Z. A. Latif

Successful Long-Term Survival of Pancreatic Islet Allografts in Spontaneous or Pancreatectomy-induced Diabetes in Dogs: Cyclosporine-induced Immune Unresponsiveness

Nineteen pancreatectomized beagles and three spontaneously diabetic dogs were recipients of canine islet allografts from one or more unrelated donors. The islets, enriched 30–45-fold for endocrine cells and contained in a packed cell volume of <1.5 ml, were engrafted in the livers of recipient animals. Treatment of diabetic recipients with cyclosporine (CsA) was begun 3–5 days before islet transplantation and the initial dosage was adjusted to attain and maintain CsA serum trough levels between 400 and 600 ng/ml. Five dogs with CsA levels less than this (155 ± 35 SEM ng/ml) at the time of transplantation promptly rejected their grafts, whereas rejection was encountered in only 1 of 17 diabetic animals in which the initial level exceeded 400 ng/ml. CsA was discontinued 30, 60, or 90 days after continuous therapy in 10 animals. Graft failure was observed 2 mo after stopping CsA in 1 animal and 5 mo in the other. Eight other islet allograft recipients have sustained fasting euglycemia for 7 and 8 mo in 2 and for at least 2 mo in the remainder. These results demonstrate that short-term CsA therapy prolongs survival of islet allografts and induces a state of immune unresponsiveness to islet alloantigens in dogs with experimental and spontaneous diabetes. The findings are unique for a nonrodent mammal and thus hold promise that similar results may be achieved for islet allografts of other mammalian species, including humans.

Natural history of multiple intrahepatic canine islet allografts during and following administration of cyclosporine

Cyclosporine (CsA) prevented acute rejection of intra-hepatic canine islet allografts (IHIA) in 5/5 pancreatectomized dogs. Normal fasting blood glucose levels were sustained in these dogs for 210±78 days (mean ± SEM) following withdrawal of CsA. We tested whether combining islets from more than one pancreas would improve function and prolong islet allograft survival during and following administration of CsA. Areas under the glucose disappearance (GDA) and C-peptide response (CPA) curves following i.v. glucose (0.5 g/kg), 1 month posttransplant were not significantly different using islets from 1 or 2 pancreases, whereas GDA and CPA approached normal if islet yields from 3 or more pancreases were combined. Mean islet allograft survival following interruption of CsA decreased with an increase in the number of donor pancreases (one: 210±78 days, vs.two: 113±23 days, vs. >2: 57±5 days). These studies demonstrate that: (1) IHIA uniformly resulted in fasting euglycemia in 36 of 38 diabetic dogs treated with CsA; (2) normal i.v. glucose metabolism required the combined islet yield of 3 or more donor pancreases, suggesting that a substantial number of intrahepatic islet cells are functionally lost despite effective CsA-induced immunosuppression; (3) use of multiple donors to accumulate an increased mass of islets may immunologically compromise allograft survival following discontinuation of CsA (these experiments, however, do not exclude a direct relationship between the duration of CsA therapy and the duration of immune unresponsiveness following interruption of CsA in multidonor islet allografts as an independent variable); and (4) unmodified islets obtained from multiple donors seem to require continuous immunosuppression to prevent rejection.

Effect of anti-Ia antibodies, culture, and cyclosporin on prolongation of canine islet allograft survival.

Evidence in rodents suggests that islet pretreatment to reduce islet immunogenicity will also require some form of immunosuppression of the recipient for islet allograft acceptance in highly reactive donor-recipient pairs. We attempted to ascertain whether outbred dogs would also require treatment of both donor islets and the recipient to prolong islet allograft survival. Untreated canine islets are uniformly rejected in 6-10 days in beagles. Tissue culture alone, at 37°C for 7 days, or treatment of freshly prepared islets with antila monoclonal antibodies (MoAbs) (B1F6 + 7.2) did not prolong canine islet allograft survival. Treatment of culture-maintained canine islets with anti-la MoAbs plus complement resulted in prolongation of islet allograft survival for 188 and 368 days in two of seven pancreatectomized nonimmunosuppressed beagles. The administration of low doses of cyclosporin A (CsA) intramuscularly, to recipients of untreated canine islet allografts had no effect on graft survival. By contrast, six of nine CsA-treated recipients of islets that were also treated with anti-la MoAbs (B1F6 + 7.2) plus complement showed prolongation of graft survival. Euglycemia was sustained for 19, 34, 89, and 300 days after the CsA was discontinued (day 30) in four of these animals. Two animals had unstable grafts from the beginning that failed 23 and 29 days after transplantation. Our results indicate that simple maneuvers like short-term tissue culture at 37°C and treatment of freshly isolated islets with anti-la MoAbs and complement are inadequate to prevent rejection in outbred pancreatectomized beagles. In contrast, low-dosage CsA acts synergistically with the in vitro treatment of islets with anti-la MoAbs to prolong islet allograft survival in outbred dogs with induced diabetes mellitus.

Immunochemical studies of infectious mononucleosis. IX. Heterophile antigen associated with a glycoprotein from the bovine erythrocyte membrane

A glycoprotein was isolated from the membrane of the bovine erythrocyte by refluxing the acetone‐ and ethanol‐extracted stroma residue with 75% ethanol. The glycoprotein was purified by phosphocellulose chromatography, ethanol precipitation, lipid‐solvent extraction and DEAE chromatography. The glycoprotein appeared to have two serological determinants, both reactive with antibodies present in the sera of patients with infectious mononucleosis. One of the determinants is similar to the Paul‐Bunnell heterophile antigen found on sheep erythrocytes. It is dependent on carbohydrate, including sialic acid residues. Another specificity, seemingly not shared by sheep erythrocytes to any great extent, is resistance to neuraminidase and to alkaline borohydride treatment and thus it may be located either on the polypeptide portion of the molecule or on an alkali‐stable oligosaccharide. The purified glycoprotein comprises 73% amino acids. Carbohydrate components and their molar ratios were sialic acid (1.0): galactose (1.5): N‐acetylglucosamine (1.1): N‐acetylgalactosamine (0.5): mannose (0.1).

Human pancreatic cell surface antigens with homologous expression in liver, sweat glands, and other exocrine tissues.

We have generated two panels of monoclonal antibodies (MoAbs) that represent a unique array of immunolabeling reagents specific for diverse cell surface and intracellular antigens of ductal epithelial cells (DEC) and of acinar cells (AC) within the human pancreas. Eight of the MoAbs are specific within the pancreas for the DEC plasma membrane (PM), three for DEC intracellular antigens, seven for AC-PM, and one for AC secretory granules. The MoAbs with PM reactivities have permitted us to substantially enhance our assessment of exocrine contamination in our human islet preparations. However, we have been unable to demonstrate consistently the effectiveness of these MoAbs in complement-mediated cytotoxicity protocols designed to improve the purity of our islet preparations by exocrine cytolysis. It is possible that primary cell isolates of pancreas or epithelial organs in general may lack inherent susceptibility to complement-mediated cytolysis or, instead, that this particular panel of MoAbs lacks the appropriate characteristics needed to achieve consistent complement-mediated cytolysis. We have also begun to explore the potentially broader applicability of the anti-DEC MoAbs as immunocytochemical tools and have observed intriguing patterns of cross-reactivities in other exocrine tissues, particularly bile duct epithelial cells in the liver and functionally discrete subsets of DEC in the eccrine sweat gland and the parotid gland. These homologous antigenic patterns, by virtue of their specificities for ductal tissue, most likely reflect the molecular bases for functions common to these tissues, e.g., active ion transport and much secretion. In addition, the anti-DEC and the anti-AC MoAbs exhibit patterns of binding specificity for human cell lines derived from pancreatic adenocarcinoma that suggest the potential value of these MoAbs in tumor diagnosis and therapy. We believe that these panels of MoAbs will find broad utility in immunopathology and in experimental approaches to questions regarding the development, normal function, and pathogenetic mechanisms in the human pancreas and other related organs as well.

Immunochemical studies of infectious mononucleosis—XI. Comparison of heterophile antibody inhibitors from the erythrocyte membranes of four mammalian species

Immunochemical comparisons were made of the reactivity of membrane glycoproteins from horse, bovine, sheep and goat erythrocytes with heterophile antibodies of infectious mononucleosis. The four receptors were tested as competitive inhibitors of a sandwich-type solid-phase radioimmunoassay and of agglutination of glycoprotein-latex reagents by infectious mononucleosis serum. The results of this study showed that the bovine glycoprotein had a superior reactivity with this heterophile antibody system and sheep erythrocyte glycoprotein was the least reactive. The latter had negligible ability to displace 125I-bovine glycoprotein and was a very poor inhibitor of the agglutination of a bovine glycoprotein-latex reagent by infectious mononucleosis serum. Horse and goat glycoproteins were more efficient inhibitors than sheep glycoprotein but less active than the preparation from bovine red cells. All of the inhibitory activity of sheep, horse and goat glycoproteins, and a major portion of that of the bovine glycoprotein was destroyed by neuraminidase treatment. We have termed this receptor--shared by all four species--the Paul-Bunnell receptor, since by definition Paul-Bunnell antibody is a sheep erythrocyte agglutinin which is also reactive with horse, bovine and goat erythrocytes. The neuraminidase (and alkaline borohydride) resistant receptor of bovine glycoprotein has been designated the Bo receptor because it is not common to the other three species.