The process of programmed cell death, or apoptosis, is generally characterized by distinct morphological characteristics and energy - dependent biochemical mechanisms. Apoptosis is a widely conserved phenomenon helping many processes, including normal cell turnover, proper development and functioning of the immune system, hormone dependent atrophy etc. Inappropriate apoptosis (either low level or high level) leads to many developmental abnormalities like, neurodegenerative diseases, ischemic damage, autoimmune disorders and many types of cancer. To use cells for therapeutic purposes through generating cell lines, it is critical to study the cell cycle machinery and signalling pathways that controls cell death and apoptosis. Apoptotic pathways provide a fundamental protective mechanism that decreases cellular sensitivity to damaging events and allow proper developmental process in multi-cellular organisms. Major mediator of apoptosis is a family of proteins known as caspases. There are mainly fourteen types of caspases but out of them only ten caspasese have got essential role in controlling the process of apoptosis. These ten caspases have been categorized into either initiator caspases (caspase 2, 8, 9, 10) or executioner caspases (caspase 3, 6, 7). Although various types of caspases have been identified so far, the exact mechanisms of action of these groups of proteins is still to be fully understood. The aim of this review is to provide a detail overview of role of different caspases in regulating the process of apoptosis.

OBJECTIVE To investigate the anti-inflammatory and immunomodulatory properties of tulathromycin in vitro and in experimental models of Actinobacillus pleuropneumoniae–induced pleuropneumonia and zymosan-induced pulmonary inflammation in pigs. ANIMALS Blood samples from six 8- to 30-week-old healthy male pigs for the in vitro experiment and sixty-five 3-week-old specific pathogen–free pigs. PROCEDURES Neutrophils and monocyte-derived macrophages were isolated from blood samples. Isolated cells were exposed to tulathromycin (0.02 to 2.0 mg/mL) for various durations and assessed for markers of apoptosis and efferocytosis. For in vivo experiments, pigs were inoculated intratracheally with A pleuropneumoniae, zymosan, or PBS solution (control group) with or without tulathromycin pretreatment (2.5 mg/kg, IM). Bronchoalveolar lavage fluid was collected 3 and 24 hours after inoculation and analyzed for proinflammatory mediators, leukocyte apoptosis, and efferocytosis. RESULTS In vitro, tulathromycin induced time- and concentration-dependent apoptosis in neutrophils, which enhanced their subsequent clearance by macrophages. In the lungs of both A leuropneumoniae– and zymosan-challenged pigs, tulathromycin promoted leukocyte apoptosis and efferocytosis and inhibited proinflammatory leukotriene B4 production, with a concurrent reduction in leukocyte necrosis relative to that of control pigs. Tulathromycin also attenuated the degree of lung damage and lesion progression in A pleuropneumoniae–inoculated pigs. CONCLUSIONS AND CLINICAL RELEVANCE Tulathromycin had immunomodulatory effects in leukocytes in vitro and anti-inflammatory effects in pigs in experimental models of A pleuropneumoniae infection and nonmicrobial-induced pulmonary inflammation. These data suggested that in addition to its antimicrobial properties, tulathromycin may dampen severe proinflammatory responses and drive resolution of inflammation in pigs with microbial pulmonary infections.

OBJECTIVE To compare effectiveness of glycerol, dimethyl sulfoxide (DMSO), and hydroxyethyl starch (HES) solutions for cryopreservation of avian RBCs. SAMPLE RBCs from 12 healthy Ameraucana hens (Gallus gallus domesticus). PROCEDURES RBCs were stored in 20% (wt/vol) glycerol, 10% (wt/vol) DMSO freezing medium, or various concentrations of HES solution (7.5%, 11.5%, and 20% [wt/vol]) and frozen for 2 months in liquid nitrogen. Cells were then thawed and evaluated by use of cell recovery and saline stability tests, cell staining (7-aminoactinomycin D and annexin V) and flow cytometry, and scanning electron microscopy. RESULTS Percentage of RBCs recovered was highest for 20% glycerol solution (mean ± SE, 99.71 ± 0.04%) and did not differ significantly from the value for 7.5% HES solution (99.57 ± 0.04%). Mean saline stability of RBCs was highest for 10% DMSO (96.11 ± 0.25%) and did not differ significantly from the value for 20% HES solution (95.74 ± 0.25%). Percentages of cells with 7-aminoactinomycin D staining but without annexin V staining (indicating necrosis or late apoptosis) were lowest for 10% DMSO freezing medium (3%) and 20% glycerol solution (1%) and highest for all HES concentrations (60% to 80%). Scanning electron microscopy revealed severe membrane changes in RBCs cryopreserved in 20% HES solution, compared with membrane appearance in freshly harvested RBCs and RBCs cryopreserved in 10% DMSO freezing medium. CONCLUSIONS AND CLINICAL RELEVANCE Cryopreservation of avian RBCs with HES solution, regardless of HES concentration, resulted in greater degrees of apoptosis and cell death than did cryopreservation with other media. Transfusion with RBCs cryopreserved in HES solution may result in posttransfusion hemolysis in birds.

The objective of this study was to develop a murine retinal/choroidal/scleral explant culture system to facilitate the intravitreous delivery of vectors. Posterior segment explants from adult mice of 2 different age groups (4 wk and 15 wk) were cultured in serum-free medium for variable time periods. Tissue viability was assessed by gross morphology, cell survival quantification, activated caspase-3 expression, and immunohistochemistry. To model ocular gene therapy, explants were exposed to varying transducing units of a lentiviral vector expressing the gene for green fluorescent protein for 48 h. Explant retinal cells remained viable for approximately 1 wk, although the ganglion cell layer developed apoptosis between 4 and 7 d. Following vector infusion into the posterior segment cups, viral transduction was noted in multiple retinal layers in both age groups. An age of donor mouse influence was noted and older mice did not transduce as well as younger mice. This explant offers an easily managed posterior segment ocular culture with minimum disturbance of the tissue, and may be useful for investigating methods of enhancing retinal gene therapy under controlled conditions.