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The cyclin-dependent kinase inhibitor 2A (CDKN2A) gene on chromosome 9p21 encodes p16 (INK4A), the inhibitor of the CDK4/retinoblastoma (Rb) cell proliferation pathway, as well as p14 (ARF), which controls p53-dependent pathways. Inactivation of p16 has previously been associated with the prognostically unfavourable primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL, LT). In this work, we analysed 22 tumors [nine primary cutaneous follicle centre lymphomas (PCFCL), seven primary cutaneous marginal zone lymphomas (PCMZL) and six PCLBCL, LT] not only for alterations of the p16 gene but also for p14, p53 and Rb by fluorescence in situ hybridization (FISH) and immunohistochemistry. In most PCLBCL, LT (4/6) alterations of CDKN2A (two biallelic deletions, one monoallelic deletion and one trisomy 9) and in addition the highest frequency of deletions of p53 (3/6) and Rb (3/6) were detected. p16 was not expressed but very high levels of phosphorylated Rb, indicating a functional effect of genomic CDKN2A alterations on the protein level in PCLBCL, LT. Regarding the p14/p53 axis, PCLBCL, LT showed a variable expression. Neither PCFCL nor PCMZL showed alterations of CDKN2A and also deletions of p53 or Rb were extremely rare in these subtypes. Exclusively in PCMZL, p53 protein was consistently lacking. In conclusion, only PCLBCL, LT is characterized by a high frequency of aberrations of the CDKN2A network components in both important tumor suppressor pathways regulated by the CDKN2A gene. Moreover, PCLBCL, LT appears to be distinguishable from PCMZL not only by its level of p53 expression but also by its stage of Rb phosphorylation. The latter may also apply to a subgroup of PCFCL.

Background Chronic venous leg ulcers (CVU) cause considerable burden of disease for the patients as well as enormous costs for health care systems. The pathophysiology of CVU is complex and not entirely understood. So far reliable pathogenic and/or prognostic parameters have not been identified. Objectives We studied the role of thrombophilia in patients referred to a University dermatology department for treatment of CVU. Patients and methods A cohort of 310 patients with active chronic venous leg ulcers (CEAP 6) was stratified into two comparably large groups according to the presence or absence of post-thrombotic syndrome (PTS+; PTS−) as determined using duplex scan and/or phlebography. In addition, several thrombophilia parameters were assessed. Results The prevalence of protein S deficiency and factor V Leiden mutation was significantly higher in PTS+ patients compared with the PTS− group. However, patients in both subgroups revealed high prevalences of thrombophilia (antithrombin deficiency, protein C deficiency, protein S deficiency, activated protein C resistance, factor V mutation or elevated homocysteine). Conclusion Based on these data, it is conceivable that thrombophilia contributes to the pathogenesis of CVU, possibly through induction of microcirculatory dysregulations.

Background Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare entity characterized by a CD4+/CD56+/CD123+ immunophenotype and a fatal clinical course. The average survival of 12–14 months may be prolonged by allogeneic bone marrow transplantation (BMT). Objectives We report about a male patient who suffered from BPDCN with a typical histology and co-expression of CD4/CD123 and a CD56 expression by 80% of the tumour cells. The cutaneous tumour relapse after chemotherapy and allogeneic BMT was completely negative for CD56. Methods We performed interphase fluorescence in situ hybridization (FISH) analysis of tumour tissue, asserved before and after BMT, using specific probes for chromosome 11, which encompass the CD56 gene region. Results The tumour cells revealed a partial loss of 11q as well as a monosomy of chromosome 11. Conclusion This case demonstrates for the first time that loss of CD56 expression can also occur as a secondary event after chemotherapy and BMT. In our case, DNA loss of 11q23 could be responsible for the negativity of 20% of tumour cells as observed before chemotherapy. However, the complete loss of CD56 expression in the relapsed tumour cannot be explained by the loss of 11q23 alone. Additional factors such as chemotherapy-induced mutations might also have contributed.

The optimal processing for the pathology of sentinel lymph nodes of patients with melanoma is still a matter of debate. We compared two protocols of sentinel lymph node processing, which were consecutively applied. For the first protocol, the sentinel lymph nodes were cut into 1-2 mm thick slices. From each slice, 12 microtome sections were stained (multiple slices protocol). For the second protocol, which is a modification of the recent European Organisation for Research and Treatment of Cancer protocol, the sentinel lymph nodes were bivalved. Five consecutive series of microtome sections, with gaps of 50 microm between them, were prepared from each cut surface (bivalving protocol). H&E and immunohistochemical staining were integral elements of both protocols. A total of 584 sentinel lymph nodes (1.8+/-0.9 per patient) were examined. The percentages of micrometastases (29 versus 27%) and of capsular naevi (13 versus 15%) detected were very similar for both protocols. As shown by multivariate logistic regression, Breslow thickness (P=0.003) and younger age (P=0.01) correlated with nodal metastasis. The type of histological preparation, ulceration and sex were not significant. The multiple slices protocol produced, on average, 4 paraffin blocks and 46 microtome sections per node. The bivalving protocol constantly produced 2 paraffin blocks and 42 microtome sections. For technical processing, the multiple slices protocol required, on average, 38 min per sentinel lymph node, whereas the bivalving protocol required 55 min. Both protocols yielded excellent detection rates with a similar amount of work being required on the part of the pathologist. Compared with the bivalving protocol, the multiple slices protocol was less labor intensive for the technical staff.

We analyzed the value of digital epiluminescence microscopy (DELM) for the long-term follow-up of atypical nevi. Patients (n=530) were prospectively categorized into defined melanoma risk groups and followed by clinical and epiluminescence microscopy (ELM) examinations. Atypical nevi (n=7001) were additionally followed by DELM. During follow-up (median 32.2 months), we detected 53 melanomas among 637 excised lesions (8.3% overall chance of success). The chance of success for melanoma detection among lesions suspicious by ELM criteria was increased to 17% when additional DELM-documented changes were present. Moreover, 18 of the 53 melanomas were exclusively identified by DELM-documented changes, indicating that DELM increased the sensitivity of the ELM analysis by identifying additional melanomas. However, for lesions exclusively excised due to DELM changes, the chance of success was lower than for ELM (5.2 vs 11.8%). Excisions due to mere DELM changes detected 66.7% of melanomas in familial atypical mole and multiple melanoma (FAMMM) and 32.5% of melanomas in atypical mole syndrome (AMS) patients. We conclude that DELM is a valuable tool for the long-term follow-up of atypical nevi, especially in the high-risk groups of FAMMM and AMS patients. Randomized controlled trials are needed to validate the data from this clinical trial.

The variola virus was declared eradicated by the World Health Organization in 1980 but human infections by cowpox virus, another member of the genus Orthopoxvirus, are still observed, mainly in European countries. We report a woman who presented with two umbilicated vesicles surrounded by an indurated erythematous edema within cat scratch injuries on her thigh. The diagnosis of an Orthopoxvirus infection was based on the visualization of characteristic virus particles by electron microscopy and the detection of the A27L gene (14-kd fusion protein gene) of the genus Orthopoxvirus by polymerase chain reaction from a lesional skin biopsy specimen. Differential diagnoses of cat scratch disease, pustula maligna, and bullous impetigo were excluded by microbiologic investigation of the biopsy specimen. Both lesions scarred after 6 weeks of a continuous local antiseptic treatment.

Psoriasis is a chronic inflammatory skin disorder characterized by accumulation of Th1-type T cells and neutrophils, regenerative keratinocyte proliferation and differentiation, and enhanced epidermal production of antimicrobial peptides. The underlying cause is unknown, but there are some similarities with the immunologic defense program against bacteria. Development of psoriasiform skin lesions has been reported after administration of granulocyte colony-stimulating factor (G-CSF), a cytokine induced in monocytes by bacterial antigens. To further investigate the relation between this type of cytokine-induced dermatitis and psoriasis, we analyzed the cutaneous cytokine profile [tumor necrosis factor-alpha (TNF-alpha), interferon-gamma, transforming growth factor-beta1 (TGF-beta1), interleukin-10 (IL-10), IL-12p35 and p40, and IL-8] and expression of markers of epidermal activation [Ki-67, cytokeratin-16, major histocompatibility complex (MHC) class II, intercellular adhesion molecule-1 (ICAM-1)] in a patient who developed G-CSF-induced psoriasiform dermatitis by using quantitative real-time reverse transcriptase-polymerase chain reaction and immunohistology. The histologic picture resembled psoriasis with regard to epidermal hyperparakeratosis and the accumulation of lymphocytes in the upper corium. CD8(+) T cells were found to infiltrate the epidermis which was associated with an aberrant expression of Ki-67, cytokeratin-16, MHC class II, and ICAM-1 on adjacent keratinocytes. As compared to normal skin (n = 7), there was an increased expression of TNF-alpha, IL-12p40, and IL-8, a decreased expression of TGF-beta1, and a lack of IL-10, similar to the findings in active psoriasis (n = 8). Therefore, G-CSF may cause a lymphocytic dermatitis that, similar to psoriasis, is characterized by a pro-inflammatory Th1-type cytokine milieu and an epidermal phenotype indicative of aberrant maturation and acquisition of non-professional immune functions.

Background There is evidence that IL-16, a cytokine that induces chemotactic responses in CD4 + T cells, eosinophils, and dendritic cells, plays an important role during different types of cutaneous inflammatory responses, including allergic contact dermatitis (ACD) and atopic dermatitis (AD). Objectives We sought to test for association between a promoter polymorphism in the IL16 gene (T to C transition at position –295) and ACD and AD, respectively. Methods IL16 –295 genotypes were determined in samples from 2 separate case-control studies with white individuals. The first study included healthy individuals (n = 310) and patients with ACD (n = 86). These patients were polysensitized as defined by a contact sensitization to para-substituted aryl compounds and at least one other structurally unrelated allergen. The second study comprised healthy subjects (n = 214) and patients with AD (n = 94). Results IL16 –295 genotypes were differently distributed among polysensitized and healthy control subjects ( P = .0021). In particular, the IL16 –295∗C/C genotype was overrepresented among polysensitized individuals (7.0% vs 1.0% in the control group; odds ratio, 7.68; 95% CI, 1.59-48.12). In contrast, there was no evidence for an association between the IL16 –295 polymorphism and AD. Conclusion The IL16 –295 promoter polymorphism might influence susceptibility to contact allergy.

SummaryBackground Atopic dermatitis (AD) and psoriasis are genetically determined inflammatory skin disorders characterized by abnormal cytokine production. From association studies there is evidence that functionally relevant cytokine gene polymorphisms contribute to the genetic basis of psoriasis. Association studies in AD have mostly been limited to polymorphisms of T-helper 2-type cytokines, which dominate in acute AD lesions. Unexpectedly, the results of recent genome scans indicate linkage of AD to psoriasis susceptibility loci. Therefore, AD may also be influenced by genes that modulate cutaneous inflammation independently from atopic mechanisms. Objectives To investigate further the role of cytokine gene polymorphisms in AD. Methods Polymorphisms in the genes encoding tumour necrosis factor-α (TNFA−238 G/A, −308 G/A), interleukin (IL)-1β (IL1B−511 T/C, +3953 T/C), IL-6 (IL6−174 C/G), IL-10 (IL10−1082 A/G) and the IL-1 receptor antagonist (IL1RN intron 2) were investigated in German patients with AD (n = 94) and in healthy nonatopic individuals (n = 214) by polymerase chain reaction-based methods and direct cycle sequencing. Results No association was found between AD and any of the polymorphisms analysed. This is in contrast to the recently described association between psoriasis and the TNFA−238 and IL1B−511 polymorphisms. Conclusions Our data indicate that cytokine gene polymorphisms may act as specific markers of inflammatory skin diseases rather than contribute to a general disposition towards cutaneous inflammation.

Malignant melanoma has been shown to be susceptible to T cell-mediated immunity and, therefore, is a candidate for vaccination approaches. Clinical trials using dendritic cells (DC) loaded with peptides corresponding to tumor antigens are ongoing in several institutions, and some promising results have already been published. However, every single peptide-based vaccine can only be used in a patient with a given single HLA type, and this strategy is not appropriate for patients with rare HLA types or with tumors without defined antigens. A clinical pilot study in patients with disseminated melanoma refractory to standard therapy was initiated using a different approach. The authors generated autologous monocyte-derived DC and fused these DC with gamma-irradiated primary autologous tumor cells by incubation in polyethylene glycol. In previous experiments, the authors had shown that these fused cell products are potent inducers of a T-cell response in a mixed lymphocyte tumor cell culture. Seventeen patients were immunized with the cell product by s.c. injection in monthly intervals without any serious side effects. Of these patients, one had a partial response with decrease in size of all evaluable tumor manifestations. In one patient, some of the metastases were regressing despite an overall progressive disease, and one patient achieved disease stabilization for six months. In the responding patient, in parallel to tumor regression, circumscript hair depigmentation occurred. These data show, that a hybrid vaccine of DC and tumor cells can be safely applied and can induce tumor regressions, however, the clinical efficacy of the approach in its present form is insufficient.

The psoriatic inflammatory process is characterized by an overexpression of pro-inflammatory cytokines such as tumor necrosis factor-alpha and interleukin-1beta compared with a relative deficiency of anti-inflammatory factors such as interleukin-10 and the interleukin-1 receptor antagonist (interleukin-1Ra). Gene polymorphisms that affect cytokine production may contribute to the disease-associated cytokine imbalance and influence susceptibility to psoriasis. Here, we investigated the relationship between polymorphisms in the genes encoding for tumor necrosis factor-alpha (G-238A, G-308A), interleukin-1beta (C-511T, T+3953C), and interleukin-1Ra (intron 2), and cytokine production in peripheral blood mononuclear cells of healthy donors, and analyzed the distribution of these polymorphisms in patients with psoriasis vulgaris (n = 231) and healthy controls (n = 345). Carriage of tumor necrosis factor A-238 allele 2 (-238*A) was associated with increased production of tumor necrosis factor-alpha in response to lipopolysaccharide in vitro, and with early onset disease (40 y), especially in male patients with psoriasis [32% vs 7% in male controls; odds ratio = 6.78, 95% confidence interval = (3.18-15.15), p(adjusted) = 2 x 10(-7)]. Carriage of the interleukin-1B-511*1 (-511*C) homozygous genotype was associated with increased production of interleukin-1Ra in response to lipopolysaccharide and interleukin-10, and with late onset psoriasis [or = 40 y; 61% vs 44% in controls; odds ratio = 2.04, 95% confidence interval = (1.19-3.53), p(adjusted) = 0.0419]. These findings indicate that gene polymorphisms associated with altered cytokine responses in vitro may modify age of onset of psoriasis. They also provide further evidence that patients with early and late onset psoriasis differ in their genetic background.

Wegener's granulomatosis is a rare disease classically characterized by a necrotizing and granulomatous inflammation of the upper and lower respiratory tract, necrotizing glomerulonephritis and systemic vasculitis of small and medium sized vessels. Cutaneous manifestations are observed in 20-50% of cases often resembling pyoderma. We report here on a 72 years old female patient in whom the cutaneous symptom of a histological confirmed leukocytoclastic vasculitis led to the diagnosis of Wegener's granulomatosis. She had also developed the characteristic symptom of a saddle nose and suffered from relapsing keratitis. Clinical symptoms, diagnostic procedure and therapeutic options are discussed.

Although smallpox was eradicated 20 years ago, other members of the genus Orthopoxvirus (OPV), such as cowpox virus (CPXV) or monkeypox virus, are still a threat to humans. Because human CPXV infection is rare, it is seldom suspected on clinical grounds only. We report a boy who presented with two necrotic ulcers with surrounding erythema. Infection with OPV was suspected, as antibiotic treatment had not produced improvement and smears were negative for anthrax. An OPV was isolated and an OPV-specific polymerase chain reaction combined with a subsequent restriction enzyme fragment length polymorphism assay confirmed infection by CPXV. Although the patient's cat had had no skin lesions, OPV-specific antibodies were found at a titre of 1 : 8 in a plaque reduction assay, suggesting that the cat had transmitted the virus to the boy.

The present study addresses the question whether an extended ilioinguinal dissection as compared to an only superficial inguinal dissection improves survival and/or local tumour control after the appearance of palpable melanoma metastases to the groin. We retrospectively analysed the data of 104 patients with 69 ilioinguinal and 35 superficial inguinal dissections (median follow up 127 months). Prognostic factors of survival and groin recurrence were assessed using Kaplan-Meier estimation and Cox proportional hazards model. By multifactorial analysis, metastatic involvement of two lymph nodes or less was associated with a significantly better survival rate than involvement of > 2 or pelvic nodes (p = 0.0002). After radical ilioinguinal dissection, patients with extremity-located primaries had a better prognosis than patients with truncal primaries (p = 0.03). Tumour infiltration of the ilio-obturator compartment was found to be an independent factor of poor prognosis (p = 0.0009). The probability of recurrence in the dissected groin paralleled the number of positive nodes and significantly increased if intransits were observed (p = 0.0002). The extent of surgery, Breslow thickness, epidermal ulceration, sex, age and adjuvant chemotherapy neither significantly influenced survival nor local control rates. In summary, when metastatic inguinal nodes become palpable, the presence of pelvic metastases indicates systemic disease. After therapeutic groin dissection, local recurrence and survival depend rather on regional tumour burden than on the extent of surgery.

Sensitization to arylamines such as p-phenylenediamine is frequently diagnosed in patients with allergic contact dermatitis. Reactive metabolites of p-phenylenediamine might be produced in the skin by O-acetylation of N-hydroxylamines catalysed by local N-acetyltransferases (NATs). In this study, we tested whether genetic polymorphisms of NATs, which are known to affect enzyme activity, may influence the susceptibility to para-substituted arylamine-induced contact eczema. Using polymerase chain reaction and restriction enzyme analysis, the distribution of polymorphisms of NAT1 and NAT2 was investigated in 88 patients sensitized to para-substituted aryl compounds and 123 healthy controls. NAT2 rapid acetylators, i.e. carriers of the NAT2*4 wild-type allele, were more common in the contact allergy (44%) than in the healthy control group [30%; P = 0·042, odds ratio 1·9 (95% confidence interval, CI 1·05–3·27)]. Slow acetylators carrying the NAT2*5b/2*6a genotype were significantly less frequent among patients [13% vs. 38% in controls; P = 0·009, odds ratio 0·39 (95% CI 0·19–0·78)]. The carriage rate of the NAT1*10 allele, which is supposed to encode for a rapid NAT1 phenotype, was not significantly different between patients and controls [43% vs. 36%; odds ratio 1·5 (95% CI 0·88–2·68)]. Interactions between NAT2*4 and NAT1*10 were suggested by the increased frequency of the NAT2*4/NAT1*10 haplotype in patients (27%) compared with controls [15%; P = 0·039, odds ratio 2·1 (95% CI 1·04–4·04)]. As the NAT1 and NAT2 encoding genes are located in close proximity on chromosome 8p22, the latter finding could at least partly be due to genetic linkage. In fact, a linkage disequilibrium between NAT2*4 and NAT1*10 was observed in the contact allergy (P = 0·0025) and in the control group (P = 0·042). Our data indicate an association between the NAT2*4/NAT1*10 haplotype and contact sensitization to para-substituted aryl compounds. Therefore, acetylation may either enhance contact sensitization or NAT2*4 and NAT1*10 might be linked to an unknown susceptibility factor.

Paraneoplastic pemphigus (PP) is an autoimmune disease, which is frequently associated with non-Hodgkin's lymphoma. Autoantibodies against components of the cytoplasmic plaque of epithelial desmosomes are usually present in the sera and are believed to play a major pathogenic part in acantholysis and suprabasal epidermal blistering. However, another typical histological feature of PP, interface dermatitis with keratinocyte dyskeratosis, is shared with skin diseases that involve epithelial damage mediated by T cells. Here, we present the detailed characterization of the cutaneous T-cell response in a patient with PP and demonstrate a selective epidermal accumulation of activated CD8+ T cells together with an increased local production of interferon-gamma and tumour necrosis factor-alpha, and a strong expression of HLA-DR and ICAM-1 on keratinocytes. Apoptosis was identified as a key mechanism of keratinocyte death, and appeared independent of the FAS/FAS ligand (FAS-L) pathway, as epidermal expression of FAS was not increased compared with normal skin, and FAS-L was undetectable on the protein and mRNA level. Triple therapy with high-dose corticosteroids, cyclophosphamide and intravenous immunoglobulins reduced levels of pemphigus-like autoantibodies and reversed the cutaneous inflammatory reaction leading to long-standing clinical remission. Our findings support the concept of a major contribution of cytotoxic T lymphocytes to the immunopathology of paraneoplastic pemphigus.

Epidermal cytokines such as interleukin (IL)-1β, tumor necrosis factor-α, and IL-12 have been described to play a crucial role in the induction and elicitation phase of allergic contact dermatitis upon exposure to haptens. In this study we asked whether these cytokines may also play a role in the epidermis of patients with atopic dermatitis after the application of house dust mite antigens (HDM) to their skin. Epidermal samples were collected by scraping healthy appearing skin of atopic patients and healthy individuals 8 h after the application of an extract of HDM. Sodium lauryl sulfate and saline served as controls. Reverse transcriptase-polymerase chain reaction was performed for IL-1β, tumor necrosis factor-α, IL-12 p35, and IL-12 p40. Exposure to HDM led to a significant upregulation of mRNA of these cytokines in atopic patients only. Whereas IL-1β and tumor necrosis factor-α also showed an upregulation in part of these patients after exposure to the irritant sodium lauryl sulfate, IL-12 p40 mRNA was exclusively enhanced by the application of the allergen. In contrast to IL-12 p40, IL-12 p35 mRNA was not detectable in significant amounts. Interestingly, also in untreated, normal appearing skin of atopic individuals (n = 16), the levels of these cytokines were higher than in normal individuals (n = 8), possibly explaining the increased skin irritability of atopic individuals. Finally, comparing epidermal cytokines in the skin of patients who developed a positive allergen patch test to those who stayed negative, suggests that only expression of IL-1β mRNA may be a predictive marker for the development of a positive patch test reaction to HDM.

Wir berichten uber Mutter und Sohn mit Erythrokeratodermia progressiva symmetrica Darier-Gottron. Beide Patienten entwickelten im Alter von 6 Monaten symmetrische erythematosquamose Plaques an den Extremitaten und im Gesicht. Beim Sohn kam es mit 2 1/2 Jahren zu einer raschen Ausbreitung mit Befall des gesamten Integuments. Die Mutter berichtet uber einen ahnlichen Verlauf, jedoch mit spontaner Regression ab dem 10. Lebensjahr. Die Klinik dieses generalisierten Zustandes war identisch mit dem Befund einer kongenitalen lamellaren Ichthyose. Lichtmikroskopisch ergaben sich unspezifische Veranderungen mit Orthohyperkeratose, fokalen Parakeratosearealen und Akanthose. Elektronenmikroskopisch fanden sich im Stratum granulosum eine hohe Keratinosomenanzahl, Keratinosomenlamellen in den Interzellularraumen und teils vermehrtes Keratohyalin mit Verklumpung. Weiterhin waren kurze Tonofilamentbundel mit Verklumpungen im Stratum spinosum auffallig. Systemische Retinoide brachten dem Sohn eine mehrere Monate anhaltende Befundbesserung. Die Mutter berichtet uber ahnlich gute Erfolge unter einer Retinoidintervalltherapie. Die Beobachtung zeigt die Schwierigkeit, die Erythrokeratodermia progressiva symmetrica als eigentlich lokalisierte Verhornungsstorung bei jedoch zwischenzeitlich ausgedehntem, generalisiertem Zustand mit den derzeitig zur Verfugung stehenden klinischen und ultrastrukturellen Kriterien zuverlassig von anderen Verhornungsstorungen abzugrenzen.

Six patients with granuloma faciale, including patients with multiple lesions, were treated successfully with cryosurgery. Granuloma faciale is known to be difficult to treat. Cryosurgery is an effective and minimally invasive therapy for this granulomatous inflammation of the skin. It should be considered as an alternative to dapsone.

A 40-year-old patient with low-grade B-NHL developed a generalized macular-papular rash following the first cycle of fludarabine treatment which progressed to a complete epidermal necrolysis following the second cycle. Clinical symptoms and the results of the direct and indirect immunofluorescence were consistent with a mucocutaneous autoimmune syndrome (pemphigus). Immunohistochemical analysis demonstrated a dense epidermal infiltration of CD8+ lymphocytes associated with the histological features of single-cell necrosis of keratinocytes. Early and aggressive immunosuppressive treatment with steroids, cyclophosphamide, and high-dose immunoglobulins resulted in regression of symptoms and complete reconstitution of epidermal integrity. The malignant lymphoma has completely regressed. The findings suggest a fludarabine-induced defect in immunosurveillance – resulting in the uncontrolled activation of autoaggressive T-cell clones – as a pathogenetic mechanism of this life-threatening dermatological complication.

Background : Nonmelanoma carcinomas of the skin represent the most frequent cancers among the Caucasian population worldwide. They occur with high frequency in renal allograft recipient patients after prolonged immunosuppression. Purpose : We analyzed tumors obtained from both immunosuppressed and nonimmunosuppressed patients for human papillomavirus (HPV) DNA. Methods : Twenty-nine specimens of nonmelanoma carcinomas of the skin were obtained from 19 renal allograft recipient patients ; these included 20 specimens of squamous cell carcinoma (SCC) from 11 patients, five specimens of basal cell carcinoma (BCC) from four patients, and four specimens of carcinoma in situ (CIS) from four patients. Forty-one specimens of nonmelanoma carcinomas of the skin were obtained from 32 nonimmunosuppressed patients ; these included 26 SCC specimens from 19 patients, 11 BCC specimens from nine patients, and four keratoacanthoma (benign epithelial tumor) specimens from four patients. A polymerase chain reaction method involving use of degenerate oligonucleotide primers, in which the conserved region of the open reading frame of the HPV L1 (major capsid protein) gene is amplified, was used to amplify total cellular DNA purified from individual tumors. The DNA of each specimen was subjected to 16 different amplification reactions ; different primer combinations were used in order to increase the sensitivity and specificity of HPV detection. Resulting products were probed with a radioactively labeled, degenerate oligonucleotide. HPV-specific DNA was either sequenced directly after elution from the gel or amplified with semi-nested, degenerate primers, after which the products were cloned and sequenced. Sequences were compared with all known papillomavirus sequences. Results : Thirteen (65%) of the 20 SCC specimens and three of the five BCC specimens from immunosuppressed (renal allograft recipient) patients contained identifiable HPV-related sequences, among them 13 putative novel HPV genomes. In addition, all other malignant tumor specimens from this patient group revealed faint signals upon amplification and hybridization ; the origin of these signals has not been identified in the present study. In nonimmunosuppressed patients, eight (31%) of 26 SCC specimens and four (36%) of 11 BCC specimens contained sequences of HPV types. Two putative novel HPV sequences could be identified in this group. Faint signals of yet undetermined origin were observed in eight of the SCC specimens and in two of the BCC specimens. Two of four keratoacanthoma specimens contained sequences of known HPV type. (Keratoacanthoma is a nonmalignant lesion for which the natural history has not been defined.) The spectrum of HPV types in both groups of patients differed substantially. Conclusions : These data point to the frequent presence of HPV sequences in SCCs and BCCs of the skin. The etiologic relationship of these infections to the respective malignant tumors remains to be evaluated. Implications : The presence of HPV DNA in a large percentage of specimens of nonmelanoma carcinomas of the skin from immunosuppressed patients, as well as from nonimmunosuppressed patients, renders a papillomavirus infection as a possible factor in the etiology of this disease

The outbreak of Shiga toxin producing E.coli O104:H4 in northern Germany in 2011 was one of the largest worldwide and involved mainly adults. Post-diarrheal hemolytic uremic syndrome (HUS) occurred in 22% of STEC positive patients. This study's aim was to assess risk factors for HUS in STEC-infected patients and to develop a score from routine hospital parameters to estimate patient risks for developing HUS. In a cohort analysis, adult patients with STEC infection were included in five participating hospitals in northern Germany between May and July 2011. Clinical data were obtained from questionnaires and medical records, laboratory data were extracted from hospitals' electronic data systems. HUS was defined as thrombocytopenia, hemolytic anemia and acute renal dysfunction. Random forests and multivariate logistic regression were used to identify risk factors for HUS and develop a score using the estimated coefficients as weights. Among 259 adults with STEC infection, vomiting (OR 3.48,95%CI 1.88-6.53), visible blood in stools (OR 3.91,95%CI1.20-16.01), age above 75 years (OR 3.27, 95%CI 1.12-9.70) and elevated leukocyte counts (OR 1.20, 95%CI 1.10-1.31, per 1000 cells/mm(3)) were identified as independent risk factors for HUS. A score using these variables has an area under the ROC curve of 0.74 (95%CI 0.68-0.80). Vomiting, visible blood in stools, higher leukocyte counts, and higher age indicate increased risk for developing HUS. A score using these variables might help to identify high risk patients who potentially benefit from aggressive pre-emptive treatment to prevent or mitigate the devastating consequences of HUS.

OBJECTIVES: Shiga-toxin producing O157:H7 Entero Haemorrhagic E. coli (STEC/EHEC) is one of the most common causes of Haemolytic Uraemic Syndrome (HUS) related to infectious haemorrhagic colitis. Nearly all recommendations on clinical management of EHEC infections refer to this strain. The 2011 outbreak in Northern Europe was the first to be caused by the serotype O104:H4. This EHEC strain was found to carry genetic features of Entero Aggregative E. coli (EAEC) and extended spectrum β lactamase (ESBL). We report symptoms and complications in patients at one of the most affected centres of the 2011 EHEC O104 outbreak in Northern Germany. METHODS: The courses of patients admitted to our hospital due to bloody diarrhoea with suspected EHEC O104 infection were recorded prospectively. These data include the patients' histories, clinical findings, and complications. RESULTS: EHEC O104 infection was confirmed in 61 patients (female = 37; mean age: 44±2 years). The frequency of HUS was 59% (36/61) in our cohort. An enteric colonisation with co-pathogens was found in 57%. Thirty-one (51%) patients were treated with plasma-separation/plasmapheresis, 16 (26%) with haemodialysis, and 7 (11%) with Eculizumab. Patients receiving antibiotic treatment (n = 37; 61%) experienced no apparent change in their clinical course. Twenty-six (43%) patients suffered from neurological symptoms. One 83-year-old patient died due to comorbidities after HUS was successfully treated. CONCLUSIONS: EHEC O104:H4 infections differ markedly from earlier reports on O157:H7 induced enterocolitis in regard to epidemiology, symptomatology, and frequency of complications. We recommend a standard of practice for clinical monitoring and support the renaming of EHEC O104:H4 syndrome as "EAHEC disease".

In atopic individuals, allergen-specific CD4+ T lymphocytes often belong to the T-helper 2 (Th2) subset as they secrete the marker cytokines interleukin-4 (IL-4) and IL-5 but not interferon-gamma (INF-gamma). IL-10 is a cytokine the production of which, in the mouse system has been described to be restricted to the Th2 subset, but in the human was found to be produced by both Th1 and Th2 T cell clones (TCC). We have recently shown that house dust mite antigen (Dermatophagoides pteronyssinus)-specific TCC isolated from skin of patients with atopic dermatitis have a more polarized Th2 cytokine production profile than TCC obtained from the peripheral blood of these patients. In this study, we report that skin-derived TCC secrete more IL-10, IL-4 and IL-5, than TCC isolated from the blood of the same individual (p < 0.05). The difference was more significant with specific TCC than with non-specific TCC. Furthermore, there was a positive correlation between the production of IL-10 and that of IL-4 and IL-5, respectively. In addition, the amount of IL-4 and IL-5 secreted by specific TCC from the skin correlated positively. These results were confirmed by the detection of mRNA by PCR. Finally, our data confirm that in human blood-derived TCC IL-10 secretion is not related to a particular cytokine production profile. We suggest that the skin of AD provides an unique environment for the development of aTh2-like secretion pattern not only with respect to IL-4 and IL-5 but also regarding IL-10.

To the Editor: Kirkwood et al 1 recently published the results of the intergroup trial E1694, designed to test the efficacy of the GMK ganglioside vaccine versus high-dose interferon alfa-2b (HDI) in malignant melanoma. Analyzing a heterogeneous population of high-risk patients (141 T4N0M0 patients with no lymph node surgery, 56 T4N0M0 patients with negative sentinel nodes, 315 patients with microscopic lymph node metastasis, and 306 patients with clinically detectable node metastasis), they found a minor overall survival benefit for HDI of 5% by 2 years (median duration of follow-up, 16 months). The recurrence-free survival rate was significantly improved by HDI treatment (13% by 2 years). However, only the subset of T4N0M0 patients benefited significantly from HDI treatment. As shown by studies dealing with sentinel-node dissection, 2,3 this group consists of two subpopulations with quite different prognosis: patients with occult micrometastases to their regional lymph nodes and patients with tumor-free regional nodes. The 3-year survival of sentinel-negative T4N0M0 patients was recently observed to be as high as 89.8%. 2 Thus, the inclusion of these patients in trial E1694 is questionable. Moreover, epidermal ulceration has been found to be a significant prognostic factor in T4N0M0 patients with thick primary tumors 2,3 as well as in node-positive patients. 4 Unfortunately, in trial E1684 this feature was not considered in the multivariate analysis. Thus, an alternative interpretation of trial E1684 could be that ulceration or occult regional lymph node involvement were not equally distributed among two therapy groups in the subsets of T4N0M0 patients because of low case numbers in this heterogeneous stratum. Therefore, it would be important to assess the frequency of recurrence to the regional lymph nodes in the T4N0M0 patients who did not receive a sentinel biopsy. If the T4N0M0 patients with HDI fared better because of a lower proportion of recurrences to their regional lymph nodes, a lower proportion of micrometastases to their sentinel nodes at the time of randomization is the most likely explanation. In T4N0M0 patients, the most frequent pathway of first recurrence is locoregional. Because skin and lymph node metastases can be easily treated by surgery with low morbidity, the effect of HDI treatment on the development of visceral metastases and on overall survival seems to be more important. Therefore, a longer duration of follow-up is mandatory to assess the real clinical benefit. In trial E1694, the survival benefit of HDI in node-negative patients was so strong that it outweighed the missing benefit in node-positive patients. Consequently, the authors considered HDI treatment significant for the whole study population. Regarding node-positive patients, some restrictions have to be made as both patients with micro- and macrometastasis were put together in the same strata. However, the clinical stage of the disease (micro- v macrometastases) has been shown to be an independent predictor of survival, besides the number of positive nodes. 5 As illustrated by our own findings, 6 the different time points of diagnosis in patients with micro- and macrometastases result in significantly different survival rates because of the so called lead time bias. In patients with delayed lymph node dissection, we observed an apparent survival benefit of 27.1% by 2 years if the survival rate as calculated from primary tumor excision (when micrometastases are expected to be present) was compared with the survival rate as calculated from the appearance of palpable nodes. Taken together, it would be interesting to know whether the overall survival benefit of HDI for the whole study population holds true in a multifactorial analysis taking into consideration clinical stage, ulceration, the number of positive nodes, Breslow thickness, age, and sex. In conclusion, mixing sentinel node‐negative patients and patients with unknown lymph node status as well as patients with micro- and macrometastasis in the same strata makes it difficult to draw conclusions. In future studies, the regional lymph nodes should be generally staged by sentinel-node biopsy. Moreover, the prognosis of sentinel-negative patients needs further exploration to answer the question if systemic therapies, associated with significant toxicity as HDI, are justified for these patients.

Please cite this paper as: Enhanced T-cell activation by immature dendritic cells loaded with HSP70-expressing heat-killed melanoma cells. Experimental Dermatology 2010; 19: 108–116. Abstract: Vaccination protocols that utilize dendritic cells (DCs) to elicit therapeutic immunity against tumors are the subject of intense research. Given that the capacity of DCs to cross-present antigens is physiologically low, there is considerable interest to develop strategies that enhance that pathway. In order to best exploit the enhanced cross-presentation of antigens bound to heat shock protein 70 (HSP70), we analysed melanoma cell preparations for their HSP70 expression. Western blotting revealed strong upregulation of HSP70 after heat-killing in contrast to UV-B irradiation. When the uptake of heat-killed necrotic cells by DCs at various levels of maturation was assessed, 61 ± 7% of immature DCs (iDCs) internalized fluorescence-labelled necrotic material. Apoptotic material from UV-B-irradiated cells was internalized by only 48 ± 5% of iDCs. Maturation-inducing cytokines did not affect the uptake when added simultaneously with the tumor cell preparations. Loading DCs with heat-necrotic or apoptotic melanoma cells slightly reduced CD83 expression while leaving CD208 (DC-LAMP) expression unchanged. As determined by IFN-γ-detecting enzyme-linked-immunospot assays, iDCs loaded with heat-killed melanoma cells activated autologous T cells most effectively when used without any further maturation, whereas DCs loaded with apoptotic material required maturation. In conclusion, HSP70-expressing melanoma cells could be generated by heat-killing. Loading iDCs with heat-killed melanoma cells resulted in a superior priming of autologous T cells in vitro.

Blastic plasmacytoid dendritic cell (BPDC) neoplasm, formerly called blastic natural killer cell lymphoma or CD4+/CD56+ hematodermic neoplasm, is a rare tumor entity, now regarded to be derived from the plasmacytoid dendritic cell (PDC) lineage. Because over 90% of patients present with skin lesions usually early in their disease, dermatologists have to be familiar with the specific diagnostic features and the clinical course of this devastating disease. We present a woman with a long standing solitary skin tumor of BPDC neoplasm, who experienced a deleterious clinical course, which is typical for this disease. Phenotypic and karyotypic characteristics distinguishing this tumor from myelomonocytic leukemia with skin involvement are presented.

Background Epidermolysis bullosa acquisita (EBA) is an autoimmune bullous disorder with mucocutaneous involvement, skin fragility, and tendency to scarring. The mechanobullous form of EBA has a chronic relapsing course and is difficult to treat. We describe herein the therapeutic response of 2 patients with recalcitrant mechanobullous EBA to combined treatment with immunoadsorption and rituximab, an anti-CD20 monoclonal antibody that induces depletion of B cells in vivo. Observations Two patients with mechanobullous EBA received combined treatment with immunoadsorption and rituximab, resulting in an almost complete clinical remission in one patient and stable disease in the other patient. In the patient with complete remission, prolonged B-cell depletion and clinical improvement with disappearance of mucocutaneous erosions paralleled the decline in titers of circulating anti–basement membrane zone autoantibodies. In the other patient, combined treatment with immunoadsorption and rituximab reduced the de novo appearance of blisters but did not lead to significant improvement of gingivitis, despite depleted B cells for 6 months that remained at 5% 12 months after the last administration of rituximab, as well as a reduction in autoantibody titers. Conclusion The patients' response suggests that combined treatment with immunoadsorption and rituximab may be a valuable adjuvant treatment regimen for severe mechanobullous EBA, which is in line with recently observed beneficial effects in inflammatory EBA.

In-transit metastasis is an important morbidity factor after sentinel lymphonodectomy (SLNE). So far, factors posing an increased risk after SLNE have not been adequately analyzed. Using Kaplan-Meier estimations and the Cox proportional hazards model, we analyzed the risk of developing in-transit metastases after SLNE for 328 consecutive patients (median tumor thickness, 2.0 mm; median follow-up period, 40 months). The 5-year probability of developing in-transit metastases as a first recurrence was 11.2%. After negative and positive SLNE, the probabilities were 6.3% and 24%, respectively. Patients in whom satellite metastases were excised concurrently with the primary tumor had a probability of recurrence with in-transit metastases of 41%. In sentinel lymph node (SLN)-negative patients with primary tumors having a thickness of more than 4 mm, the probability was 22.1%. Among the group of SLN-positive patients, significantly increased in-transit probabilities were observed in those with primary tumors that were thicker than 4 mm (41.8%), with tumors located on the distal extremities (42.1%), and with penetration of the nodal metastasis of >1 mm into the SLN (36%) and in patients with capsular breakthrough (63.3%). By using multifactorial analysis, the SLN status (P = .005), Breslow thickness (P = .0009), and extremity location of the primary melanoma (P = .005) significantly predicted the risk of in-transit recurrence. Satellite metastasis (P < .089), Clark level, and ulceration did not reach significance. Subgroups of patients can be identified who seem to have an increased risk of developing in-transit metastases as a first recurrence after SLNE. Individualized therapeutic strategies should be developed for these patients.

Individuals with the rare DNA repair deficiency syndrome xeroderma pigmentosum (XP) are sensitive to the sun and exhibit a 1000-fold increased risk for developing skin cancers, including cutaneous melanoma. Inherited polymorphisms of XP genes may contribute to subtle variations in DNA repair capacity and genetic susceptibility to melanoma. We investigated the role of three polymorphic alleles of the DNA repair gene XPC in a hospital-based case-control study of 294 Caucasian patients from Germany who had cutaneous melanoma and 375 healthy cancer-free sex-matched Caucasian control subjects from the same area. We confirmed that the XPC intron 9 PAT+, intron 11 -6A, and the exon 15 2920C polymorphisms are in a linkage disequilibrium. Only 1.6% of the 669 donors genotyped were discordant for these three polymorphisms. The allele frequencies (cases: controls) were for intron 9 PAT+ 41.7%:36.9%, for intron 11 -6A 41.8%:37.0% and for exon 15 2920C 41.3%:37.3%. Using multivariate logistic regression analyses to control for age, skin type and number of nevi, the three polymorphisms were significantly associated with increased risks of melanoma: OR 1.87 (95% CI: 1.10-3.19; P = 0.022), OR 1.83 (95% CI: 1.07-3.11; P = 0.026), and OR 1.82 (95% CI: 1.07-3.08; P = 0.026), respectively. Exploratory multivariate analyses of distinct subgroups revealed that these polymorphisms were associated with increased risks for the development of multiple primary melanomas (n = 28). The results of our case-control study support the hypothesis that the intron 9 PAT+, intron 11 -6A and exon 15 2920C haplotype may contribute to the risk of developing cutaneous melanoma by increasing the rate of an alternatively spliced XPC mRNA isoform that skips exon 12 and leads to reduced DNA repair. Our results should be validated in independent samples in order to guard against false positive findings.

Dermatomyositis/polymyositis (DM/PM) and Lambert-Eaton myasthenic syndrome (LEMS) are two autoimmune disorders that have very rarely been reported to occur together in the same patient. We report on two patients with DM who were later diagnosed with concomitant LEMS, and point out diagnostic challenges in identifying LEMS in patients with DM/PM. As specific treatment for LEMS is available, it is important to identify those DM/PM patients who suffer from concomitant LEMS.

The diagnostic and prognostic importance of recurrent chromosomal aberrations in systemic B cell lymphoma is well documented. In contrast, limited data exist on genetic changes in primary cutaneous B cell lymphoma. In this study we investigated chromosomal aberration patterns in two types of primary cutaneous B cell lymphoma with a different clinical behavior. Twenty-two primary cutaneous B cell lymphomas, including nine follicle center cell lymphomas and 13 large B cell lymphomas of the leg, were analyzed by comparative genomic hybridization and in part by fluorescence in situ hybridization. The most frequent imbalances detectable were gains in 18q (eight of 22), 1q (six of 22), 7 (six of 22), 12q (six of 22), or Xp (four of 22), and losses in 6q (four of 22). In contrast to large B cell lymphomas of the leg, primary cutaneous follicle center cell lymphomas had fewer imbalances and lacked translocations affecting the IGH locus. Gains in 18q (eight of 13) and losses in 6q (four of 13) as well as breakpoints within the IGH locus (six of 11) were restricted to the large B cell lymphomas of the leg subtype. Translocation t(14; 18) was excluded in 16 primary cutaneous B cell lymphomas of both subtypes that were studied by fluorescence in situ hybridization. These results suggest that primary cutaneous follicle center cell lymphoma and large B cell lymphoma of the leg are characterized by different chromosomal aberration patterns, which in part might determine the different clinical course of these malignancies.

The three peroxisome proliferator-activated receptor (PPAR) subtypes alpha, delta (beta), and gamma belong to the group of nuclear receptors that act as ligand-activated transcription factors. Recently, expression of PPAR alpha and gamma in keratinocytes has been demonstrated, and ligands of PPAR alpha and gamma have been found to enhance epidermal maturation and protect against cutaneous inflammation. There is evidence for a possible role of PPARs in psoriasis, as the expression of PPAR alpha and gamma is decreased in lesional skin and treatment with PPAR gamma agonists improves psoriatic keratinocyte pathology in vitro and in vivo. We performed a case-control study to search for possible associations between variations in the genes encoding PPAR alpha and gamma and psoriasis. Seven variations in these genes were analyzed in 192 patients with chronic plaque-type psoriasis and 330 healthy controls by PCR-based methods. No association between any of the investigated PPAR variants and psoriasis was found. Our findings argue against a significant contribution of the investigated PPAR variations to the genetic basis of psoriasis.

A 42-year-old man had a large speckled lentiginous nevus on the left side of his trunk. The involved area was painful when touched and paresthetic. Moreover, the ipsilateral half of his body showed a pronounced hyperhidrosis. This case can be categorized as a typical example of speckled lentiginous nevus syndrome, a recently recognized phenotype characterized by a speckled lentiginous nevus of the papular type and ipsilateral neurological abnormalities in the form of dysesthesia, muscular weakness or hyperhidrosis. Speckled lentiginous nevus syndrome represents a mosaic phenotype. Most likely it originates from loss of heterozygosity occurring in a heterozygous embryo at an early developmental stage.

We describe a new family with the rare genodermatosis keratosis punctata palmo-plantaris Buschke-Fischer-Brauer (keratoma disseminatum). In all, 3 family members in 3 generations were affected, a pattern consistent with autosomal dominant inheritance. Clinical symptoms started in the third decade with disseminated, small, round, hyperkeratotic papules on the palms and soles. Punctate keratoses coalesced into hyperkeratotic plaques on pressure points. Identification of additional families is necessary to permit definitive genetic classification of this genodermatosis.

BACKGROUND Dermabrasion is one approach to the treatment of treating giant melanocytic congenital nevi. Treatment is recommended to reduce the risk of spontaneous malignant transformation of giant nevi into malignant melanomas that usually occur in childhood. OBJECTIVE To describe the development of a multicentric malignant melanoma in a giant melanocytic congenital nevus after dermabrasion. METHODS We report about a 46-year-old male patient who developed a multicentric malignant melanoma in a giant melanocytic congenital nevus. The nevus was located on his left shoulder extending to his neck and chest. Previously, dermabrasion of the nevus was performed twice at the ages of 26 and 28. RESULTS To our knowledge, this is the first report of malignant transformation of a giant nevus into a multicentric malignant melanoma diagnosed 20 years after the procedure of dermabrasion. CONCLUSION We conclude that a close follow-up of such patients is mandatory.

Background: The factors controlling infiltration of inflammatory cells into atopic dermatitis (AD) lesions remain to be fully explored. Recently, epidermal cells in lesional AD were reported to contain increased messenger (m)RNA levels of IL-16, a cytokine that induces chemotactic responses in CD4 + T cells, monocytes, and eosinophils. Objectives: We sought to determine the expression of IL-16 in epidermal cells in normal skin and skin from AD lesions and to investigate whether Langerhans cell (LC)-derived IL-16 may contribute to the initiation of atopic eczema. Methods: The cutaneous expression of IL-16 was investigated by in situ hybridization and immunohistochemistry. Expression of IL-16 was also investigated in freshly isolated LCs and in keratinocytes by intracellular cytokine staining, quantitative real-time RT-PCR, and ELISA. Results: Low levels of IL-16 mRNA, but no stored IL-16 protein, were detected in keratinocytes and LCs isolated from normal skin. Synthesis, storage, and secretion of IL-16 could be induced in LCs, but not keratinocytes, by activation with phorbol ester and ionomycin. In normal skin (n = 10) neither keratinocytes nor LCs expressed IL-16. In contrast, IL-16 was contained in approximately 40% of CD1a + LCs in patients with active AD (n = 16). IL-16 expression in LCs in patients with AD correlated with the number of infiltrating CD4 + cells ( r = .72, P = .0017) and was completely downregulated parallel to the clinical response of AD lesions to topical treatment with FK506. Conclusion: LC-derived IL-16 may participate in the recruitment and activation of inflammatory cells in AD. (J Allergy Clin Immunol 2002;109:681-7.)

Recent studies of the cytokine pattern in skin lesions of patients with cutaneous T-cell lymphoma (CTCL) have shown that interleukin-4 (IL-4) and Il-10, both cytokines produced by T-helper type 2 cells, domi- nate in these lesions. Also, in single studies, interferon- A (IFN-A ), a major cytokine of Th-1-cells, has been found to be absent. Consequently, it has been hypothesized that immune-suppressive Th-2 cytokines may promote local growth of the malignant lymphocyte clone. How- ever, there is so far no evidence for T-cells as the source of the Th-2 cytokines in CTCL skin lesions nor have these cytokines been investigated at a clonal T-cell level. We established a total of 120 T-cell clones (TCCs) from lesional skin and 54 TCCs from the blood of four pa- tients with mycosis fungoides. Epidermal TCCs (mostly CD8-positive) and dermal TCCs (mostly CD4-positive) were stimulated by the mitogen concanavalin A and, seeking a polarized cytokine pattern, the supernatants were assessed by ELISA. We showed that the vast ma- jority of TCCs were able to secrete IFN-A and IL-4. IFN-A-deficient TCCs occurred only in the epidermis. Some (18) TCCs were found to be either negative for IL-10 production or to produce low levels only. No sig- nificant differences were observed between blood- and skin-derived TCCs. Thus a polarized Th-2 cytokine pattern was not detectable among cultured skin-infil- trating nonmalignant T-cells (TILs) isolated from early mycosis fungoides. It therefore appears unlikely that Th-2-mediated immune suppression is a major mecha- nism operating in early CTCL. However, this does not exclude its role in late-stage disease.

Environmental and genetic factors are thought to interact in the manifestation of psoriasis, but knowledge about the involved genes and antigens is incomplete. This study has focused on the association between psoriasis and inherited variations in xenobiotic metabolism and cytokine production as two components that may influence cutaneous immune responses to foreign substances. Polymorphisms of N-acetyltransferase 2, glutathione S-transferases T1 and M1, and promoter polymorphisms of the genes encoding for tumor necrosis factor-alpha and interleukin-10 were investigated in 151 Caucasian patients with psoriasis (100 with type I and 51 with type II psoriasis) and in 123 healthy controls. Polymorphisms were detected by polymerase chain reaction-based methods, restriction enzyme analysis, and direct sequencing. There were no significant differences in the distribution of enzyme polymorphisms or point mutations at position -1082 of the interleukin-10 promoter between the psoriasis groups and the control group. The G--A polymorphism at position -238 of the tumor necrosis factor-alpha promoter (TNF alpha-238*A allele) was more common in type I psoriasis (27%) than in the controls [9.8%; odds ratio 3.4 (95% confidence interval 1.6-7.2); p = 0.0012; pcorr = 0.018]. Surprisingly, this overrepresentation of the tumor necrosis factor alpha-238*A allele was observed in male patients [4.1 (1.5-11.0); p = 0.0046; pcorr = 0.064] but not in female patients [1.8 (0.5-6.5); p = 0.5]. The G--A polymorphism at position -308 of the tumor necrosis factor-alpha promoter was less frequent in type I psoriasis (23%) compared with controls (35.7%), although the negative association was weak [0.54 (0.3-0.97); p = 0.041; pcorr = not significant]. The distribution of the TNF alpha-238*A and TNF alpha-238*A alleles was similar in type II patients and controls. Our results suggest that male carriers of the G--A polymorphism at position -238 of the tumor necrosis factor-alpha promoter are at an increased risk to develop early-onset psoriasis.

Background: Specific T-cell responses to food antigens have been described in children withatopic dermatitis (AD). However, a subgroup of adolescent and adult patients still experiences food-responsive AD. Objective: This study was designed to evaluate lymphocyte responses to bovine casein in adultpatients with AD. Methods: The stimulatory capacity of lipopolysaccharide-depleted bovine casein was tested in proliferation assays and in limiting dilution assays. Casein-specific T-cell clones (TCCs) were generated and characterized. Results: Higher proliferative responses to casein and modulation of cytokine receptors wereobserved in patients with milk-responsive Ad compared with individuals without clinical reactions on milk ingestion. The results did not correlate with the amount of casein-specific IgE in the serum. The frequencies of T cells that grew in the presence of casein or house dust mite antigens were similar. Only 27% of CD4 + casein-specific TCCs from these patients, but the majority of house dust mite-specific TCCs, produced IL-4 on mitogen stimulation. Interferon-γ, on the other hand, was produced by the majority of TCCs with both specificities. Conclusion: A specific T-cell-mediated immune response to casein can be found in the blood of adolescent and adult patients with milk-related exacerbation of AD. In contrast to house dust mite-specific T cells, casein-specific T cells of adult patients who respond to cow's milk with worsening of AD produce little or no IL-4.

The mechanism underlying positive patch tests with house dust mite-allergen, Dermatophagoides pteronyssinus (Der p), in patients with atopic dermatitis was investigated by isolating T cells from the test sites of two patients. Eighty-five T cell clones (TCC) were established from the epidermis and dermis of lesional skin by the limiting-dilution method with Der p and interleukin (IL)-2. With restimulation assays, 29 of 60 TCCs tested demonstrated specific proliferation; 85% were of the CD3+, CD2+, and CD4+ phenotype. Der p-specific T cells constituted 0.4% to 2.7% of lesional T cells, and they were more frequent in the skin than in the blood of the patients by one order of magnitude. The mitogen-stimulated lymphokine profile of 55 TCCs was assessed; 42% (11/26) of the allergen-specific TCCs secreted IL-4 but almost no interferon-gamma, as described for the Th2 subset of the mouse. Also, six selected TCCs supported IgE secretion by autologous lymphocytes. Only three of 26 allergen-specific, skin-derived TCCs demonstrated a Th1-like lymphokine profile. These results support the specific nature of Der p-induced patch test lesions in patients with atopic dermatitis, and the results demonstrate also that a considerable proportion of lesional T cells are allergen-specific, IL-4-producing T cells that are capable of enhancing IgE production.

In patients suffering from primary cutaneous lymphomas, secondary malignancies of various origin may develop. However, the frequency of a second neoplasm deriving from another lymphoid lineage is still unclear and may be underestimated. We screened all our patients with primary cutaneous lymphomas from a 4-year recruitment period for a coexisting secondary lymphoproliferative disorder. The cohort comprised of a total of 82 patients with primary cutaneous lymphomas, 62 with primary cutaneous T-cell lymphoma (CTCL), 18 with primary cutaneous B-cell lymphomas, and two with CD4+/CD56+ hematodermic neoplasm/blastic lymphomas. Seven patients (8.5%) were identified with a coexisting lymphoma of a different lymphoid lineage. Four patients with Sezary syndrome (SS) suffered from systemic B-cell lymphoma. Two of these developed SS after chemotherapy of their B-cell lymphoma. The other three patients with various types of skin lymphomas (SS, Mycosis fungoides [MF], primary cutaneous marginal zone lymphoma) developed Hodgkin’s disease (hairy cell leukemia). Our data indicate that patients with primary cutaneous lymphomas have an elevated risk for the development of a secondary lymphoproliferative disorder even without previous chemotherapy. Possible explanations for this association include a genetic predisposition, alterations in early progenitor cells, underlying viral infections, and/or stimulation of a B-cell clone by the malignant helper T cells of the primary CTCL and vice versa.

Therapy related acute myelogenous leukemia (t-AML) is frequently observed among patients who undergo a high dose chemotherapy and its incidence varies between 5 to 15% in non-myeloablative and myeloablative treatment protocols. Deficiencies in individual DNA-repair systems seem to be involved in t-AML leukemogenesis since indirect genetic markers of impaired DNA repair, e.g. microsatellite instability (mismatch repair) and polymorphisms of XRCC genes (base/nucleotide excision repair), are frequent in these patients. To directly determine the activity of different DNA repair systems we developed a modified host cell reactivation (HCR) assay which is applicable for the analysis of primary hematopoietic cells of clinical samples. This assay determines the ability of transfected host cells to repair damaged plasmid DNA as reflected in the recovery of luciferase activity. Depending on the type of DNA damage introduced to the plasmid prior to transfection, different DNA repair systems can be analysed, e.g. nucleotide excision repair (NER) or double strand break repair. We performed the HCR assay using 75 to 250 ng luciferase reporter gene plasmid (pCMV-Luc). Lymphocytes and cord blood CD34+-progenitor cells from healthy donors were collected according to the convention of Helsinki. 200,000 to 2,000,000 cells were cryopreserved, thawed and transfected using DEAE-dextran at a concentration of 0,1 mg/ml in a transfection volume of 250 μl. We obtained luciferase activities of 350-fold above background in CD34+ progenitor cells (1000-fold in lymphocytes) rendering these cells applicable for DNA repair analysis. In addition, we evaluated the normalized NER capacity (1000 J/m2 UVC irradiated plasmid vs. unirradiated control) of lymphocytes and two AML cell lineages, Kasumi-1 and HL60. Kasumi-1 and HL60 cells exhibited a significantly reduced NER capacity compared to lymphocytes (6.15% +/− 1.57% and 6.5% +/− 1.59% vs. 12.3% +/− 3.2%). Clinical AML samples are currently been investigated. Our modified HCR can be used for functional DNA repair analysis in fresh and cryopreserved patient samples of pre- and post-leukemic conditions as well as in leukemic blasts to elucidate the role of defective DNA repair during t-AML leukemogenesis. Furthermore, the modified HCR may also be used to determine the individual susceptibility for therapy related myeloid leukemia prior to chemotherapy.