Human Skin as an Ex Vivo Model for Maintaining Mycobacterium leprae and Leprosy Studies.

The in vitro cultivation of M. leprae has not been possible since it was described as causing leprosy, and the limitation of animal models for clinical aspects makes studies on leprosy and bacteria-human host interaction a challenge. Our aim was to standardize the ex vivo skin model (hOSEC) to maintenance and study of M. leprae as an alternative animal model. Bacillary suspensions were inoculated into human skin explants and sustained in DMEM medium for 60 days. Explants were evaluated by RT-PCR-16SrRNA and cytokine gene expression. The viability and infectivity of bacilli recovered from explants (D28 and D60) were evaluated using the Shepard's model. All explants were RT-PCR-16SrRNA positive. The viability and infectivity of recovered bacilli from explants, analyzed after 5 months of inoculation in mice, showed an average positivity of 31%, with the highest positivity in the D28 groups (80%). Furthermore, our work showed different patterns in cytokine gene expression (TGF-ß, IL-10, IL-8, and TNF-α) in the presence of alive or dead bacilli. Although changes can be made to improve future experiments, our results have demonstrated that it is possible to use the hOSEC to maintain M. leprae for 60 days, interacting with the host system, an important step in the development of experimental models for studies on the biology of the bacillus, its interactions, and drug susceptibility.

Peculiar Histological Features of Oral Intravascular Papillary Endothelial Hyperplasia.

A 55-year-old male patient with single and well-circumscribed nodule in the lower lip. Accurate diagnosis is based only on histopathological examination using hematoxylin and eosin and immunohistochemical approach, which a large, organized thrombus within the dilated lumen of a poorly demarcated vein, associated with papillary projections of endothelial proliferation occupying vascular spaces. The final diagnosis was intravascular papillary endothelial hyperplasia (IPEH) associated with a thrombus. Oral IPEH is rare and has historically been difficult to diagnose due to its resemblance to other oral lesions. However, the distinctive histological features of oral IPEH associated with a thrombus now allow for its diagnosis through hematoxylin and eosin staining alone, without the need for additional techniques. Therefore, it is crucial for pathologists to be familiar with these unique morphological features to accurately diagnose oral IPEH and differentiate it from more common benign, malignant, or reactive vascular lesions in the oral cavity.

Prediction of the occurrence of leprosy reactions based on Bayesian networks.

Introduction: Leprosy reactions (LR) are severe episodes of intense activation of the host inflammatory response of uncertain etiology, today the leading cause of permanent nerve damage in leprosy patients. Several genetic and non-genetic risk factors for LR have been described; however, there are limited attempts to combine this information to estimate the risk of a leprosy patient developing LR. Here we present an artificial intelligence (AI)-based system that can assess LR risk using clinical, demographic, and genetic data. Methods: The study includes four datasets from different regions of Brazil, totalizing 1,450 leprosy patients followed prospectively for at least 2 years to assess the occurrence of LR. Data mining using WEKA software was performed following a two-step protocol to select the variables included in the AI system, based on Bayesian Networks, and developed using the NETICA software. Results: Analysis of the complete database resulted in a system able to estimate LR risk with 82.7% accuracy, 79.3% sensitivity, and 86.2% specificity. When using only databases for which host genetic information associated with LR was included, the performance increased to 87.7% accuracy, 85.7% sensitivity, and 89.4% specificity. Conclusion: We produced an easy-to-use, online, free-access system that identifies leprosy patients at risk of developing LR. Risk assessment of LR for individual patients may detect candidates for close monitoring, with a potentially positive impact on the prevention of permanent disabilities, the quality of life of the patients, and upon leprosy control programs.

Patologia da hanseníase e a importância do diagnóstico histopatológico: uma entrevista com Dr. Cleverson Teixeira Soares / Pathology of leprosy and the importance of histopathological diagnosis: an interview with Dr. Cleverson Teixeira Soares

A revista Hansenologia Internationalis tem a honra de entrevistar o Dr. Cleverson Teixeira Soares, médico patologista, responsável pelo Laboratório de Anatomia Patológica do Instituto Lauro de Souza Lima, Bauru, São Paulo, Brasil. Dr. Cleverson é autor do livro Histopathological Diagnosis of Leprosy, publicado em 2021 pela editora Bentham Books, além de relevante publicação de artigos, em periódicos científicos nacionais e internacionais, com impacto nas áreas de patologia e hansenologia. Ele descreve sobre sua trajetória profissional, os desafios para o entendimento da hanseníase em suas múltiplas formas clínicas e como a patologia clássica e molecular tem contribuído para a construção do conhecimento sobre esta doença tão complexa.

Prediction of the occurrence of leprosy reactions based on Bayesian networks

Introduction: Leprosy reactions (LR) are severe episodes of intense activation of the host inflammatory response of uncertain etiology, today the leading cause of permanent nerve damage in leprosy patients. Several genetic and non-genetic risk factors for LR have been described; however, there are limited attempts to combine this information to estimate the risk of a leprosy patient developing LR. Here we present an artificial intelligence (AI)-based system that can assess LR risk using clinical, demographic, and genetic data. Methods: The study includes four datasets from different regions of Brazil, totalizing 1,450 leprosy patients followed prospectively for at least 2 years to assess the occurrence of LR. Data mining using WEKA software was performed following a two-step protocol to select the variables included in the AI system, based on Bayesian Networks, and developed using the NETICA software. Results: Analysis of the complete database resulted in a system able to estimate LR risk with 82.7% accuracy, 79.3% sensitivity, and 86.2% specificity. When using only databases for which host genetic information associated with LR was included, the performance increased to 87.7% accuracy, 85.7% sensitivity, and 89.4% specificity. Conclusion: We produced an easy-to-use, online, free-access system that identifies leprosy patients at risk of developing LR. Risk assessment of LR for individual patients may detect candidates for close monitoring, with a potentially positive impact on the prevention of permanent disabilities, the quality of life of the patients, and upon leprosy control programs.

B lymphocytes deficiency results in altered immune response and increased susceptibility to Mycobacterium leprae in a murine leprosy model

Leprosy is a chronic and infectious disease that primarily affects the skin and peripheral nervous system, presenting a wide spectrum of clinical forms with different degrees of severity. The distinct host immune response patters developed in the response to the bacillus Mycobacterium leprae, the leprosy etiologic agent, are associated with the spectral clinical forms and outcome of the disease. In this context, B cells are allegedly involved in the disease immunopathogenesis, usually as antibody-producing cells, but also as potential effector or regulatory elements. In order to determine the regulatory B cells role in experimental leprosy, this study evaluated the outcome of M. leprae infection in B cell deficient mice (BKO) and WT C57Bl/6 control, by means of microbiological/bacilloscopic, immunohistochemical and molecular analysis, performed 8 months after M. leprae inoculation. The results demonstrated that infected BKO showed a higher bacilli number when compared with WT animals, demonstrating the importance of these cells in experimental leprosy. The molecular analysis demonstrates that the expression of IL-4, IL-10 and TGF-ß was significantly higher in the BKO footpads when compared to WT group. Conversely, there was no difference in IFN-γ, TNF-α and IL-17 expression levels in BKO and WT groups. IL-17 expression was significantly higher in the lymph nodes of WT group. The immunohistochemical analysis revealed that M1 (CD80+) cells counts were significantly lower in the BKO group, while no significant difference was observed to M2 (CD206+) counts, resulting a skewed M1/M2 balance. These results demonstrated that the absence of B lymphocytes contribute to the persistence and multiplication of M. leprae, probably due to the increased expression of the IL-4, IL-10 and TGF-ß cytokines, as well as a decrease in the number of M1 macrophages in the inflammatory site.

Association of CD209 (DC-SIGN) rs735240 SNV with paucibacillary leprosy in the Brazilian population and its functional effects.

BACKGROUND: Leprosy, caused by Mycobacterium leprae, is a public health problem in Brazil that affects peripheral nerves, resulting in physical disabilities. During host-pathogen interactions, the immune response determines leprosy outcomes from a localised (paucibacillary) form to a disseminated (multibacillary) form. The recognition of M. leprae involves the DC-SIGN receptor, which is present on the dendritic cells (DCs) and participates in immune activation. OBJECTIVES: To evaluate the association of polymorphisms in the promoter region of the gene encoding DC-SIGN (CD209) and the clinical form of leprosy, and to investigate its functional effects. METHODS: The study population included 406 leprosy patients from an endemic area in Brazil [310 multibacillary (MB); 96 paucibacillary (PB)]. A functional evaluation based on the effects of the single nucleotide variant (SNV) associated with PB leprosy on the specific immune response was also performed. RESULTS: The GA genotype and the presence of the A allele of rs735240 (-939G>A) were associated with PB leprosy [OR: 2.09 (1.18-3.69) and 1.84 (1.07-3.14), respectively]. Carriers of the A allele showed reduced expression of CD209 and TGF-ß1 in leprosy lesions in comparison with individuals with GG genotype, in addition to a higher response to the Mitsuda test. CONCLUSION: These data suggest that rs735240 influences the immune response against M. leprae and clinical presentation of leprosy.

IFN-γ Mediated Signaling Improves Fungal Clearance in Experimental Pulmonary Mucormycosis.

We established three immunocompetent murine models of pulmonary mucormycosis to determine the involvement of the adaptive immune response in host resistance in pulmonary mucormycosis, a rapidly fatal disease caused mainly by Rhizopus spp. Immunocompetent inbred (C57BL/6, BALB/c) and outbred (Swiss) strains of mice were inoculated with R. oryzae via the intratracheal route. The inoculation resulted in a disseminated infection that spread to the brain, spleen, kidney, and liver. After 7 and 30 days of R. oryzae infection, BALB/c mice showed the lowest fungal load and highest production of IFN-γ and IL-2 by splenocytes. Swiss mice showed a higher fungal load 30 days p.i. and was associated with a weak development of the Th-1 profile. To confirm our findings, R. oryzae-infected IFN-γ-/- mice were evaluated after 60 days, where the mice still showed viable fungi in the lungs. This study showed, for the first time, that pulmonary mucormycosis in three widely used mouse strains resulted in an acute fungal dissemination without immunosuppression whose outcome varies according to the genetic background of the mice. We also identified the partial role of IFN-γ in the efficient elimination of R. oryzae during pulmonary infection.

Association of CD209 (DC-SIGN) rs735240 SNV with paucibacillary leprosy in the Brazilian population and its functional effects

BACKGROUND Leprosy, caused by Mycobacterium leprae, is a public health problem in Brazil that affects peripheral nerves, resulting in physical disabilities. During host-pathogen interactions, the immune response determines leprosy outcomes from a localised (paucibacillary) form to a disseminated (multibacillary) form. The recognition of M. leprae involves the DC-SIGN receptor, which is present on the dendritic cells (DCs) and participates in immune activation. OBJECTIVES To evaluate the association of polymorphisms in the promoter region of the gene encoding DC-SIGN (CD209) and the clinical form of leprosy, and to investigate its functional effects. METHODS The study population included 406 leprosy patients from an endemic area in Brazil [310 multibacillary (MB); 96 paucibacillary (PB)]. A functional evaluation based on the effects of the single nucleotide variant (SNV) associated with PB leprosy on the specific immune response was also performed. RESULTS The GA genotype and the presence of the A allele of rs735240 (-939G>A) were associated with PB leprosy [OR: 2.09 (1.18-3.69) and 1.84 (1.07-3.14), respectively]. Carriers of the A allele showed reduced expression of CD209 and TGF-β1 in leprosy lesions in comparison with individuals with GG genotype, in addition to a higher response to the Mitsuda test. CONCLUSION These data suggest that rs735240 influences the immune response against M. leprae and clinical presentation of leprosy.

IFN-y mediated signaling improves fungal clearance in experimental pulmonary mucormycosis

We established three immunocompetent murine models of pulmonary mucormycosis to determine the involvement of the adaptive immune response in host resistance in pulmonary mucormycosis, a rapidly fatal disease caused mainly by Rhizopus spp. Immunocompetent inbred (C57BL/6, BALB/c) and outbred (Swiss) strains of mice were inoculated with R. oryzae via the intratracheal route. The inoculation resulted in a disseminated infection that spread to the brain, spleen, kidney, and liver. After 7 and 30 days of R. oryzae infection, BALB/c mice showed the lowest fungal load and highest production of IFN-γ and IL-2 by splenocytes. Swiss mice showed a higher fungal load 30 days p.i. and was associated with a weak development of the Th-1 profile. To confirm our findings, R. oryzae-infected IFN-γ−/− mice were evaluated after 60 days, where the mice still showed viable fungi in the lungs. This study showed, for the first time, that pulmonary mucormycosis in three widely used mouse strains resulted in an acute fungal dissemination without immunosuppression whose outcome varies according to the genetic background of the mice. We also identified the partial role of IFN-γ in the efficient elimination of R. oryzae during pulmonary infection.

Classification and general aspects of leprosy

Leprosy is a chronic infectious disease whose etiological agent is Mycobacterium leprae. Recently, Mycobacterium lepromatosis is also implicated as a causative agent and has been identified in different forms of the disesase. Leprosy is a comples disease from a clinical histopathological, and molecular point of view. The wide complex disease from a clinical, histopathological characteristics observed throughout the disease spectrum and reactions render it a challenging disease in clinical and pathological practice. This chapter discusses the main aspects of the disease and its hispathological classification. An important approach to the bacilloscopic examination, which is fundamental for the histopathological classification of the disease, showing its quantitative and qualitative aspects, is discussed. the various photographic panels demonstrate the bacillus' ability to parasitize different types of tissues and cells of the skin and other organs of the human body...

Indeterminate leprosy

Leprosy in its determinate from (I) is a clinical presentation of the disease preceding the forms described in the Ridley and Jopling (R & J) classification and any other special forms of leprosy or the reactions. In this chapter, the histopathological and bacilloscopic characteristics of the I form of leprosy are described, and the main differential diagnoses are discussed. The histopathological criteria that distinguish the I form from the other forms of leprosy and the reaction processes that may occur during the disease course are also discussed. The identification of the histopathological characteristics of I leprosy is of great importance with respect to the selection of the treatment. I leprosy should not be confused with other forms of leprosy, especially the multibacillary forms, wich require more prolonged treatment and wich can develop reaction phenomena, causing permanent sequelae.

Polar forms (TT and LL)

Leprosy is a spectral disease. Its two polar forms, tuberculoid (TT) and lepromatous (LL), are distinct presentations of the disease, both from a clinical and histopathological/bacilloscopic point of view. In this chapter, the histopathological characteristics that define the two polar forms (TT and LL), are presented, and their main differential diagnoses are discussed. These two forms also have significant differences in their treatment protocol. Histopathological recognition of both forms of the disease is important for choosing the correct treatment. Also, there are a large numbre of disease that can have a clinical presentation similar to the TT and LL forms of leprosy. In this context, histopathological examination is essential for defining the diagnosis of leprosy.

Intermediate or Borderline forms (BT, BB, and BL)

Leprosy is a long-term spectrum disease and can present various clinical and histopathological aspects. Between the two poles of leprosy, there is a wide range of types, consisting of intermediate or borderline forms. In this chapter, the clinical, histopathological, and bacilloscopic characteristics of the intermediate forms (borderlibe-tuberculoid [BT], borderline-borderline [BB], and borderline lepromatous [BL]) are presented and discussed. The main clinical and pathological characteristics that allow the diagnosis and classification of leprosy among the different borderline forms are described and illustrated in panel form, as well as their most significant clinical and histopathological differential diagnoses are also discussed. The clinical-pathological classification of this disease has important implications in the choice of the correct treatment, the understanding of the pathophysiology, and the development of the reaction phenomena typical of leprosy,.

Type 1 reaction (T1R)

A type 1 reaction (T1R) is also known as a reversal reaction. This phenomenon involves exarcebation of the immune system or delayed-type hypersensitivity in response to the antigens of Mycobacterium leprae present in parasitized tissues. It occurs in mnost patients of the tuberculoid and borderline forms of the Ridley & Jopling classification for leprosy. It is an important phenomenon that can occur before, during, or after leprosy treatment and can be destructive, causing tissue damage mainly in the nerves, as well as irreversible sequelae. The recognition of T1R in histological sections may be notified prior to clinical presentation. Histopathological recognition is vital in defining or confirming the presence of T1R, guiding the treatment of the reaction process, avoiding or reducing the possibiliy of serious sequelae, correcting possible mistakes in the classification of patients within the spectrum of leprosy, and ruling out other diseases that can clinically simulate a T1R. In this chapter, the histopathological characteristics that allow the recognition of T1R, various histopathological aspects of the common forms of leprosy, and histopathological differential diagnoses are discussed.

Type 2 reaction (T2R)

Type 2 reaction (t2R), also called erythema nodosum leprosum (ENL), is a reactional phenomenon that occurs in response to Mycobacterium leprae antigens in patients with borderline lepromatous our lepromatous forms of leprosy. T2R usually occurs after starting treatment and can affect any parasitic tissue in the body, causing neuritis, arthritis, painful lymphadenitis, buccopharyngeal lesions, laryngitis, hepatomegaly, splenomegaly, bone injuries, iridocyclitis, uveitis, orchitis, glomerulitis with proteinuria, and hematuria. Recognition of the histopathological characteristics of T2R is important for guiding early treatmen tof the reaction process, decreasing the likelihood of developing serious sequelae, especially when T2R affects the nerves, and to exclude different diseases that can simulate T2R over leprosy lesions during the T2R diagnosis are described, and some of its differential clinicopathological diagnoses and their possible pathophysiological mechanisms are discussed.