Flow cytometric analysis of cellular infiltrate from American tegumentary leishmaniasis lesions

BACKGROUND: CD4+ and CD8+ T lymphocytes play different roles in the outcome of leishmaniasis. However, T-cell distribution in lesions shows significant variability in in situ immunocytochemical studies. OBJECTIVES: In this report flow cytometry was used to determine the predominant T-cell subsets in leishmaniasis lesions, and their relationship with Leishmania-responsive circulating T cells. PATIENTS AND METHODS: Mononuclear cells from lesions or peripheral blood (PBMC) of 34 cutaneous (CL), four mucosal (ML) and four disseminated leishmaniasis were phenotypically characterized by flow cytometry. Leishmania-responsive T cells were obtained after in vitro stimulation of PBMC with leishmanial antigens. RESULTS/CONCLUSIONS: Variable amounts of gammadelta lymphocytes were present in all lesions, with no association with duration of illness. The highest percentages of interleukin-2R- and interferon-gammaR-positive cells were observed in ML lesions and could render these T cells more susceptible to the effects of these cytokines. The distribution of intralesional T-lymphocyte subsets was quite variable (CD4+ > CD8+ = 18 cases, CD8+ > CD4+ = 12 cases and CD4+ congruent with CD8+ = 4 cases) without any association with clinical parameters, and could explain the controversy regarding proportions of these T-cell subsets in leishmaniasis lesions. Low percentages of Leishmania-reactive CD8+ T cells were observed in blood while an enrichment of CD8+ cells was shown in the inflammatory infiltrate, suggesting that local immunoregulatory factors could favour the recruitment and/or proliferation of local CD8+ lymphocytes. Increased percentages of CD8+ cells observed in older lesions are consistent with the hypothesis that they can mediate healing, although their involvement in tissue damage cannot be ruled out. It is possible that these mechanisms can influence the clinical outcome or even the response to therapy.     

Serotonin transporter protein overexpression and association to Th 17 and T regulatory cells in lupoid leishmaniasis

The immunopathogenesis of chronic non-healing Old World cutaneous leishmaniasis is challenging. There is a bidirectional communication between the nervous and immune systems, serotonin being an important mediator in this respect. Our aim was to study the role of the serotonin transporter protein (SERT) and its relation to T cell-related immune responses in lupoid leishmaniasis. Paraffin-embedded skin biopsies of 12 cases of lupoid and 12 cases of usual types of cutaneous leishmaniasis were investigated using immunohistochemistry regarding expression of SERT, Th1, Th2, Th17 and T regulatory cell (Treg) markers. SERT as well as Tregs and interleukin (IL)-17 positive cells were more prevalent while IL-5 (Th2) and interferon (IFN)-γ (Th1) expressing cells were less numerous in the lupoid tissue compared to those from the usual type of leishmaniasis. The majority of the SERT⁺ cells were also tryptase⁺ (mast cells). There was a positive correlation between a higher number of SERT⁺ and IL-17⁺ cells in the lupoid type, while lower numbers of SERT⁺ cells were significantly related to lower percentages of CD25⁺ cells in the usual type of leishmaniasis. These results might indicate a role for SERT, Th17 and Tregs in the pathogenesis of lupoid leishmaniasis.     

Evidence of in situ cytotoxicity in American cutaneous leishmaniasis

The role of cytotoxicity in the defense mechanisms or pathogenesis of human cutaneous leishmaniasis is not yet well known. In the present work we assessed the presence of NK, CD8+ and CD45RO+ T cells, as well as the expression of a molecule associated with cytotoxic properties (TIA-1) in the lesions of cutaneous leishmaniasis. CD8+ T cells, NK and activated T cells were found within the dermal cell infiltrate. We found a heterogeneous but usually strong expression of TIA-1, a marker of cytotoxic granules of T and NK cells, in human cutaneous leishmaniasis lesions. These data suggest that cytotoxic activity occurs in situ in American cutaneous leishmaniasis and that both NK cells and activated CD8+ T cells are involved in this reaction.     

Intermediate or chronic cutaneous leishmaniasis: leukocyte immunophenotypes and cytokine characterisation of the lesion

The American cutaneous forms of leishmaniasis include immune-responder individuals with localised cutaneous leishmaniasis (LCL) and non-responder individuals with diffuse cutaneous leishmaniasis (DCL). Patients with intermediate or chronic cutaneous leishmaniasis (ICL) have increased morbidity due to the length of their illness, atypical forms and areas of compromise. In the present study, we evaluated the expression of the leukocyte antigens (CD4, CD8, CLA: cutaneous lymphocyte antigen, CD69, CD83 and CD1a) and cytokines (IFN-gamma, IL-4, IL-10 and TGF-beta 1) in the lesions of patients with ICL (n = 18) using an immunocytochemical procedure. ICL results were compared with the information for LCL (n = 19) and DCL (n = 4). The numbers of CD4+ and CD8+ T cells in ICL were similar to those of LCL lesions, but significantly different (P < or = 0.05) from DCL lesions. LCL lesions have about half the numbers of early activated CD69+ cells as ICL, but most are CLA+ skin homing memory T cells, whereas ICL lesions have the highest number of CD69+ T cells, but about one-third of these cells expressed CLA. This suggests that the granuloma of ICL patients contains many activated T cells that are unprimed to cutaneous-launched antigens, thus contributing to an aberrant immune response. In contrast, DCL granulomas presented the lowest numbers of activated CD69+ and CLA+ cells, associated with the characteristic tolerogenic state of these patients. The immunolocalisation of cytokines showed a mixed cytokine pattern in ICL lesions with many positive cells for IL-10, TGF-beta 1, IL-4 and IFN-gamma, with a preponderance of the first two, and different from the prevalent Th1 and Th2 responses associated with LCL and DCL lesions, respectively. CD1a+ Langerhans cells were decreased (P < or = 0.05) in both ICL (271 +/- 15 cells/mm2) and DCL (245 +/- 19 cells/mm2) as compared to LCL (527 +/- 54 cells/mm2) epidermis. The percentage of IL-10+ epidermal Langerhans cells in ICL (33.69), from the total CD1a+ population, was higher than in LCL (17.45). In addition, fewer CD83+ primed Langerhans cells were present in ICL epidermis. The diminished participation of epidermal Langerhans cells, causing a defective signalling by the epidermis, in ICL lesions may account for the tissue-damaging state observed in these patients.     

Comparative study of cutaneous leishmaniasis in human immunodeficiency virus (HIV)-infected patients and non-HIV-infected patients in French Guiana

BACKGROUND: Few data are available on cutaneous leishmaniasis caused by dermotropic species in human immunodeficiency virus (HIV)-infected patients. OBJECTIVES: To describe nine cases of cutaneous leishmaniasis in HIV+ patients and to compare their clinical features and their response to treatment with those of HIV- patients with the forms of leishmaniasis commonly found in French Guiana. METHODS: A case-control study was carried out between July 1994 and December 2000 in French Guiana. We compared the following variables in nine HIV-infected patients with leishmaniasis and 27 matched controls: clinical type of leishmaniasis, number of lesions, presence of lymphangitis and adenopathy, the rate of recovery after treatment, and recurrence or reinfection. RESULTS: Eight of the HIV-infected patients had localized cutaneous leishmaniasis and one had mucocutaneous leishmaniasis. All of the controls had localized cutaneous leishmaniasis. Leishmania guyanensis was the only species isolated from HIV-infected subjects. HIV-Leishmania coinfected patients had a higher rate of recurrence or reinfection (P < 0.02) and a lower rate of recovery after one treatment cycle with pentamidine (P < 0.02) than did HIV- subjects. The CD4+ lymphocyte counts exceeded 200 mm(-3) in all HIV+ patients at the time of the diagnosis with leishmaniasis. CONCLUSIONS: In French Guiana, cutaneous leishmaniasis in moderately immunosuppressed HIV-infected subjects (> 200 CD4+ T cells mm(-3)) is characterized by a higher rate of recurrence or reinfection and is more difficult to treat than that in HIV- subjects.     

Primary Cutaneous CD8+ CD30+ Anaplastic Large Cell Lymphoma: An Unusual Case with a High Ki-67 index—A Short Review

Primary cutaneous anaplastic large cell lymphoma (PCALCL) is a part of the spectrum of CD30⁺ cutaneous lymphoproliferative disorder, characterized by variable degrees of CD2, CD3, CD4 and CD5 expression by lymphoid cells. PCALCLs with an expression of cytotoxic phenotype (CD8⁺) and cytotoxic proteins are uncommon. Cutaneous CD8⁺ CD30⁺ lymphoproliferative lesions are difficult to classify, diagnose and may be the cause of misdiagnose. CD8⁺ PCALCL must be distinguished from CD8⁺ mycosis fungoides, lymphomatoid papulosis type D and primary cutaneous aggressive epidermotropic CD8⁺ T-cell lymphoma. Usually CD8⁺ PCALCL is an indolent disease with a favorable prognosis, except few cases can show poor outcomes. The high Ki-67 index points toward advanced PCALCL. Treatment modalities include surgical excision, radiotherapy and clinical monitoring. Chemotherapy is reserved for disseminated disease. We report a 59-year-old male presented with rapid development of multiple painful reddish-brown plaques and nodular ulcerative skin lesions over the left thigh region since 2 months. A diagnosis of CD8⁺ PCALCL with a high Ki-67 index was made on the basis of histology and immunohistochemistry, in co-relation with clinical presentation.     

In situ depletion of CD4<Superscript>+</Superscript> T cells in human skin by Zanolimumab

CD4⁺ T cells, in activated or malignant form, are involved in a number of diseases including inflammatory skin diseases such as psoriasis, and T cell lymphomas such as the majority of cutaneous T cell lymphomas (CTCL). Targeting CD4 with an antibody that inhibits and/or eliminates disease-driving T cells in situ may therefore be a useful approach in the treatment of inflammatory and malignant skin diseases. Depletion of CD4⁺ T cells in intact inflamed human skin tissue by Zanolimumab, a fully human therapeutic monoclonal antibody (IgG1, κ) against CD4, was studied in a human psoriasis xenograft mouse model. Zanolimumab treatment was shown to induce a significant reduction in the numbers of inflammatory mononuclear cells in upper dermis. This reduction in inflammatory mononuclear cells in situ was primarily due to a significant reduction in the numbers of skin-infiltrating CD4⁺, but not CD8⁺ CD3⁺ T cells. The capacity of Zanolimumab to deplete the CD4⁺ T cells in the skin may be of importance in diseases where CD4⁺ T cells play a central role. Indeed, in a phase II clinical trial Zanolimumab has shown a dose-dependent clinical response in patients with CTCL and the antibody is currently in a phase III clinical trial for CTCL, a disease for which there is no safe and effective treatment available today.     

OX40+ T lymphocytes and IFN-γ are associated with American tegumentary leishmaniasis pathogenesis

BACKGROUND

Leishmaniases are zoonoses considered a public health problem, representing a complex group of diseases with a broad clinical spectrum and epidemiological diversity. Leishmaniasis is caused by several species of protozoa of the genus Leishmania. The evolution of the pathology and the resolution of the leishmaniasis are dependent mainly on the Leishmania species involved, although the cytokine profile plays an important role in the development of the immune response.

OBJECTIVES

The purpose of our study was to evaluate the immune response of patients affected by lesions of cutaneous leishmaniasis by immunostaining of the OX40, CD20, IFN-γ and IL-4 proteins.

METHODS

The tissue samples were collected from indolent skin ulcers confirmed as cutaneous leishmaniasis of 41 patients aged between six and 90 years. The lesions were submitted to OX40, CD20, INF-γ and IL-4 immuno-labeling.

RESULTS

We observed a statistically significant higher expression of IFN-γ compared with IL-4 (p=0.009). Besides, OX40 had higher expression when compared with CD20 (p<0.001).

CONCLUSION

The present study indicates that the immune response in lesions of cutaneous leishmaniasis is associated with a healing process, which can be explained by the higher expression of IFN-γ when compared with IL4 protein levels.     

Unusual manifestations of tegumentary leishmaniasis in AIDS patients from the New World

Background  Comorbidity from tegumentary leishmaniasis and AIDS is poorly characterized.
Objectives  To describe a series of patients coinfected with Leishmania and human immunodeficiency virus (HIV).
Methods  Clinical records from patients were analysed by demographic data, clinical manifestations, diagnoses, treatments and outcomes.
Results  Fifteen cases of AIDS/tegumentary leishmaniasis were found. The diagnosis of leishmaniasis was confirmed by the detection of Leishmania amastigotes or antigens from the cutaneous or mucosal lesions. The mean CD4+ T-cell count was 84 cells mm⁻³ (range 8–258) and all patients were classified as having AIDS according to the Centers for Disease Control and Prevention. A wide range of manifestations was found, varying from a single ulcer to multiple and polymorphic lesions. Mucosal lesions were present in 80% and cutaneous lesions in 73% of patients (53% with mucocutaneous form), disseminated lesions in 60% and genital lesions in 27% of patients. All patients received anti- Leishmania therapy and 53% showed relapses. Sixty-seven per cent received highly active antiretroviral therapy but showed no difference in outcomes and relapses compared with those not using medication. Forty per cent died during the study period. In these patients, the anti- Leishmania antibody and Montenegro skin test were useful in the diagnosis of leishmaniasis, probably because leishmaniasis preceded immunosuppression due to HIV infection.
Conclusions  Clinical manifestations of tegumentary leishmaniasis in HIV-infected patients are diverse. Our data emphasize possible unusual manifestations of this disease in HIV-infected patients, particularly in severely immunosuppressed cases (< 200 CD4+ cells mm⁻³).     

Lymphomatoid papulosis in association with mycosis fungoides: A clinical and histopathologic review of five Taiwanese cases

Background/ObjectivesLymphomatoid papulosis (LyP) is a cutaneous CD30+ lymphoproliferative disorder characterized by recurrent, self-healing lesions with a chronic clinical course. Approximately 10–20% of the patients have lymphomas, including mycosis fungoides (MF). LyP in association with MF is not well documented in Taiwan. We aimed to describe the clinicopathologic characteristics of LyP with MF in a Taiwanese case series of LyP.MethodsA retrospective clinicopathologic study was performed on cases of LyP with MF diagnosed in our Department during the period 1990–2012. The diagnosis of LyP and MF were based on their characteristic clinical and pathologic features as well as correlation with the clinical course of the specific skin lesions.ResultsA total of 24 cases of LyP (10 males and 14 females, age 18–63 years, mean 40.4 years) were included. Multiple biopsies were often done in individual patients during the clinical course to establish the diagnosis of LyP and MF. LyP was further classified pathologically as type A (n = 16), B (n = 3), C (n = 3), and mixed type with A&B (n = 1) and A&C (n = 1). Five cases (21%) also had MF; two had juvenile-onset LyP and three had juvenile-onset MF (one with hypopigmented MF, one with hyperpigmented MF, two with CD8+ LyP, and two with CD8+ MF). In the case of juvenile-onset hypopigmented CD8+ MF, the patient developed CD8+ LyP 25 years after the onset of MF and died of aggressive epidermotropic CD8+ lymphoma involving the skin and lung.ConclusionMF occurred in five of the 24 cases (21%) in the present series of LyP. These five cases had several unusual clinical and pathologic features, including subtle or uncommon skin manifestation of MF and more frequent juvenile-onset and CD8 phenotype of LyP and/or MF lesions. Long-term follow-up and repeated biopsy of selected skin lesions are necessary for correct diagnosis and proper treatment of both diseases.     

Treatment of cutaneous leishmaniasis with thermotherapy in Brazil: an efficacy and safety study

Background:Pentavalent antimonials remain as the standard drugs in the treatment of cutaneous leishmaniosis. The high cost, difficult administration, long treatment time, toxicity and increasing morbidity are factors that limit the use of these drugs.Objectives:To describe the response to radiofrequency thermotherapy in the treatment of localized cutaneous leishmaniasis in Brazil, and to evaluate its safety and tolerability.Methods:We conducted a non-comparative open trial with a total of 15 patients confirmed to have cutaneous leishmaniasis on parasitological examination. A single radiofrequency thermotherapy session at 50°C for 30 seconds was applied to the lesion and its edges. In patients with more than one lesion, only the largest one was treated initially. If after 30 days there was no evidence of healing, the smaller lesion was also treated with thermotherapy. Clinical cure was defined as visible healing for three months after treatment. The patients were followed-up for six months and there was no follow-up loss.Results:Of all 23 lesions, only two evolved to complete healing without the need of treatment. Of 21 lesions, 18 (85.7%) achieved full healing. The main observed side effects were itching, burning sensation, pain and blisters.Study limitations:Sample with a small number of patients and short follow-up.Conclusion:Thermotherapy can be considered a therapeutic alternative in localized cutaneous leishmaniasis, especially in cases of single cutaneous lesions and with formal contraindications to conventional treatment with pentavalent antimonials.     

Brote de leishmaniasis cutánea en el municipio de Fuenlabrada

IntroductionLeishmaniasis, an endemic infection in Spain, is caused by protozoan parasites of the Leishmania genus. Between 2010 and 2012, there was an outbreak of cutaneous and visceral leishmaniasis in Fuenlabrada, Madrid.ObjectivesTo describe the cases of cutaneous leishmaniasis diagnosed over a 17-month period at the dermatology department of Hospital de Fuenlabrada.Material and methodsWe analyzed the epidemiological, clinical, histological, and microbiological features of each case and also evaluated the treatments administered and outcomes.ResultsWe studied 149 cases. The incidence of cutaneous leishmaniasis showed a peak in the age range between 46 and 60 years and was similar in men and women. At the time of consultation, the lesions had been present for between 2 and 6 months in the majority of patients. The most common clinical presentation was with erythematous plaques and papules without crusts (52% of cases). Lesions were most often located in sun-exposed areas and were multiple in 57% of patients. In 67% of cases, the histological study showed non-necrotizing granulomatous dermatitis with no evidence of parasites using conventional staining methods. Diagnosis was confirmed by polymerase chain reaction (PCR) in 98% of patients. In the remaining cases, the histological study revealed Leishman-Donovan bodies in the skin. Intralesional pentavalent antimonials were the most commonly used drugs (76% of cases) and produced satisfactory results.ConclusionsWe have presented a large series of cases of cutaneous leishmaniasis diagnosed in the context of an outbreak. Multiple papules were the most common clinical presentation, with histology that showed non-necrotizing granulomatous dermatitis with no evidence of parasites. PCR of skin samples was the test that most frequently provided the diagnosis.     

Co-culture of healthy human keratinocytes and T-cells promotes keratinocyte chemokine production and RORγt-positive IL-17 producing T-cell populations

BackgroundBoth keratinocytes and T-cells are crucial players in cutaneous immune responses. We hypothesized that direct interactions between keratinocytes and T-cell subsets could shape the nature or strength of the local immune response.ObjectiveWe investigated direct interactions between keratinocytes and T-cell subsets, focused on keratinocyte chemokine production and T-cell phenotype and cytokine production.MethodsA newly developed in vitro serum free co-culture model using primary keratinocytes and T-cells subsets from healthy human donors was used. Keratinocyte chemokine production was analyzed with luminex, T-cell phenotype and cytokine production were analyzed with flow cytometry.ResultsOur data show that upon co-culture with CD4^pos or CD8^pos T-cells primary human keratinocytes increased production of functionally active chemokines CCL2, CCL20 and CXCL10 and that regulatory T-cells did not regulate keratinocyte chemokine production. Next to that, we found that keratinocytes skewed CD4^pos and CD8^pos T-cell populations toward an IL-17^pos CCR6^pos RORγt^pos phenotype in a cell–cell contact independent manner, and that Treg were able to decrease the absolute number of IL-17 producing T-cells in keratinocyte/T-cell co-cultures. Correspondingly, freshly isolated skin-derived T-cell populations contained relatively high percentages of IL-17^pos cells.ConclusionWe provide evidence that keratinocyte/T-cell communication may regulate leukocyte influx in the skin, and that keratinocytes enrich T-cell populations for Th17/Tc17 cells. Accumulation of Th17/Tc17 cells, but not chemokine production, appears under the control of regulatory T-cells. Dysregulation of these processes may well contribute to the pathophysiology of inflammatory skin diseases.     

Erfahrungen mit der kutanen Leishmaniasis

Background

The German S1 guidelines from 2009 contain a variety of recommendations for the treatment of cutaneous leishmaniasis.

Patients and methods

We report the results of our diagnostic procedures and treatment of 32 international patients in autumn 2010 in northern Afghanistan.

Results

Giemsa stain confirmed the clinical diagnosis within 24 hours. Eleven simple lesions and one larger ulcer responded well to cryotherapy and intralesional sodium stibogluconate. More complex lesions in 19 patients responded well to oral miltefosine. One patient refused outpatient therapy.

Conclusions

Cryotherapy and intralesional antimony compounds showed good results in early lesions of cutaneous leishmaniasis in northern Afghanistan. Outpatient treatment of complex lesions with miltefosine was successful in all cases.     

银屑病皮损CD8αα+ T细胞表型鉴定

目的 探究银屑病皮损局部浸润的CD8+ T细胞表型及其在银屑病发病中的作用。方法 收集2017年1 - 12月第四军医大学西京皮肤医院明确诊断的8例进行期斑块状银屑病患者皮损,男女各4例,年龄24 ~ 50岁。8例健康对照皮肤来源于整形外科手术剩余皮肤,男女各4例,年龄23 ~ 46岁。采用免疫荧光技术检测皮损CD8+ T细胞的分布及亚群比例,鉴定其免疫学表型及炎症因子白细胞介素（IL）17A的表达。结果 8例银屑病皮损真、表皮均可见CD8+ T细胞浸润,其中CD8αα+ T细胞表型占88.48% ± 7.39%,8例健康人皮肤局部仅有个别CD8+ T细胞浸润,其中CD8αα+ T细胞表型占14.43% ± 13.14%,两组比较,t = 11.5,P < 0.01。银屑病皮损表皮CD8αα+ T细胞表达组织局部定植标志CD103,真皮CD8αα+ T细胞不表达CD103。银屑病皮损CD8αα+ T细胞表达CD45RA-CCR7-效应记忆性T细胞表型,不表达CD8+ 调节性T细胞标志Foxp3、CD25和CD122。银屑病皮损CD8αα+ T细胞中分泌IL?17A的细胞比例为24.85% ± 4.25%,对照组CD8αα+ T细胞不分泌IL?17A,两组差异有统计学意义（t = 5.853,P < 0.01）。结论 银屑病皮损浸润的CD8αα+ T细胞是效应记忆性T细胞,可能通过分泌IL?17A参与银屑病的发生发展。     

Exploring the imbalance of circulating follicular helper CD4+ T cells in sarcoidosis patients

BackgroundSarcoidosis is a systemic granulomatous disease characterized by the combination of Th1 and Th17 responses. Recently, several arguments have suggested a potential involvement of B cells as well as T cells in the pathogenesis of sarcoidosis. Follicular helper CD4⁺ T (T_FH) cells are specialized in interacting with and helping B cells, and play a crucial role in the formation of germinal centers.ObjectiveWe sought to explore the status of T_FH cells and investigate their possible pathogenic role in sarcoidosis.MethodsT_FH cells and B cells in peripheral blood were examined by flow cytometry, and serum samples were studied by cytokine arrays. Immunohistochemistry was performed to check for the presence of T_FH cells in sarcoidosis skin lesions. Gene expression in isolated T_FH cells was analyzed by quantitative RT-PCR.ResultsThe proportion of circulating T_FH cells was decreased. CD4⁺CXCR5⁺ T_FH cells were observed in cutaneous lesions in sarcoidosis. Gene expression in circulating T_FH cells and serum cytokine concentrations related to Th17 were increased in sarcoidosis patients. Gene expressions of B cell differentiation cytokines in T_FH cells were not altered in sarcoidosis patients.ConclusionWe herein describe a decrease of circulating T_FH cells and their migration to affected tissues. Circulating T_FH cells are one of the potential cell types capable of producing IL-17 and enhancing Th17 responses, and may promote the chronic inflammation. We could not demonstrate a direct linkage between the imbalance of T_FH cells and abnormal B cell differentiation in sarcoidosis.     

In‐situ‐topoproteome analysis of cutaneous lymphomas: Perspectives of assistance for dermatohistologic diagnostics by Multi Epitope Ligand Cartography (MELC)

Background: Immunophenotyping is essential for diagnostics of cutaneous lymphomas. In this regard we present a skin tissue‐adapted application platform of MELC technology. Patients and Methods: This topoproteome analysis allows the subcellular colo‐calization of at least n = 100 epitopes in situ. For this purpose the specimen is processed by a Toponome Imaging Cycler^® for a n‐fold repetition of the following cycle: 1) staining with a fluorophore‐labeld antibody, 2) fluorescence‐imaging, and 3) photobleaching. Overlay and binarization of fluorescence images lead to combinatorial molecular phenotypes (CMP), which relate to a pixel or microtopographic unit (450 × 450 nm², 20× objective). Skin biopsies were derived from patients with mycosis fungoides (patch/plaque lesions), psoriasis, atopic eczema and from healthy skin donors. Results: In orientation to the WHO‐EORTC‐classification of cutaneous lym‐phomas a MELC‐library of 23 markers was established. According to an inaugurative detailed procedure the CMP frequency was determined in a normalization to 100 μm horizontal skin width. By a TopoMiner strategy mycosis fungoides could be separated from the other states with a maximum of significance (p ≤ 0.03) by at least 10‐fold overexpression of the following tumor cell‐representative CMP‐motif: CD3+/CD4+/CD1a‐/CD7‐/CD8‐/CD45R0+/CD45RA‐/CD11a+. Conclusions: The skin tissue‐adapted MELC‐application‐platform extends substantially conventional lymphoma diagnostics by an unprecedented dimension of in‐situ‐analysis of marker combinatorics including its exact quantification and visualization.     

Papuloerythroderma‐like cutaneous involvement of a CD62L− subclone of T‐cell prolymphocytic leukemia

We report the case of an 88‐year‐old Japanese man with erythrodermic involvement of T‐cell prolymphocytic leukemia (T‐PLL). He had a history of pharyngeal diffuse large B‐cell lymphoma successfully treated with polychemotherapy including cyclophosphamide and epirubicin, 6 years before the current illness. He presented with numerous reddish, coalescing, flat‐topped papules on the trunk and extremities, sparing the skin folds of the abdomen, the features of which mimicked those of papuloerythroderma. Immunohistochemistry showed perivascular and epidermotropic infiltration of CD3⁺ CD4⁺ T cells in the cutaneous lesion. However, flow cytometric analysis revealed that the skin infiltrating T cells were negative for surface CD4, and that CD3⁺ CD4^? CD8^? cells made up 92% of the T‐cell fraction of peripheral blood. The circulating atypical T cells had a round or oval nucleus and prominent nucleoli, and the deletion of chromosomes 6q, 13 and 17. These cytological profiles were consistent with those of T‐PLL and distinct from those of Sézary cells. The same T‐cell clone was detected in the cutaneous lesion and peripheral blood, but the expression of CD62L was absent in the skin infiltrates and present in the circulating cells. No specific mutation was detected in STAT3 or STAT5B. Although low‐dose oral etoposide had a beneficial effect on the skin rash, a fatal crisis of marked leukocytosis (169 × 10³/μL) occurred 19 months after the illness onset. CD62L‐leukemic cells of T‐PLL may infiltrate the skin to form papuloerythroderma‐like cutaneous lesions.     

Pyogenic lymphoma of the skin: a peculiar variant of primary cutaneous neutrophil-rich CD30+ anaplastic large-cell lymphoma. Clinicopathological study of four cases and review of the literature

Systemic anaplastic large-cell lymphoma (ALCL) in human immunodeficiency virus (HIV)-infected individuals showing an extensive infiltrate of neutrophils has been reported and referred to as 'neutrophil-rich' CD30+ ALCL. Secondary cutaneous involvement has been found in a subset of these cases. We report the clinicopathological features of four immunocompetent patients with primary cutaneous neutrophil-rich ALCL and present a new histological subtype with a dissolute growth pattern of CD30+ tumour cells. Four HIV-negative patients presented with rapidly growing solitary or multiple tumours located on the face. Ulceration of the lesions with purulent discharge was a typical finding. Various inflammatory dermatoses were considered clinically in all cases. The histological hallmark was a large number of neutrophils in the infiltrate that masked neoplastic CD30+ anaplastic cells. In two cases, a dissolute growth pattern of anaplastic tumour cells was observed. In two cases, a strong correlation between tumour growth and interleukin (IL)-8 cytokine pattern as well as the production of IL-8 by tumour cells was demonstrated. The diagnosis of neutrophil-rich ALCL is challenging clinically and histologically as the tumour cell compartment is masked by an extensive inflammatory infiltrate of neutrophils and other reactive cells such as histiocytes which may be mainly due to release of IL-8 by tumour cells. The term 'pyogenic' designates the typical feature of this distinct neutrophil-rich ALCL, namely abscess formation ('pyo-') by cytokines (IL-8) produced by tumour cells ('-genic'). The clinical behaviour of this type is the same as in primary cutaneous CD30+ ALCL with classical histological presentation.     

Expression of Foxp3, TGF-β and IL-10 in American cutaneous leishmaniasis lesions

Regulatory T cells (Tregs) are a unique population of CD25+CD4+ T cells that regulate innate and adaptive immune responses and have the ability to control the excessive or misdirected effects of the immune system. This modulation involves different mechanisms, such as the suppression of T cell proliferation and cytokine production, the secretion of suppressive cytokines (IL-10 and TGF-β) and the induction of effector T cell apoptosis in humans with infectious diseases such as Leishmania infections. The aim of this study was to evaluate the expression of Foxp3, IL-10 and TGF-β through immunohistochemistry in 22 skin biopsies of patients with localized cutaneous leishmaniasis (LCL) caused by Leishmania (Viannia) spp. from an endemic area in pre-Amazonian area of Maranhão State, Brazil. The density of these markers was also analyzed according to the species of parasite and the progression of the disease. The cellular density was 234 cells/mm² for Foxp3+ cells, 357 cells/mm² for TGF-β+ cells and 648 cells/mm² for IL-10+ cells in the studied skin lesions. The analysis of the cellular density of these immunological markers in relation to the species of Leishmania demonstrated that lesions caused by L. (V.) braziliensis had a lower density of Foxp3+ cells than lesions caused by L. (Viannia) spp. The expression of IL-10 was also lower in lesions caused by L. (V.) braziliensis. There were no significant differences in TGF-β expression between the two groups. The evaluation of these markers according to the progression of the disease did not reveal any significant differences. These findings suggest that Treg Foxp3+ cells, IL-10, and TGF-β play important roles in the immunopathogenesis of LCL and that these roles differ depending on the causal Leishmania species.