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Background: Waning of immunoglobulin G (IgG) antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) complicates the diagnosis of past infection. The durability of T-cell memory against SARS-CoV-2 remains unclear, and most current T-cell protocols are unsuited for large-scale automation., Methods: Whole-blood samples from 31 patients with verified past coronavirus disease 2019 (COVID-19) and 46 controls, of whom 40 received COVID-19 vaccine, were stimulated with peptides spanning the nucleocapsid (NC) or spike 1 (S1) regions of SARS-CoV-2 and analyzed for interferon γ in supernatant plasma. Diagnostic accuracy of these assays was evaluated against serum anti-NC and anti-receptor-binding domain S1-IgG., Results: Induction of interferon γ in whole blood by NC or S1 peptides diagnosed past COVID-19 with high accuracy (area under the receiver operating characteristic curve, 0.93 and 0.95, respectively). In accordance with previous studies, NC-IgG levels rapidly waned with only 5 of 17 patients (29%) remaining seropositive >180 days after infection. By contrast, NC peptide-induced T-cell memory responses remained in 13 of 17 study participants (76%) >180 days after infection (P = .01 for comparison with NC-IgG; McNemar test). After 2 vaccine doses, all 18 donors exhibited S1-specific T-cell memory., Conclusions: Cytokine release assays for the monitoring of T-cell memory in whole blood may be useful for evaluating complications following unverified past COVID-19 and for long-term assessment of vaccine-induced T-cell immunity., Clinical Trials Registration: EudraCT 2021-000349-42., (© The Author(s) 2022. Published by Oxford University Press for the Infectious Diseases Society of America.)

The formation of insulin amyloid can dramatically impact glycemic control in patients with diabetes, making it an important therapeutic consideration. In addition, the cost associated with the excess insulin required by patients with amyloid is estimated to be $3K per patient per year, which adds to the growing financial burden of this disease. Insulin amyloid has been observed with every mode of therapeutic insulin administration (infusion, injection and inhalation), and the number of reported cases has increased significantly since 2002. The new cases represent a much broader demographic, and include many patients who have used exclusively human insulin and human insulin analogs. The reason for the increase in case reports is unknown, but this review explores the possibility that changes in patient care, improved differential diagnosis and/or changes in insulin type and insulin delivery systems may be important factors. The goal of this review is to raise key questions that will inspire proactive measures to prevent, identify and treat insulin amyloid. Furthermore, this comprehensive examination of insulin amyloid can provide insight into important considerations for other injectable drugs that are prone to form amyloid deposits.

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.

Amyloid fibrils are highly ordered protein assemblies known to contribute to the pathology of a variety of genetic and aging-associated diseases. More recently, these fibrils have been shown to be useful as structural scaffolds in both natural biological systems and nanotechnology applications. The intense interest in amyloid fibrils has led to the investigation of well-characterized proteins, such as hen egg white lysozyme (HEWL), as model systems to examine structural and mechanistic principles that may be generally applicable to all amyloid fibrils. The purpose of this review is to critically examine the fibril-formation literature of proteins in the lysozyme family with respect to the known structure and folding properties of these proteins. The goal is to identify similarities and differences within the family, examine general misfolding / aggregation principles, and identify key areas of importance for future work on the fibril formation of these proteins.

Amyloid fibrils are typically rigid, unbranched structures with diameters of approximately 10 nm and lengths up to several micrometres, and are associated with more than 20 diseases including Alzheimer's disease and type II diabetes. Insulin is a small, predominantly alpha-helical protein consisting of 51 residues in two disulfide-linked polypeptide chains that readily assembles into amyloid fibrils under conditions of low pH and elevated temperature. We demonstrate here that both the A-chain and the B-chain of insulin are capable of forming amyloid fibrils in isolation under similar conditions, with fibrillar morphologies that differ from those composed of intact insulin. Both the A-chain and B-chain fibrils were found to be able to cross-seed the fibrillization of the parent protein, although these reactions were substantially less efficient than self-seeding with fibrils composed of full-length insulin. In both cases, the cross-seeded fibrils were morphologically distinct from the seeding material, but shared common characteristics with typical insulin fibrils, including a very similar helical repeat. The broader distribution of heights of the cross-seeded fibrils compared to typical insulin fibrils, however, indicates that their underlying protofilament hierarchy may be subtly different. In addition, and remarkably in view of this seeding behavior, the soluble forms of the A-chain and B-chain peptides were found to be capable of inhibiting insulin fibril formation. Studies using mass spectrometry suggest that this behavior might be attributable to complex formation between insulin and the A-chain and B-chain peptides. The finding that the same chemical form of a polypeptide chain in different physical states can either stimulate or inhibit the conversion of a protein into amyloid fibrils sheds new light on the mechanisms underlying fibril formation, fibril strain propagation and amyloid disease initiation and progression.

Amyloid fibrils are ordered aggregates of peptides or proteins that are fibrillar in structure and contribute to the complications of many diseases (e.g., type 2 diabetes mellitus, Alzheimer's disease, and primary systemic amyloidosis). These fibrils can also be prepared in vitro and there are three criteria that define a protein aggregate as an amyloid fibril: green birefringence upon staining with Congo Red, fibrillar morphology, and beta-sheet secondary structure. The purpose of this review is to describe the techniques used to study amyloid fibril formation in vitro, address common errors in the collection and interpretation of data, and open a discussion for a critical review of the criteria currently used to classify a protein aggregate as an amyloid fibril.

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.

Islet amyloid polypeptide (IAPP), or 'amylin', is co-stored with insulin in secretory granules of pancreatic islet beta-cells. In Type 2 diabetes, IAPP converts into a beta-sheet conformation and oligomerizes to form amyloid fibrils and islet deposits. Granule components, including insulin, inhibit spontaneous IAPP fibril formation in vitro. To determine the mechanism of this inhibition, molecular interactions of insulin with human IAPP (hIAPP), rat IAPP (rIAPP) and other peptides were examined using surface plasmon resonance (BIAcore), CD and transmission electron microscopy (EM). hIAPP and rIAPP complexed with insulin, and this reaction was concentration-dependent. rIAPP and insulin, but not pro-insulin, bound to hIAPP. Insulin with a truncated B-chain, to prevent dimerization, also bound hIAPP. In the presence of insulin, hIAPP did not spontaneously develop beta-sheet secondary structure or form fibrils. Insulin interacted with pre-formed IAPP fibrils in a regular repeating pattern, as demonstrated by immunoEM, suggesting that the binding sites for insulin remain exposed in hIAPP fibrils. Since rIAPP and hIAPP form complexes with insulin (and each other), this could explain the lack of amyloid fibrils in transgenic mice expressing hIAPP. It is likely that IAPP fibrillogenesis is inhibited in secretory granules (where the hIAPP concentration is in the millimolar range) by heteromolecular complex formation with insulin. Alterations in the proportions of insulin and IAPP in granules could disrupt the stability of the peptide. The increase in the proportion of unprocessed pro-insulin produced in Type 2 diabetes could be a major factor in destabilization of hIAPP and induction of fibril formation.

We have investigated the chemical modification of insulin under conditions that promote the conversion of the soluble protein into amyloid fibrils. The modifications that are incorporated into the fibrils include deamidation of Asn A21, Asn B3, and Gln B4. In order to prepare fibrils with minimal deamidation of these residues, the kinetics of aggregation were accelerated by seeding with aliquots of a solution containing preformed fibrils. The resulting fibrils were then reincubated to determine the extent to which chemical modification occurs in the fibril itself. The deamidation of Asn A21 in particular could be followed in detail. Deamidation of this residue in the fibrillar form of insulin was found to occur in only 52 +/- 5% of molecules. This result indicates that there are at least two different packing environments of insulin molecules in the fibrils and suggests that the characterization of chemical modifications may be a useful probe of the environment of polypeptide chains within amyloid fibrils.

Lactoferrin has previously been identified in amyloid deposits in the cornea, seminal vesicles, and brain. We report in this paper a highly amyloidogenic region of lactoferrin (sequence of NAGDVAFV). This region was initially identified by sequence comparison with medin, a 5.5 kDa amyloidogenic fragment derived from lactadherin. Subsequent characterization revealed that this peptide forms amyloid fibrils at pH 7.4 when incubated at 37 degrees C. Furthermore, although full-length lactoferrin does not by itself form amyloid fibrils, the protein does bind to the peptide fibrils as revealed by an increase in thioflavin T fluorescence and the presence of enlarged fibrils by transmission electron microscopy and polarized light microscopy. The binding of lactoferrin is a selective interaction with the NAGDVAFV fibrils. Lactoferrin does not bind to insulin or lysozyme fibrils, and the NAGDVAFV fibrils do not bind to soluble insulin or lysozyme. The lactoferrin appears to coat the peptide fibril surface to form mixed peptide/protein fibrils, but again there is no evidence for the formation of lactoferrin-only fibrils. This interaction, therefore, seems to involve selective binding rather than conventional seeding of fibril formation. We suggest that such a process could be generally important in the formation of amyloid fibrils in vivo since the identification of both full-length protein and protein fragments is common in ex vivo amyloid deposits.

The polypeptide hormone amylin forms amyloid deposits in Type 2 diabetes mellitus and a 10-residue fragment of amylin (amylin(20-29)) is commonly used as a model system to study this process. Studies of amylin(20-29) and several variant peptides revealed that low levels of deamidation can have a significant effect on the secondary structure and aggregation behavior of these molecules. Results obtained with a variant of amylin(20-29), which has the primary sequence SNNFPAILSS, are highlighted. This peptide is particularly interesting from a technical standpoint. In the absence of impurities the peptide does not spontaneously aggregate and is not amyloidogenic. This peptide can spontaneously deamidate, and the presence of less than 5% of deamidation impurities leads to the formation of aggregates that have the hallmarks of amyloid. In addition, small amounts of deamidated material can induce amyloid formation by the purified peptide. These results have fundamental implications for the definition of an amyloidogenic sequence and for the standards of purity of peptides and proteins used for studies of amyloid formation.

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., (Copyright 2001 Academic Press.)

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., (Copyright 1999 Academic Press.)

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.

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.

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.

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.

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.

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.