Healing process of venous ulcers: the role of microcirculation.

In order to describe adequately the process of healing in the intermediate degrees, we investigated microcirculatory changes in the venous ulcers at well-defined stages of wound repair. We investigated dynamic changes in microcirculation during the healing process of venous ulcers. Ten venous ulcers were investigated in three consecutive clinical stages of wound healing: non granulation tissue (NGTA), GTA and scar. Subpapillary microcirculation was measured by laser Doppler perfusion (LDP) imaging and expressed using LDP values in arbitrary units. Nutritive perfusion by capillary microscopy and expressed as capillary density (CD) - the number of capillaries per square millimetre. Before the development of GTA the LDP was low (median 1·35; lower-upper quartiles 0·71-1·83) accompanied with zero CD in all but one patient who had a density of 1. With the first appearance of GTA in the same area, the LDP was improved (2·22; 1·12-2·33; P = 0·0024) when compared with NGTA, in combination with a significant increase in CD (1·75; 0-3; P = 0·0054). In scar, the LDP was similar to that in the NGTA (1·03; 0·77-1·83; P = 0·278), combined with the highest CD (5·75; 4·5-8) in comparison with the previous stages of the area (for both pairs, P < 0·0001). Venous ulcers are caused by poor nutritive and subpapillary perfusion. Subpapillary perfusion plays a major role in the formation of GTA. In a scar, the increased nutritive perfusion is sufficient to cover the blood supply and keep skin viable while subpapillary perfusion is low.

Expression of FcRn, the MHC class I-related receptor for IgG, in human keratinocytes.

The neonatal Fc receptor (FcRn) for IgG has been shown to be responsible for IgG transport and to be involved in IgG catabolism. In this study, we show expression of FcRn in normal human epidermal keratinocytes. By RT-PCR, we demonstrate the FcRn alpha-chain mRNA obtained from cultured keratinocytes creating a 457 bp product as confirmed by sequence analysis. Northern blot analysis shows a 1.5 kb transcript. Real-time PCR reveals consistent expression of FcRn alpha-chain mRNA in human keratinocytes from different donors. Anti-FcRn alpha2-extracellular domain and anti-FcRn cytoplasmic tail antibody (Ab) directed against defined antigenic targets were generated and used for immunoblotting and immunoprecipitation revealing protein expression of the 46 kDa FcRn alpha-chain. By immunofluorescence microscopy, we find granular-vesicular staining for FcRn alpha-chain in keratinocytes. Fluorescence-activated cell sorting analysis gives predominantly an intracellular distribution of FcRn in keratinocytes. Biochemically, we demonstrate Fc-dependent binding of human IgG at acidic pH. In normal human epidermis, we find a cytoplasmic vesicular staining of predominantly basal and suprabasal keratinocytes. In summary, we demonstrate expression of a functional FcRn in normal human epidermal keratinocytes. These findings further emphasize the role of keratinocytes as immunomodulating cells in inflammatory and immunologic processes of the skin.

RasGAP-like protein IQGAP1 is expressed by human keratinocytes and recognized by autoantibodies in association with bullous skin disease.

Autoantibodies in patients with autoimmune bullous skin diseases, such as pemphigus or bullous pemphigoid are of diagnostic value and might play a part in the pathogenic scenario. In this study we present five patients with erythematous plaques, subepidermal blister formation of the skin, and the presence of circulating autoantibodies directed against a so far unrecognized 190 kDa antigen in human keratinocytes. Amino acid sequence analysis identified the protein as IQGAP1, a recently described human Ras GTPase-activating-like protein suspected to act as an effector molecule for Cdc42 and Rac1, members of the Rho small GTPase family and to play a key part in regulating E-cadherin-mediated cell adhesion. The protein is selectively recognized by a monoclonal anti-IQGAP1 antibody on western blots and immunoprecipitates from keratinocyte extracts. Indirect immunofluorescence locates IQGAP1 within individual keratinocytes in a cytoplasmic pattern and along the cell periphery at adhesive sites. Our results demonstrate IQGAP1, a newly described multifunctional protein, to be constitutively expressed in human keratinocytes where it may contribute to the integrity of the epidermal layer. Furthermore, we found autoantibodies reacting with IQGAP1 in patients with bullous skin eruptions most apparently belonging to the spectrum of bullous pemphigoid.

RPE65 of retinal pigment epithelium, a putative receptor molecule for plasma retinol-binding protein, is expressed in human keratinocytes.

Retinoids are important modulators for cell growth and differentiation of normal skin. In plasma, retinol is transported coupled to plasma retinol-binding protein. In this study, we investigated gene and protein expression of RPE65, a putative receptor for plasma retinol-binding protein in human epidermal keratinocytes. We performed real-time PCR analysis to evaluate expression of RPE65 mRNA in proliferating and differentiating keratinocytes. Immunoblotting with anti-RPE65 antibody shows distinct reactivity to a 61-kDa protein. Indirect immunofluorescence on normal human epidermis reveals cell surface labeling of keratinocytes. Laser scan microscopy exhibits colocalization of plasma retinol-binding protein and RPE65 on cultured keratinocytes. Internalization experiments with [3H]retinoic acid-retinol-binding protein complex in the presence and absence of excess of retinol-binding protein indicates receptor-dependent uptake of retinoids. We further show isolation of RPE65 protein by affinity chromatography from lysates of keratinocytes using a retinol-binding protein-matrix gel column. In summary, we demonstrate mRNA and protein expression of RPE65 in epidermal keratinocytes. Colocalization of plasma retinol-binding protein with RPE65 and affinity binding suggest a direct interaction of RPE65 with plasma retinol-binding protein in cultured human keratinocytes that might be involved in retinoid uptake of keratinocytes.

FcgammaRIII expression on cultured human keratinocytes and upregulation by interferon-gamma.

Keratinocytes of human epidermis are actively involved in inflammatory and autoimmune reactions of the skin and interact with resident or infiltrating immunocompetent cells via cytokines, chemokines, and intercellular adhesion mechanisms. Most immunocompetent cells have been reported to express Fcgamma receptors (FcgammaR), which are important for immunoregulatory functions. In this study we investigate FcgammaRIII expression on cultured human keratinocytes and upregulation by interferon-gamma. By real-time polymerase chain reaction, we show basal mRNA expression of both subclasses FcgammaRIIIA and FcgammaRIIIB, but after interferon-gamma treatment mRNA of FcgammaRIIIA and FcgammaRIIIB is increased 4.4 and 6.5 times, respectively. FcgammaRIII protein expression and its increase after interferon-gamma treatment were shown on cultured human keratinocytes by indirect immunofluorescence. In immunoblotting experiments, a bonified anti-CD16 antibody revealed reactivity to a polypeptide of 50-65 kDa on lysates of treated and untreated keratinocytes. In summary, we demonstrate expression of mRNA specific for the FcgammaRIIIA and FcgammaRIIIB subclasses and their upregulation by interferon-gamma on human keratinocytes in vitro, and confirm FcgammaRIII protein expression by indirect immunofluorescence and by immunoblotting experiments.

Unusual clinical manifestation of linear IgA dermatosis: a report of two cases.

Linear IgA dermatosis is a rare autoimmune bullous skin disease with subepidermal blister formation and linear IgA deposits along the basement membrane zone. We describe two female patients showing erythematous annular plaques with scaling at the margin, strictly localized to the palms in one patient, and also found on the soles and buttocks in the second patient. Histology showed numerous neutrophils in the dermis with an admixture of eosinophils, some subepidermal clefting, and occasional papillary microabscesses. Direct immunofluorescence and immunoelectron microscopy revealed in vivo IgA deposition along the basement membrane zone. One patient cleared after treatment with dapsone. The second patient did not respond to dapsone alone and various immunosuppressive treatment regimens. Considerable improvement was achieved with intravenous immunoglobulin therapy combined with corticosteroid and dapsone.

Intravenous anti TNF-alpha antibody therapy leads to elevated triglyceride and reduced HDL-cholesterol levels in patients with rheumatoid and psoriatic arthritis.

BACKGROUND & AIMS: We investigated the effect of Infliximab, an anti TNF-alpha antibody, on plasma lipids and lipoproteins in patients with rheumatoid arthritis and psoriatic arthritis. METHODS: Five male and 10 female patients with a mean age of 56.7 years were included in this study. Seven of the patients were diagnosed with rheumatoid arthritis and 8 patients with psoriatic arthritis. All patients received infusions of 3 mg/kg Infliximab (at week 0, 2 and 6). Lipids, lipoproteins and standard clinical parameters were assessed at baseline (0 week), after 2 weeks, and in 4 patients after 6 weeks. RESULTS: There was a significant increase in triglyceride levels during treatment with Infliximab (112 +/- 48 versus 133 +/- 53 mg/dl, p < 0.01). In contrast, HDL-cholesterol levels were significantly lowered (56 +/- 12 versus 50 +/- 13 mg/dl, p < 0.006) by the treatment. There was no significant difference in total cholesterol (209 +/- 25 versus 205 +/- 36 mg/dl) or in LDL-cholesterol (131 +/- 24 versus 118 +/- 43 mg/dl) before and after treatment. Similarly, lipoprotein(a) levels did not alter during treatment (median: 1.1 versus 1.4 mg/dl). CONCLUSION: This study shows that intravenous Inflixmab therapy leads to changes in plasma lipid and lipoprotein levels in patients with rheumatoid and psoriatic arthritis and may result in a more atherogenic lipid and lipoprotein profile. Although larger patient numbers need to be studied to confirm our findings, these results suggest that lipid levels should be checked and monitored in patients receiving infliximab therapy, particularly in patients with vascular disease.

Treatment of psoriatic arthritis and psoriasis vulgaris with the tumor necrosis factor inhibitor infliximab.

OBJECTIVE: The aim was to evaluate the efficacy and safety of multiple infusions with achimeric, anti-tumor necrosis factor (TNF)alpha monoclonal antibody (infliximab) in patients with psoriatic arthritis (PsA) and psoriasis vulgaris. METHODS: Over 22 weeks, nine patients with both active psoriasis and PsA received five infusions of 3 mg/kg infliximab. The endpoints included changes in the swollen and tender joints counts, American College of Rheumatology (ACR) preliminary criteria for improvement response rates 20, 50, and 70, and improvement in the psoriasis area and severity index (PASI). RESULTS: The swollen count (SJC) and tender joint count (TJC) fell from means of 5.33+/-2.22 and 17.80+/-4.21 to 1.44+/-1.09 and 9.77+/-0.92, respectively, by week 2 ( P=0.02, P=0.02). This benefit was sustained through week 22 (2.00+/-1.12/7.77+/-3.68, P=0.05/ P=0.002). The ACR 20/50/70 response was achieved in 89%/56%/22% of cases. The mean PASI score improved from 19.04+/-5.41 to 4.91+/-2.51 ( P=0.002). CONCLUSION: Multiple infusions of infliximab were effective and well tolerated in patients with active psoriasis and PsA.

Internalization via plasmalemmal vesicles: a route for antidesmoplakin autoantibodies into cultured human keratinocytes.

Recently, autoantibodies to desmoplakin I and II have been identified in a subset of patients with a severe form of erythema multiforme. These autoantibodies recognize a specific peptide sequence at the carboxy terminal domain of desmoplakin I and II responsible for interaction with keratin filaments. Desmoplakins are major constitutive proteins of the inner dense desmosomal plaque of keratinocytes and are entirely localized within the cells. With the assumption of pathogenecity for circulating autoantibodies, the question arose how antidesmoplakin autoantibodies enter keratinocytes. Utilizing immunhistochemical procedures for cell motility and time kinetic studies at the light- and electron-microscopic level, we found that autoantibodies are bound at the cell surface of cultured human keratinocytes, internalized via plasmalemmal vesicles, and are found consecutively within tubulovesicular structures inside the cells. At the same time, a fraction of antibodies can be detected at the inner dense desmosomal plaques. Immunogold labeling reveals internalization of autoantibodies in small non-coated plasmalemmal vesicles positive for caveolin. These observations indicate that vesicular transport may represent a relevant biological mechanism for antidesmoplakin autoantibodies to enter keratinocytes and allow access to their corresponding antigenic target in vivo.