Whole blood transcriptomics reveals the enrichment of neutrophil activation pathways during erythema nodosum leprosum reaction.

Introduction: Patients with the multibacillary form of leprosy can develop reactional episodes of acute inflammation, known as erythema nodosum leprosum (ENL), which are characterized by the appearance of painful cutaneous nodules and systemic symptoms. Neutrophils have been recognized to play a role in the pathogenesis of ENL, and recent global transcriptomic analysis revealed neutrophil-related processes as a signature of ENL skin lesions. Methods: In this study, we expanded this analysis to the blood compartment, comparing whole blood transcriptomics of patients with non-reactional lepromatous leprosy at diagnosis (LL, n=7) and patients with ENL before administration of anti-reactional treatment (ENL, n=15). Furthermore, a follow-up study was performed with patients experiencing an ENL episode at the time of diagnosis and after 7 days of thalidomide treatment (THAL, n=10). Validation in an independent cohort (ENL=8; LL=7) was performed by RT-qPCR. Results: An enrichment of neutrophil activation and degranulation-related genes was observed in the ENL group, with the gene for the neutrophil activation marker CD177 being the most enriched gene of ENL episode when compared to its expression in the LL group. A more pro-inflammatory transcriptome was also observed, with increased expression of genes related to innate immunity. Validation in an independent cohort indicated that S100A8 expression could discriminate ENL from LL. Supernatants of blood cells stimulated in vitro with Mycobacterium leprae sonicate showed higher levels of CD177 compared to the level of untreated cells, indicating that the leprosy bacillus can activate neutrophils expressing CD177. Of note, suggestive higher CD177 protein levels were found in the sera of patients with severe/moderate ENL episodes when compared with patients with mild episodes and LL patients, highlighting CD177 as a potential systemic marker of ENL severity that deserves future confirmation. Furthermore, a follow-up study was performed with patients at the time of ENL diagnosis and after 7 days of thalidomide treatment (THAL, n=10). Enrichment of neutrophil pathways was sustained in the transcriptomic profile of patients undergoing treatment; however, important immune targets that might be relevant to the effect of thalidomide at a systemic level, particularly NLRP6 and IL5RA, were revealed. Discussion: In conclusion, our study reinforces the key role played by neutrophils in ENL pathogenesis and shed lights on potential diagnostic candidates and novel therapeutic targets that could benefit patients with leprosy.

Whole blood transcriptomics reveals the enrichment of neutrophil activation pathways during erythema nodosum leprosum reaction

Introduction: Patients with the multibacillary form of leprosy can develop reactional episodes of acute inflammation, known as erythema nodosum leprosum (ENL), which are characterized by the appearance of painful cutaneous nodules and systemic symptoms. Neutrophils have been recognized to play a role in the pathogenesis of ENL, and recent global transcriptomic analysis revealed neutrophil-related processes as a signature of ENL skin lesions. Methods: In this study, we expanded this analysis to the blood compartment, comparing whole blood transcriptomics of patients with non-reactional lepromatous leprosy at diagnosis (LL, n=7) and patients with ENL before administration of anti-reactional treatment (ENL, n=15). Furthermore, a follow-up study was performed with patients experiencing an ENL episode at the time of diagnosis and after 7 days of thalidomide treatment (THAL, n=10). Validation in an independent cohort (ENL=8; LL=7) was performed by RT-qPCR. Results: An enrichment of neutrophil activation and degranulation-related genes was observed in the ENL group, with the gene for the neutrophil activation marker CD177 being the most enriched gene of ENL episode when compared to its expression in the LL group. A more pro-inflammatory transcriptome was also observed, with increased expression of genes related to innate immunity. Validation in an independent cohort indicated that S100A8 expression could discriminate ENL from LL. Supernatants of blood cells stimulated in vitro with Mycobacterium leprae sonicate showed higher levels of CD177 compared to the level of untreated cells, indicating that the leprosy bacillus can activate neutrophils expressing CD177. Of note, suggestive higher CD177 protein levels were found in the sera of patients with severe/moderate ENL episodes when compared with patients with mild episodes and LL patients, highlighting CD177 as a potential systemic marker of ENL severity that deserves future confirmation. Furthermore, a follow-up study was performed with patients at the time of ENL diagnosis and after 7 days of thalidomide treatment (THAL, n=10). Enrichment of neutrophil pathways was sustained in the transcriptomic profile of patients undergoing treatment; however, important immune targets that might be relevant to the effect of thalidomide at a systemic level, particularly NLRP6 and IL5RA, were revealed. Discussion: In conclusion, our study reinforces the key role played by neutrophils in ENL pathogenesis and shed lights on potential diagnostic candidates and novel therapeutic targets that could benefit patients with leprosy.

Patient with McCune albright syndrome: Case report and 10 Years of follow-up imaging examination.

The McCune Albright syndrome (MAS) is a rare, multi-system disease composed of the triad of polyostotic fibrous dysplasia of bone (PFDB), café-au-lait skin hyperpigmentation, and endocrine disorders. The diagnosis involves clinical, biochemical and imaging findings, with dentistry playing an important role in MAS, since many patients present DFPO in the craniofacial bones, including the maxilla and mandible, and in view of their dental needs, the correct management of these patients is not only an essential but important area to be investigated. This report presents a case of a patient with McCune Albright Syndrome, the behavior of the disease over a period of 10 years and how imaging exams such as scintigraphy and tomography were important for planning the dental treatment of this patient, since they are fundamental allies for identification and evaluation of the progression and/or stability of the disease. Key words:Craniofacial fibrous dysplasia, cone-beam computed tomography, scintigraphy, imaging diagnosis.

Mycobacterium leprae Induces Neutrophilic Degranulation and Low-Density Neutrophil Generation During Erythema Nodosum Leprosum.

Erythema Nodosum Leprosum (ENL) is a recurrent acute inflammatory complication of leprosy affecting up to 50% of all Borderline Lepromatous and Lepromatous Leprosy (BL/LL) patients. Although ENL is described as an immune reaction mediated by neutrophils, studies demonstrating the direct role of neutrophils in ENL are still rare. One subpopulation of low-density neutrophils (LDNs), present within the fraction of peripheral blood mononuclear cells (PBMC), has been associated with the pathogenesis and severity of diseases like sepsis, lupus, and tuberculosis. We herein analyzed LDNs and high-density neutrophils (HDNs) in terms of frequency, phenotype, and morphology. Serum levels of MMP-9 (a neutrophilic degranulation marker) were evaluated by ELISA; and LDNs were generated in vitro by stimulating healthy-donor, whole-blood cultures. PBMC layers of ENL patients presented segmented/hypersegmented cells that were morphologically compatible with neutrophils. Immunofluorescence analyses identified LDNs in ENL. Flow cytometry confirmed the elevated frequency of circulating LDNs (CD14-CD15+) in ENL patients compared to healthy donors and nonreactional Borderline Tuberculoid (BT) patients. Moreover, flow cytometry analyses revealed that ENL LDNs had a neutrophilic-activated phenotype. ENL patients under thalidomide treatment presented similar frequency of LDNs as observed before treatment but its activation status was lower. In addition, Mycobacterium leprae induced in vitro generation of LDNs in whole blood in a dose-dependent fashion; and TGF-ß, an inhibitor of neutrophilic degranulation, prevented LDNs generation. MMP-9 serum levels of BL/LL patients with or without ENL correlated with LDNs frequency at the same time that ultrastructural observations of ENL LDNs showed suggestive signs of degranulation. Together, our data provide new insights into the knowledge and understanding of the pathogenesis of ENL while enriching the role of neutrophils in leprosy.

Erythema Nodosum Leprosum Neutrophil Subset Expressing IL-10R1 Transmigrates into Skin Lesions and Responds to IL-10.

Erythema nodosum leprosum (ENL) is an inflammatory complication in leprosy. Yet, the involvement of ENL neutrophils in the inflammatory response against Mycobacterium leprae remains poorly explored. Our primary aim was to investigate the utility of the surface expression of neutrophil IL-10R1 as an ENL biomarker and, secondarily, to evaluate whether leprosy or healthy M. leprae-stimulated neutrophils produce cytokines and are able to respond to IL-10. We, in this study, describe a subpopulation of circulating neutrophils of ENL patients that exclusively expressed IL-10R1, providing evidence that IL-10R1+ neutrophils are present in ENL lesions. It was also found that ENL neutrophils, but not those of nonreactional leprosy controls, were able to secret detectable levels of TNF ex vivo and the addition of IL-10 blocked TNF release. It was likewise observed that M. leprae-stimulated, healthy neutrophils expressed IL-10R1 in vitro, and ENL-linked cytokines were released by M. leprae-cultured neutrophils in vitro. Moreover, consistent with the presence of a fully functional IL-10R, the addition of IL-10 prevented the release of M. leprae-induced cytokines. Most importantly, dead M. leprae revealed its superior capacity to induce CCL4 and IL-8 in primary neutrophils over live Mycobacterium, suggesting that M. leprae may hamper the inflammatory machinery as an immune escape mechanism.

Leptolegnia chapmanii como alternativa biológica para el control de Aedes aegypti

Leptolegnia chapmanii is a facultative pathogen of many species of mosquitoes, among which species of the genus Aedes, Culex and Anopheles stand out for their medical and sanitary importance. The potential of L. chapmanii as an alternative to control lies in its virulence, pathogenicity and specificity against the larval stages of mosquitoes, and because of its harmlessness to non-target species such as fish and amphibians, among others. The natural presence of L. chapmanii had been reported in Argentina, Brazil and the United States. Its presence is possible in other countries throughout the American continent. The development of protocols to produce, formulate, store and apply products based on this microorganism is one of the objectives proposed for the group of Entomopathogenic Fungi at the Centro de Estudios Parasitológicos y de Vectores, Universidad Nacional de La Plata. The efficacy of L. chapmanii as controller is affected by external factors such as temperature, pH, salinity and radiation among others. The process of transfer from the research centers to industry implies many phases. In this way, our project with L. chapmanii is in an initial phase, where we are working on a laboratory scale in proof of concept. We hope to begin soon with the efficacy, efficiency, stability and ecotoxicological safety tests, at the laboratory, semi-field and field scale.

Leptolegnia chapmanii es un microorganismo patógeno facultativo de diversas especies de mosquito, entre las que se destacan, por su importancia médica y sanitaria, especies de los géneros Aedes, Culex y Anopheles. El potencial de L. chapmanii como alternativa de control radica en la virulencia, capacidad patógena y grado de especificidad que presenta hacia los estadios larvales de las diferentes especies de mosquito, y por su inocuidad frente a organismos acuáticos no blanco como, por ejemplo, peces y anfibios. Su presencia natural ha sido reportada en Argentina, Brasil, y Estados Unidos, pensándose como posible en otros países dentro del continente americano. La eficacia de L. chapmanii como controlador se ve influenciada por factores externos, como la temperatura, la radiación y el pH, entre otros. Uno de los objetivos de trabajo del Grupo de Hongos Entomopatógenos del Centro de Estudios Parasitológicos y de Vectores de la Universidad Nacional de La Plata, corresponde al desarrollo de protocolos para la producción, formulación, almacenamiento y aplicación de productos basados en este microorganismo. Con este referente, estamos desarrollando un proyecto con L. chapmanii que se encuentra en la fase inicial, en la que se está trabajando la prueba de concepto a escala de laboratorio. Se espera continuar en el futuro con estudios de eficacia, eficiencia, estabilidad y seguridad ecotoxicológica, a diferentes escalas.

Leptolegnia chapmanii como alternativa biológica para el control de Aedes aegypti / Leptolegnia chapmanii as a biological alternative to control Aedes aegypti

Leptolegnia chapmanii es un microorganismo patógeno facultativo de diversas especies de mosquito, entre las que se destacan, por su importancia médica y sanitaria, especies de los géneros Aedes, Culex y Anopheles. El potencial de L. chapmanii como alternativa de control radica en la virulencia, capacidad patógena y grado de especificidad que presenta hacia los estadios larvales de las diferentes especies de mosquito, y por su inocuidad frente a organismos acuáticos no blanco como, por ejemplo, peces y anfibios. Su presencia natural ha sido reportada en Argentina, Brasil, y Estados Unidos, pensándose como posible en otros países dentro del continente americano. La eficacia de L. chapmanii como controlador se ve influenciada por factores externos, como la temperatura, la radiación y el pH, entre otros. Uno de los objetivos de trabajo del Grupo de Hongos Entomopatógenos del Centro de Estudios Parasitológicos y de Vectores de la Universidad Nacional de La Plata, corresponde al desarrollo de protocolos para la producción, formulación, almacenamiento y aplicación de productos basados en este microorganismo. Con este referente, estamos desarrollando un proyecto con L. chapmanii que se encuentra en la fase inicial, en la que se está trabajando la prueba de concepto a escala de laboratorio. Se espera continuar en el futuro con estudios de eficacia, eficiencia, estabilidad y seguridad ecotoxicológica, a diferentes escalas.

Leptolegnia chapmanii is a facultative pathogen of many species of mosquitoes, among which species of the genus Aedes, Culex and Anopheles stand out for their medical and sanitary importance. The potential of L. chapmanii as an alternative to control lies in its virulence, pathogenicity and specificity against the larval stages of mosquitoes, and because of its harmlessness to non-target species such as fish and amphibians, among others. The natural presence of L. chapmanii had been reported in Argentina, Brazil and the United States. Its presence is possible in other countries throughout the American continent. The development of protocols to produce, formulate, store and apply products based on this microorganism is one of the objectives proposed for the group of Entomopathogenic Fungi at the Centro de Estudios Parasitológicos y de Vectores, Universidad Nacional de La Plata. The efficacy of L. chapmanii as controller is affected by external factors such as temperature, pH, salinity and radiation among others. The process of transfer from the research centers to industry implies many phases. In this way, our project with L. chapmanii is in an initial phase, where we are working on a laboratory scale in proof of concept. We hope to begin soon with the efficacy, efficiency, stability and ecotoxicological safety tests, at the laboratory, semi-field and field scale.

Neutrophils in Leprosy.

Leprosy is an infectious disease caused by the intracellular bacillus Mycobacterium leprae that mainly affects the skin and peripheral nerves. One of the most intriguing aspects of leprosy is the diversity of its clinical forms. Paucibacillary patients are characterized as having less than five skin lesions and rare bacilli while the lesions in multibacillary patients are disseminated with voluminous bacilli. The chronic course of leprosy is often interrupted by acute episodes of an inflammatory immunological response classified as either reversal reaction or erythema nodosum leprosum (ENL). Although ENL is considered a neutrophilic immune-complex mediated condition, little is known about the direct role of neutrophils in ENL and leprosy disease overall. Recent studies have shown a renewed interest in neutrophilic biology. One of the most interesting recent discoveries was that the neutrophilic population is not homogeneous. Neutrophilic polarization leads to divergent phenotypes (e.g., a pro- and antitumor profile) that are dynamic subpopulations with distinct phenotypical and functional abilities. Moreover, there is emerging evidence indicating that neutrophils expressing CD64 favor systemic inflammation during ENL. In the present review, neutrophilic involvement in leprosy is discussed with a particular focus on ENL and the potential of neutrophils as clinical biomarkers and therapeutic targets.

Manipulação gengival para harmonia gengivo-dental em reabilitação unitária anterior com implante û relato de caso / Gingival management for gingival and dental harmony in anterior single rehabilitation with implant û case report

A harmonia gengivo-dental define estética nas reabilitações do arco superior. Em situações, nas quais ocorre perda óssea alveolar no sentido vestíbulo-lingual e ápico-coronal, é necessária associação de técnicas reconstrutivas, visando recuperar volume tecidual. Paciente do gênero masculino, 32 anos, perdeu elemento 11 devido à reabsorção radicular após trauma na região anterior. Foi feita extração dentária, seguida por enxerto ósseo em bloco, instalação de implante e restauração adesiva com dente de estoque. Após o período de osseointegração foi realizado enxerto conjuntivo subepitelial tanto para ganho no sentido vestíbulo-lingual, como ápico-coronal. Imediatamente após o enxerto, iniciou-se o condicionamento gengival com a própria prótese adesiva com pôntico em formato convexo. Em seguida, foi feita a reabertura do implante, instalação de intermediário e confecção de provisório, preservando a arquitetura gengival conseguida. O procedimento provou ser eficiente, garantindo margem gengival e volume de tecido muito próximos do dente adjacente e satisfazendo a expectativa estética do paciente.

The aesthetics of maxillary restorations is defined by the harmony between gingiva and teeth. gingival-dental harmony defines the aesthetics in the upper arch rehabilitation. In cases of alveolar bone loss in buccolingual and apical-coronal direction an association of techniques is required to recover the tissues volume. Male patient, 32 years old, lost element 11 due to radicular resorption, after trauma in the anterior region. Tooth extraction, block bone graft, implant installation and adhesive restoration with a stock tooth were performed. After osseointegration period, a subepithelial connective tissue graft was made to have gain in both directions, buccolingual and apical-coronal. Immediately after the graft, gingival conditioning started with the same adhesive prosthesis with convex shape pint. Then implant was reopened, abutment was installed and the preparation of provisional was executed, preserving the achieved gingival architecture. The procedure proved to be efficient, ensuring gingival margin and tissue volume very close to the adjacent tooth and satisfying the aesthetic expectations of the patient.