Effects of differential pulse frequencies of chicken gonadotrophin-releasing hormone-I (cGnRH-I) on laying hen gonadotrope responses in vitro

The aim of this work was to determine the effects of cGnRH I pulse frequencies on FSH and LH release and the changes in features and number of cultured laying hen FSH-cells and LH-cells in vitro. Primary adenohypophyseal cell cultures taken from laying hens were stimulated by four 5 min pulses using 1 or 10 nM cGnRH, administered with interpulses between pulses at 15, 30 or 60 min. Pulse frequencies and dose dependent effects were examined in six separate experiments including two controls. After the last interpulse time, the supernatants were collected and stored at –70º C until the performance of an indirect enzyme-linked immunosorbent assay (ELISA) using chicken LH and chicken FSH antisera at 1:1000 and 1:2000 dilutions, respectively. Supernatants were coated in duplicate on the inner surface of Immulon 2 plates and later blocked with the optimal solutions. They were incubated with each antiserum and subsequently with isotype-specific peroxidase-labeled anti-rabbit antibodies. Hydrogen peroxide/o-phenylenediamine was added as substrate/chromogen and the optical density (OD) was determined at 492 nm. The ABC immunocytochemical method was performed to characterize and re-count the gonadotropes employing anti-chicken FSH and anti-chicken LH as primary antibodies. The number of FSH-LH cells was obtained using stereological analysis and the data were statistically processed. The ODs obtained for each anti-hormone were compared with the control groups and with each other. Significant differences were found in number of aggregated-positive LH cells, which decreased with 1 nM cGnRH-I, 15 vs. 30 min pulses, increased with 30 vs. 60 min pulses, and also with 10 nM cGnRH-I, 30 vs. 60 min pulses. Aggregated positive FSH cells, however, did not show significant differences in percentage at any GnRH dose or pulse frequencies, but did show activity at low pulse frequencies of 15 and 30 min. The results suggest that LH cells varied in percentage in a dose dependent manner at higher pulse frequency (15 min) and were dose independent at low pulse frequency (60 min) and showed inactive features; while FSH cell numbers were unaffected showing features of activity at low pulse frequencies. High and moderate pulse frequencies of cGnRH-I (15-30 min) increased the FSH release in dose independent manner without changes in features or percentage of FSH cells. Low pulse frequency (60 min) of cGnRH-I increased LH release dose independently disminished LH cell percentage and showed changes in cells’ features. These results in avian cells showed differences in responses to GnRH pulse frequencies from those reported earlier in mammals.Fil: Soñez, María Cristina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Histología y Embriologías; ArgentinaFil: Soñez, Carlos Alberto. Universidad Nacional de Río Cuarto. Facultad de Agronomía y Veterinaria. Departamento de Anatomía Animal; ArgentinaFil: Mugnaini, María Teresa. Universidad Nacional de Río Cuarto. Facultad de Agronomía y Veterinaria. Departamento de Anatomía Animal; ArgentinaFil: Haedo, Mariana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Romera, Sonia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología e Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Lombardo, Daniel Marcelo. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Histología y Embriologías; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Delhon, Gustavo Alfredo. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Histología y Embriologías; Argentin

Coxsackievirus B3 Infection Leads to the Generation of Cardiac Myosin Heavy Chain-α-Reactive CD4 T Cells in A/J Mice

Enteroviruses like coxsackievirus B3 (CVB3) are common suspects in myocarditis/dilated cardiomyopathy patients. Autoimmunity has been proposed as an underlying mechanism, but direct evidence of its role is lacking. To delineate autoimmune response in CVB3 myocarditis, we used IAk dextramers for cardiac myosin heavy chain (Myhc)-α 334–352. We have demonstrated that myocarditis-susceptible A/J mice infected with CVB3 generate Myhc-α-reactive CD4 T cells and such a repertoire was absent in naïve mice as measured by proliferative response to Myhc-α 334–352 and IAk dextramer staining. We also detected Myhc-α 334–352 dextramer+ cells in the hearts of CVB3-infected mice. The autoreactive T cell repertoire derived from infected mice contained a high frequency of interleukin-17-producing cells capable of inducing myocarditis in naïve recipients. The data suggest that CVB3, a bona fide pathogen of cardiovascular system that primarily infects the heart can lead to the secondary generation of autoreactive T cells and contribute to cardiac pathology

Identification of T-cell epitopes in African swine fever virus CD2v and C-type lectin proteins.

African swine fever (ASF) is an emerging disease threat for the swine industry worldwide. No ASF vaccine is available, and progress is hindered by lack of knowledge concerning the extent of ASF virus (ASFV) strain diversity and the viral antigens conferring type-specific protective immunity in pigs. Previously, we demonstrated that ASFV serotype-specific proteins CD2v (EP402R) and/or C-type lectin (EP153R) are important for protection against homologous ASF infection. Here, we identified six discrete T-cell epitope regions present on CD2v and C-type lectin using IFN-γ ELISpot assay and PBMCs from ASF immune animals, indicating cellular reactivity to these proteins in the context of ASFV infection and protective immunity. Notably, three of the epitope regions map to previously described serotype-specific signature regions of these proteins. Improved understanding of ASFV protective antigens, relevant epitopes and their diversity in nature will facilitate ASFV subunit vaccine design and development

Branched chain α-ketoacid dehydrogenase kinase 111–130, a T cell epitope that induces both autoimmune myocarditis and hepatitis in A/J mice

Introduction: Organ-specific autoimmune diseases are believed to result from immune responses generated against self-antigens specific to each organ. However, when such responses target antigens expressed promiscuously in multiple tissues, then the immune-mediated damage may be wide spread. Methods: In this report, we describe a mitochondrial protein, branched chain α-ketoacid dehydrogenase kinase (BCKDk) that can act as a target autoantigen in the development of autoimmune inflammatory reactions in both heart and liver. Results: We demonstrate that BCKDk protein contains at least nine immunodominant epitopes, three of which, BCKDk 71–90, BCKDk 111–130 and BCKDk 141–160, were found to induce varying degrees of myocarditis in immunized mice. One of these, BCKDk 111–130, could also induce hepatitis without affecting lungs, kidneys, skeletal muscles, and brain. In immunogenicity testing, all three peptides induced antigen-specific T cell responses, as verified by proliferation assay and/or major histocompatibility complex class II/IAk dextramer staining. Finally, the disease-inducing abilities of BCKDk peptides were correlated with the production of interferon-γ, and the activated T cells could transfer disease to naive recipients. Conclusions: The disease induced by BCKDk peptides could serve as a useful model to study the autoimmune events of inflammatory heart and liver diseases