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Islet transplantation is a promising treatment for diabetes but long-term success is limited by progressive graft loss. Aggregates of the beta cell peptide islet amyloid polypeptide (IAPP) promote beta cell apoptosis and rapid amyloid formation occurs in transplanted islets. Porcine islets are an attractive alternative islet source as they demonstrate long-term graft survival. We compared the capacity of transplanted human and porcine islets to form amyloid as an explanation for differences in graft survival. Human islets were transplanted into streptozotocin-diabetic immune-deficient mice. Amyloid deposition was detectable at 4 weeks posttransplantation and was associated with islet graft failure. More extensive amyloid deposition was observed after 8 weeks. By contrast, no amyloid was detected in transplanted neonatal or adult porcine islets that had maintained normoglycemia for up to 195 days. To determine whether differences in IAPP sequence between humans and pigs could explain differences in amyloid formation and transplant viability, we sequenced porcine IAPP. Porcine IAPP differs from the human sequence at 10 positions and includes substitutions predicted to reduce its amyloidogenicity. Synthetic porcine IAPP was considerably less amyloidogenic than human IAPP as determined by transmission electron microscopy, circular dichroism, and thioflavin T binding. Viability assays indicated that porcine IAPP is significantly less toxic to INS-1 beta cells than human IAPP. Our findings demonstrate that species differences in IAPP sequence can explain the lack of amyloid formation and improved survival of transplanted porcine islets. These data highlight the potential of porcine islet transplantation as a therapeutic approach for human diabetes.

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The role of islet amyloidosis in the onset and progression of Type 2 diabetes remains obscure. Islet amyloid polypeptide is a 37 amino-acid, beta-cell peptide which is co-stored and co-released with insulin. Human islet amyloid polypeptide refolds to a beta-conformation and oligomerises to form insoluble fibrils; proline substitutions in rodent islet amyloid polypeptide prevent this molecular transition. Pro-islet amyloid polypeptide (67 amino acids in man) is processed in secretory granules. Refolding of islet amyloid polypeptide may be prevented by intragranular heterodimer formation with insulin (but not proinsulin). Diabetes-associated abnormal proinsulin processing could contribute to de-stabilisation of granular islet amyloid polypeptide. Increased pro-islet amyloid polypeptide secretion as a consequence of islet dysfunction could promote fibrillogenesis; the propeptide forms fibrils and binds to basement membrane glycosamino-glycans. Islet amyloid polypeptide gene polymorphisms are not universally associated with Type 2 diabetes. Transgenic mice expressing human islet amyloid polypeptide gene have increased islet amyloid polypeptide concentrations but develop islet amyloid only against a background of obesity and/or high fat diet. In transgenic mice, obese monkeys and cats, initially small perivascular deposits progressively increase to occupy 80% islet mass; the severity of amyloidosis in animal models is related to the onset of hyperglycaemia, suggesting that islet amyloid and the associated destruction of islet cells cause diabetes. In human diabetes, islet amyloid can affect less than 1% or up to 80% of islets indicating that islet amyloidosis largely results from diabetes-related pathologies and is not an aetiological factor for hyperglycaemia. However, the associated progressive beta-cell destruction leads to severe islet dysfunction and insulin requirement.

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The polypeptide hormone amylin forms amyloid deposits in patients with type 2 diabetes mellitus. Amyloid-forming peptides are often very difficult to synthesize and purify. Amylin and fragments of amylin are no exception. In this paper we describe the efficient synthesis and purification of two amyloidogenic fragments of human amylin. One fragment corresponds to residues 17 to 37 of the full-length hormone and the other corresponds to residues 24 to 37. These fragments have previously been identified in vivo and have been shown to form amyloid in vitro. The strategy used to elucidate appropriate conditions for the synthesis and purification of these peptides is generally applicable to other peptides that are difficult to synthesize. These peptides were prepared using solid-phase peptide synthesis with Fmoc alpha-amino protection. The effects of varying the solvent, side-chain-protecting group and choice of cleavage conditions were examined. The use of NMP as the main solvent and cleavage with trifluoroacetic acid, phenol, ethanedithiol, thioanisole, and water proved to be optimal. 1,1,1,3, 3,3-Hexafluoro-2-propanol (HFIP) was found to be the best solvent for solubilizing the crude peptides. A wide range of HPLC conditions for the purification of the peptides were examined and an acetonitrile-based solvent system with HCl as the ion pairing agent provided efficient purification.

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Human amylin is the primary component of amyloid deposits found in the pancreatic beta-cells of patients with type 2 diabetes mellitus. Recently, two fragments of amylin have been identified in vivo. One fragment contains residues 17 to 37 of human amylin (AMYLIN17-37) and the other contains residues 24 to 37 (AMYLIN24-37). The secondary structure and amyloid forming ability of each peptide was determined at pH 5.5(+/-0.3) and pH 7.4(+/-0.3). Results at these two values of pH were very similar. Both peptides are predominantly unstructured in solution (CD) but adopt a significant amount of beta-sheet secondary structure upon aggregation (FTIR). Transmission electron microscopy (TEM) confirmed the presence of amyloid fibrils. AMYLIN24-37 was further dissected by studying peptides corresponding to residues 24 to 29 and 30 to 37. The AMYLIN30-37 peptide forms amyloid deposits. Samples of the 24 to 29 fragment which had TFA as the associated counterion formed ordered deposits but samples associated with HCl did not. Residues 20 to 29 are traditionally thought to be the amyloidogenic region of amylin, but this study demonstrates that peptides derived from other regions of amylin are capable of forming amyloid, and hence indicates that these regions of amylin can play a role in amyloid formation.

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The antibody responsiveness to and the specific vaccination effect of rabies virus infection were investigated in high- and low-responder lines of mice produced by two-way selective breedings for quantitative production of antibodies to flagellar (H/f and L/f lines) or somatic (H/s and L/s lines) antigens of salmonellae. After specific immunization, both high lines were more resistant to rabies virus infection than were the low lines, and the protector effect was related to the level of antibody produced, as demonstrated by neutralizing serum activity. The present findings confirm the nonspecific genetic modification of the general antibody responsiveness induced in high- and low-responder lines selected for quantitative antibody production.

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Forty two saliva samples from rabid dogs were examined by intracerebral inoculation of weanling and suckling mice. Although rabies virus assay were successful in all of the samples in both groups of mice used, a significant higher death proportion (p < 0.01) were observed in the suckling mice group.

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Rabies virus was isolated from the brain, salivary and interscapular (brown fat) glands, heart, lungs and testis of naturally infected vampire bat Desmodus rotundus found paralyzed in the day at Barueri, São Paulo State. The rabies virus isolations were made by intracerebral inoculation in 4-5 days and 30 days old mice. The virus strain was identified as rabies virus by the Sellers and Faraco (Mann) techniques, the fluorescent antibody test and intracerebral inoculation of mice. The isolation of virus from lungs and testis was made only in suckling mice. Only one of eight and two of eight mice inoculated died with rabies.

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Rabies virus was isolated from insectivorous bat Molossus obscurus found in a semi-paralyzed condition, in broad daylight, in Campinas, São Paulo State. Suckling and adult mice inoculated intracerebrally with a 20% suspension of bat brain showed typical rabies symptoms within eight days. The mortality of inoculated mice was 100%. Negri bodies were seen in the brains of infected mice by Sellers and Fraco's methods. Rabies antigens was found in the brains of inoculated mice by fluorescent antibodies test.

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Complement-fixation test based in 50% hemolytic end point was applied to investigate the immune status to rabies of dogs vaccinated with heigh egg-passage Flury vaccine. The complement-fixation titer was compared with serum neutralization results. Twenty-five sera was employed and the complement fixation titer varied of 0 to 256. Three sera was anticomplementary. The results indicated a lack of quantitative correlation, but was found a qualitative correlation between the two methods.

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Five livers of equine fetuses, aborted due to the action of equine abortion virus, five livers from men, two of whom died of epidemic hepatitis and three obtained by needle biopsies, 5 livers of dogs with infectious canine hepatitis and 7 livers of ducklings that had hepatitis, were studied histopathologically. The foals' livers were studied by several staining methods and the others by H. E. only. The results indicate that the lesions are quite similar in the four species with the appearance of nuclear inclusion bodies only in foals and dogs. The strong staining properties of the nuclear inclusion bodies in infectious canine hepatitis and the weak staining properties of the equine virus abortion reveal that the protein-DNA association is different resulting in a different electropolarity. The lesions in foals are of two main types, one a Necrotic-Mosaic Type in which the hepatocyte degeneration is irregularly distributed within the hepatic lobules and the other an Hyperplastic Type in which marked regeneration occurs. In the Hyperplastic Type the practical absence of plasmocytes in foals' livers might suggest that if the newborn is a female, abortions may occur later in life because the virus remained alive in colts which were born in an immune tolerance state.Histologically the picture in the livers of aborted foals assume features of a viral hepatitis similar to the viral hepatitis in men, dogs and ducklings.

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