Characterization of monoclonal antibodies against porcine pulmonary alveolar macrophages of gnotobiotic miniature swine.

There is no sufficient porcine specific antibody available to investigate the ontogeny and development of porcine pulmonary alveolar macrophage (PAM). Therefore, several mouse anti-porcine macrophage mAbs have been produced and characterized in this study. First, the monoclonal antibody PM18-7, an IgG1, kappa isotype, bound to 91% of PAM, 6% of monocytes, and 2% of granulocytes indicating PM18-7 was found to be PAM specific. Monocyte derived macrophages could not be induced to express the PM18-7 antigen by culture. PM18-7 was immunoprecipitated with a molecule of 260 kDa under non-reducing conditions and with that of 130 kDa under reducing conditions in SDS-PAGE. Second, the monoclonal antibody PM3-15, an IgG1, kappa isotype, bound to 92% of PAM, 86% of monocytes, and 3% of granulocytes indicating PM3-15 was mononuclear phagocyte specific. PM3-15 was immunoprecipitated with a molecule of 245 kDa under non-reducing conditions and those of 150, 95 kDa under reducing conditions in SDS-PAGE. Third, the monoclonal antibody PM16-6, an IgM isotype, bound to 82% of PAM, 89% of monocytes, and 82% of granulocytes indicating PM16-6 recognizes those cells of myeloid lineage such as macrophages, monocytes and granulocyte. The antigen immunoprecipitated by PM16-6 was 120 kDa under non-reducing conditions and was 75, 25 kDa under reducing conditions. Finally, the antigen bound to PM18-7 was identified as ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1, CD203a), PM3-15 was figured out as integrin alpha M beta 2 precursor (ITGaM, CD11b) and PM16-6 was identified as Thimet oligopeptidase (THOP-1) by the LC-MS/MS protein sequencing method.

Establishment of major histocompatibility complex homozygous gnotobiotic miniature swine colony for xenotransplantation.

To overcome shortages of human donor organs for organ failure patients, we made a commitment to develop gnotobiotic miniature swine as an alternative organ donor source for xenotransplantation. For this, we have constructed an absolute barrier-sustained gnotobiotic facility. Pregnant sows of gnotobiotic miniature swine, were procured and germfree piglets were obtained by hysterectomy. These were maintained in germfree isolators for about 4 weeks, deprived of colostrum and were fed sterilized soybean milk. They were associated with di-flora, anaerobic Lactobacillus sp. and Streptococcus sp. After confirmation of successful associations, gnotobiotic piglets were transferred into the facility aseptically. The piglets are maintained on high-efficiency particle air-filtered air in and out; maintaining constant room air pressure of 33 ± 3 mmAq, and sterile water and diet. In 10 sessions of hysterectomy, 18 male and 32 female piglets were obtained of which piglets (M six, F eight) died within 5 days. Among live piglets, piglets (M eight, F 12) were confirmed to be germfree by microbiological monitoring. For research of xenotransplantation, one consistent experimental result was essential. Therefore, major histocompatibility complex class II which related innate immunity, homozygotic gnotobiotic miniature swine was developed. As a result, genotyping revealed 14 individuals to be homozygous for major histocompatibility complex class II (DRB, DQB) as 0301, three individuals were homozygous as 0201 and each of two were homozygous for DQB as 0701 and DRB as 0404, respectively. Genetic modifications and immunological research for ideal alternative organ sources are in progress.

CM1 ligation initiates apoptosis in a caspase 8-dependent manner in Ramos cells and in a mitochondria-controlled manner in Raji cells.

Burkitt lymphoma (BL) is a tumor with the characteristics of germinal center B cells. We previously reported that the CM1 (centrocyte/-blast marker 1) molecule is expressed only in germinal center B cells, specifically, in a subpopulation of centroblasts and centrocytes. In the present study, we investigated the apoptosis induced by anti-CM1 in the Ramos and Raji human BL cell lines. The Ramos is protected from apoptosis by the crosslinking of sIgM and the calcium ionophore by the ligation of CD40 with anti-CD40 monoclonal antibodies (mAb) or soluble CD40 ligand (sCD40L). In this investigation on the effect of CM1 on apoptosis in BL cell lines, we found that cellular signaling by CM1 induces apoptosis and decreases cell viability, in BL cell lines cultured for 24 hours with protein-G agarose beads conjugated anti-CM1 mAb. Stimulation by CD40 ligated with sCD40L protected Raji cells from CM1-induced apoptosis, but did not protect Ramos cells. Furthermore, after anti-CM1 mAb stimulation, CD95 expression was upregulated and CD40 expression was unaltered or slightly decreased in Ramos cells, whereas CD95 was downregulated and CD40 was slightly upregulated in Raji cells. The engagement of CD40 by sCD40L enhanced CD95 expression, but the level of CM1 expression was unchanged in Ramos. However, sCD40L downregulated both CD95 and CM1 expression in Raji. In addition, the caspase-8 specific inhibitor blocked CM1-induced apoptosis in Ramos cells, but not in Raji cells. Increased mitochondrial membrane permeabilization was observed only in Raji cells. Moreover, the effector caspase inhibitor, z-DEVD, blocked CM1-mediated apoptosis in both cell lines. We found that CM1-induced apoptosis is achieved via different initiation pathways, which are cell-type dependent.

Establishment and characterization of endothelial cell lines from the aorta of miniature pig for the study of xenotransplantation.

Pig endothelial cells are the first cells to interact with human immune components after organ xenotransplantation, which is a procedure currently considered to be the best treatment option for end-stage organ failure. It is, therefore, essential to study the mechanisms of molecular interaction between pig endothelial cells and human immune components, in order to overcome xenograft rejection. The aim of this study was to establish immortalized pig aortic endothelial cell lines, in order to facilitate future in vitro studies of human anti-pig immune responses. Endothelial cell lines were established following the transfection of primary endothelial cells isolated from the aortas of the Minnesota miniature pig with plasmid pRNS-1 carrying genes for neomycin resistance and the SV40 large T antigen. The immortalized cell lines showed a relatively rapid doubling time (17.6h) and the endothelial cell phenotype, as indicated by the formation of typical cobblestone monolayers and by the constitutive expression of PECAM-1 and the von Willebrand factor. Flow cytometric analysis demonstrated the constitutive expression of SLA class I and CD86, whereas the expression of E-selectin and SLA class II was only induced after stimulation with human TNF-alpha and pig IFN-gamma, respectively. On the other hand, no CD80 expression was detected in the primary cells or cell lines in the presence or absence of either human TNF-alpha or pig IFN-gamma. A vigorous human T cell proliferation against these cell lines was observed in the mixed lymphocyte-endothelial cell culture. These results suggest that pig endothelial cells, immortalized by the introduction of SV40 T, retain their original characteristics, except for the acquired property of immortalization, and that they may be useful for future in vitro studies of xenogeneic human anti-pig immune responses.

Thomsen-Friedenreich (T) antigen expression increases sensitivity of natural killer cell lysis of cancer cells.

In this study, we demonstrate a correlation between T antigen expression on a panel of human carcinoma cell lines and their sensitivity to porcine NK cell lysis. Specifically, the more T antigen is expressed, the more sensitive the cancer cells are to porcine NK cell lysis. Furthermore, this correlation also exists for these cells and their ability to induce tumors in vivo. In this porcine animal model, the less T antigen is expressed, the more prolific the tumor growth in vivo and vice versa. Using the human colorectal adenocarcinoma cell line SW-48, we used limiting dilution to clone 2 populations of cells, one expressing high and the other low levels of T antigen, clones 143 and 111, respectively. In these cloned cells, the clone that expressed more T antigen was more NK-sensitive in vitro and weakly induced tumor growth in vivo. Inversely, the clone that expressed less T antigen clone was more NK-resistant in vitro and grew more prolific tumors in vivo. Using soluble T antigen in a competitive inhibition assay, there was a decrease in porcine NK cell killing of the T antigen+ human cell line Colo 320HSR. Taken together, these findings suggest a novel role for T antigen in the NK cell recognition of cancer cells, specifically as markers for NK sensitivity in carcinoma cell lines. The significance of T antigens as targets for NK cell-mediated lysis is novel and identifies NK cell-T antigen interactions as potentially significant in the immunotherapy of cancer and its associated metastases.