Jorge Lobo's disease: immunohistochemical characterization of dendritic cells in cutaneous lesions.

BACKGROUND: Jorge Lobo's disease (JLD) is a cutaneous chronic mycosis caused by Lacazia loboi. We studied Factor XIIIa + dermal dendrocytes (FXIIIa + DD), Langerhans cells (LC) through the expression of langerin and the expression of S100 protein. METHODS: A total of 41 biopsies and 10 normal skins (control) were developed with a polymer-based immunohistochemical method. RESULTS: Lesions presented infiltrate comprising macrophages, some asteroid corpuscles, lymphocytes, multinucleated giant cells and a large number of fungi. LCs presented short dendrites and were scarcely distributed. Dermal langerin + cells were detected in nine JLD lesions. FXIIIa + DD were hypertrophic, visualized in the inflammatory infiltrate of JLD lesions. Cells S100+ were present in JLD and control group with a similar number of cells. A total of 14 specimens did not express FXIIIa, and this considerable number probably contributed to the statistical similarity with the control group. CONCLUSIONS: The results indicate that LCs are present in the immune response against Lacazia loboi. Some dermal langerin + cells could be another subset of dendritic cells. Our data indicate changes of LCs in JLD cutaneous lesions and present, for the first time, results that show langerin + cells in the dermis and corroborate previous observations on the participation of FXIIIa + DD in the in situ immune response in JLD.

Type I reaction in leprosy--a histopathological analysis.

Even though type 1 lepra reaction (TIR) is a commonly encountered clinical problem, its histology has not yet been clearly delineated. This study attempts to enumerate the most sensitive parameters for the histological diagnosis of TIR. Case records between March 2007 and September 2007 of patients with TIR were reviewed and the biopsies were evaluated by a pathologist blinded to the previous diagnoses. Twenty three patients were included in the study. The most sensitive parameters in our study were dermal edema, intra-granuloma edema and giant cell size. Though clinical findings should remain the mainstay of diagnosis of TIR, the above mentioned parameters should be evaluated in biopsies of leprosy to look for signs of reaction which might otherwise be missed.

Metabonomics reveals drastic changes in anti-inflammatory/pro-resolving polyunsaturated fatty acids-derived lipid mediators in leprosy disease.

Despite considerable efforts over the last decades, our understanding of leprosy pathogenesis remains limited. The complex interplay between pathogens and hosts has profound effects on host metabolism. To explore the metabolic perturbations associated with leprosy, we analyzed the serum metabolome of leprosy patients. Samples collected from lepromatous and tuberculoid patients before and immediately after the conclusion of multidrug therapy (MDT) were subjected to high-throughput metabolic profiling. Our results show marked metabolic alterations during leprosy that subside at the conclusion of MDT. Pathways showing the highest modulation were related to polyunsaturated fatty acid (PUFA) metabolism, with emphasis on anti-inflammatory, pro-resolving omega-3 fatty acids. These results were confirmed by eicosanoid measurements through enzyme-linked immunoassays. Corroborating the repertoire of metabolites altered in sera, metabonomic analysis of skin specimens revealed alterations in the levels of lipids derived from lipase activity, including PUFAs, suggesting a high lipid turnover in highly-infected lesions. Our data suggest that omega-6 and omega-3, PUFA-derived, pro-resolving lipid mediators contribute to reduced tissue damage irrespectively of pathogen burden during leprosy disease. Our results demonstrate the utility of a comprehensive metabonomic approach for identifying potential contributors to disease pathology that may facilitate the development of more targeted treatments for leprosy and other inflammatory diseases.

Wade histoid leprosy: histological and immunohistochemical analysis.

Histoid leprosy is a rare multibacillary form that presents with disseminated papule-nodular cutaneous lesions. To study the inflammatory infiltrate of the histoid form and to compare it with other lepromatous forms, we performed histological and immunohistochemical analysis on skin biopsies. Fifteen patients were included for histopathological analysis (10 histoid and five lepromatous) via the haematoxylin-eosin and Ziehl-Neelsen-Faraco stains. Thus, immunohistochemical techniques using immunoperoxidase assay were performed for: anti-BCG, anti-M. leprae, anti-CD8, anti-CD3, anti-CD20, anti-S100, anti-CD1a, anti-CD68 and antivimentin. Spindle cells were present in all histoid patients. A pseudocapsule was observed in half of both studied forms. A comparison using the Ziehl-Neelsen-Faraco stain to evaluate anti-BCG and anti-M.leprae showed no major differences. The CD3+ cells were more pronounced in the histoid form than the lepromatous form. There was greater immunoreactivity toward CD8+ cells in the histoid form, as well as the CD20+ cell count. A similar count of S100+ cells in the epidermis of both leprosy forms was observed. There was a slight increase of dendritic cells in the histoid patients in the superficial and deep dermis. For CD1a marker, we observed expression in the epidermis and superficial dermis in both forms. A diffuse and intense infiltrate of CD68+ cells was also observed in the histoid and lepromatous forms. The high positivity for vimentin did not allow for a positive cell count. We concluded that the activation of both the cellular and humoral response is more pronounced in the histoid form because the T and B cells showed greater infiltration than those in the lepromatous form. The activation of dendritic and Langerhans cells is similar in both forms. The spindle cells likely belong to the macrophage population, thus maintaining phagocytic ability. The quantities of pseudocapsules and bacilli are similar and cannot serve as criteria for diagnosis.

Leprosy patients: neurotrophic factors and axonal markers in skin lesions.

Neurotrophins are growth factors with crucial roles in neural pathophysiology. These mediators functionally modulate nociceptive fibers, and changes in neurotrophins expression have been correlated with early loss of nociception in leprosy. This study investigated the expression of NGF, BDNF, and NT3 in dermal nerves of leprosy patients. Characterization of Remak bundles was achieved by p75(NTR), and axonal markers NF-L and PGP 9.5 immunostaining. Clinical parameters of neural impairment have been evaluated by Semmes-Wenstein monofilaments. Our findings demonstrated decrease of NGF in borderline leprosy, when compared to control specimens. Similar results were observed in PGP 9.5 expression (borderline: p<0.001 and lepromatous: p<0.05) and NF-L (lepromatous: p<0.05), suggesting advanced Remak bundles degeneration in multibacillary leprosy. It has also been observed positive correlation between p75(NTR) and PGP 9.5, indicating association between Schwann cells and axons in Remak bundles. Present data indicate that neurotrophins imbalance may participate in the establishment of peripheral nerve damage.

Leprosy patients: neurotrophic factors and axonal markers in skin lesions / Pacientes com hanseníase: fatores neurotróficos e marcadores axonais em lesões de pele

Neurotrophins are growth factors with crucial roles in neural pathophysiology. These mediators functionally modulate nociceptive fibers, and changes in neurotrophins expression have been correlated with early loss of nociception in leprosy. This study investigated the expression of NGF, BDNF, and NT3 in dermal nerves of leprosy patients. Characterization of Remak bundles was achieved by p75NTR, and axonal markers NF-L and PGP 9.5 immunostaining. Clinical parameters of neural impairment have been evaluated by Semmes-Wenstein monofilaments. Our findings demonstrated decrease of NGF in borderline leprosy, when compared to control specimens. Similar results were observed in PGP 9.5 expression (borderline: p<0.001 and lepromatous: p<0.05) and NF-L (lepromatous: p<0.05), suggesting advanced Remak bundles degeneration in multibacillary leprosy. It has also been observed positive correlation between p75NTR and PGP 9.5, indicating association between Schwann cells and axons in Remak bundles. Present data indicate that neurotrophins imbalance may participate in the establishment of peripheral nerve damage.

Neurotrofinas são fatores de crescimento com papel fundamental na fisiopatologia neural. Esses mediadores modulam funcionalmente fibras nociceptivas. Mudanças em sua expressão têm sido relacionadas à perda precoce da nocicepção na hanseníase. Este estudo investigou a expressão de NGF, BDNF e NT3 em nervos dérmicos de pacientes hansenianos. A caracterização de fibras nervosas não mielinizadas foi feita por p75NTR e marcadores axonais NF-L e PGP 9.5. Os parâmetros clínicos de dano neural foram avaliados por monofilamentos Semmes-Wenstein. Nossos achados demonstram diminuição de NGF nos pacientes dimorfos em comparação aos controles. Resultados similares foram observados para PGP 9.5 (dimorfos: p<0,001; virchowianos: p<0,05) e NF-L (virchowianos: p<0.05), sugerindo degeneração avançada das terminações nervosas na hanseníase multibacilar. Foi observada correlação positiva entre p75NTR e PGP 9.5, indicando associação entre células de Schwann e axônios em fibras nervosas não mielinizadas. Os resultados indicam que o desequilíbrio na expressão das neurotrofinas pode participar do dano neural periférico.

High matrix metalloproteinase production correlates with immune activation and leukocyte migration in leprosy reactional lesions.

Gelatinases A and B (matrix metalloproteinase 2 [MMP-2] and MMP-9, respectively) can induce basal membrane breakdown and leukocyte migration, but their role in leprosy skin inflammation remains unclear. In this study, we analyzed clinical specimens from leprosy patients taken from stable, untreated skin lesions and during reactional episodes (reversal reaction [RR] and erythema nodosum leprosum [ENL]). The participation of MMPs in disease was suggested by (i) increased MMP mRNA expression levels in skin biopsy specimens correlating with the expression of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha), (ii) the detection of the MMP protein and enzymatic activity within the inflammatory infiltrate, (iii) increased MMP levels in patient sera, and (iv) the in vitro induction of MMP-9 by Mycobacterium leprae and/or TNF-alpha. It was observed that IFN-gamma, TNF-alpha, MMP-2, and MMP-9 mRNA levels were higher in tuberculoid than lepromatous lesions. In contrast, interleukin-10 and tissue inhibitor of MMP (TIMP-1) message were not differentially modulated. These data correlated with the detection of the MMP protein evidenced by immunohistochemistry and confocal microscopy. When RR and ENL lesions were analyzed, an increase in TNF-alpha, MMP-2, and MMP-9, but not TIMP-1, mRNA levels was observed together with stronger MMP activity (zymography/in situ zymography). Moreover, following in vitro stimulation of peripheral blood cells, M. leprae induced the expression of MMP-9 (mRNA and protein) in cultured cells. Overall, the present data demonstrate an enhanced MMP/TIMP-1 ratio in the inflammatory states of leprosy and point to potential mechanisms for tissue damage. These results pave the way toward the application of new therapeutic interventions for leprosy reactions.

Exploratory multivariate spectroscopic study on human skin.

BACKGROUND/AIMS: Spectroscopy on human skin is a field that is being adopted increasingly because of its rapidity and high reproducibility. Infrared reflectance (IR), near-infrared reflectance (NIR), and fluorescence spectroscopy have previously been applied to human skin in vivo to compare healthy and sick skin, including skin cancer, atopy, and leprosy. Exploratory data analysis/chemometrics is a tool for evaluating multivariate data such as spectroscopic measurements. The objective of this study was to explore the spectral variance spanned by people with normal integument, and to demonstrate the advantages of multivariate analysis to skin research. METHODS: IR, NIR and fluorescence spectroscopy have been carried out in vivo on 216 volunteers' forearms before and after four tape strippings. The subjects were asked to fill in a questionnaire regarding factors suspected to influence the measurement results. Principal Component Analysis (PCA) was used to investigate whether the population can be divided into groups on the basis of their skin chemistry. Unless otherwise stated, the results are from the measurements prior to stripping. RESULTS: In contrast to IR and fluorescence spectra, NIR spectra proved able to detect gender differences. By use of PCA, classifications on male and female subjects were observed from the IR and NIR measurements, and as an indication from the fluorescence measurements. The NIR and fluorescence measurements varied between elderly and young subjects. The largest variance in the fluorescence landscapes was seen between pigmented and non-pigmented skin. No connection was found between the spectroscopic measurements and smoking or drinking habits. CONCLUSIONS: Future spectroscopic skin investigations should be balanced as regards to gender and age, as these can possibly affect the measurement results. Chemometrics proved to be superior to traditional attempts of interpreting the spectra.

Analise de aspectos afetivo-familiares de mulheres portadoras de alopecia areata / Analysis of affective-familial aspects of women with alopecia areata

O objetivo deste trabalho é levantar e analisar os fragmentos da historia de vida de mulheres portadoras de alopecia areata, em relacao as experiencias afetivas, com enfase em aspectos da relacao parental e conjugal. Entrevistamos, individualmente, cinco mulheres adultas, encaminhadas pelo Ambulatorio de Tricoses (dermatoses do cabelo). Elas preenchem o requisito de terem experienciado relacionamentos conjugais, pelo menos em algum periodo da vida, sejam elas casadas, viuvas, divorciadas ou amasiadas. Utiliamos a analise interpretativa e qualitativa ao identificar caracteristicas unicas e singulares. Aborsdamos as entrevistas, enquanto pessoas distintas uma das outras, e, em alguns momentos, destacamos aspectos que se conjugam quanto ao conteudo dos depoimentos. Constatamos que, no decorrer das historias de vida, as experiencias afetivas sao revividas intensamente, com sentimentos de abandono, rejeicao e carencia afetiva, embora perpetuem-se na fase adulta e se atualizem no convivio conjugal. A aderencia anormal ao passado impede a exteriorizacao de afetos nos relacionamentos conflitivos atuais que sao apenas descrits e pouco vivenciados por elas. Verificamos que a queda de cabelos desenvolve-se com expressao, mediante representacoes afetivas que essasmulheres estabelecem e mantem com os pais e marido, vistos como principais figuras de apego. A doenca manifesta-se em meio as condicoes de esvaziamento da plenitude afetiva, como expressao do afeto reprimido por ocasiao de acontecimentos traumaticos, acompanhada de outras desorganizacoes somaticas. Nossas entrevistas apresentam sentimentos auto-depreciativos, inseguranca, empobrecimento nas trocas interpessoais e dificuldade de interiorizar os sentimentos afetivos de maneira nao traumatica. Alem de reviver psiquicamente as experiencias de afeto e as circunstancias de maneira insatisfatoria, estabelecem uma relacao causal entre os eventos do passado e do presente. Atribuem suas insatisfacoes afetivas e prejuizos...

Anti-inflammatory drugs block cytokine mRNA accumulation in the skin and improve the clinical condition of reactional leprosy patients.

The aim of this study was to investigate in what ways in vivo anti-inflammatory treatment affects cytokine mRNA expression in situ in both erythema nodosum leprosum and reversal reaction patients. Serial biopsies were collected from the patients undergoing leprosy reactions before and during pentoxifylline (n = 7) or thalidomide (n = 3) treatment for erythema nodosum leprosum and prednisone (n = 3) for reversal reaction. Clinical evolution of the skin lesion was assessed during the study and semiquantitative reverse transcription-polymerase chain reaction was used to investigate cytokine mRNA expression at the lesion site. Results showed expression of interferon-gamma, interleukin-6, interleukin-10, interleukin-12 p40, and tumor necrosis factor-alpha in all patients tested at the onset of reactional episodes, but interleukin-4 mRNA was rarely detected in the lesions (n = 4). Follow-up analysis showed that, irrespective of the drugs used, tumor necrosis factor-alpha mRNA was diminished in 10 of the 13 patients tested. A concomitant decrease of mRNA accumulation was also observed for interferon-gamma (nine of 11 patients), interleukin-6 (nine of 11), and interleukin-12 p40 (six of eight). An inhibitory effect on interleukin-10 mRNA was likewise seen after thalidomide and pentoxifylline, but not subsequent to prednisone treatment. The data also demonstrated that cytokine mRNA inhibition correlates to the resolution of the inflammatory response in situ (n = 10), whereas the persistence/enhancement of cytokine message expression after treatment was associated with worsening of the skin condition, as seen in three erythema nodosum leprosum patients whose maintenance of local inflammation was accompanied by the appearance/persistence of interleukin-4 gene expression in situ subsequent to anti-inflammatory treatment. In summary, the participation of cytokines in leprosy inflammatory episodes seems to be directly associated with the patients' clinical evolution following therapy for reaction.

Comparison of the T cell patterns in leprous and cutaneous sarcoid granulomas. Presence of Valpha24-invariant natural killer T cells in T-cell-reactive leprosy together with a highly biased T cell receptor Valpha repertoire.

The T-cell-reactive (eg, tuberculoid and reversal) forms of leprosy represent a well-defined granulomatous reaction pattern against an invading pathogen. The immune response in cutaneous sarcoidosis is a granulomatous condition that pathologically is very similar to T-cell reactive leprosy. However, it lacks a defined causative agent. In view of the role of NKT cells in murine granulomas induced by mycobacterial cell walls, we have searched for the presence of NKT cells in the cutaneous lesions of both leprosy and sarcoidosis. These cells were present in T-cell-reactive leprosy but were undetectable in cutaneous sarcoidosis. We have also studied the TCR Valpha repertoire in the two diseases. In addition to Valpha24(+) NKT cells, all patients with T-cell-reactive leprosy showed a very restricted T-cell-reactive Valpha repertoire with a strong bias toward the use of the Valpha6 and Valpha14 segments. Valpha6 and Valpha14(+) T cells were polyclonal in terms of CDR3 length and Jalpha usage. In contrast, most sarcoidosis patients showed a diverse usage of Valpha chains associated with clonal or oligoclonal expansions reminiscent of antigen-driven activation of conventional T cells. Thus the origin and perpetuation of the two kinds of granulomatous lesions appear to depend on altogether distinct T-cell recruiting mechanisms.

Hansen's disease in a patient with a history of sarcoidosis.

We report a rare case of concomitant Hansen's disease (HD) and sarcoidosis. Reticulin staining may be a helpful diagnostic tool in establishing the diagnosis of sarcoidosis in skin lesions. The diagnosis of HD can be established despite negative polymerase chain reaction results for the detection of Mycobacterium leprae DNA. Finally, a well-established diagnosis of sarcoidosis does not preclude the development of another granulomatous disorder. Hence, when new lesions developed in a patient with sarcoidosis despite appropriate therapy, other concurrent diagnoses should be pursued.

Immunohistochemical detection of PGL-1, LAM, 30 kD and 65 kD antigens in leprosy infected paraffin preserved skin and nerve sections.

A panel of lipid, carbohydrate and protein antibodies were optimized for use in detecting M. leprae antigens in paraffin embedded material. Skin and nerve biopsies from 13 patients across the leprosy spectrum were studied. All antibodies detected antigen in tissues with a BI > 1. Phenolic-glycolipid was not detected in bacteriologically negative tissue but lipoarabinomanan (LAM) and protein antigens were detected. Staining with LAM was strongest and gave least background. The transfer of this immunohistochemical technique to paraffin embedded material will allow examination of tissue with better morphology and from clinics without access to tissue freezing facilities.

Nodular post-kala-azar dermal leishmaniasis: a distinct histopathological entity.

Post-kala-azar dermal leishmaniasis (PKDL) is an infrequently occurring sequel to treated visceral leishmaniasis. Diagnosis, particularly in non-endemic areas, is difficult because the clinical appearances may be subtle and simulate lepromatous leprosy. The histopathology of the condition has been a neglected subject. Nodular lesions constitute one of the large variety of lesions that can be seen in PKDL. This paper describes the histopathology of such lesions in 26 patients seen over a period of approximately 8 years in a non-endemic setting. All the biopsies had strikingly similar light microscopic features with characteristic findings: a dense lymphohistiocytic infiltrate beneath an atrophic epidermis, pronounced follicular plugging, vascular hyalinization and collagen changes and negative Fite stain. These allow a definite diagnosis of PKDL even in the absence of demonstrable Leishman-Donovan (L-D) bodies.

Recognition of lipid antigens by sera of mice infected with Mycobacterium lepraemurium.

Lipids extracted from mouse tissues infected with Mycobacterium lepraemurium (MLM) were analyzed by thin-layer chromatography. Although the extracted lipids were heterogeneous in polarity, the lipids of intermediate polarity were the ones that predominated. All of the lipids of intermediate polarity were glycosylated species. There were also lipids of low and high polarity, the latter being glycolipids. Compared to lipids extracted from normal tissue (mostly to lipids of high and low polarity), all of the additional lipids extracted from the infected tissue corresponded to lipids present in the purified bacteria. Enzyme-linked immunoassays (ELISAs) were then performed with the whole lipids extracted from purified bacilli, the lipids of high, intermediate and low polarity, and the sera from 20 normal and 20 MLM-infected mice. Lipids of intermediate polarity were specifically recognized by MLM-infected mice. Neither sera (diluted 1:500) from normal mice nor infected mice reacted with the lipids of high or low polarity, but a higher concentration (sera diluted 1:100) of some sera from mice in both groups reacted significantly with these lipids. In the ELISAs the whole-lipid extract and the lipids of intermediate polarity were similarly recognized by the sera of the infected mice. Thus, as observed in human leprosy, the mycobacterial disease in the mouse (murine leprosy) is also accompanied by the development of antibodies to the glycolipids of the infecting microorganism.

CD26 expression in leprosy and other granulomatous diseases correlates with the production of interferon-gamma.

BACKGROUND: Leprosy represents a spectrum of clinical manifestations that reflect the immune response to antigens of Mycobacterium leprae. The tuberculoid form of leprosy, which is characterized by an organized development of granulomas, has recently been correlated with a Th1-like immune response. The lepromatous form of leprosy, with a characteristic lack of cellular immunity, has been correlated with a Th2-like immune response to mycobacterial antigens. Dipeptidylpeptidase IV (CD26) is an ectopeptidase that is expressed in various tissues; in the hemopoietic system, it is predominantly expressed by T cells. EXPERIMENTAL DESIGN: We stained frozen sections of skin biopsies obtained from patients with different forms of leprosy, sarcoidosis, and Piringer's lymphadenitis. Sections were stained for interferon-gamma (IFN-gamma) and CD26 with the alkaline phosphatase anti-alkaline phosphatase technique and in two-color stainings by immunofluorescence. RESULTS: We found strong signals for IFN-gamma and for CD26 in all investigated cases of tuberculoid leprosy. In contrast, in all biopsies taken from patients with lepromatous leprosy, we found no or very weak signals for these antigens. By immunofluorescence double-labeling, we could show that IFN-gamma and CD26 were expressed by the identical cell population. We confirmed this correlation of CD26 expression and IFN-gamma production in other granulomatous inflammatory reactions such as sarcoidosis and Piringer's lymphadenitis. CONCLUSIONS: From our results, we conclude that a high expression of CD26 may be suggestive of Th1-like immune reactions.

Biometals in skin and sera of leprosy patients and their correlation to trace element contents of M. leprae and histological types of the disease; a comparative study with cutaneous tuberculosis.

The present study has provided information on the biometal contents of killed and dried Mycobacterium leprae as well as dermal granulomas induced by the invading mycobacteria in various histological types of leprosy patients. For comparison, the biometal contents of the contralateral leprosy-unaffected skin of the same patients also were measured. The study also reports changes of serum levels of the biometals in these patients which were compared with those in healthy control subjects and patients with skin tuberculosis. These data show that M. leprae is rich in zinc. During the course of the evolution of the disease there is gross alteration of the dynamics of the inflammatory cell population that infiltrates into leprosy granulomas, resulting in the alterations of trace element contents of the disease-affected skin lesions. Interestingly, the changes of the biometal contents in the granulomas of the patients with skin tuberculosis are similar to those in leprosy patients. It is postulated that the significant decrease of the contents of copper, zinc, iron, calcium and magnesium in the disease-affected skin in comparison to that of the contralateral healthy skin is a local effect, perhaps due to erosion or influx of biometal-deficient inflammatory cells into the affected skin with eventual loss of connective tissue of skin and mobilization of tissue-bound microelements into the vascular compartment. On the contrary, the changes in biometal levels in the sera of leprosy patients appear to be a general effect perhaps due to the release of interleukin-1, a product of inflammatory cells, causing hypercupremic, hypozincemic and hypoferremic responses in the hosts. Moreover, growth and multiplication of M. leprae, especially in polar lepromatous leprosy patients with a high bacillary load, demand essential biometals which may be mobilized into the bacterial bodies from the hosts. This perhaps results in the change in the homeostasis of the essential biometals in the hosts.