Inactivation of viruses infecting ectothermic animals by amphibian and piscine antimicrobial peptides

The ability of five purified amphibian antimicrobial peptides (dermaseptin-1, temporin A, magainin I, and II, PGLa), crude peptide fractions isolated from the skin of Rana pipiens and R. catesbeiana, and four antimicrobial peptides (AMPs) from hybrid striped bass (piscidin-1N, -1H, -2, and -3) were examined for their ability to reduce the infectivity of channel catfish virus (CCV) and frog virus 3 (FV3). All compounds, with the exception of magainin I, markedly reduced the infectivity of CCV. In contrast to CCV, FV3 was 2- to 4-fold less sensitive to these agents. Similar to an earlier study employing two other amphibian peptides, the agents used here acted rapidly and over a wide, physiologically relevant, temperature range to reduce virus infectivity. These results extend our previous findings and strongly suggest that various amphibian and piscine AMPs may play important roles in protecting fish and amphibians from pathogenic viruses.     

Chemosensory Cell-Derived Acetylcholine Drives Tracheal Mucociliary Clearance in Response to Virulence-Associated Formyl Peptides

1. Download : Download high-res image (202KB)
2. Download : Download full-size image

     

Ontogeny of guanylin-immunoreactive cells in rat salivary glands

Guanylin-like peptides regulate electrolyte/water transport through the epithelia. Moreover, these peptides possess antiproliferative activity and regulate the turnover of epithelial cells. In an earlier study we localized guanylin immunoreactivity in secretory ducts of adult rodent salivary glands. In this study we investigated the appearance and distribution pattern of this peptide during the development of rat salivary glands. Guanylin immunoreactivity appeared at the beginning of cell differentiation from solid bud, on embryonic day 17 in the submandibular and sublingual glands and after day 18 in the parotid gland. Guanylin immunoreactivity appeared first in ductal and acinar anlage: its cell distribution pattern and fate differed in these two compartments. In the duct cells guanylin immunoreactivity spread after the duct system developed, whereas in acinar cells it disappeared after cell differentiation. The guanylin immunoreactivity we detected in adult salivary duct cells accords with guanylins role in regulating electrolyte and water transport through the various epithelia. It does so by activating guanylate cyclase-C receptor, increasing intracellular cGMP concentration, and phosphorylating the cystic fibrosis transmembrane conductance regulator (CFTR) protein by the cGMP-dependent protein kinase II. This signaling cascade couples to the ductal electrolyte/water secretion and modulates finally the electrolyte composition of the saliva. On the other hand, CFTR is also involved in mechanisms of cell growth, by regulating apoptosis, and promoting cell differentiation. The early diffuse guanylin immunoreactivity we observed in ducts and acinar anlage, before the secretory set is operative, suggests guanylin has a role in cell differentiation.     

Differential regulation of Th1 and Th2 cells by p91–110 and p21–40 peptides of the 16-kD α-crystallin antigen of Mycobacterium tuberculosis

Permissively recognized peptides which can activate lymphocytes from subjects with a variety of class II HLA types are interesting diagnostic and vaccine candidates. In this study we generated T helper clones reactive to the permissively recognized p21–40 and p91–110 peptides of the 16-kD heat shock protein of Mycobacterium tuberculosis. All the clones specific for p91–110 secreted interferon-gamma (IFN-γ) and were of the Th1 phenotype. By contrast, the p21–40 peptide favoured the generation of IL-4-producing clones. Antibody blockade established that the peptide-specific Th clones could either be DR-, DP- or DQ-restricted. Thus, two permissively recognized sequences p21–40 and p91–110 from the same mycobacterial antigen can drive the differentiation of functionally distinct T helper subsets. Attempts to immunize against tuberculosis should bear in mind epitope specificity if a favourable Th subtype response is to be generated.     

Peptides derived from the whole sequence of BCR-ABL bind to several class I molecules allowing specific induction of human cytotoxic T lymphocytes

Chronic myeloid leukemia (CML) is characterized cytogenetically by a t(9;22) translocation which generates a hybrid bcr-abl gene, encoding a p210^bcr-abl fusion protein. The induction in vitro of leukemia-specific T cells reactive with p210^bcr-abl is a strategy developed for an immunological therapeutic approach in CML. Peptides from the junction region of this chimeric protein have been considered as potential targets for a cytotoxic response against leukemic cells. However, only a few peptides encompassing the two p210^bcr-abl breakpoints have been shown to bind to the most common HLA class I molecules, which limits the number of patients who could benefit from this approach. We assume that the presence of chimeric BCR-ABL protein in leukemic cells may affect processing and delivery of peptides, possibly giving rise to new epitopes at the cell surface. We selected 162 peptides from the whole sequence of this protein, including 14 peptides of the b2a2 and b3a2 junctions, which had an anchor motif for a common HLA class I molecule. We tested their ability to bind to eight HLA class I molecules (HLA-A1, -A2, -A3, -A11, -B7, -B8, -B27, -B44). We identified 48 peptides from outside the junction region, with intermediate or strong binding capacities to these HLA class I molecules contrasting with only six junction peptides with a moderate binding capacity to HLA-A3/A11, -B8, or -B44 molecules. Moreover, cytotoxic T lymphocyte lines specific for various peptides outside the junction were generated from peripheral blood mononuclear cells of HLA-A2 or -B7 healthy donors and from one CML patient. These results contribute to evaluation of immunity to the BCR-ABL chimeric protein. Further studies are required to investigate whether such epitopes are correctly processed and presented by leukemic cells.     

Epitope analysis of HLA-DR-restricted helper T-cell responses to Der p II, a major allergen molecule of Dermatophagoides pteronyssinus

T-cell epitopes of Der p II, a major allergen of Dermatophagoides pteronyssinus , were analyzed by using human T-cell clones. We tested 38 cloned T cells from two Japanese patients with allergic rhinitis, and identified at least two peptides (K33-T47 and 158-C73) as helper T-cell epitopes. The former epitope was shown to be restricted by HLA-DRB1* 1502, and the latter by HLA-DRB1* 0405, both of which are typical Japanese HLA-DR alleles, suggesting that those T-cell epitopes might be important for the onset of house-dust mite allergy in the Japanese population. We prepared 15 analog peptides of the HLA-DRB1* 1502-restricted 15-mer peptide. Of those 15 residues, five (F35, L37, A39, F41, and E42) were critical for the epitope activity, and three residues (F35, A39, and E42) seemed to be included in anchor motifs for HLA-DRB1* 1502. The epitope peptide was also recognized by HLA-DRB1* 1502-positive healthy donors; however, only allergic T cells showed Th2 functions. Antigen-presenting cells of nonallergic donors were able to activate allergic T cells to express Th2 function. This seemed to suggest that antigen recognition of T cells, as well as additional unknown factors which promote Th2, rather than Th1, responses, might be important for the onset of house-dust mite allergy.     

Inhibition of heparin/protamine complex-induced complement activation by Compstatin in baboons

Complement activation products are major components of the inflammatory response induced by cardiac surgery and cardiopulmonary bypass which contribute to postoperative organ dysfunction, fluid accumulation, and morbidity. Activation of the complement system occurs during extracorporeal circulation, during reperfusion of ischemic tissue, and after the formation of heparin-protamine complexes. In this study we examine the efficacy of Compstatin, a recently discovered peptide inhibitor of complement, in preventing heparin/protamine-induced complement activation in baboons. The study was performed in baboons because Compstatin binds to baboon C3 and is resistant to proteolytic cleavage in baboon blood (similar to humans); Compstatin inhibits only the activation of primates' complement system. After testing various doses and administration regimens, Compstatin produced complete inhibition at a total dose of 21 mg/kg when given as a combination of bolus injection and infusion. Compstatin completely inhibited in vivo heparin/protamine-induced complement activation without adverse effects on heart rate or systemic arterial, central venous, and pulmonary arterial pressures. This study indicates that Compstatin is a safe and effective complement inhibitor that has the potential to prevent complement activation during and after clinical cardiac surgery. Furthermore, Compstatin can serve as the prototype for designing an orally administrated drug.     

Invriant chain peptides enhancing or inhibiting the presentation of antigenic peptides by major histocompatibility complex class II molecules

Two soluble invariant chain (Ii) peptides with overlapping sequences had contrasting effects on the presentation of antigenic peptides by murine A^d, A^k, E^d, and E^k major histocompatibility complex (MHC) class II molecules. Naturally produced class II-associated invariant chain peptides human (h)Ii81–104/murine (m)Ii80–103 inhibited antigen presentation on these MHC class II alleles in a manner consistent with competitive inhibition. The Ii-4 peptides hIi77–92/mIi76–91 enhanced presentation of antigenic peptides on I-E class II alleles by promoting the exchange of peptides at the cell surface. Treatment of antigenpresenting cells (APC) with Ii-4 before the addition of antigenic peptide greatly enhanced subsequent T cell responses, while treatment of APC with Ii–4 after antigenic peptide binding decreased subsequent T cell responses. The hIi81–104 and mIi80–103 peptides inhibited T cell responses in both types of assays. The binding of biotinylated antigenic peptide to MHC class II-transfected L cells, as measured by flow cytometry, was inhibited by mIi80-103 and enhanced by mIi-4. Segments of Ii fragments remaining associated with MHC class II, or released Ii peptides, appear to regulate the formation of stable antigenic peptide/MHC class II complexes either positively or negatively through interactions at or near the antigenic peptide binding site. These findings open a pathway for the design of novel therapeutics based on the structure and function of natural and rationally designed fragments of Ii.     

Effect of μ-Opiate Receptor Agonist Tetrapeptide A10 on DNA Synthesis and Protein Content in the Myocardium of Albino Rats

Effect of intraperitoneal injection of tetrapeptide A10 (H-Tyr-D-Orn-Phe-Gly-OH), selective -opiate receptor agonist, synthetic analog of dermorphine, in a dose of 100 g/kg on DNA synthesis and protein content in the myocardium was studied in albino rats. Five injections of tetrapeptide on days 2-6 after birth caused no changes in DNA synthesis 17 days after the last injection, i. e. in 24-day rats. The number of nucleoli and their area increased. In adult males long-term (3-week) treatment with tetrapeptide A10 increased the number of nucleoli and the mean and integral optical density of isolated cardiomyocytes stained with amido black B, which probably attested to activation of protein synthesis in the myocardium. Simultaneously, the content of catecholamines in the heart increased. These data are comparable with delayed effects of k-opiate receptor agonist dinorphine A1-13 and indicate that morphogenetic properties of opioid peptides in rat myocardium are realized via the same routes.     

Identification of an HLA-DQ2 peptide binding motif and HLA-DPw3-bound self-peptide by pool sequencing

Molecules of the major histocompatibility complex (MHC) present antigenic peptides to T cells. Sequencing peptide pools eluted from MHC class I molecules has established allele-specific peptide binding motifs. We applied pool sequencing to analyze human MHC class II-bound peptides and found that HLA-DQ2-eluted peptides predominantly contained lysine, isoleucine, and phenylalanine at relative position i, i + 3 and i + 8, respectively. These residues putatively represent anchor residues for MHC binding. Analysis of a heterogeneous HLA-DPw3/DPw4-eluted peptide pool yielded a sequence matching an epitope from the endogeneous enzyme glyceraldehyde-3-phosphate dehydrogenase. This self-peptide and a partially identical, known allo-epitope bound specifically to DPw3 and DR13 molecules, suggesting the sharing of a binding motif. In particular, the presence of an arginine at relative position 4 appeared important for binding to these HLA class II specificities. Thus, pool sequencing is applicable for the analysis of MHC class II-eluted peptides.