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Im Fruhsommer 2011 wurde Norddeutschland vom bis dato grosten Ausbruch einer Infektion mit EHEC O104:H4 heimgesucht. Im Unterschied zu fruheren EHEC-Ausbruchen betraf die Erkrankung vorwiegend junge Erwachsene und fuhrte in unserem Kollektiv bei 72% unserer 61 hospitalisierten Patienten zu teils schwerwiegenden Komplikationen. Aufgrund der genetischen und klinischen Eigenstandigkeit der Infektion schliesen wir uns dem Vorschlag der von Brzuszkiewicz et al. an, die Erkrankung als EAHEC-Syndrom („Entero-Aggregative-Haemorrhagic Escherichia coli“) zu bezeichnen. Die Wechselhaftigkeit und Vielfalt der Verlaufe zwingt zu einem engmaschigen klinischen Monitoring; wir schlagen den „Altona EAHEC Monitoring Standard“ vor. Einen ungunstigen Einfluss der Gabe von Antibiotika auf den Krankheitsverlauf konnten wir – im Gegensatz zu fruher publizierten Bedenken – nicht beobachten.

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.

Chronic venous leg ulcers are common and cause considerable burden of disease for affected patients with significant costs for health care systems worldwide. The complex pathophysiology of chronic venous leg ulcers is still not entirely understood. In addition, reliable pathogenic and/or prognostic parameters are not known. Published data suggest that patients with chronic venous leg ulcers reveal congenital or acquired thrombophilia. We examined the serum Lipoprotein (a) [Lp(a)] level, a proatherogenic and prothrombotic risk factor, in patients with chronic venous leg ulcers (n=210, stratified into patients with postthrombotic syndrome or without) and in a healthy control group (n=341). Forty-two percent of all patients, compared with 20% of healthy controls, revealed significantly increased Lp(a) serum concentrations above 0.3 g/L. Furthermore, 49% without postthrombotic syndrome but only 35% with postthrombotic syndrome showed increased Lp(a) levels. The increase of Lp(a) level was significantly different between all three groups (p

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.

Zusammenfassung Hintergrund: Im Vergleich zur alleinigen Vitalfarbung der Lymphabflusswege bei der Sentinel-Lymphknotendissektion hat die intraoperative Anwendung einer Gamma-Detektionssonde die Identifikationsraten verbessert. In der vorliegenden retrospektiven Studie wird der Einfluss der Gamma-Detektion hinsichtlich weiterer Aspekte ausfuhrlich analysiert. Patienten und Methodik: 81 Patienten, bei denen zur intraoperativen Detektion des Sentinel-Lymphknotens (SLK) ausschlieslich Patentblau zum Einsatz kam, wurden mit 247 Patienten verglichen, bei denen zusatzlich eine Gamma-Sonde verwendet wurde. Ergebnisse: Die Einfuhrung der Gamma-Sonde erbrachte eine Steigerung der SLK-Detektionsrate von 87,7 % auf 99,2 % (P

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.

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.

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.

Wir berichten uber ein Madchen mit auffallend vielen grosknotigen kongenitalen Xanthogranulomen. Die eineiige Zwillingsschwester war erscheinungsfrei. Die bis zu 1,5 cm grosen, halbkugeligen am Kopf und der oberen Korperhalfte lokalisierten Tumoren waren bei Geburt vorhanden und bestanden aus CD68+ histiozytaren Non-Langerhanszellen. In einem Nachbeobachtungszeitraum von 18 Monaten traten keine neuen Tumoren auf, die bestehenden Tumoren zeigten Regression. Extrakutane Manifestationen bestanden nicht. Die differentialdiagnostische Abgrenzung vor allem gegenuber Langerhanszell-Histiozytosen ist unerlaslich zur richtigen Einschatzung der Prognose.

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.

Erosive mucosal lichen planus is a painful and disabling inflammatory skin disease that is highly resistant to topical treatment. We report on six patients with severe recalcitrant erosive mucosal lichen planus who benefited from topical application of tacrolimus ointment. After 4 weeks of treatment, complete resolution was observed in three cases, and substantial improvement was achieved in the other three patients. In these cases, prolonged treatment resulted either in further improvement or in complete healing. All patients reported rapid relief from pain and burning. No severe side-effects were observed.

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.

Glomustumoren (Glomangiome) sind gutartige, von den Glomuszellen ausgehende Tumoren. Multiple Glomangiome sind seltener und weniger schmerzhaft als die meist subungual lokalisierten, solitaren Glomangiome. Sie konnen aber bei Druckeinwirkung und Temperaturwechsel Beschwerden bereiten. Wegen ihrer Vielzahl stellen die multiplen Glomangiome ein therapeutisches Problem dar. Die Sklerosierung bietet eine therapeutische Alternative bzw. Erganzung zur operativen Entfernung oder zu kryochirurgischen Verfahren. Wir berichten uber die Sklerosierungstherapie bei einer 35jahrigen Patientin mit multiplen familiaren Glomangiomen.

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.

Summary A reliable and early diagnosis of viral infections and rejection episodes is a major goal of immunologic monitoring in transplantation. Soluble markers like the cytokines, neopterin or serum amyloid A are frequently recommended as diagnostic parameters. However they are not often used routinely in transplant medicine. The study investigates the diagnostic value of plasma (P), serum (S), and urine (U) levels of neopterin (S-/U-NEOP), serum amyloid A (SAA), tumor necrosis factor-α, (P-/U-TNF-α), soluble interleukin-2 receptor (P-/U -sIL-2R), and interleukin-6 (U -IL-6) in transplant monitoring and describes an approach for their use in the clinical routine decision making. In 29 renal transplant patients blood and urine samples were collected daily during the posttransplant course on ward. The cytokines were measured by ELISAs, neopterin by RIA, and SAA by immunonephelometry. Descriptive statistics, sensitivity and specificity, day of first significant parameter increase/decrease, receiver operating characteristic curves, and post-test probabilities were calculated for each parameter. 12 acute rejection episodes were diagnosed. As rejection markers, S-NEOP and P-TNF-α had the highest sensitivity, U-IL-6 the highest specificity. 8 viral infections occurred. U-NEOP showed values higher than 1000 μmol/moICrea. It did not exceed this threshold in case of rejection episodes. SAA and U-IL-6 showed peak. levels during rejections but not during episodes of viral infections. Using the calculated likelihood ratio formulas the probability for the occurrence of an acute rejection could be estimated for the individual, daily parameter measurement. Adding the physician IS rating for rejection posttest probabilities were computed. Immunologic monitoring is possible in transplant medicine. Using daily measurements of serum amyloid A and neopterin facilitates the differential diagnosis of acute rejection episodes and viral infections. The likelihood ratio approach permits an application of the parameter monitoring in the clinical routine.

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.

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".

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.

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.

Background: Maturation of dendritic cells (DCs) influences important DC functions such as production of cytokines. Recently, DCs were identified as a source of interleukin-16 (IL-16), a chemotactic factor for DCs themselves, CD4+ T cells, and eosinophils. There is evidence that DC-derived IL-16 may contribute to the pathogenesis of atopic dermatitis (AD). Objective: To investigate the production of IL-16 during differentiation of monocytes into DCs in healthy individuals and patients with AD. Methods: IL-16 production was investigated by quantitative real-time RT-PCR, intracellular cytokine staining, immunoblotting, and ELISA. Results: DCs generated from peripheral monocytes by 5-day culture in the presence of IL-4 and granulocyte/macrophage colony-stimulating factor acquired the capability to synthesize, store, and secrete IL-16. Storage and release of IL-16 was further enhanced during final DC maturation induced by additional 3-day culture with tumor necrosis factor-α (TNF-α) and monocyte-conditioned medium. Maturation, as determined by up-regulation of CD83 and CD86 surface expression, and production of IL-16, but not production of IL-10 and IL-12p40 was impaired in day 8 DCs derived from AD patients compared to those from healthy donors. Stimulation of day 8 DCs from AD patients with TNF-α and IL-1β enhanced the expression of CD83 and CD86 and restored the production of IL-16. Conclusions: Signals involved in the activation and maturation of DCs enhance their capacity to produce IL-16. Functional abnormalities present in patients with AD at the monocyte level may account for impaired maturation and IL-16 production of monocyte-derived DCs.

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.

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.

The analysis of cytokine profiles plays a central part in the characterization of disease-related inflammatory pathways and the identification of functional properties of immune cell subpopulations. Because tissue biopsy samples are too small to allow the detection of cytokine protein, the detection of mRNA by RT-PCR analysis is often used to investigate the cytokine milieu in inflammatory lesions. RT-PCR itself is a qualitative method, indicating the presence or absence of specific transcripts. With the use of internal or external standards it may also serve as a quantitative method. The most widely accepted method is quantitative competitive RT-PCR, based on internal shortened standards. Recently, online real-time PCR has been introduced (LightCycler), which allows quantitation in less than 30 min. Here, we have tested its use for the analysis of cytokine gene expression in different experimental in vitro and ex vivo settings. First, we compared quantitative competitive RT-PCR with real-time RT-PCR in the quantitation of transcription levels of the CD4(+) cell-specific chemoattractant Interleukin-16 during the maturation of monocyte-derived dendritic cells, and found a good correlation between both methods. Second, differences in the amounts of IL-16 mRNA in synovial tissue from patients with rheumatoid arthritis and osteoarthritis as assessed by real-time RT-PCR paralleled differences in the level of IL-16 protein in the synovial fluid. Finally, we employed real-time RT-PCR to study the cutaneous expression of several cytokines during experimental immunomodulatory therapy of psoriasis by Interleukin-10, and demonstrate that the technique is suitable for pharmacogenomic monitoring. In summary, real-time RT-PCR is a sensitive and rapid tool for quantifying mRNA expression even with small quantities of tissue. The results obtained do not differ from those generated by quantitative competitive RT-PCR.

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.

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.

Objective To evaluate the effect of different treatment strategies on enterohaemorrhagic Escherichia coli O104:H4 induced haemolytic uraemic syndrome. Design Multicentre retrospective case-control study. Setting 23 hospitals in northern Germany. Participants 298 adults with enterohaemorrhagic E coli induced haemolytic uraemic syndrome. Main outcome measures Dialysis, seizures, mechanical ventilation, abdominal surgery owing to perforation of the bowel or bowel necrosis, and death. Results 160 of the 298 patients (54%) temporarily required dialysis, with only three needing treatment long term. 37 patients (12%) had seizures, 54 (18%) required mechanical ventilation, and 12 (4%) died. No clear benefit was found from use of plasmapheresis or plasmapheresis with glucocorticoids. 67 of the patients were treated with eculizumab, a monoclonal antibody directed against the complement cascade. No short term benefit was detected that could be attributed to this treatment. 52 patients in one centre that used a strategy of aggressive treatment with combined antibiotics had fewer seizures (2% v 15%, P=0.03),fewer deaths (0% v 5%, p=0.029), required no abdominal surgery, and excreted E coli for a shorter duration. Conclusions Enterohaemorrhagic E coli induced haemolytic uraemic syndrome is a severe self limiting acute condition. Our findings question the benefit of eculizumab and of plasmapheresis with or without glucocorticoids. Patients with established haemolytic uraemic syndrome seemed to benefit from antibiotic treatment and this should be investigated in a controlled trial.

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.