both T cell subsets secreted similar amounts of IFN-gamma. We conclude that the protocol permits rapid evaluation of cytokine production by different T cell populations. The method can also be used to define immune status in non-infected and contact individuals.

Induction of apoptosis in monocytes by Mycobacterium leprae in vitro: a possible role for tumour necrosis factor-alpha.

A diverse range of infectious organisms, including mycobacteria, have been reported to induce cell death in vivo and in vitro. Although morphological features of apoptosis have been identified in leprosy lesions, it has not yet been determined whether Mycobacterium leprae modulates programmed cell death. For that purpose, peripheral blood mononuclear cells obtained from leprosy patients were stimulated with different concentrations of this pathogen. Following analysis by flow cytometry on 7AAD/CD14+ cells, it was observed that M. leprae induced apoptosis of monocyte-derived macrophages in a dose-dependent manner in both leprosy patients and healthy individuals, but still with lower efficiency as compared to M. tuberculosis. Expression of tumour necrosis factor-alpha (TNF-alpha), Bax-alpha, Bak mRNA and TNF-alpha protein was also detected in these cultures; in addition, an enhancement in the rate of apoptotic cells (and of TNF-alpha release) was noted when interferon-gamma was added to the wells. On the other hand, incubation of the cells with pentoxifylline impaired mycobacterium-induced cell death, the secretion of TNF-alpha, and gene expression in vitro. In addition, diminished bacterial entry decreased both TNF-alpha levels and the death of CD14+ cells, albeit to a different extent. When investigating leprosy reactions, an enhanced rate of spontaneous apoptosis was detected as compared to the unreactive lepromatous patients. The results demonstrated that M. leprae can lead to apoptosis of macrophages through a mechanism that could be at least partially related to the expression of pro-apoptotic members of the Bcl-2 protein family and of TNF-alpha. Moreover, while phagocytosis may be necessary, it seems not to be crucial to the induction of cell death by the mycobacteria.

Induction of apoptosis in monocytes by Mycobacterium leprae in vitro: a possible role for tumour necrosis factor-alpha

A diverse range of infectious organisms, including mycobacteria, have been reported to induce cell death in vivo and in vitro. Although morphological features of apoptosis have been identified in leprosy lesions, it has not yet been determined whether Mycobacterium leprae modulates programmed cell death. For that purpose, peripheral blood mononuclear cells obtained from leprosy patients were stimulated with different concentrations of this pathogen. Following analysis by flow cytometry on 7AAD/CD14+ cells, it was observed that M. leprae induced apoptosis of monocyte-derived macrophages in a dose-dependent manner in both leprosy patients and healthy individuals, but still with lower efficiency as compared to M. tuberculosis. Expression of tumour necrosis factor-alpha (TNF-alpha), Bax-alpha, Bak mRNA and TNF-alpha protein was also detected in these cultures; in addition, an enhancement in the rate of apoptotic cells (and of TNF-alpha release) was noted when interferon-gamma was added to the wells. On the other hand, incubation of the cells with pentoxifylline impaired mycobacterium-induced cell death, the secretion of TNF-alpha, and gene expression in vitro. In addition, diminished bacterial entry decreased both TNF-alpha levels and the death of CD14+ cells, albeit to a different extent. When investigating leprosy reactions, an enhanced rate of spontaneous apoptosis was detected as compared to the unreactive lepromatous patients. The results demonstrated that M. leprae can lead to apoptosis of macrophages through a mechanism that could be at least partially related to the expression of pro-apoptotic members of the Bcl-2 protein family and of TNF-alpha. Moreover, while phagocytosis may be necessary, it seems not to be crucial to the induction of cell death by the mycobacteria.