Gulnar M. Shivji

The role of cis-urocanic acid in UVB-induced suppression of contact hypersensitivity

Ultraviolet light B (UVB) is well recognized to suppress the contact hypersensitivity (CHS) response and it has been postulated that cis-urocanic acid (UCA) is a mediator of the immunosuppression. This study was designed to examine the effect of UCA on CHS and to clarify its role in UVB-induced immunosuppression in C57BL/6 mice. Intradermal injection of 0.5-50 micrograms cis-UCA into the ear 2 h before DNFB sensitization resulted in a 60-70% reduction of CHS assessed by ear swelling, whereas trans-UCA did not have a significant effect on CHS except at a high dose (50 micrograms) which showed a 20-40% suppression. Intraperitoneal injection of anti-cis-UCA antibody before administration of cis-UCA abrogated the suppression. To examine the effect of UCA on UVB-induced immunosuppression, some mice were pre-treated with anti-cis-UCA antibody and then exposed to UVB (960 J/m2). After 3 days the mice were sensitized either on the irradiated abdominal skin or on the unirradiated dorsal surface of the right ear followed by the challenge on the left ear. The CHS response was significantly suppressed in UVB-irradiated mice both locally (abdominal sensitization, suppression was 45%, P < 0.001) and systemically (ear sensitization, suppression was 53%, P < 0.0025). The CHS response was partially restored in both abdominal sensitized mice and ear sensitized mice by pre-treatment with anti-cis-UCA antibody. These results confirmed the immunosuppressive effects of cis-UCA on CHS and suggest that cis-UCA plays a role in UVB-induced local and systemic immunosuppression.

Synthetic Peptide Immunogens Elicit Polyclonal and Monoclonal Antibodies Specific for Linear Epitopes in the D Motifs of Staphylococcus aureus Fibronectin-Binding Protein, Which Are Composed of Amino Acids That Are Essential for Fibronectin Binding

ABSTRACT A fibronectin (Fn)-binding adhesin of Staphylococcus aureus contains three tandem 37- or 38-amino-acid motifs (D1, D2, and D3), which function to bind Fn. Plasma from patients with S. aureus infections contain antibodies that preferentially recognize ligand induced binding sites in the D motifs and do not inhibit Fn binding (F. Casolini, L. Visai, D. Joh, P. G. Conaldi, A. Toniolo, M. Höök, and P. Speziale, Infect. Immun. 66:5433–5442, 1998). To eliminate the influence of Fn binding on antibody development, we used synthetic peptide immunogens D121–34 and D320–33, which each contain a conserved pattern of amino acids that is essential for Fn binding but which cannot bind Fn without N- or C-terminal extensions. The D320–33 immunogen promoted the production of polyclonal antibodies that were 10-fold more effective as inhibitors of Fn-binding to the D3 motif than antibodies obtained by immunizing with an extended peptide D316–36, which exhibits functional Fn binding. The D320–33 immunogen also facilitated the production of a monoclonal antibody, 9C3, which was highly specific for the epitope SVDFEED, and abolished Fn binding by the D3 motif. When mixed with polyclonal anti-D121–34 immunoglobulin G, 70% inhibition of Fn binding to the three tandem D motifs was achieved compared to no more than 30% inhibition with either antibody preparation alone. Therefore, by immunizing with short synthetic peptides that are unable to bind Fn, we have effectively stimulated the production of antibodies specific for epitopes comprised of amino acids that are essential for Fn binding. Although these epitopes occur within a conserved pattern of amino acids that is required for Fn binding, the antibodies recognized specific linear epitope sequences and not a conserved structure common to all repeated motifs.

The role of CD4 molecules in the induction phase of contact hypersensitivity cytokine profiles in the skin and lymph nodes

We have previously demonstrated that CD4 gene‐targeted mutant mice (CD4− mice) demonstrate hyporesposiveness in contact hypersensitivity (CHS) suggesting that CD4 molecules are required for optimal induction of CHS. In the present study, we wished to examine the mechanisms of this hyporesponsiveness, in particular, we examined whether cytokines were altered in the skin and lymph nodes of CD4− mice following exposure to the contact allergen dinitrofluorobenzene (DNFB). Cytokine mRNA in the ear skin and draining lymph nodes (DLN) were examined by reverse transcription–polymerase chain reacion (RT–PCR) at various times after sensitization. Skin cytokine patterns revealed that in normal mice, interleukin (IL)‐2, interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α mRNA levels increased at 12 hr sensitization, whereas these cytokines were below the level of detection in CD4− mice. In the DLN of normal mice following the hapten application, sequential upregulation of cytokine mRNA including IL‐1α, IL‐1β, IL‐2, IL‐4, IL‐10, IFN‐γ and TNF‐α was found. No change was seen for IL‐1α, IL‐1β, IL‐10 and TNF‐α and IL‐2, IL‐4 and IFN‐γ mRNA levels were below the level of detection in DLN from CD4– mice following the hapten application. However, IL‐1β, IL‐2 and TNF‐α mRNA levels of lymph node cells from CD4− mice could be upregulated by phorbol myristate acetate in vitro. Flow cytometry study has revealed that the number of Langerhans’ cells (LC) in DLN of CD4− mice was similar to that of normal mice, thus, inferring that the alterations of cytokine milieu in the ear skin did not have a significant effect on LC migration to DLN. These results suggest that CD4 molecules are crucial for the induction of certain cytokines in the skin and in inducing sequential cytokine signals in DLN required for optimal development of CHS, but that these changes in cytokines do not effect LC migration.

Effects of contact sensitizers neomycin sulfate, benzocaine and 2,4-dinitrobenzene 1-sulfonate, sodium salt on viability, membrane integrity and IL-1α mRNA expression of cultured normal human keratinocytes

The toxic effect of three potential contact sensitization chemicals [the aminoglycosidic antibiotic neomycin sulfate, the local anaesthetic benzocaine and the primary sensitizer 2,4-dinitrobenzene 1-sulfonate, sodium salt (DNBS)], on cultured human keratinocytes was examined. The three chemicals were compared with respect to their cytotoxic potential (determined by crystal violet staining assay), their membrane disruptive potential ([3H]arachidonic acid release assay), and their effects on interleukin 1 alpha (IL-1 alpha) mRNA expression [reverse transcription-polymerase chain reaction (RT-PCR)]. At the concentrations used, neomycin sulfate (0.004-0.32%) and benzocaine (0.0165-0.165%) did not show relevant cytotoxicity or membrane perturbation. On the other hand, DNBS (0.001-1%) caused a significant dose-dependent cytotoxic response at concentrations higher than 0.1%, while the [3H]arachidonic acid release assay indicated absence of membrane perturbation activity in all the range of DNBS concentrations examined. The effects of the three sensitizers on IL-1 alpha mRNA expression were varied; neomycin sulfate caused a dose-dependent induction of IL-1 alpha mRNA, benzocaine did not significantly affect its signal, and DNBS suppressed IL-1 alpha gene expression.

Cutaneous Toxicity of Surfactants in Normal Human Keratinocytes Assessed by Cytotoxicity, Arachidonic Acid Release, and Regulation of Interleukin-lα mRNA

Development and validation of in vitro methods for cutaneous irritation testing is aimed at establishing simple and reliable assays that eliminate the need for animals in toxicity testing. It is therefore important to identify in vitro end points that are predictive of in vivo toxicity. Identification of proinflamma-tory mediators and cytokines in keratinocytes may represent an early event in cutaneous inflammation. Three end points were evaluated: cytotoxicity [(crystal violet staining (CVS)], the release of [3H]arachidonic acid (AAR), and the regulation of the proinflammatory cytokine interleukin-1α (IL-lα) message in keratinocytes as a potential in vitro assay for surfactants. Cultured normal human epidermal keratinocytes were treated with various concentrations of three surfactants (SDS, Triton X-100, and Tween 20) of different in vivo potencies. The CVS50 1 h (surfactant concentrations yielding 50% viability) and AAR50 (surfactant concentrations causing 50% release of labelled arachidonic acid), both...