Topo-proteomic in situ analysis of psoriatic plaque under efalizumab treatment.

In a pilot study 6 psoriasis patients were treated over 12 weeks with efalizumab targeting the CD11a subunit of LFA-1. The treatment was well tolerated. Five of these patients proved to be responders with an average decrease in psoriasis area and severity index (PASI) from 21.3 +/- 5.4 (day 0) to 3.9 +/- 0.6 (week 12). The nonresponder was subsequently successfully treated with cyclosporin. Skin biopsies were taken before and after efalizumab treatment and subjected to Multi-Epitope Ligand Cartography (MELC) robot microscopy. A MELC library of 46 antibodies including FITC-labeled efalizumab was chosen focusing upon inflammatory epitopes. Quantification of marker expression was performed using a special adaptation to the needs of skin tissue in terms of pixel events normalized to a standardized horizontal skin width of 100 mum. The before-versus-after comparison for the responders revealed at the 'single epitope level' of MELC analysis a significant decrease (p < 0.05) in epidermal thickness (represented by pan-cytokeratin, CD71, CD138), of the expression of common leukocyte antigen (CD45), T-cell markers (CD2, CD4, CD8, CD45R0), CD11a, efalizumab binding site (EfaBS), and CD58. At the 'EfaBS-centered, double colocation level' a corresponding decrease was observed for CD2, CD3, CD4, CD8, CD11a, CD13, CD26, CD44, CD45, CD45R0, CD54, CD62L, HLA-DR, and TIA-1. MELC analysis at the 'multicombinatorial level' revealed predominant combinatorial molecular phenotype (CMP) motifs, which showed an efalizumab treatment-dependent significant decrease. These CMP motifs were defined as toponomic combinations of lead markers for (i) leukocytes in general (CD45), (ii) T cells (CD2, CD3, CD4, CD45R0, CD45RA), (iii) macrophages (CD68), (iv) cell activation (CD13, CD26, HLA-DR), and (v) cell adhesion (CD11a, EfaBS). Thirty-five of the most relevant 50 CMP motifs were directly related to the T-cell type. A descriptive statistical analysis of the nonresponder before treatment showed a below-responder range degree of expression for CD4, CD8, CD44 (H-CAM), CD56, CD62L, HLA-DQ, and also for these epitopes in colocation with EfaBS. In the nonresponder and before treatment we observed an above-responder range degree of expression for CD54 (ICAM-1) as LFA-1 ligand. In conclusion, the topo-proteomic data provide new diversified insights into the pleiotropic cellular dynamics in psoriatic skin lesions under effective efalizumab treatment. Moreover, the data may be relevant to the future development of possible strategies for individual prediction of efalizumab treatment response or nonresponse.

The CD11a binding site of efalizumab in psoriatic skin tissue as analyzed by Multi-Epitope Ligand Cartography robot technology. Introduction of a novel biological drug-binding biochip assay.

Efalizumab (Raptiva) is an immunomodulating recombinant humanized IgG1 monoclonal antibody that binds to CD11a, the alpha-subunit of leukocyte function antigen-1 (LFA-1). By blocking the binding of LFA-1 to ICAM-1, efalizumab inhibits the adhesion of leukocytes to other cell types and interferes with the migration of T lymphocytes to sites of inflammation (including psoriatic skin plaques). Analysis of the response in patients treated with efalizumab to date shows that distinct groups of responders and nonresponders to the drug exist. It would therefore be of great practical value to be able to predict which patients are most likely to respond to treatment, by identifying key parameters in the mechanism of action of efalizumab. Detailed investigation and detection of multiple epitopes in microcompartments of skin tissue has until recently been restricted by the available technology. However, the newly developed technique of Multi-Epitope Ligand Cartography (MELC) robot technology combines proteomics and biomathematical tools to visualize protein networks at the cellular and subcellular levels in situ, and to decipher cell functions. The MELC technique, which is outlined in this paper, was used to help characterize the binding of efalizumab to affected and unaffected psoriatic skin as compared to normal control skin under ex vivomodel conditions. Efalizumab was labeled with fluorescein isothiocyanate and integrated into a MELC library of more than 40 antibodies. These antibodies were selected for their potential to detect epitopes which may be indicative of (a) various cell types, (b) structural components of the extracellular matrix, or (c) the processes of cell proliferation, activation and adhesion. Efalizumab bound to CD11a in affected psoriatic skin by a factor 15x and 32x higher than in unaffected psoriatic skin and normal control skin, respectively. CD11a and the efalizumab binding site were primarily expressed in the extravascular dermis, whereas CD54 (ICAM-1) as its ligand was most prevalent in the dermal vessels. T lymphocytes (for which the markers were CD3, CD8, CD4, and CD45R0) were the major cellular targets of efalizumab. In contrast, NK cells were only a minor target of efalizumab. Our study demonstrated that efalizumab represents a treatment for psoriasis that primarily targets memory CD4+ and CD8+ T cells and has a high specificity for psoriatic disease activity. Moreover, we hereby introduce the novel principle of a biological drug-binding biochip assay being especially useful for the future monitoring of psoriatic skin lesions under efalizumab treatment conditions.

Interferon-gamma-dependent in vitro model for the putative keratin 17 autoimmune loop in psoriasis: exploration of pharmaco- and gene-therapeutic effects.

In 1999, A.S. Gudmundsdottir et al. have envisaged an epitope on keratin 17 (K17) as a putative psoriasis major autoantigen recognized by T cells. In a HaCaT keratinocyte model, we now demonstrate that IFN-gamma and to a less extent also TNF-alpha and TGF-alpha are able to induce K17 protein expression, in contrast to IL-1alpha, IL-1beta, IL-6, IL-8 and IL-18. This supports our hypothesis of an existing proinflammatory cytokine/K17 autoimmune loop as a presumptive positive feedback mechanism driving psoriasis etiopathogenesis. K17 overexpression was now found to also coincide with suppression of keratinocyte proliferation, e.g. induced by NF-kappa B inhibitors (Bay 11-7082 and Bay 11-7085), and thereby correlated hyperapoptosis to be encountered in psoriatic epidermis. Acitretin as an established antipsoriatic drug and the tyrosine kinase inhibitor imatinib decreased, whereas hydrocortisone as well as dexamethasone increased the IFN-gamma-induced K17 overexpression. The latter might be another mechanism explaining the well-known rebound phenomena after abrupt withdrawal of corticosteroids in psoriasis treatment. Finally, we defined a K17-directed and effective antisense oligodesoxynucleotide which may hold promise for future gene-therapeutic approaches in psoriasis.

Prognostic significance of detecting micrometastases by tyrosinase RT/PCR in sentinel lymph node biopsies: lessons from 322 consecutive melanoma patients.

This prospective study was performed to determine the prognostic value of tyrosinase mRNA detection in sentinel lymph nodes (SLN) of melanoma patients. About 847 SLNs from 322 consecutive patients were assessed by histopathology and immunohistochemistry as well as tyrosinase-reverse transcriptase-polymerase chain reaction (RT/PCR) for the presence of micrometastases. The results were correlated with the prognostic parameters employing a multivariate analysis after a median follow-up of 37 months. Histopathological analysis revealed metastases in 34/322 patients (10.6%). Among the 288 patients with histopathologically negative SLN, tyrosinase-mRNA was detected in 39 patients. A relapse of the tumour occurred in 44.1% of the patients with histopathologically positive SLN, in 25.6% with histopathologically negative, but tyrosinase-RT/PCR-positive SLN, and 8.0% with "double-negative" SLN. A multivariate analysis identified tumour thickness, the histopathological SLN status, and the ulceration of the primary tumour as independent prognostic factors. Thus, by assessing tyrosinase mRNA in the SLN of melanoma patients, we identified a subgroup with histopathologically negative, but Tyr-RT-PCR-positive SLN who have a high risk of disease relapse.

UV erythema reducing capacity of mizolastine compared to acetylsalicylic acid or both combined in comparison to indomethacin.

UV light exerts hazardous effects such as induction of skin cancer and premature skin aging. In this study we evaluated an assumptive anti-inflammatory effect of the nonsedative histamine H1-receptor antagonist, mizolastine, on UV-induced acute sunburn reaction. Therefore, a clinical, randomized, double-blind, four-arm, crossover study was conducted in healthy young female volunteers (skin type II) comparing the UV sensitivity under mizolastine, acetyl-salicylic acid (ASA), indomethacin or a mizolastine/ASA combination. Moreover, HaCaT keratinocytes were incubated with mizolastine under various UV treatment modalities in vitro to study its effect on the release of inflammatory cytokines, i.e. interleukin (IL)-1 alpha, IL-6 and tumor necrosis factor alpha (TNF-alpha). All three drugs were effective in suppressing the UVB-, UVA- and combined UVA/UVB-erythema. However, the strongest effects were observed using the combined treatment with both 250 mg ASA and 10 mg mizolastine. An inhibitory effect in vitro of 10 nM mizolastine upon UV-induced cytokine release from HaCaT keratinocytes was observed for IL-1 alpha at 24 h after 10 J/cm2 UVA1, for IL-6 at 48 h after 10 J/cm2 UVA1 and 30 mJ/cm2 UVB, and also for TNF-alpha at 4 h after 10 J/cm2 UVA, 10 J/cm2 UVA1 and 30 mJ/cm2 UVB, respectively. The combination of mizolastine and ASA can be strongly recommended as a protective measure against UV erythema development with a lower unwanted side effect profile than that of the hitherto treatment modality, i.e. indomethacin.

Dithranol and dimethylfumarate suppress the interferon-gamma-induced up-regulation of cytokeratin 17 as a putative psoriasis autoantigen in vitro.

In psoriasis an etiopathogenetic vicious circle has been hypothesized in which disease manifestation is triggered by skin-specific autoantigen structures. Autoreactive T cells are supposed to mediate inflammation and hyperproliferation in the epidermopapillary compartment, positively feeding back the expression and accessibility of decisive antigen structures. Recently an epitope within cytokeratin 17 (K17) has been described as such a putative psoriasis autoantigen, which is moreover known to be up-regulated under the influence of proinflammatory interferon-gamma (IFN-gamma), which is abundantly detected in psoriatic plaques. The present study proposes an in vitro model for this presumptive IFN-gamma/K17 autoimmune loop, i.e. the incubation of hyperproliferative human HaCaT keratinocytes with 25 U IFN-gamma/ml for 72 h. This treatment led to a significant up-regulation of K17 protein expression (> or =300%) measured by flow immunocytometry as compared to the untreated control (100%, p < or = 0.05). Preincubation with a subcytotoxic and antiproliferative dithranol concentration as low as 0.3 microM for 2 h prior to the IFN-gamma exposure resulted in a K17 expression that was significantly lower than the IFN-gamma-induced K17 expression reference level. The IFN-gamma-induced K17 expression was also significantly lowered by coincubation with a subcytotoxic and nonantiproliferative concentration of 3 microM dimethylfumarate. The data indicate for dithranol and dimethylfumarate that a part of their antipsoriatic mode of action may be related to a direct down-regulation of putative psoriasis autoantigen structures.

Suprabasal overexpression of the hsRPB7 gene in psoriatic epidermis as identified by a reverse transcriptase-polymerase chain reaction differential display model comparing psoriasis plaque tissue with peritonsillar mucosa.

In psoriasis an etiopathogenetic vicious circle is nowadays hypothesized that the disease is triggered by skin-specific autoantigen structures, the expression and accessibility of which are positively correlated with the intensity of the hyperproliferation and inflammation in the epidermopapillary compartment driven by autoreactive T cells. Despite the close microanatomical relation between skin and mucosa, clinicians have always been intrigued by the observation that psoriatic affection of the mucosa, if at all existing, is only seen as very rare events in the lips and tongue sparing buccopharyngeal sites. This prompted us to establish an experimental model system comparing psoriatic-involved skin and peritonsillar mucosa from tonsillectomies by a reverse transcriptase-polymerase chain reaction/differential display strategy. Among more than 60 cDNA species to be displayed in psoriasis, but missing in peritonsillar mucosa, one species was identified as coding for the RNA polymerase IIA seventh subunit (hsRPB7 gene) as a most critical factor for DNA to RNA transcription. Immunohistochemistry showed a hitherto unknown, distinctive pattern of hsRPB7 expression that was 1) tissue type-dependent with a surplus in skin keratinocytes and a near absence in peritonsillar mucosa, 2) tightly regulated by the keratinocyte differentiation process with a sharp suprabasal up-regulation in contrast to a basal down-regulation, and 3) substantially augmented in psoriatic-involved skin as compared to normal and psoriatic uninvolved skin. Keratinocytes of actinic keratoses also showed a strong hsRPB7 expression that however did not strictly spare the basal cell layer presumably reflecting the disturbed intraepidermal stratification because of the premalignant status of these precancerous lesions.

Influence of nitric oxide synthase activity on amino acid concentration in the quinolinate lesioned rat striatum: a microdialysis and histochemical study.

The influence of nitric oxide synthase (NOS) activity on the KCl-evoked amino acid concentrations was investigated by in vivo microdialysis in the striatum in a rat model of excitotoxic lesion. Basal microdialysate levels of amino acids decreased during the quinolinic acid-induced neurodegeneration process, except for glutamine that increased initially and returned to control values 30 days after quinolinic acid exposure. KCl-evoked increase of extracellular amino acid concentration was reduced due to NOS activity in the striatum of both controls and lesioned animals, except for 120 days after quinolinic acid injection. These changes of amino acid concentrations in microdialysates correlated with the known biochemistry of the consecutive domineered cell types during the lesion process as revealed by histochemistry for NOS, NADPH-diaphorase, GFAP and isolectin B4. The present data provide direct evidence that NOS activity can modulate extracellular amino acid concentrations in the striatum not only under physiological conditions, but also during a pharmacologically induced lesion process and, thus, suggests that nitric oxide affects neurodegeneration via this pathway.

Optimized visualization and PCR reamplification of differentially displayed cDNA bands detected by silver staining in polyacrylamide gels as established in the model of dithranol-treated keratinocytes.

The differential display of cDNA species defined by a combination of so-called anchored and arbitrary primers has been acknowledged as a powerful complex strategy to identify differences in gene expression, and depends in its original version, inaugurated by P. Liang and A. B. Pardee in 1992, on the use of radioactive-labelled nucleotides. As a non-radioactive methodological alternative, we established the use of polyesterfilm-backed 10% polyacrylamide gels for horizontal differential-display electrophoresis under non-denaturing conditions, with subsequent detection of cDNA bands by an optimized, semi-automated silver staining omitting any fixation step. Polyacrylamide gel slices carrying the silvered cDNA species of interest were cut out, chopped, squashed and incubated in an ammonium acetate/EDTA solution at 37 degrees C overnight under vigorous shaking. This procedure resulted in a 70% average success rate for subsequent PCR reamplification with regard to the number of cDNAs harvested from the differential-display gel. Novel sequence data of three cDNA clones are communicated, which under these methodological conditions were selected to be up- or down-regulated, respectively, by antipsoriatic dithranol in cultured HaCaT keratinocytes.

Potassium-stimulated taurine release and nitric oxide synthase activity during quinolinic acid lesion of the rat striatum.

The microdialysis technique was used to study the effect of nitric oxide synthase (NOS) activity on taurine release. Taurine release was characterized in rat striatum that was excitotoxically lesioned compared to normal conditions. The basal taurine level of the dialysate decreased during quinolinate (QUIN) lesion in parallel to the cell degeneration process. The K+-stimulated taurine concentration also decreased during QUIN-lesion, but to an extent that was different from that of basal values. K+-stimulated taurine levels were further markedly lowered by coapplication of the NOS inhibitor L-NAME in control and in lesioned animals up to 30 days after QUIN-injection. Postdegenerative tissue did not show any NOS-dependency in K+-induced taurine release. We conclude that a substantial part of K+-induced taurine release depends on NOS-activity both in normal brain tissue and in excitotoxically induced neurodegeneration. The main source of K+-induced taurine release in control rats are neurons but in lesioned animals are activated astroglial cells.

NADPH-diaphorase/nitric oxide synthase containing neurons in normal and Parkinson's disease putamen.

Nitric oxide (NO) is thought to be involved in neurodegenerative processes. Concerning Parkinson's disease (PD) it remains to be elucidated, if NO contributes to pathological alterations in the striatum. The present study evaluates the post-mortem putamen of PD patients and control subjects for distribution patterns of NO-synthase containing neurons, using the NADPH-diaphorase technique. The ratio of positively stained neurons and the total number of cells (control: 1,120 +/- 69 per mm2, n = 5; PD: 575 +/- 164mm2, n = 5) shows striking differences between controls and PD patients. Our findings give reason to conclude that NADPH-diaphorase positive structures may have pathogenetic importance in degenerative processes in PD putamen.