Co-expressing GroEL-GroES, Ssa1-Sis1 and Bip-PDI chaperones for enhanced intracellular production and partial-wall breaking improved stability of porcine growth hormone.

Porcine growth hormone (pGH) is a class of peptide hormones secreted from the pituitary gland, which can significantly improve growth and feed utilization of pigs. However, it is unstable and volatile in vitro. It needs to be encapsulated in liposomes when feeding livestock, whose high cost greatly limits its application in pig industry. Therefore we attempted to express pGH as intracellular soluble protein in Pichia pastoris and feed these yeasts with partial wall-breaking for swine, which could release directly pGH in intestine tract in case of being degraded in intestinal tract with low cost. In order to improve the intracellular soluble expression of pGH protein in Pichia pastoris and stability in vitro, we optimized the pGH gene, and screened molecular chaperones from E. coli and Pichia pastoris respectively for co-expressing with pGH. In addition, we had also explored conditions of mechanical crushing and fermentation. The results showed that the expression of intracellular soluble pGH protein was significantly increased after gene optimized and co-expressed with Ssa1-Sis1 chaperone from Pichia pastoris. Meanwhile, the optimal conditions of partial wall-breaking and fermentation of Pichia pastoris were confirmed, the data showed that the intracellular expression of the optimized pGH protein co-expressed with Ssa1-Sis1 could reach 340 mg/L with optimal conditions of partial wall-breaking and fermentation. Animal experiments verified that the optimized pGH protein co-expression with Ssa1-Sis1 had the best promoting effects on the growth of piglets. Our study demonstrated that Ssa1-Sis1 could enhance the intracellular soluble expression of pGH protein in Pichia pastoris and that partial wall-breaking of yeast could prevent pGH from degradation in vitro, release targetedly in the intestine and play its biological function effectively. Our study could provide a new idea to cut the cost effectively, establishing a theoretical basis for the clinic application of unstable substances in vitro.

CpG oligodeoxynucleotide protect neonatal piglets from challenge with the enterotoxigenic E. coli.

CpG motifs activates mammalian lymphocytes and macrophages to produce cytokines and polyclonal Ig. These include IFN-γ, IL-12, TNF-a, which are important in the control of bacterial infection. But thus far, the innate immunostimulatory effects of CpG ODN against pathogen have been established mainly in mouse, monkey, sheep, chicken, but not in neonatal piglets. The purpose of this study is to determine the potential protection of CpG ODN against enterotoxigenic Escherichia coli (ETEC) (with which neonatal piglets were susceptible to infection in our lab) in neonatal piglets. Here, we show intranasal (IN)-mucosal and intramuscularly (IM) systemic administration of CpG ODN could enhance innate cellular (cytokine) immunity in the sera and intestine mucosa post challenge, and thereafter the development of antigen-specific antibodies in piglets. IN and IM immunizations of neonatal piglets without antigen both reduced the ETEC excretion and alleviated diarrhoea symptoms upon challenge, and IN route had better protection effects than IM route. Protection in this study was linked to induction of a Th1 response which induced by CpG ODN. Co-delivery with Emulsigen (EM), could improve protection mediated by CpG ODN. These observations indicate that IN administration of 100 µg/kg CpG ODN with 20% EM codelivery may represent a valuable strategy for induction of innate immunity against ETEC infection in neonatal piglets.

Immunization of aged pigs with attenuated pseudorabies virus vaccine combined with CpG oligodeoxynucleotide restores defective Th1 immune responses.

BACKGROUND AND AIMS: Attempts to immunize aged subjects often result in the failure to elicit a protective immune response. Murine model studies have shown that oligonucleotides containing CpG motifs (CpG ODN) can stimulate immune system in aged mice as effectively as in young mice. Since many physiological and pathophysiological data of pigs can be transferred to humans, research in pigs is important to confirm murine data. Here we investigated whether immunization of aged pig model with attenuated pseudorabies virus vaccine (PRV vaccine) formulated with CpG ODN could promote a successful development of immune responses that were comparable to those induced in young pigs in a similar manner. METHODOLOGY: Young and aged pigs were immunized IM with PRV vaccine alone, or in combination with CpG ODN respectively. At days 3, 7, 14 post immunization sera were assayed by ELISA for IgG titres, at day 7 for IgG1 and IgG2 subtypes titres. All blood samples collected in evacuated test tubes with K-EDTA at day 7 were analyzed for flow cytometer assay. Blood samples at day 7 collected in evacuated test tubes with heparin were analysed for antigen-specific cytokines production and peripheral blood mononuclear cells (PBMCs) proliferative responses. RESULTS: CpG ODN could enhance Th1 responses (PRV-specific IgG2/IgG1 ratio, proliferative responses, Th1 cytokines production) when used as an adjuvant for the vaccination of aged pigs, which were correlated with enhanced CD4+ T cells percentage, decreased CD4+CD8+CD45RO+ T cells percentage and improved PRV-specific CD4+ T cells activation. CONCLUSIONS: Our results demonstrate a utility for CpG ODN, as a safe vaccine adjuvant for promoting effective systemic immune responses in aged pig model. This agent could have important clinical uses in overcoming some of age-associated depressions in immune function that occur in response to vaccination.

Age-related impaired Th1 responses to PRV vaccine in vivo in aged pigs.

Murine model studies have shown that function of the immune system declines with aging, but data on aged pigs are scarce. Many physiological and pathophysiological data of pigs can be transferred to human, research in pigs is important to confirm murine data, therefore, aged pigs were chosen as an aged animal model. In this study, we demonstrated an age-related decline in Th1 responses in vivo to PRV vaccine in the pig model, and this decline in type 1 immune responses was associated with reduced PRV-specific T cell proliferation, IgG2/IgG1, and Th1 cytokines production. More importantly, these impaired Th1 responses correlated with reduced CD4(+) T cells and markedly increased CD4(+)CD8(+) T cells. Taken together, these data demonstrated that there was a decline in Th1 immune responses to PRV vaccine with aging in pigs, which may help to explain the age-related decline in vaccine efficacy and increase in morbidity and mortality of infectious diseases.

Obesity and joint replacement surgery in Canada: findings from the Canadian Joint Replacement Registry (CJRR).

Obesity has been strongly implicated as a risk factor for knee osteoarthritis and, in some studies, for osteoarthritis of the hip. Osteoarthritis is the most commonly reported diagnosis for joint replacement patients. In this study, we conducted analyses based on data from the Canadian Joint Replacement Registry (CJRR) to estimate the relationships between overweight and obesity and rates of joint replacement surgery in Canada. Obese persons were over three times as likely and overweight persons were one and a half times more likely to undergo joint replacement surgery, compared to those in the acceptable weight category in 2003-04. This study provides evidence of a clinically relevant association between obesity and joint replacement surgery.