Implanted skeletal myoblasts form viable grafts in infarcted myocardium, resulting in enhanced post-MI exercise capacity and contractile function and attenuated ventricular dilation. These data illustrate that syngeneic myoblast implantation after MI improves both in vivo and ex vivo indexes of global ventricular dysfunction and deleterious remodeling and suggests that cellular implantation may be beneficial after MI.

These findings represent demonstration of autologous myoblast cell survival in human heart. The implanted skeletal myoblasts formed viable grafts in heavily scarred human myocardial tissue. These results establish the feasibility of myoblast transplants for myocardial repair in humans.

The higher level of alphaGal-reactive IgM suggests that xenoreactive NAbs may be the product of germ-line genes. Two-dimensional gel analysis of affinity-purified alphaGal-reactive NAb from two donors provided evidence suggesting that IgM from this subpopulation of NAb were restricted in protein charge heterogeneity.

Even if hyperacute rejection, which is mediated by human natural antibodies (nAb) and complement, could be prevented, xenoreactive human anti‐pig cellular responses may lead to delayed and/or chronic xenograft rejection. Among the cell populations participating in such rejection, NK cells have been proposed as an important component. In this study we report the in vitro cytotoxic activity of natural killer (NK) cells obtained from healthy human donors against porcine target cells. Freshly isolated peripheral blood mononuclear cells (PBMC) and purified NK cells (CD16+/CD56+, CD3‐, CD20‐, CD33‐) exhibited little or no cytotoxic activity when tested on porcine phytohemagglutinin (PHA)‐stimulated lymphoblasts or bone marrow‐ or aortic‐derived endothelial cell lines in the presence of serum‐free medium. Killing was considerably higher in the presence of human decomplemented plasma, containing xenoreactive nAb, or purified Gal(α1,3)Gal‐reactive antibodies, suggesting that antibody dependent cell‐mediated cytotoxicity (ADCC) mediated by NK cells is an important mechanism involved in xenogeneic cytotoxicity. After incubation of human PBMC for 6 days in the presence of irradiated xenogeneic porcine or allogeneic stimulator cells, or in the presence of exogenous interleukin 2 (IL‐2), the cytotoxic activity of the bulk cultures as well as that of isolated NK cells (separated from stimulated bulk cultures) against xenogeneic targets increased considerably, and corresponded to an increased NK‐specific lysis of K562 target cells. Cell surface staining and flow cytometry showed that CD16+/CD56+, CD3‐ NK cells composed ca. 25% of short‐term (6 days) xenogeneic, allogeneic, or IL‐2 stimulated bulk cultures. In summary, these data suggest that, in contrast to allogeneic cell‐ mediated killing, xenogeneic human anti‐porcine cytotoxicity includes an important contribution from NK 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.

Treatment of esophageal disease can necessitate resection and reconstruction of the esophagus. Current reconstruction approaches are limited to utilization of an autologous conduit such as stomach, small bowel, or colon. A tissue engineered construct providing an alternative for esophageal replacement in circumferential, full thickness resection would have significant clinical applications. In the current study, we demonstrate that regeneration of esophageal tissue is feasible and reproducible in a large animal model using synthetic polyurethane electro-spun grafts seeded with autologous adipose-derived mesenchymal stem cells (aMSCs) and a disposable bioreactor. The scaffolds were not incorporated into the regrown esophageal tissue and were retrieved endoscopically. Animals underwent adipose tissue biopsy to harvest and expand autologous aMSCs for seeding on electro-spun polyurethane conduits in a bioreactor. Anesthetized pigs underwent full thickness circumferential resection of the mid-lower thoracic esophagus followed by implantation of the cell seeded scaffold. Results from these animals showed gradual structural regrowth of endogenous esophageal tissue, including squamous esophageal mucosa, submucosa, and smooth muscle layers with blood vessel formation. Scaffolds carrying autologous adipose-derived mesenchymal stem cells may provide an alternative to the use of a gastro-intestinal conduit for some patients following resection of the esophagus.

Rheumatoid arthritis (RA) represents a heterogenous disease characterized by chronic polyarthritis. Most patients with adult RA inherit HLA-DR4 or -DR1 major histocompatibility complex (MHC) genes. While the molecular basis for this genetic predisposition is unknown, the major function of these MHC-encoded molecules is to present peptides to T lymphocytes. It is hypothesized that an endogenous or environmental antigen initiates a MHC-restricted immune response mediated by T lymphocytes, which is followed by a chronic inflammatory reaction involving many cell types. In chronic RA, previous or ongoing antigenic activation might result in detectable skewing of the peripheral alpha/beta T cell receptor (TCR) repertoire. Here we demonstrate a marked expansion of V alpha 12.1-bearing CD8+ T cells in the peripheral blood (mean, 22%; range, 10-43%) of > 15% of RA patients. A major proportion of these patients shared HLA-DQ2 in addition to the expected high frequency DR1 and DR4 alleles. Detailed molecular analysis in three of the RA patients with elevated V alpha 12.1+ T cells identified repeated TCR alpha chain sequences consistent with clonal V alpha 12.1+,CD8+ T cell expansion. In addition to shared TCR V alpha 12.1 germline gene usage among unrelated subjects, a conserved J alpha motif was also detected. Together, these results suggest an antigen-driven mechanism of T cell expansion in these patients and may offer a new approach in examining specific antigen that stimulate T cells in RA.

SummaryT cells bearing the T cell receptor ot//$ (TCR-cdB) are the predominant lymphocyte population in the human intestinal epithelium. To examine if normal intestinal intraepithdial lymphocytes (IEL) have a TCR repertoire distinct from the TCR-cffB repertoire in peripheral blood lymphocytes (PBL), comparative analysis of relative VB gene usage in IEL and PBL was performed by quantitative polymerase chain reaction. In each of the six individuals examined, one to three V/~ families made up more than 40% of the total V/$ transcripts detected in the IEL, whereas there was a more even distribution of V/$ gene usage in the paired PBL. The predominant V/$ families, especially VB1, V/~2, V/~3, and V~6, were frequently shared among IEL of different individuals. PCR cloning and sequence analysis of the predominant V/86 family in two individuals revealed an identical V-D-J-C sequence in 13 of 21 dones obtained from one donor, and a different repeated sequence in 18 of 27 clones examined in the second donor. These data suggest that the V/~ skewing in IEL is due to an oligoclonal T cell expansion and may reflect the response of the intestinal mucosal immune system to a restricted set of as yet undefined antigens present in the gut.ttle is known about the function of lymphocytes at epithelial sites. Intestinal intraepithelial lymphocytes (IEL) 1 are a predominantly CD4-8 + subset of T cells localized throughout the epithelial lining of the gut (1). In the mouse, the majority of lymphocytes at epithelial surfaces such as the intestine use the TCR-3,/& Murine 3'/8 IEL express two major TCR V3"/V8 pairs (V3'5/V~4,6) and have extensive junctional diversity. As in the mouse, human TCR-3'/8 cells preferentially localize within the gut epithelium rather than in the lamina propria. However, the dramatic numerical epithelial predominance of 3,/8 cells in mice (50-75 % of the total IEL population) has not been observed in humans, where 3,/8 T cells make up *10% (range, 5-20%) of small bowel IEL and, according to one recent report, 37% (range, 13-87%) of large intestine IEL (2, 3). Thus, the majority of T cells in the adult human gut epithdium express the TCR-a/~