Porcine IFN-gamma Research Articles

Comparative Physicochemical Characterization and Bioavailability of Nano-Liposomes Formed by Mixing Lipids at Different Ratios

Liposomes are small lipid vesicles that mimic biological membranes and have been spotlighted in the clinical field due to their ability to enclose a biologically active substance of any structure and to release it into the host's body. This study compares the physicochemical properties and biological activity of nano-liposomes with different compositions to determine the most effective formulation for further in vivo application. Nano-scale liposomes composed of different ratios of 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), dihexadecyl phosphate (DCP), and cholesterol (Chol): DMPE, DMPE/DCP, DMPE/Chol, and DMPE/DCP/Chol were produced. The thermal phase transition was assessed via differential scanning calorimetry (DSC); the particle size, via dynamic light scattering (DLS); the colloidal stability, via the zeta potential; the direct morphological characterization, via transmission electron microscopy (TEM); and the protein encapsulation efficiency. The bioavailability was also investigated with respect to the immunological responses via porcine interferon gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay in peripheral blood mononuclear cells (PBMC) of immunocompetent pigs. All the liposomes can be expected to be stable in an in vivo physiological temperature, and the liposomes that were prepared from DMPE/DCP showed the best efficiency in the in vitro model that mimicked the release of a bioactive substance in vivo. In the result of DLS and the zeta potential for the investigation of the colloidal stability in the system, DMPE/DCP/Chol appeared better than the other formulations. The porcine IFN-gamma ELISPOT assay results postulated that DMPE/DCP most potently induced the IFN-gamma secretion by PBMC, followed by DMPE/DCP/Chol and DMPE alone, in that order.

Construction and immunogenicity of recombinant adenovirus expressing ORF2 of PCV2 and porcine IFN gamma

The capsid structural protein encoded by the gene ORF2 of Porcine Circovirus Type 2 is critical for eliciting the broad protective immunity. We constructed and characterized the recombinant adenovirus encoding the capsid and porcine IFN gamma, designated as rAd-ORF2-IFN-γ. The construct was further confirmed by the enzyme digestion, PCR, sequencing and transfection. The humoural immunity to the capsid protein, induced by the recombinant in the mice, was tested by ELISA. Notably, this recombinant induced a much better ORF2-specific antibody response than that of rAd-ORF2 alone or the adenovirus itself. Clearly, the porcine IFN gamma could strongly enhance the immunogenicity of ORF2. The results showed that the recombinant adenovirus might be an attractive candidate vaccine for preventing the disease associated with PCV2 infection.

Characterization of porcine UL16‐binding protein 1 endothelial cell surface expression

Natural killer (NK) cells participate in the immune response against solid organ allo- and xenografts and are tightly regulated through signals mediated by inhibiting and activating receptors expressed on their cell surface. Human NK cytotoxicity against porcine endothelial cells (pEC) is mediated by the interaction of the activating human NK receptor hNKG2D and its corresponding ligand on pEC, porcine UL-16 binding protein 1 (pULBP1). The aim of the present study was to characterize the regulation of pULBP1 cell-surface expression on primary porcine aortic endothelial cells (PAEC). A monoclonal antibody (mAb; aE5-63) directed against pULBP1 was generated by immunizing C57BL/6 mice with the pEC line PEDSV.15, and used in cellular ELISA to determine pULBP1 cell surface expression. PAEC were either left untreated or stimulated with human or porcine cytokines [interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha)], human serum, cultured under hypoxic conditions, or infected with human or porcine cytomegalovirus (CMV). Neither human nor porcine IFN-gamma stimulation changed pULBP1 expression, whereas both human and porcine TNF-alpha stimulation as well as human and porcine CMV infection significantly decreased pULBP1 expression on PAEC. Coculture of PAEC with human serum strongly increased pULBP1 expression depending on the binding of human anti-pig antibodies. Exposure of PAEC to hypoxia only slightly increased pULBP1 expression. In conclusion, (i) the novel anti-pULBP1 IgM mAb aE5-63 represents a useful tool to study pULBP1/hNKG2D-mediated responses in xenotransplantation, and (ii) the expression of pULBP1, a human-pig cross-species functional hNKG2D ligand, on the surface of PAEC is modulated by various stimuli associated with transplantation.

Synergistic inhibition of pseudorabies virus replication by porcine alpha/beta interferon and gamma interferon in vitro.

Interferon (IFN) is crucial for initiating the innate immune response and for the generation of the adaptive response. IFN, in most species, comprises IFN-alpha (IFN-alpha), IFN-beta (IFN-beta) and IFN-gamma (IFN-gamma). In this study, we compared the capacity of porcine IFN-alpha, -beta and -gamma, or a combination of them, to protect IBRS-2 cells (porcine kidney cells) from infection with pseudorabies virus (PRV). The results demonstrated that porcine IFN-beta (PoIFN-beta) was the most efficient of the three IFNs in conferring resistance PRV infection; 100 U/mL PoIFN-beta inhibited PRV plaque formation 5.3-fold. Compared with PoIFN-beta, porcine IFN-gamma (PoIFN-gamma) was less capable of inhibiting PRV plaque formation (3.3-fold inhibition). Porcine IFN-alpha (PoIFN-alpha) had the least capability of the three PoIFNs, and inhibited PRV plaque formation only 1.26-fold. The inhibitory capacity increased to only 2.3-fold with a treatment of 12,800 U/mL PoIFN-alpha. A combination of PoIFN-gamma and PoIFN-alpha or PoIFN-beta inhibited PRV plaque formation 12.8-fold or 100-fold, respectively. Treatment of IBRS-2 cells with PoIFN-alpha/beta and PoIFN-gamma inhibited PRV replication 29- or 146-fold. Additionally, real-time PCR analyses of the PRV immediate early (IE) gene revealed that IE mRNA expression was profoundly decreased in cells stimulated with PoIFN-alpha/beta and PoIFN-gamma (23.8-133.0-fold) compared with vehicle-treated cells. All the findings indicate that PoIFN-gamma acts synergistically with other PoIFNs (PoIFN-alpha and -beta) to potently inhibit PRV replication in vitro.

T‐cell responses during pig‐to‐primate xenotransplantation

Xenotransplantation using porcine organs may resolve a chronic shortage of donor organs for clinical transplantation if significant immunological barriers can be overcome. To determine the potential role of T lymphocytes in Xenograft (Xg) rejection, we transplanted transgenic hCD46 porcine hearts heterotopically into baboon recipients. Recipients were treated to deplete anti-Gal antibody with a non-antigenic alpha-Gal polyethylene glycol polymer (TPC) (n = 2), TPC plus rituximab (anti-CD20) (n = 1) or were untreated (n = 1). None of the recipients received T-cell immunosuppression. All Xgs failed within 7 days and showed evidence of a mixed humoral and cellular rejection process. Cellular infiltration consisting primarily of CD4+ T cells and few CD8+ T cells. Proliferation and cytotoxicity assays showed sensitization of CD4+ and CD8+ T cells that reacted with porcine IFN-gamma (pIFN-gamma)-stimulated porcine aortic endothelial cells (PAEC). The CD4+ lymphocytes displayed greater cytotoxicity than CD8+ cells. An increased frequency of PAEC-specific interleukin (IL) 2 and IFN-gamma-secreting T cells was observed, suggesting a Th1 cytokine bias. An increase in the percentage of circulating CD4+CD28- cells was observed at the time of rejection and over 50% of the CD4+ cells recovered from residual pig tissue at necropsy lacked CD28 expression. These findings show that lymphocytes are efficiently stimulated by PAEC antigens and can mediate direct tissue destruction. These studies (1) provide an insight into the potential of cellular-mediated cardiac Xg rejection, (2) show for the first time the induction of cytotoxic pig-specific CD4+CD28- lymphocytes and (3) provide a rational basis for determining different modes of immunosuppression to treat Xg rejection.

Enhancement of Porcine Circovirus 2 Replication in Porcine Cell Lines by IFN-γ Before and After Treatment and by IFN-α After Treatment

Stimulation of the porcine immune system causes increased replication of porcine circovirus 2 (PCV2) in vivo. In the present study, we investigated whether various cytokines (interleukin-1 [IL-1], IL-6, IL-10, tumor necrosis factor-alpha [TNF-alpha], interferon-alpha [IFN-alpha], and IFN-gamma) are able to influence PCV2 infection in vitro. No changes were observed in IL-1, IL-6, TNF-alpha, or IL-10-treated cells. However, it was demonstrated that IFN- alpha and IFN-gamma influenced PCV2 infection in porcine kidney cells (PK-15) and porcine monocytic cells (3D4/31). IFN-gamma added to the culture medium before, during, or after inoculation increased the number of PCV2 antigen- positive cells, respectively, by 418%, 171%, and 691% in PK-15 cells and by 706%, 114%, and 423% in 3D4/31 cells. IFN-alpha pretreatment decreased the number of infected PK-15 cells. When it was added after inoculation, IFN-alpha enhanced PCV2 infection by 529% in PK-15 cells and by 308% in 3D4/31 cells. The effect of both IFNs on PCV2 infection was dose dependent and could be blocked with IFN-alpha or IFN-gamma neutralizing antibodies. Leukocyte-derived porcine IFN-gamma induced a similar effect on PCV2 infection. Treatment of PK- 15 cultures with IFN-gamma caused a 20 times higher production of progeny virus. Confocal microscopy studies showed that the enhancing effect of IFN-gamma on PCV2 infection was achieved by increased internalization of PCV2 virionlike particles (VLPs). Binding of the VLPs to the cell or expression kinetics of PCV2 proteins in infected cells were not altered by IFN-gamma treatment. To our knowledge, this study reports the first enhancement of a viral infection by treatment with type I or type II IFNs.

Porcine Reproductive and Respiratory Syndrome (PRRS) Virus-Specific Interferon-γ+T-Cell Responses After PRRS Virus Infection or Vaccination with an Inactivated PRRS Vaccine

Although field studies have found porcine reproductive and respiratory syndrome (PRRSV) inactivated vaccines to be beneficial in reducing losses linked to PRRSV infection, immune mechanisms induced by these vaccines need better understanding. In the study reported here, we examined the interferon-gamma(+) (IFNgamma(+)) PRRS-specific T cell responses induced after infection and vaccination with an inactivated PRRS vaccine. Autologous monocyte-derived dendritic cells loaded with the PRRSV P120 strain were used to re-stimulate ex vivo T cells that had been primed in vivo by either the virus or the vaccine, or both. Virus-specific IFNgamma(+) T cells were quantified by using a porcine IFNgamma- ELISpot assay. A specific but low live virus-induced response was observed between days 35 and 70 for most of the pigs tested, while a significant inactivated vaccine-induced PRRSV-specific IFNgamma(+) T-cell response was measured soon after vaccination. Moreover, we observed that vaccination of pre-challenged pigs clearly favoured the PRRSV-specific cell-mediated immunity primed by the live virus. To characterize further the nature of the PRRSV-specific T cells, the different T-cell subsets involved in PRRSV immunity were analyzed by flow cytometry. We showed that the inactivated vaccine was able to prime both CD4(+)CD8(int+) and CD8(high) virus-specific T cells and that CD4(+)CD8(int+) were preferentially recalled by the live virus.

Immune responses of swine following DNA immunization with plasmids encoding porcine reproductive and respiratory syndrome virus ORFs 5 and 7, and porcine IL-2 and IFNγ

In the present study, five eukaryotic double-gene expression plasmids containing porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 and ORF7 genes combined with cDNAs encoding porcine IFNgamma and IL-2 were constructed for evaluation as PRRSV vaccine candidates. After immunization and viral challenge, two of three pigs immunized with pIRESorf5/IFNgamma, one of three pigs immunized with pIRESorf5/IL-2 and one of three pigs immunized with pIRESorf7/IL-2 were protected from lung lesions that were present in other vaccinated and control animals. Virus replication was reduced but not completely prevented in organs of the DNA-vaccinated animals as compared to controls. Therefore, the porcine cytokines IFNgamma and IL-2, delivered in combination with ORF5 or ORF7, may improve the immune efficacy of DNA vaccines against PRRSV.

Fetal pig beta cells are resistant to the toxic effects of human cytokines.

The cytokine tumour necrosis factor-alpha (TNF-alpha) is thought to be responsible for primary nonfunction of islets when transplanted. This, and two other cytokines, interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) are also implicated in the autoimmune destruction of beta cells. It is unknown if the fetal pig beta cell, which is being transplanted as a treatment for type 1 diabetes, is affected by these cytokines. We compared the effects of the cytokines on the function and viability of adult and fetal pig beta cells. The cells were cultured for up to 3 days in the presence of 2000 pg/ml of human IL-1beta, 1000 U/ml of TNF-alpha, and 1000 U/ml of IFN-gamma, as well as 1000 U/ml of porcine IFN-gamma. Cumulative insulin levels, insulin content, metabolic activity, and viability of these cells were examined. Additionally, nitric oxide production and the activity of antioxidant enzymes in these cells were also determined. TNF-alpha and the combination of the three human cytokines caused a transient increase in cumulative insulin levels. TNF-alpha alone enhanced insulin content on day 3. There was no effect of these human cytokines on mitochondrial function and viability. In contrast, porcine IFN-gamma inhibited fetal pig beta cell function and also caused their death. Adult pig islets are sensitive to the toxic effects of human TNF-alpha, IL-1beta, the combination of the three cytokines, and porcine IFN-gamma. The activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase were significantly higher in fetal pig beta cells than in adult islets, implying that this may be the reason for the lack of adverse effects of the cytokines on the fetal beta cell. Fetal pig beta cells are resistant to the toxic effect of the human cytokines, TNF-alpha and IL-1beta, in vitro. This resistance suggests that fetal, but not adult beta cells, when transplanted into humans with type 1 diabetes may be protected from primary nonfunction and will be partially protected from autoimmune destruction.

Immortalized bone-marrow derived pig endothelial cells.

Primary cultures of porcine endothelial cells (EC) can only be maintained for a limited number of passages. To facilitate studies of xenogeneic human anti-pig immune responses in vitro, pig microvascular bone-marrow (BM) and macrovascular aortic EC were obtained from our herd of partially inbred miniature swine, homozygous for the major histocompatibility locus, and immortalized with a modified SV40 large T vector. The resulting BM-derived (2A2) and aortic (PEDSV.15) immortalized EC lines showed unlimited growth and EC phenotype as indicated by expression of von Willebrand Factor (vWF) and low density lipoprotein (LDL) receptors as well as by formation of typical cobblestone monolayers. Ultrastructural studies revealed morphological similarities in primary and immortalized EC. Flow cytometry analysis demonstrated constitutive SLA class I expression by all lines whereas SLA class II was only expressed after stimulation with porcine IFNgamma. Furthermore, pig CD34 mRNA was detected by Northern blot analysis in primary and immortalized aortic EC but not in 2A2. Both EC lines expressed a number of myeloid markers, adhesion molecules and xenoantigens, the latter being determined by binding of human natural antibodies. Gene transfer into the porcine EC lines was successfully performed by electroporation or calcium-phosphate transfection, as well as by adenoviral infection. Finally, the functional similarity between primary and immortalized EC was demonstrated in adhesion and cytotoxicity assays. Together, these results suggest that 2A2 and PEDSV. 15 represent valuable tools to study both human cellular and humoral immune responses in vitro against pig EC derived from microvascular and large vessels.

GroEL/ES chaperonins protect interferon-gamma against physicochemical stress--study of tertiary structure formation by alpha-casein quenching and ELISA.

Interferon-gamma (IFN-gamma) is a structurally labile cytokine that rapidly denatures upon exposure to acid or heat. Here we show that both acid-denatured (pH 2) and thermally inactivated (50 degrees C) porcine IFN-gamma can be rescued with the Escherichia coli GroEL/ES chaperonin system and ATP, and reassembled into bioactive dimers. At 35 degrees C, spontaneous refolding of acid-denatured IFN-gamma was found to be dependent on the presence of guanidinium hydrochloride (0.15-0.25 M) or NaCl (0.1-0.2 M). Under non-permissive reaction conditions for regain of native structure (low-ionic-strength buffer at 35 degrees C), the yield of IFN-gamma refolded with GroEL/ES/ATP increased about 30-fold above the level of spontaneous refolding. In the absence of GroES, GroEL captured IFN-gamma in a folding-competent complex. Under these conditions, both ATP and alpha-casein induced release of IFN-gamma from GroEL but with the released protein tending to partition into sedimentable aggregates. Only in the presence of GroES, did ATP induce complete discharge of IFN-gamma from GroEL, with the released protein refolded into a conformation that is (a) immunoreactive/bio-active, (b) resistant to precipitation and (c) in a dimeric configuration. Chicken egg albumin and 90-kDa heat-shock protein were inactive in the exertion of any protective effect against physicochemical stress. The precise amount of protein refolded to the native state at different times of the folding reaction was determined by alpha-casein quenching and ELISA. The former is based on the conversion by excess alpha-casein of any population of unfolded IFN-gamma into one that escapes antibody recognition by subsequent ELISA. Since the native dimers, however, are not affected by alpha-casein quenching, immunoreactivity is directly proportional to the yield of correctly refolded protein. The validity of this approach was confirmed by measurement of biological activity. GroEL/ES-meditated reactivation amounted to > 80% both by ELISA and antiviral assay.

A quantitative competitive reverse transcription-polymerase chain reaction technique to measure porcine interferon-gamma.

In swine, as in other mammals, interferon-gamma (IFN-gamma) plays an important role in the regulation of the immune response. The objective of this work was to develop a sensitive, quantitative competitive reverse transcription-polymerase chain reaction (qcRT-PCR) assay, using an internal cRNA standard, to monitor the expression of porcine IFN-gamma gene. We applied this qcRT-PCR assay to quantify the expression of IFN-gamma transcripts in peripheral blood mononuclear cells (PBMC) incubated for different times with or without concanavalin A (ConA). Results showed that the expression of the IFN-gamma transcripts in PBMC occurred within the first 2 h after mitogenic stimulation and reached a peak after 24 h of incubation with ConA. The qcRT-PCR technique provides an efficient and sensitive tool for studying the expression of porcine IFN-gamma gene under a variety of experimental conditions. This will help to understand the regulation of T cell-mediated immune responses by IFN-gamma at the gene level.

Engineering by pcr-based exon amplification of the genomic porcine interferon-gamma DNA for expression in Escherichia coli

Interferon-gamma (IFN-gamma) is coded for by a single gene containing three introns, localized within the coding region. We have previously cloned the IFN-gamma gene from a pig genomic DNA lambda library and have determined its nucleotide sequence. In order to construct the porcine IFN-gamma DNA without intervening sequence, the four exons were separately amplified by the polymerase chain reaction (PCR) using primers matching the exon-termini. From the amplified exon-fragments the complete intron-free DNA was obtained by a strategy consisting of alternate rounds of PCR and ligation. The sequence so-obtained was used for expression in E. coli. The recombinant protein appeared as inclusion bodies which were solubilized and refolded in order to obtain biologically active IFN-gamma.