Lesional Skin Sections Research Articles

Spatial transcriptomics landscape of lesions from non-communicable inflammatory skin diseases

Abundant heterogeneous immune cells infiltrate lesions in chronic inflammatory diseases and characterization of these cells is needed to distinguish disease-promoting from bystander immune cells. Here, we investigate the landscape of non-communicable inflammatory skin diseases (ncISD) by spatial transcriptomics resulting in a large repository of 62,000 spatially defined human cutaneous transcriptomes from 31 patients. Despite the expected immune cell infiltration, we observe rather low numbers of pathogenic disease promoting cytokine transcripts (IFNG, IL13 and IL17A), i.e. >125 times less compared to the mean expression of all other genes over lesional skin sections. Nevertheless, cytokine expression is limited to lesional skin and presented in a disease-specific pattern. Leveraging a density-based spatial clustering method, we identify specific responder gene signatures in direct proximity of cytokines, and confirm that detected cytokine transcripts initiate amplification cascades of up to thousands of specific responder transcripts forming localized epidermal clusters. Thus, within the abundant and heterogeneous infiltrates of ncISD, only a low number of cytokine transcripts and their translated proteins promote disease by initiating an inflammatory amplification cascade in their local microenvironment.

A novel histopathological scoring system to distinguish urticarial vasculitis from chronic spontaneous urticaria.

BackgroundUrticarial vasculitis (UV) is defined by long‐lasting urticarial lesions combined with the histopathologic findings of leukocytoclastic vasculitis. As one of the major unmet needs in UV, diagnostic criteria are rather vague and not standardized. Moreover, there seems to be considerable overlap with chronic spontaneous urticaria (CSU), particularly for the normocomplementemic variant of UV. Therefore, this study aimed to develop a diagnostic scoring system that improves the histopathologic discrimination between UV and CSU.MethodsLesional skin sections of patients with clinical and histopathologic diagnosis of UV (n = 46) and CSU (n = 51) were analyzed (blinded to the diagnosis) for the following pre‐defined criteria: presence of leukocytoclasia, erythrocyte extravasation, fibrin deposits, endothelial cell swelling, ectatic vessels, blurred vessel borders, dermal edema, intravascular neutrophil, and eosinophil numbers and numbers of dermal neutrophils, macrophages and mast cells.ResultsThe greatest differences between UV and CSU samples were observed for leukocytoclasia (present in 76% of UV vs. 3.9% of CSU samples; p < 0.0001), erythrocyte extravasation (present in 41.3% of UV vs. 2.0% of CSU samples; p < 0.0001), and fibrin deposits (present in 27.9% of UV vessels vs. 9.7% of CSU vessels; p < 0.0001). Based on these findings, we developed a diagnostic score, the urticarial vasculitis score (UVS), which correctly assigned 37 of 46 cases of UV and 49 of 51 cases of CSU to the previously established diagnosis.ConclusionOur results suggest that the UVS, a combined quantitative assessment of the three criteria leukocytoclasia, fibrin deposits and extravasated erythrocytes, distinguishes UV from CSU in skin histopathology. The UVS, if validated in larger patient samples, may help to improve the diagnostic approach to UV.

Increased systemic and epidermal levels of IL-17A and IL-1β promotes progression of non-segmental vitiligo

BackgroundNon-segmental vitiligo (NSV) results from autoimmune destruction of melanocytes. The altered levels of various cytokines have been proposed in the pathogenesis of vitiligo. However, the exact immune mechanisms have not yet been fully elucidated. ObjectivesTo investigate the role of epidermal and systemic cytokines in active and stable NSV patients. MethodsSerum levels of inflammatory cytokines were checked in 42 active and 30 stable NSV patients with 30 controls. The lesional, perilesional and normal skin sections were subjected to H&E staining. The mRNA expression of inflammatory cytokines and their respective receptors were assessed by quantitative PCR in lesional skin of both active and stable NSV skin. The MITF and IL-17A were immunolocalized in lesional, perilesional and normal skin tissue. ResultsSignificant increase in the expression of inflammatory cytokines, IL-17A, IL-1β and TGF-β was observed in active patients, whereas no change was observed in stable patients. A marked reduction in epidermal thickness was observed in lesional skin sections. Significant increase in IL-17A and significant decrease in microphthalmia associated transcription factor (MITF) expression was observed in lesional and perilesional skin sections. Moreover, qPCR analysis showed significant alterations in the mRNA levels of IL-17A, IL-1β, IFN-γ, TGF-β and their respective receptors in active and stable vitiligo patient samples. ConclusionIncreased levels of IL-17A and IL-1β cytokines and decreased expression of MITF suggested a possible role of these cytokines in dysregulation of melanocytic activity in the lesional skin and hence might be responsible for the progression of active vitiligo.

Profiles of tumor-associated macrophages in the lesional skin of mycosis fungoides at different tumor stages

Mycosis fungoides (MF) starts as an indolent disease that progresses from a patch stage to confluent plaques, and finally develops skin tumors and shows visceral involvement. Recently, several reports suggested that immunosuppressive tumor-associated M2 macrophages (TAMs) and tolerogenic dendritic cells (DCs), together with regulatory T cells (Tregs), play roles in maintaining the tumor microenvironment, including MF. The purpose of this study was to elucidate the involvement of immunosuppressive cells in the lesional skin of different stages (10 cases of patchy, 10 cases of plaque and 6 cases of tumor stages) of MF. We immunohistologically examined for the presence of CD163+ CD206+ M2 macrophages as well as their related chemokines in fixed sections of lesional skin from MF. CD163+ CD206+ M2 macrophages were prominent in all stages of MF. As previous reports suggested, the dense infiltration of CD163+M2 macrophages into the lesional skin of MF suggested a contribution of M2 macrophages to the prognosis of MF. To further investigate the functional molecules of TAMs and dendritic cells (DCs), we employed immunohistochemical staining for PD-L1 as well as CD205. Interestingly, while PD-L1+ cells were mainly detected around T cell areas in all stages, CD205+ cells were detected inside of the masses of atypical T cells. This phenomenon was especially prominent in the tumor stage of MF. These finding suggested that CD205+ DCs might correlate with the progression of MF, while PD-L1+ cells might contribute to maintaining the tumor microenvironment in the lesional skin of MF.

Hyaluronan Metabolism in Human Keratinocytes and Atopic Dermatitis Skin Is Driven by a Balance of Hyaluronan Synthases 1 and 3

Hyaluronan (HA) is a glycosaminoglycan synthesized directly into the extracellular matrix by three hyaluronan synthases (HAS1, HAS2, and HAS3). HA is abundantly synthesized by keratinocytes but its epidermal functions remain unclear. We used culture models to grow human keratinocytes as autocrine monolayers or as reconstructed human epidermis (RHE) to assess HA synthesis and HAS expression levels during the course of keratinocyte differentiation. In both the models, epidermal differentiation downregulates HAS3 mRNA expression while increasing HAS1 without significant changes in hyaluronidase expression. HA production correlates with HAS1 mRNA expression level during normal differentiation. To investigate the regulation of HAS gene expression during inflammatory conditions linked to perturbed differentiation, lesional and non-lesional skin biopsies of atopic dermatitis (AD) patients were analyzed. HAS3 mRNA expression level increases in AD lesions compared with healthy and non-lesional skin. Simultaneously, HAS1 expression decreases. Heparin-binding EGF-like growth factor (HB-EGF) is upregulated in AD epidermis. An AD-like HAS expression pattern is observed in RHE incubated with HB-EGF. These results indicate that HAS1 is the main enzyme responsible for HA production by normal keratinocytes and thus, must be considered as an actor of normal keratinocyte differentiation. In contrast, HAS3 can be induced by HB-EGF and seems mainly involved in AD epidermis.

Expression of CD39/Entpd1 on granuloma-composing cells and induction of Foxp3-positive regulatory T cells in sarcoidosis

Regulatory T cells (Tregs), together with tolerogenic dendritic cells (tDCs) are involved in maintaining peripheral tolerance. A recent report suggested both Tregs and tDCs may be pathogenic in granulomatous skin disorders. To examined the expression of CD39 on granuloma-composing cells and Foxp3-positive Tregs in the skin in two representative granulomatous diseases, sarcoidosis and granuloma annulare (GA). We immunohistologically examined expression of CD39 on granuloma-composing cells and expression of Foxp3 on CD4+ or CD25+ cells in fixed sections of lesional skin from 16 patients with sarcoidosis and five patients with GA. The granuloma-composing cells expressed CD39 in both sarcoidosis and GA. Significant numbers of CD4+ Foxp3+ Tregs were present diffusely throughout the granulomatous tissues in sarcoidosis, whereas Tregs in GA existed only at the peripheral lesion of palisading granulomatous tissue. There was infiltration of increased numbers of Foxp3+ Tregs around the CD39+ granuloma-composing cells in both GA and sarcoidosis.

Comparison of Foxp3+ Regulatory T cells and CD163+ Macrophages in Invasive and Non-invasive Extramammary Paget’s Disease

Regulatory T cells (Tregs), identified by the expression of CD4, CD25 and Foxp3, together with immunosuppressive macrophages, such as CD163+ M2 macrophages, are involved in maintaining peripheral tolerance. The aim of this study was to elucidate the involvement of Tregs and CD163+ macrophages in invasive and non-invasive extramammary Paget's disease. The presence of CD4+CD25+Foxp3+ Tregs, CD163+ M2 macrophages and matrix metalloproteinase-9+ cells was examined immunohistologically in fixed sections of lesional skin from 10 patients with non-invasive extramammary Paget's disease and 7 patients with invasive extramammary Paget's disease. Fewer CD4+CD25+Foxp3+ Tregs were observed in non-invasive extramammary Paget's disease than in invasive extramammary Paget's disease. In contrast, higher numbers of CD163+ macrophages and metalloproteinase-9+ cells were detected only in invasive extramammary Paget's disease. These findings suggest that the induction of immunosuppressive cells in extramammary Paget's disease differs according to the tumour stage.

Increased chitotriosidase activity in serum of leprosy patients: Association with bacillary leprosy

Human phagocyte-specific chitotriosidase is associated with several diseases involving macrophage activation. Since macrophage activation plays an important role in the control of Mycobacterium leprae infection, we studied the association of chitotriosidase with leprosy both in serum and in situ in lesional skin biopsies from patients. Serum samples from 78 Indonesian leprosy patients (39 non-reactional and 39 reactional leprosy patients) and 36 healthy controls (HC) from the same endemic region were investigated. The patients were classified as multibacillary (MB, n=69) or paucibacillary (PB, n=9) based on the bacterial index in slit-skin smears. Thirty-six of the reactional patients had erythema nodosum leprosum (ENL), while only 3 had reversal reaction (RR). Follow-up serum samples after corticosteroid treatment were also obtained from 17 patients with ENL and one with RR. Multibacillary (MB) patients showed increased chitotriosidase activity in serum as compared to paucibacillary (PB) patients and healthy controls. Although no significant difference was observed between reactional and the corresponding non-reactional groups, ENL showed significantly higher chitotriosidase activity as compared to HC. Furthermore, corticosteroid treatment resulted in significant decline of enzyme activity in ENL sera. Chitotriosidase activity correlated with levels of neopterin, another macrophage activation marker, but not with IL-6, IFN-gamma, TNF-alpha and IL-10. Immunohistochemical staining of 6 MB (LL=5, BL=1) lesional skin sections from stored material showed positive staining for chitotriosidase within lipid-laden macrophages suggesting that macrophages are the source of the enzyme detected in serum. Thus, serum chitotriosidase activity is potentially useful in distinguishing MB from PB leprosy and in monitoring response to therapy in ENL.

Low Strength of Correlation between the Intensity of Neutrophil Elastase Expression in Lesional Skin and the Level of Serum IgA Antibodies to Epidermal Transglutaminase in Dermatitis Herpetiformis

Whereas it has been shown that neutrophil elastase (NE) is a crucial enzyme degrading the dermal-epidermal junction (DEJ) in bullous pemphigoid (BP), experimental studies on the role of NE in dermatitis herpetiformis (DH), a disease in which, as in BP, an intra-lamina lucida blister is formed, are scanty. The aim of this study was to analyse whether there is a correlation between levels of serum IgA antibodies to the epidermal transglutaminase (TG3), an enzyme believed to be the autoantigen of DH, and expression of NE in lesional skin in DH. A series of 21 consecutive patients with DH was studied. The levels of IgA antibodies to TG3 in sera were calculated with ELISA. The expressions of NE were examined with immunohistochemical technique in sections of lesional skin using a mouse monoclonal antibody to human NE. The digital microscopic image analysis with the appropriate software was then used to measure intensities of NE expression. The correlation between the intensity of NE expression in lesional skin and the level of serum IgA anti- bodies to TG3 in DH was of low strength. Thus, it is speculated that in DH the engagement of IgA autoantibodies to the enzyme, TG3, on cutaneous neutrophils might not be a principal stimulus to releasing NE, the enzyme known to degrade DEJ in subepidermal blistering diseases with autoimmunity to DEJ structural proteins.

Soluble FAS Ligand: A Discriminating Feature between Drug-Induced Skin Eruptions and Viral Exanthemas

The clinical spectrum of cutaneous eruptions comprises benign variants like maculopapular rashes (MPRs) and potentially life-threatening events such as toxic epidermal necrolysis (TEN). Apoptosis of keratinocytes is a common histopathological feature of all these drug eruptions. As in skin lesions of TEN and Stevens-Johnson syndrome patients, apoptosis of keratinocytes is often accompanied by an only sparse cellular infiltrate, a soluble fatty acid synthetase ligand (sFASL)-mediated mechanism of keratinocyte cell death is postulated. In MPR patients, evidence for the occurrence of a similar process could not be established so far. We therefore examined sera and lesional skin sections from patients with clinical variants of drug eruptions for FASL expression using a sandwich ELISA and immunohistochemistry, respectively. As controls, healthy persons and patients with other inflammatory skin diseases such as viral exanthema were analyzed. Elevated levels of FASL were detected not only in TEN patients but also in sera and lesional skin of patients with MPR. In contrast, sFASL was repeatedly negative in all viral exanthemas and healthy controls tested. Thus, determination of sFASL serum concentration may represent a discriminating tool between drug rashes and viral exanthemas.

Abnormal accumulation of inter‐α‐trypsin inhibitor and hyaluronic acid in lichen sclerosus

Inter-alpha-trypsin inhibitor (ITI) is a recently identified extracellular hyaluronic acid (HA)-binding protein which greatly improves extracellular HA stability. In lichen sclerosus (LS), a broad hyalinized zone of superficial dermis is the most prominent pathological change. To assess the pathogenic role of ITI in accumulation of HA in a broad hyalinized zone in LS, we examined the expression and localization of ITI and HA immunohistochemically. In LS lesional skin sections, ITI staining revealed a strong, diffuse immunoreactivity predominantly in the upper dermis, whereas no staining was detected in normal skin sections. HA staining clearly showed positive reactivity in the superficial dermis, the epidermis, and occasionally the perivascular inflammatory infiltrate in LS skin sections. In normal skin, HA was present only in the epidermis. Double staining for ITI and HA demonstrated that ITI was localized in the areas where HA was abnormally deposited in the superficial dermis of LS. Other HA-binding proteins, CD44 and versican, did not show enhanced staining in the upper dermis of LS compared to normal skin specimens. These findings strongly suggest that ITI is closely implicated in the accumulation of HA in a broad hyalinized zone of the superficial dermis of LS.

Immunopathology of vesicular cutaneous lupus erythematosus in the rough collie and Shetland sheepdog: a canine homologue of subacute cutaneous lupus erythematosus in humans.

Clinical and histological features of an erosive disease in the rough collie and Shetland sheepdog are most consistent with a vesicular variant of cutaneous lupus erythematosus (VCLE). This paper reports the immunopathological findings of canine VCLE using samples from 17 affected dogs. Lesional skin sections were stained with monoclonal antibodies specific for CD3 (11 dogs) or a panel of monoclonal antibodies specific for leukocyte antigens (two dogs). Apoptotic cells were detected using the TUNEL method in 12 cases. Direct (14 dogs) and indirect immunofluorescence tests (five dogs) were also performed. Circulating antibodies to extractable nuclear antigens (ENA) were surveyed in 11 dogs by immunoblotting and ELISA. The predominant cells at the dermal-epidermal interface were identified as CD3(+) T lymphocytes expressing CD4 or CD8 and CD1(+) dendritic antigen presenting cells. In 7/12 dogs (58%), apoptosis of basal keratinocyte nuclei was present. Up-regulation of MHCII and ICAM-1 was observed on basal keratinocytes from the two dogs examined. Direct immunofluorescence revealed deposition of immunoglobulins bound to the cytoplasm of keratinocytes (6/14 dogs; 43%), to the dermal-epidermal junction (7/14 dogs; 50%), or to superficial dermal venules (13/14 dogs; 93%). Circulating IgG auto-antibodies targeting one or more ENA were detected in nine (82%) and eight (73%) of 11 dogs by immunoblotting and ELISA, respectively. These auto-antibodies recognized Ro/SSA and/or La/SSB in four (36%) and six (55%) of 11 dogs respectively by these two methods. Altogether, results of these studies provide evidence supporting the hypothesis that canine VCLE is an immunological homologue of subacute cutaneous lupus erythematosus in humans.

Apoptosis in different cutaneous manifestations of lupus erythematosus.

It has been shown that dysregulation of apoptosis may play an important part in the development of autoimmune diseases such as lupus erythematosus (LE). The aim of the study was to investigate whether apoptosis may contribute to the pathogenesis of cutaneous changes in LE and whether certain apoptosis-related markers such as Fas antigen, Fas ligand (FasL), Bcl-2 and Bax proteins take part in this process. Cryostat sections of lesional skin from 31 patients with LE-specific skin lesions [discoid (DLE) n = 17, subacute cutaneous (SCLE) n = 10 and acute cutaneous (ACLE) n = 4] and normal skin samples (n = 10) were stained with monoclonal antibodies against Fas antigen, FasL, Bcl-2 and Bax proteins. For the detection of apoptotic nuclei, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling technique (TUNEL) was employed. The keratinocytes of the epidermis and mononuclear cells of the dermal infiltrate in LE showed a significant increase in Fas antigen expression as compared with normal skin. The anti-Bcl-2 staining intensity within the epidermis was markedly decreased, while the mononuclear infiltrate stained for this protein just like single lymphocytes scattered in normal skin. There was an increase of FasL and/or Bax-positive cells within the dermal infiltrate, but not in the epidermis or in the hair follicles. Accumulation of FasL-bearing cells around hair follicles was a hallmark of DLE. Numerous cells with pyknotic, TUNEL-positive nuclei were demonstrated in the epidermis, in hair follicles and among the cells of the dermal infiltrate. The extent of apoptosis in lesional skin was greater in acute and subacute cutaneous than in discoid forms of LE. The exact stimulus triggering apoptosis in cutaneous LE still remains unknown but the present study provides strong evidence that the reduction of Bcl-2 expression in the basal cells is associated with the overexpression of Fas antigen and correlates directly with the extent of apoptosis in the epidermis. Furthermore, the current study indicates that the extent of apoptosis in both the epidermis and dermal infiltrate may correlate with the chronological development of LE skin lesions.

Mast cells in psoriatic skin are strongly positive for interferon-gamma.

The increased number and early activation of cutaneous mast cells is a typical feature of psoriatic inflammation. Interferon-gamma (IFN-gamma) is believed to be one of the important mediators in the cytokine cascade of psoriasis. Human mast cells have been previously reported to release various cytokines upon stimulation including interleukin (IL) -4, IL-5, IL-6, IL-8, IL-13 and tumour necrosis factor-alpha. Here we report that human mast cells synthesize also IFN-gamma at mRNA and protein level and that the number of IFN-gamma producing mast cells is significantly increased in the psoriatic skin. IFN-gamma immunoreactivity in mast cells was demonstrated by staining non-lesional and lesional skin sections from 21 patients with psoriasis. Ten patients with atopic dermatitis (AD) and five healthy persons served as control groups. The percentage (mean +/- SD) of IFN-gamma + mast cells in lesional compared with non-lesional psoriatic skin was 67 +/- 18% vs. 44 +/- 17% (P < 0.0001, paired t-test), respectively, but only 9 +/- 6% vs. 10 +/- 7% in corresponding skin samples of AD. In the skin of healthy controls, only 12 +/- 12% of the mast cells were IFN-gamma +. Using immunoelectron microscopy, we confirmed the ultrastructural localization of IFN-gamma within the granules of mast cells in psoriatic skin. In addition, stimulation of a human mast cell line HMC-1 with phorbol myristate acetate (PMA) (100 nmol/L) for periods of 2-24 h induced expression of IFN-gamma mRNA, which peaked at 24 h. When HMC-1 cells were stimulated with PMA (100 nmol/L) for periods of 0-3 days, the cells released IFN-gamma protein, peaking on day 1. These results provide further evidence for the important role of mast cells in the pathogenesis of psoriasis.

No Evidence of Human Herpesvirus 8 Infection in Patients with Paraneoplastic Pemphigus, Pemphigus Vulgaris, or Pemphigus Foliaceus

Paraneoplastic pemphigus has been associated with both malignancies and multicentric Castleman's disease; the latter is a rare angiolymphoproliferative disorder that has also been linked with human herpesvirus 8 (HHV8) infection. Other diseases definitively associated with HHV8 include Kaposi's sarcoma and primary effusion lymphoma. In a search for additional HHV8-associated diseases, patients with paraneoplastic pemphigus, as well as patients with pemphigus vulgaris and pemphigus foliaceus, were studied. Using an immunofluorescence assay able to specifically detect antibodies directed against lytically induced HHV8 antigens, HHV8 antibodies were not detected in sera from 24 patients with paraneoplastic pemphigus (including 10 with concomitant Castleman's disease) nor from 19 patients with pemphigus vulgaris. Sera from patients with Kaposi's sarcoma and from healthy U.S. blood donors were positive (25 of 26) and negative (none of 20), respectively. In addition, HHV8 DNA was not found in frozen lesional skin of five patients with pemphigus vulgaris and five patients with pemphigus foliaceus by nested polymerase chain reaction (lower limit of detection = 10 copies viral DNA per microg total cellular DNA). Finally, tissue sections of lesional skin from 10 patients with pemphigus vulgaris were negative for HHV8 by in situ hybridization, using probes able to detect both latently and lytically expressed HHV8 genes in Kaposi's sarcoma tissue. In summary, no evidence of HHV8 infection was found in all types of pemphigus using a variety of methods. These findings do not support a general role for HHV8 in skin diseases associated with immunosuppression.

Mast cells of psoriatic and atopic dermatitis skin are positive for TNF-alpha and their degranulation is associated with expression of ICAM-1 in the epidermis.

The release of cytokines from cutaneous cells may be of major importance in the initiation and development of many inflammatory skin disorders. For example, tumor necrosis factor-alpha (TNF-alpha), which in healthy skin is found preformed only in mast cells, is able to induce the expression of several adhesion molecules including intercellular adhesion molecule-1 (ICAM-1). Increased expression of ICAM-1 occurs in keratinocytes in lesional skin of psoriasis and atopic dermatitis (AD) and it is considered to be an important initiator of leucocyte/keratinocyte interactions in skin inflammation. We counted the mast cells showing TNF-alpha immunoreactivity using a double-staining method in nonlesional and lesional skin sections from 12 patients with AD and 12 patients with psoriasis. The percentage of TNF-alpha+ mast cells in lesional and nonlesional AD skin was 36 +/- 22% and 21 +/- 15% (P < 0.018, paired t-test), respectively, and in psoriatic skin was 16 +/- 25% and 15 +/- 15%, respectively (P < 0.89, paired t-test). We also cultured whole skin biopsies taken from the healthy-looking skin of psoriatic and AD patients in the presence of mast cell degranulator compound 48/80, which resulted in focal expression of ICAM-1 in the epidermis. In cultured keratinocytes, both histamine and an extract of a human mast-cell line (HMC-1) induced ICAM-1 immunostaining only in occasional cells, but the combination of histamine and the HMC-1 extract resulted in intense ICAM-1 staining in numerous cells. This enhancement of ICAM-1 staining was abolished by preincubation of the HMC-1 extract with anti-TNF-alpha antibody. These results suggest that the degranulation of mast cells induces the expression of ICAM-1 in keratinocytes probably via TNF-alpha and histamine.

Correlation of soluble adhesion molecules in the peripheral blood of scleroderma patients with their in situ expression and with disease activity

To correlate serum levels of the soluble adhesion molecules intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), E-selectin, and P-selectin with (a) clinical disease activity and progression and (b) the in situ expression and distribution of these adhesion molecules in lesional skin, in patients with systemic sclerosis (SSc). Serum samples from 12 SSc patients and 36 healthy controls were examined by enzyme-linked immunosorbent assay. Immunohistologic staining was carried out on cryostat sections of lesional skin. Patients whose SSc was in the early inflammatory stage or who had prominent disease progression showed elevated serum levels of soluble adhesion molecules. Serum levels correlated positively with the expression of these molecules on endothelial cells and fibroblasts in lesional skin. Serum levels of soluble ICAM-1, VCAM-1, P-selectin, and, to a lesser degree, E-selectin correlate well with their in situ activity and with clinical disease activity. These parameters therefore provide a useful tool for the characterization of disease stage, progression, and prognosis in SSc.

Mast cells are one major source of interleukin-4 in atopic dermatitis

Several reports have shown the presence of T-helper lymphocytes with Th2 characteristics in the skin lesions of atopic dermatitis (AD). However, Th2 cells themselves require an exogenous pulse of IL-4 to initiate their differentiation and synthesis of IL-4. As mast cells have recently been shown to contain IL-4, this finding prompted us to investigate IL-4 in mast cells of AD lesions, to determine if they might provide the IL-4 pulse needed by the Th2 cells. In this study, we measured IL-4 immunoreactivity in mast cells of non-lesional and lesional skin sections from 20 patients with AD. Ten patients with nummular eczema (NE) without any atopic features or background, and five healthy subjects, served as the control groups. Mast cells were first identified using an enzyme--histochemical staining method for tryptase. Subsequently, the sections were photographed, the tryptase stain was removed, and IL-4 was demonstrated with a polyclonal antibody. The sections were photographed again, and the percentage of IL-4 positive mast cells was calculated. The percentage of mast cells exhibiting IL-4 immunoreactivity in the upper dermis in lesional vs. non-lesional skin was 66 +/- 18% vs. 37 +/- 18% in AD (P < 0.0001, paired t-test), but only 46 +/- 19% vs. 31 +/- 22% in NE. In the skin of healthy controls, only 23 +/- 25% of the mast cells were positive for IL-4. In addition, a significant difference was found between lesional skin of AD vs. NE patients (P < 0.008, unpaired t-test).(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclosporine in atopic dermatitis: Modulation in the expression of immunologic markers in lesional skin

In previous studies, oral cyclosporine was highly effective in the treatment of patients with severe atopic dermatitis. In this study seven patients with severe and therapy-resistant atopic dermatitis underwent therapy with cyclosporine, 5 mg/kg/day, for 6 weeks. The effect of cyclosporine on the expression of cytokines, which probably play a role in this disease, was examined. The study was performed with a panel of antibodies as markers of inflammatory cells, adhesion molecules, and cytokines (interferon-gamma [IFN-gamma], tumor necrosis factor-alpha [TNF-alpha] and interleukins 1 alpha, 1 beta, and 8 [IL-1 alpha, IL-1 beta, and IL-8, respectively]). They were visualized by indirect immunoperoxidase techniques. After 2 weeks of cyclosporine therapy, a reduction of 60% in the disease (severity and extent) was observed. This reduction was 89% after 4 weeks and 90% after 6 weeks of therapy. Results of indirect immunoperoxidase stains performed on lesional skin sections after 2 weeks of treatment showed statistically significant reduced numbers of CD14+, CD25 (IL-2R+) and IL-8+ inflammatory cells in the dermis and CD36(OKM5)+ cells in both the epidermis and dermis. The number of cells expressing IFN-gamma and TNF-alpha, assumed to be the products of the helper T-cell (TH)1 subset, was unaltered despite the impressive clinical benefit observed. Keratinocytes in lesional atopic skin did not express intercellular adhesion molecule type 1 (ICAM-1). The expression of the adhesion molecules ICAM-1, lymphocyte function-associated (LFA) type 1, and LFA-3 on inflammatory cells also remained unaffected by cyclosporine treatment. A statistically significant reduction in the number of activated T cells and in the number of cells expressing the IL-2 receptor (CD25) paralleled a marked improvement in the disease and supports the view that atopic dermatitis is based on a T-cell-mediated immune inflammation.

Unilateral Cutaneous Emboli of Aspergillus

A 40-year-old white woman with acute nonlymphocytic leukemia, which relapsed despite bone marrow transplantation and various chemotherapeutic regimens, developed fever and neutropenia. Her fever was unresponsive to broad-spectrum antibiotics, and on hospital day 53 she developed purpuric macules with necrotic centers on her left hand and forearm. Frozen sections of lesional skin were stained with Grocott's methenamine-silver and showed hyphae consistent with a species of Aspergillus; culture of the skin biopsy specimen yielded a pure culture of Aspergillus flavus. Localization of the emboli to the left upper extremity was subsequently explained by magnetic resonance imaging scan of the chest demonstrating invasion of the left subclavian artery by a pulmonary aspergilloma.