The in vitro protection of human decay accelerating factor and hDAF/heme oxygenase-1 transgenes in porcine aortic endothelial cells against sera of Formosan macaques.

To mitigate hyperacute rejection, pigs have been generated with alpha-Gal transferase gene knockout and transgenic expression of human decay accelerating factor (hDAF), MCP, and CD59. Additionally, heme-oxygenase-1 (HO-1) has been suggested to defend endothelial cells. Sera (MS) (0%, 1%, 5%, 10%, and 15%) from Formosan macaques (Macaca cyclopis, MC), an Old World monkey wildly populated in Taiwan, was used to test the protective in vitro, effects of hDAF or hDAF/hHO-1 on porcine aortic endothelial cells (pAEC) derived from hDAF(+), hDAF(+)/hHO-1(+), and hDAF(+)/hHO-1(-) and 1 nontransgenic pAEC. Ten percent human serum (HS) served as a positive control. When MS addition increased to 10% or 15%, all transgenic pAEC exhibited a greater survival than nontransgenic pAEC. Noticeably, 15% MS reduced survived to <10% versus >40% in nontransgenic and transgenic pAEC, respectively. These results revealed that hDAF exerted protective effects against MC complement activation. However, comparing with 10% MS and HS in pAEC of nontransgenic pigs, the survivability was higher in HS, suggesting that complement activation by MS was more toxic than that by HS. Furthermore, hDAF(+)/hHO-1(+) showed no further protection against effects of MS on transgenic pAEC.

The impact of human ABO blood groups on human xenoreactive antibody-mediated cytotoxicity and the protective effect of human decay-accelerating factors exogene on swine endothelium.

UNLABELLED: Swine tissue can express antigens similar to human A/B blood types. We evaluated whether the variation in human blood type influences the human xenoreactive antibody-mediated cytotoxicity and modifies the protective effect of human decay-accelerating factor (hDAF) exogene, a complement activation regulator, on swine endothelium. METHODS: Pig aortic endothelial cells were harvested form normal and hDAF transgenic pigs. Cellular viability was evaluated with an MTT assay. RESULTS: As compared with that of other human blood types, human serum from blood type O donors induced more prominent cytotoxicity on swine endothelial cells both from hDAF transgenic or normal pigs (P < .05). In addition, this difference of xenoreactive antibody-induced cytotoxicity between treatment with O and other human blood type sera was more evident in hDAF transgenic swine endothelial cells than those of normal pigs (P < .05). The hDAF exogene can significantly protect the endothelial cells from human xenoreactive antibody-mediated cytotoxicty when treated with human serum from AB blood type (P < .05). Our data demonstrated that human ABO blood type significantly affected human xenoreactive antibody-induced cytotoxicity, which may modulate the protective effect of hDAF exogene expression on swine endothelial cells.

Long-term survival of HLA-DR15+ pig skin in SCID mice after reconstitution with human peripheral blood mononuclear cells and under short-term immunosuppression.

In allotransplantation, donor-recipient human leukocyte antigen (HLA) matches improve graft survival. For studies of the role of donor-recipient HLA II matching on xenotransplantation, we successfully generated HLA-DR15+ transgenic pigs the the skins of which were transplanted to SCID mice, which were thereafter reconstituted with HLA-DR15+ or -DR15(-) hPBMC. Cyclosporine was given intraperitoneally to SCID mice for 12 days. Human T cells were observed in SCID mice after reconstitution. Mixed lymphocytes responses showed greater responses by HLA-DR15(-) human peripheral blood mononuclear cells (hPBMC) against HLA-DR15+ porcine PBMC. HLA-DR15+ porcine skins survived more than 100 days in all SCID mice. HLA-DR15+ porcine skins were rejected in all non-SCID (Balb/c) mice. The histologic pictures of transplanted HLA-DR15+ porcine skins showed surviving porcine epithelium in remodeling murine dermis and little lymphocyte infiltration into the murine dermis. The long-term survival of HLA-DR15+ pig skin in all hPBMC-SCID mice might be due to poor engraftment or function of reconstituted T cells. Further studies are needed to clarify the role of donor-recipient matching of HLA-DR15.

The hDAF exogene protects swine endothelial and peripheral blood mononuclear cells from xenoreactive antibody mediated cytotoxicity in hDAF transgenic pigs.

Xenoreactive antibody-induced complement activation and cytotoxicity poses a major obstracle to xenograft survival in humans. Previously, we have generated transgenic pigs carrying the hDAF exogene to help overcome this problem. In this study, we examined whether the hDAF exogene in various swine cells shows an equally protective effect for the complement-mediated cytotoxicity induced by human xenoreactive antibodies. Pig peripheral blood mononuclear cells (PBMCs) and aortic endothelial cells (PAECs) were used as targets. Fresh human serum was harvested from a single healthy human donor as the source of human xenoreactive antibodies and complement. The target cells cocultured with medium containing various concentrations of human serum for 24 hours were evaluated using the 3-[4,5-dimethylthiaolyl]-2,5-diphenyl-tetrazolium bromide assay for cellular viability. We observed that xenoreactive antibody plus human complement-mediated cellular cytoxicity dose-dependently correlated with the concentration of human serum in the culture medium. As compared with the PBMCs from the normal pigs, PBMCs from hDAF transgenic pigs showed significantly better survival after treatment with human serum (P < .05). Similarly, the survival of PAECs from the hDAF transgenic pig were also significantly higher than that from normal pigs (P < .05). Our data demonstrated a protective effect from human xenoreactive antibodies and complement-mediated cytoxicity of hDAF exogenes in pig PBMCs and PAECs. These observations support the clinical value of the hDAF transgenic pig as an organ donor in xenotransplantation.

Mycoplasma hyorhinis in Taiwan: diagnosis and isolation of swine pneumonia pathogen.

This study attempted to determine whether one multiplex polymerase chain reaction (PCR) is an effective adjunct method for diagnosing Mycoplasma hyopneumoniae and Mycoplasma hyorhinis infection, and whether M. hyorhinis should be considered as an enzootic pneumonia or porcine respiratory disease complex pathogen in Taiwan. To our knowledge, this study is the first to isolate and identify M. hyorhinis as a porcine pathogen in Taiwan. A novel isolation method and a multiplex PCR test were applied to detect and isolate M. hyorhinis. The correlation of M. hyorhinis with swine pneumonia was also examined using a challenge test. Based on weight, 18 pigs were assigned to three groups and housed throughout the study in a specific-pathogen-free (SPF) facility and provided with aseptic feed and water. Groups 1 (n=6) and 2 (n=6) were challenged with 5mL M. hyorhinis culture via tracheal intubation on day 1. The M. hyorhinis strains ATIT-1, -3, and-7 were used to infect group 1 and the strain ATCC 27717 was used for group 2. Culture medium was replaced by phosphate-buffered saline in group 3 (n=6). All pigs were slaughtered on day 28, and their lungs were removed for examination of lesions. Of the six pigs in group 1 challenged with wild-type strains, two had typical mycoplasma pneumonia lesions. No gross lung lesions were observed in groups 2 and 3. Although further examination is necessary to confirm that wild-type strains can cause pneumonia, it appears that M. hyopneumoniae is no longer the only mycoplasma pathogen implicated in the diagnosis of swine enzootic pneumonia (SEP).

Orally administrable enterotoxigenic Escherichia coli vaccine encapsulated by ethylcellulose powder dispersion.

To overcome the limitations of injection administration to vaccinate neonatal piglets against diarrheal disease, an oral vaccine needs to be developed. Enteric microspheres of oral vaccines were developed by a co-spray drying process based on formalin-inactivated enterotoxigenic Escherichia coli antigens with various encapsulating materials. The encapsulating efficiencies of ECN7m, ECN14m and ECN22m (vaccine microsphere formulations) tested by extraction procedure are high, more than 85%. To assess enteric characteristics, an in vitro dissolution test was performed with microspheres. Formulations with ethylcellulose ECN14m and ECN22m allow controlled release in a neutral or basic environment and resisted acid damage. In all cases, 95% of the E. coli protein was released within 2 h at pH 6.8-7, but there was no release at pH 1.5-2. However, ECN7m was less acid-resistant and had lower release at low pH. In animal immunization tests, oral immunization with microspheres of formulations ECN14 and ECN22m effectively evoked both systemic IgG and mucosal IgA responses against E. coli whole cell antigens in mice. In the mice challenge test, orally administrable ECNm14 (12 mg) or ECN22m (12.6 mg) vaccine (i.e., encapsulating 3.0x10(9) cfu inactive bacterial mass) provided good protection from infection in animals.

Protective effects of oral microencapsulated Mycoplasma hyopneumoniae vaccine prepared by co-spray drying method.

The efficacy of Mycoplasma hyopneumoniae oral vaccine was investigated in microsphere dosage form. A co-spray drying process was used to apply an encapsulating material, Eudragit L30 D-55, to microspheres containing Mycoplasma hyopneumoniae antigens. The microspheres were generally effective (>93%) with protein release at pH 7.4, but almost none were released at pH 1.2, for 3 hr in an in vitro dissolution test. An SPF-swine model was used to evaluate the effectiveness of the microspheres as an oral vaccine, and the related immune responses. The serum's systemic IgG against M. hyopneumoniae was evoked by ELISA analysis, after a 2nd immunization of all pigs. The vaccinated groups' mean lesion score was significantly lower after the Mycoplasma hyopneumoniae challenge than that of the nonvaccinated/challenged groups (P<0.05). This study strongly suggests that the oral microspheres vaccine prepared by a co-spray drying method can provide effective protection against M. hyopneumoniae infection in pigs.

In vivo and in vitro comparisons of spray-drying and solvent- evaporation preparation of microencapsulated Mycoplasma hyopneumoniae for use as an orally administered vaccine for pigs.

OBJECTIVE: To evaluate the efficacy of an orally administered vaccine of Mycoplasma hyopneumoniae that was prepared by spray drying or solvent evaporation. ANIMALS: Thirty 6-week-old, crossbred, specific-pathogen-free (SPF) pigs. PROCEDURE: Pigs were randomly allocated into 5 groups and housed in an SPF facility. Pigs in 2 groups (groups AQ and CAP) were fed M hyopneumoniae enteric-coated vaccine on days 0, 10, and 20. A third group (group IM) received an IM injection of M hyopneumoniae vaccine with aluminium hydroxide as an adjuvant on days 0, 10, and 20. The last 2 groups (non-vaccinated-challenged [NV-C] and nonchallenged [NC]) were fed a sham treatment. All 24 pigs in groups AQ, CAFP IM, and NV-C were challenge exposed with 5 ml of a 10% pneumonic lung suspension administered on day 40 via intubation of the trachea. All pigs were slaughtered and the lungs removed and examined for lesions on day 68. RESULTS: In vitro studies indicated that these 2 microencapsulation techniques formed an effective shell and protected mycoplasmal antigen from gastric acid. Results of inoculation and challenge tests indicated that microencapsulated M hyopneumoniae were sufficiently potent to induce an immune response and provide good protection. CONCLUSIONS AND CLINICAL RELEVANCE: Orally administered microencapsulated M hyopneumoniae vaccines induced an immune response and reduced the severity of lung lesions in challenge-exposed pigs. Results suggest that this novel method can be applied to other antigens, because the spray-drying process yielded an orally administered M hyopneumoniae vaccine that induced a good immune response.

A recombinant chimera composed of repeat region RR1 of Mycoplasma hyopneumoniae adhesin with Pseudomonas exotoxin: in vivo evaluation of specific IgG response in mice and pigs.

Using the binding and translocation domain of Pseudomonas exotoxin A [domain III deleted PE termed PE(DeltaIII)] as a vehicle, this study characterized and evaluated a novel application of PE toxin in Mycoplasma hyopneumoniae adhesin used as an immunogen. PCR and sequence analysis revealed that 16 copies of AAKPV(E) in tandem repeat region 1 (RR1) of M. hyopneumoniae 97kDa adhesion were successfully fused to the downstream of PE(DeltaIII) to create a subunit vaccine, i.e. PE(DeltaIII)-RR1. This chimeric protein, over-expressed in inclusion bodies of E. coli BL21(DE3)pLysS, was characterized by a monoclonal antibody (MAb) F2G5 prepared against RR1 of the 97kDa adhesin and was readily purified. The data indicated that the epitope recognized by MAb F2G5 was located in the structure of PE(DeltaIII)-RR1. Using ELISA and Western blot analyses, the specific IgG immune response against RR1 and whole adhesin in mice immunized with PE(DeltaIII)-RR1 was found more marked than that in mice immunized with the M. hyopneumoniae whole cells. Similarly, PE(DeltaIII)-RR1 also stimulated a remarkable IgG response against RR1 in pigs compared to that in pigs immunized with the conventional M. hyopneumoniae vaccine. The PE(DeltaIII)-RR1 would be potentially useful for the future development of a M. hyopneumoniae adhesin vaccine.

Release characteristics of microspheres prepared by co-spray drying Actinobacillus pleuropneumoniae antigens and aqueous ethyl-cellulose dispersion.

Using formalin inactivated Actinobacillus pleuropneumoniae antigens and aqueous ethylcellulose dispersions, microspheres of oral vaccines were developed by a co-spray drying process. The present study attempted to determine whether the dosage formulations of microspheres could form enteric matrices. To assess the enteric characteristics, an in vitro dissolution test was performed with the AQ6-AP microspheres; 95% of the A. pleuropneumoniae protein was released within 3 h at pH 7, but there was no release at pH 1.5. The scanning microscopy revealed that the surface structure of AQ6-AP microspheres became porous at neutral pH. The SDS-PAGE analysis showed that the release rate of proteins from the microspheres was pH dependent not only for the AQ6-AP formulation but also when antigens of A. pleuropneumoniae were replaced with porcine serum. The results suggest that the A. pleuropneumoniae antigens were entrapped in the AQ6 microspheres under the acidic conditions. In a mouse model, oral immunization with AQ6-AP microspheres containing A. pleuropneumoniae evoked systemic IgG and mucosal IgA responses against A. pleuropneumoniae antigens. Thus, the present method may further provide an opportunity to develop oral vaccines and mucosal immunity.

Identification of the copper-zinc superoxide dismutase activity in Mycoplasma hyopneumoniae.

Copper-zinc superoxide dismutase (Cu/ZnSOD), a key enzyme in defense against toxic oxygen-free radicals, is widespread in eukaryotes and several species of gram-negative bacteria. The presence of this enzyme in Mycoplasma hyopneumoniae (M. hyopneumoniae), the primary pathogen of mycoplasmal pneumonia in pigs, was examined since the polyclonal antibody against bovine Cu/ZnSOD was dominantly cross-reactive with the M. hyopneumoniae Cu/ZnSOD from whole cellular proteins. In situ activity staining on SDS-PAGE showed that the molecular mass of M. hyopneumoniae Cu/ZnSOD in reducing form was approximately 17kDa. The presence of Cu and Zn ions at the active site of the enzyme was confirmed on the basis of inhibition by KCN and by H(2)O(2). The activity of M. hyopneumoniae Cu/ZnSOD on both SDS- and native-polyacrylamide gels was completely inhibited by 2mM KCN and the gels showed no iron-containing SOD (FeSOD) or manganese-containing SOD (MnSOD) in the crude extracts. The activity of M. hyopneumoniae Cu/ZnSOD in crude extract was 70units/mg protein and was 55% inhibited by 5mM KCN and 56% inactivated by 40mM H(2)O(2). This enzyme was growth-stage dependent and evidenced markedly higher production during the early log phase. Different expression levels of Cu/ZnSOD activity in field isolates were also detected. Taken together, the presence of Cu/ZnSOD in M. hyopneumoniae was identified for the first time.

Generation of HLA-DP transgenic pigs for the study of xenotransplantation.

The shortage of human organs has prompted scientists to seek xenogeneic sources of donors. To date, DAF, MCP, and CD59 transgenic pigs have been generated to inhibit hyperacute rejection. However, besides hyperacute rejection, acute and chronic rejection must also be considered in the use of porcine organs for xenotransplantation. The role of HLA-II in transgenic xeno-organ transplantation remains to be elucidated. By microinjecting 1655 embryos, we have generated one stillborn HLA-DR and two live HLA-DP transgenic pigs: P113-7 (male, carrying one copy of exogene) and P113-8 (female, carrying 2-3 copies of exogenes). The gene status of the live transgenic pigs was confirmed by PCR, Southern blot, and PCR product sequencing analysis. The expression of transgenes in these transgenic pigs were confirmed by RT-PCR analysis and immunohistochemical staining of frozen sections of ear tissue.

Protective effects of partially purified antigens of Actinobacillus pleuropneumoniae on experimentally infected mice.

Three different models of protection experiments in mice using partially purified Actinobacillus pleuropneumoniae antigens such as crude culture supernatant extract (CCSE) and partially purified cell extract (PPCE) were attempted. Biochemical analysis showed that these two immunogens had protein concentration of 0.17-0.2 mg/mL and pentose concentration of 0.012-0.014 mg/mL. In the first model intranasal (IN) vaccination with different doses (from 0.01-10 IN-LD50) against IN challenge with the dose of 20 IN-LD50 containing 1.2 x 10(9) colony forming unit (CFU)/50 microL showed that only those with the dose more than 1 IN-LD50 had slight protection in terms of survival index (SI). In the second model of protection experiment, in which subcutaneous vaccination (s.c.) with the immunogens plus soybean oil against IN challenge with 10 IN-LD50 containing 6 x 10(8) CFU/50 microL, showed that formalin-killed bacteria (bacterin) and CCSE plus PPCE had only a slight protection whereas vaccination with CCSE or PPCE immunogen alone had no protection. In the third model of protection experiment, in which the intramuscular (i.m.) vaccination with the immunogens plus aluminum hydroxide [Al(OH3)] gel against intraperitoneal (i.p.) challenge with the dose of either 2 or 6 i.p.-LD50 containing 1.2-3.6 x 10(8) CFU/0.5 mL of 0.3% mucin saline showed highly effective.

Sequence analysis of the nucleocapsid protein gene of the porcine reproductive and respiratory syndrome virus Taiwan MD-001 strain.

The 3'-portion of the genome from a Taiwan isolate of porcine reproductive and respiratory syndrome (PRRS) virus, strain MD-001, was cloned and sequenced. The resultant 549 nucleotides contained an open reading frame with a coding capacity of 123 amino acids (predicted Mr 13,600). The predicted protein corresponds to the nucleocapsid protein, the gene product of ORF7. Comparative sequence analysis of several known PRRSV strains indicated that this protein showed the highest degree of amino acid similarity to the US VR2332 and the Canadian IAF-Exp91 strains (92.7%) and the least to the Dutch Lelystad strain (56.5%). The phylogenic trees constructed on the basis of the known PRRSV nucleotide sequences indicated that MD-001 strain belongs to the North American strain cluster and that it is distinct from the European virus.

Identification of a novel adhesin-like glycoprotein from Mycoplasma hyopneumoniae.

This study identifies an adhesin-like glycoprotein, which was a 110 kDa protein (P110) under HPLC-GPC assay. This adhesin consisted of one P54 and two P28 subunits. In addition, N-glycosidase F could cleave all N-linked oligosaccharides on the P54 subunit. Experimental results indicated that P110 with native conformations significantly inhibited the adherence of biotin-labeled porcine tracheal epithelial cell extracts to the intact M. hyopneumoniae cells (p < 0.01). Furthermore, the biotin-labeled porcine tracheal epithelial cell extracts specifically bound to P54 and P28 subunits. This binding could be competitively inhibited by unlabeled porcine tracheal epithelial extracts and SPF porcine antisera against Mycoplasma hyopneumoniae. Both P54 and P28 subunits were constitutively expressed in different strains of M. hyopneumoniae. Their production was negligibly changed at various passages during in vitro cultivation. The significant role of this adhesin-like glycoprotein in the pathogenesis of swine pneumonia is under study.

Isolation and expression of a porcine lactoferrin gene.

OBJECTIVE: To elucidate the spatial and temporal expression of a porcine lactoferrin (LTF) gene. ANIMALS: 4 female and 4 male Large White pigs. PROCEDURES: We examined LTF expression in various organs excised from the pigs, using northern blot hybridization with a porcine LTF cDNA probe. Antibodies against porcine LTF were raised in rabbits and were used along with immunohistochemical staining to localize the LTF protein. RESULTS: High amounts of porcine LTF mRNA were detected in the secreting mammary gland and epididymis. This distribution is consistent with that of porcine LTF examined by immunohistochemistry. In female pigs, porcine LTF mRNA concentration increased remarkably in the ductal cells of the lactating mammary gland then significantly decreased at day 21 after parturition. Furthermore, specific staining for LTF was observed in the epithelial cells of the gastrointestinal tract of female pigs, but not in the uterus, ovaries, spleen, kidneys, pancreas, muscles, heart, brain, lungs, or liver of postpartum female pigs, or in the testes of male pigs. CONCLUSIONS: Gene expression of porcine LTF is closely related to lactation in the mammary gland. Distribution of LTF in the epididymis suggests that LTF may have a regulatory role in development of the reproductive tract of male pigs.

Polyethylene glycol conjugated insulin-like growth factor binding protein-1 (IGFBP-1) inhibits growth of breast cancer in athymic mice.

Insulin-like growth factor (IGF) binding protein-1 (BP-1) inhibits IGF-mediated proliferation of some breast cancer cell lines in vitro. Here we examined whether recombinant human wild-type IGFBP-1 (WT-BP-1) and IGFBP-1 conjugated with polyethylene glycol (PEG-BP-1) could inhibit breast cancer growth. Three breast cancer cell lines were used: MCF-7, MDA-MB-231 and MDA-MB-435A (ascites model). The cells were grown in agar with or without the BP-1 conjugates to investigate their effect on colony formation. Both WT-BP-1 and PEG-BP-1 inhibited anchorage-independent growth (AIG) of MCF-7 and MDA-MB-435A cells. AIG of MDA-MB-231 cells was not inhibited by PEG-BP-1, whereas WT-BP-1 significantly stimulated colony number. We also tested both forms of BP-1 in xenograft tumour models. Two solid breast tumour models were studied using MCF-7 and MDA-MB-231 cell lines, and one ascites model using the MDA-MB-435A cell line. PEG-BP-1 inhibited malignant ascites formation in the MDA-MB-435A model. Conversely, PEG-BP-1 did not significantly inhibit MCF-7 xenograft growth. However, the MDA-MB-231 tumour growth curves were significantly different by a constant amount, suggesting that PEG-BP-1 treatment inhibited early tumour growth of this cell line. In contrast, WT-BP-1 was ineffective in the MDA-MB-231 tumours. These data show that anti-IGF strategies can be used to inhibit breast cancer cell growth. Since PEG-BP-1 inhibited the in vivo, but not in vitro, growth of MDA-MB-231, we speculate that PEG-BP-1 may block host IGF functions required for optimal tumorigenesis. Because PEG-BP-1 has a prolonged serum half-life compared to WT-BP-1, we conclude that improvements in BP-1 pharmacological properties enhanced its antitumour effects in vivo.