2d) was demonstrated following porcine heart transplantation. Pigs are physiologically similar to humans, easily bred and are available in unlimited numbers.3,4 The carbohydrate galactose (1,3) galactose (gal 1,3 gal epitope) expressed on pig cells initiates the humoral response and contributes to cell-mediated rejection of porcine xenografts.5C7 Humans and Old World primates do not express a functional 1,3-galactosyltransferase gene and develop TRIM13 high levels of pre-existing anti-gal xenoantibodies as a result of antigenic stimulation by gastrointestinal bacteria expressing the -gal epitope.8,9 A small number of germline progenitors encode anti-gal xenoantibodies in humans and galactosyltransferase knockout mice.10C13 IgVH genes encoding the antibody response have an evolutionarily conserved structural configuration.14,15 Their possible counterpart in non-human Diprophylline primates which represent an important preclinical model for human xenotransplantation, has not been characterized. Since the human and rhesus variable region heavy chain family 3 (VH3) homologues defined to date show similarity, the humoral immune response Diprophylline in primates may be a closely related, appropriate model for studying the human immune response to porcine xenografts.16 We report the identification of the IgVH genes encoding xenoantibody responses induced following transplantation of rhesus monkeys (IGHV3-11. The kinetics of the anti–gal xenoantibody Diprophylline responses in four monkeys exposed to porcine heart or fetal pig islet cell xenografts is similar, and is encoded Diprophylline by a restricted group of genes. Materials and methods AnimalsFour colony-bred rhesus macaques (to provide an ongoing stimulus for xenoantibody production after heart removal. Blood samples were taken prior to transplantation (d0), 4 hr, 8 hr, 24 hr, 11 days, 21 days and monthly post-transplant. Serum samples were used to characterize the xenoantibody response by ELISA. Peripheral blood lymphocytes were isolated to produce cDNA libraries from pre- and post-transplantation samples. Preparation and immunostaining of porcine islet-like cell clustersFreshly isolated fetal ICCs were grown on poly-lysine-coated coverslips for immunostaining. Cells were washed with phosphate-buffered saline (PBS) containing 2 mm MgCl2, then blocked for 1 hr with 1% bovine serum albumin (BSA) in PBS. The first antibody [guinea-pig anti-human insulin, immunoglobulin G (IgG) fragment] (Linco Research Inc., St Charles, MO) was diluted 1 : 100 in 1% BSA and was added overnight at 4. The cells were washed with PBS/002% Triton X-100. Texas-Red-conjugated donkey anti-guinea-pig IgG (Jackson Immuno Research, West Grove, PA) was added to detect the bound anti-insulin antibody and the BSA-isolectin B4 conjugated to fluorescein isothiocyanate (BS-IB4 lectin-FITC) (1 mg/ml) (Sigma Aldrich, St Louis, MO), both antibodies at a dilution of 1 1 : 50, was added for gal carbohydrate detection.18 The cell nucleus was stained with 02 l/ml 4,6-diamidino-2-phenylindol (DAPI). Anti-fading solution (V-Laboratories Inc., Covington, LA) was used for mounting. The pictures were taken at the Image Core facility at the Children’s Hospital of Los Angeles, CA. Islet cell immunizationPorcine fetal ICCs (15 106 cells) were prepared by culturing collagenase-digested pancreata from fetuses at 66 days of gestation. Cells were cultured for 1C3 weeks19 and injected via an intraperitoneal route into two monkeys. ICCs were stained with BS-IB4 lectin-FITC and an anti-insulin antibody conjugated to Texas Red20 to determine whether insulin-secreting cells expressed the gal carbohydrate after culture. Blood samples were taken prior to transplantation on day 0, then at day 8, day 12, day 20 and day 39 post-transplantation. The two monkeys were re-immunized on day 45 with 15 106 newly prepared porcine ICCs. Samples were taken prior to the second injection, on day 45, and at.