Sukhi Bansal

Mucosal tolerance and suppression of collagen-induced arthritis (CIA) induced by nasal inhalation of synthetic peptide 184-198 of bovine type II collagen (CII) expressing a dominant T cell epitope

The purpose of the study was to map the dominant T cell epitope of the CB11 sequence of CII in RT1u haplotype rats and to determine if, when used as a synthetic peptide, it would induce tolerance to protect against CIA. A dominant epitope corresponding to residues 184‐198 included in the sequence of the CB11 fragment of bovine CII was identified in proliferation assay using peptides in an epitope scanning system using synthetic peptides of 15 amino acids, overlapping by 12 amino acids. This epitope is bovine‐specific, but cross‐reacts with the corresponding rat peptide. Minor epitopes in the bovine CB11 sequence were also autoantigenic. Use of independently synthesized and purified 184‐198 peptide confirmed its dominance in the T cell responses of arthritic rats. The peptide itself was not arthritogenic. Cells from lymph nodes draining arthritic feet were particularly responsive to the dominant peptide sequence, and showed evidence of epitope spreading to include reactions to at least four subdominant epitopes. Mucosal tolerance was successfully induced by instilling CII into the nose of rats before induction of CIA; this was found to delay the onset of disease, reduce mean disease severity, shift the anti‐CII antibody response to favour antibodies of the IgGl, rather than the IgG2b isiotype, and to reduce T cell reactivity to both CII and to the 184‐198 peptide. The dominant 184‐198 peptide itself had the same tolerogenic effects when given nasally to rats daily, on the 4 days immediately preceding the induction of CIA. Two forms of CIA with acute and delayed disease onset were each modified by pre‐treatment with the peptide. This study demonstrates that mucosal tolerance to CII can be induced by delivering it nasally in a way similar to that achieved previously by oral delivery, and that the use of an immunodominant epitope contained in a synthetic peptide will also suppress the immunologic and arthritic responses to collagen.

Molecular mimicry and ankylosing spondylitis: possible role of a novel sequence in pullulanase of Klebsiella pneumoniae

Molecular mimicry has been shown between two sequences of Klebsiella pneumoniae pulD secretion protein (DRDE) with HLA‐B27 (DRED) and pulA (pullulanase) enzyme (Gly‐X‐Pro) with types I, III and IV collagen respectively. IgG antibody levels in AS patients were elevated against 16mer synthetic peptides of HLA‐B27 and pulD by enzyme linked immunosorbent assay (ELISA) compared to controls (P < 0.001). ELISA assays against K. pneumoniae grown in the absence and presence of pullulan demonstrated significant levels of IgA antibody in AS patients compared to controls (P < 0.001). Increased IgA and IgG antibody levels to pulA and types I and IV collagen were observed in AS patients compared to controls (P < 0.001). These observations could be relevant in the sequence of molecular events in AS.

Antibacterial and antipeptide antibodies in Japanese and Finnish patients with rheumatoid arthritis

It has been suggested that Proteus infection may be involved in the pathogenesis of rheumatoid arthritis (RA). Bacterial and peptide immune responses in patients with RA and other control subjects were investigated in two geographically different populations. Serum samples from Finnish patients with early (n=72) and advanced (n=27) RA and 30 Finnish healthy controls, as well as from Japanese RA patients from two different locations: Tokyo (n=30) and Otsu (n=30), 18 patients with systemic lupus erythematosus (SLE) and 23 Japanese healthy controls were all screened for the total, and class-specific (IgG, IgA and IgM) antibodies against Proteus mirabilis, Escherichia coli and Serratia marcescens by indirect immunofluorescence assay. These samples were also tested for the determination of levels of isotypic antibodies against the shared epitope involving 16-mer synthetic peptides containing the EQRRAA or ESSRAL sequences and compared to scrambled control peptide by using an enzyme-labeled immunosorbent assay method. Significantly elevated levels of IgG and IgM antibodies to P. mirabilis and antibodies against both EQRRAA and ESSRAL peptides were detected in sera of Finnish patients with early and advanced RA, and in Japanese patients from Otsu or Tokyo compared to their corresponding control groups. In contrast, no difference either in the total or in any of the isotypic antibodies were observed between these groups when serum samples were screened against each of E. coli and S. marcescens or against the control peptide. Furthermore, there was a significant correlation between the antibody levels against Proteus bacteria only and both EQRRAA and ESRRAL peptides. Our findings support the possibility for specific involvement of P. mirabilis in the etiopathogenesis of RA even in early cases.

Systematic investigations of the influence of molecular structure on the transport of peptides across cultured alveolar cell monolayers

AbstractPurpose. To determine how the structures of peptides influence theiralveolar permeability. Methods. The studies were performed using 14 synthetic ‘model’peptides, labelled with a novel, non-intrusive amino acid fluorophore, andtheir transport studied using rat alveolar cell monolayers cultured onpermeable supports. Results. The passage of the peptides across the epithelial cellmonolayers is shown to be primarily paracellular, with an inverse dependenceon molecular size, and an enhanced flux observed for cationic peptides.The apparent permeability coefficients (Papp) for the peptides(together with those for other organic solutes, taken from the literature) areshown to be well-modelled assuming two populations of ‘pores’ in themonolayers, modelled as cylindrical channels of radii 15 Å and 22nm. The former pores are shown to be numerically equatable withthe monolayer tri-junctional complexes, and the latter are taken asmonolayer defects. Conclusions. The various monolayer Papp values correlatewell with the results from in vivo transport experiments, and the conclusion isdrawn that the pulmonary delivery of peptide drugs is perfectlyexploitable.

Further characterization of a bacteriocin produced by Lactobacillus paracasei HL32

Aims:  Purification, identification and partial characterization of bacteriocin produced by Lactobacillus paracasei HL32. It has been shown to have activity against Porphyromonas sp.

Arthritis: Animal Models of Oral Tolerancea

Four experimental arthritides have been investigated through the study of induced mucosal tolerance for the control of autoimmunity. The first report of the successful control of an autoimmune disease concerned a study from the author’s group of collageninduced arthritis (CIA) in rats in which the prior oral dosing with soluble-type I1 collagen (CII) suppressed the subsequent development of arthritis that would normally have followed from immunization with CII,’ and similar results were published shortly afterwards by Thorbecke’s group on the suppression of CIA in the mouse? These experiments were done against a background of speculation that dietary antigens might influence the initiation or progression of autoimmune diseases.’ However, until that time the speculation had not been matched by experiment. In common with the many other diseases discussed in this volume, CIA has been shown to be modified in many of its aspects by oral dosing with specific antigen (CII in this case); the parallel modification of disease symptoms and immune parameters has underlined the link between the modification of immunity through transmucosal encounter with antigen and the amelioration of inflammatory autoimmune lesions. The history of the subject, of course, established that mucosal immunization had systemic effects distinct from those that ensue from parenteral immunization, and more recent approaches, also described elsewhere in this book, have led to attempts to modify human autoimmune conditions by induced mucosal tolerance. Aside from CIA in the rat and the mouse, it has been shown that adjuvant arthritis (AA) in the rap and Pristane-induced arthritis (PIA) in the mouse5 can be prevented by oral dosing with CII. These diseases have pathologies that bear some comparison to human rheumatoid arthritis (RA), but the extent to which they model RA itself is not so much the question that matters here. Rather, the question is, To what extent does their modification by oral dosing with CII predict what will happen in humans? Here we will review briefly

Systematic comparison of the mono-, dimethyl- and trimethyl 3-hydroxy-4(1H)-pyridones - Attempted optimization of the orally active iron chelator, deferiprone.

A range of close analogues of deferiprone have been synthesised. The group includes mono-, di- and tri-methyl-3-hydroxy-4(1H)-pyridones. These compounds were found to possess similar pFe(3+) values to that of deferiprone, with the exception of the 2.5-dimethylated derivatives. Surprisingly the NH-containing hydroxy-4(1H)-pyridones were found to be marginally more lipophilic than the corresponding N-Me containing analogues. This same group are also metabolised less efficiently by Phase 1 hydroxylating enzymes than the corresponding N-Me analogues. As result of this study, three compounds have been identified for further investigation centred on neutropenia and agranulocytosis.

Functional Characterization of Fluorescent Hepcidin

Hepcidin is a peptide hormone that regulates homeostasis in iron metabolism. It binds to the sole known cellular iron exporter ferroportin (Fpn), triggers its internalization, and thereby modulates the efflux of iron from cells. This functional property has been adopted in this study to assess the bioactivity and potency of a range of novel fluorescent hepcidin analogues. Hepcidin was selectively labeled with 6-carboxyfluorescein (CF) and 6-carboxytetramethylrhodamine (TMR) using Fmoc solid phase peptide chemistry. Internalization of Fpn by hepcidin was assessed by high-content microscopic analysis. Both K18- and M21K-labeled hepcidin with TMR and CF exhibited measurable potency when tested in cultured MDCK and T47D cells expressing human ferroportin. The bioactivity of the labeled hepcidin varies with the type of fluorophore and site of attachment of the fluorophores on the hepcidin molecule.

Solution‐structure of a Peptide Designed to Mimic the C‐terminal Hexapeptide of Endothelin

The peptide: Ac‐cyclo(Cys‐His‐Leu‐Asp‐Cys)‐Ile‐Trp‐OH, has been designed by computer‐aided molecular‐modelling techniques to mimic the proposed α‐helical conformation of the C‐terminal hexapeptide of endothelin.

The evaluation of a novel class of non‐toxic absorption enhancer

The delivery of peptide and protein drugs is problematic and the subject of intense research. Absorption Enhancer’s (AE’s) may be useful for delivery of these types of molecules, but their usage is limited due to toxic effects at the level of the cell membrane, (De Boer et al 1990). This report describes the evaluation of a novel class of peptidebased AE, that facilitates a reversible, dynamic absorption enhancement. A total of 12 have been synthesised so far, designated AEI -AEl2, and are currently the subject of a patent application in view of the promising results obtained so far.