Spontaneous regional heading of extensive skin lesions in diffuse cutaneous leishmaniasis (DCL)

The authors report a case of diffuse cutaneous leishmaniasis, with longstanding evolution and presenting with diffuse infiltrated lesions rich in amastigotes in the absence of mucosal involvement. In situ characterization with monoclonal antibodies revealed Leishmania amazonensis. Large regional lesions have presented spontaneous healing without specific therapy. Considering that DCL presents with a defect in the cellular immune response, thisfact demonstrate that this patient may develop a regional cellular immune response enough to destroy the parasites and to produce clearing of some lesions.

Detection of early apoptosis and cell death in T CD4+ and CD8+ cells from lesions of patients with localized cutaneous leishmaniasis

Human localized cutaneous leishmaniasis (LCL), induced by Leishmania braziliensis, ranges from a clinically mild, self-healing disease with localized cutaneous lesions to severe forms which can present secondary metastatic lesions. The T cell-mediated immune response is extremely important to define the outcome of the disease; however, the underlying mechanisms involved are not fully understood. A flow cytometric analysis of incorporation of 7-amino actinomycin D and CD4+ or CD8+ T cell surface phenotyping was used to determine whether different frequencies of early apoptosis or accidental cell death occur at different stages of LCL lesions. When all cells obtained from a biopsy sample were analyzed, larger numbers of early apoptotic and dead cells were observed in lesions from patients with active disease (mean = 39.5 ± 2.7%) as compared with lesions undergoing spontaneous healing (mean = 17.8 ± 2.2%). Cells displaying normal viability patterns obtained from active LCL lesions showed higher numbers of early apoptotic events among CD8+ than among CD4+ T cells (mean = 28.5 ± 3.8 and 15.3 ± 3.0%, respectively). The higher frequency of cell death events in CD8+ T cells from patients with LCL may be associated with an active form of the disease. In addition, low frequencies of early apoptotic events among the CD8+ T cells were observed in two patients with self-healing lesions. Although the number of patients in the latter group was small, it is possible to speculate that, during the immune response, differences in apoptotic events in CD4+ and CD8+ T cell subsets could be responsible for controlling the CD4/CD8 ratio, thus leading to healing or maintenance of disease.

Cell populations in lesions of cutaneous leishmaniasis of leishmania (L.) amazonensis- infected rhesus macaques, Macaca mulatta

The cellular nature of the infiltrate in cutaneous lesion of rhesus monkeys experimentally infected with Leishmania (L.) amazonensis was characterized by immunohistochemistry. Skin biopsies from infected animals with active or healing lesions were compared to non-infected controls (three of each type) to quantitate inflammatory cell types. Inflammatory cells (composed of a mixture of T lymphocyte subpopulations, macrophages and a small number of natural killer cells and granulocytes) were more numerous in active lesions than in healing ones. T-cells accounted for 44.7 ± 13.1% of the infiltrate in active lesions (versus CD2+= 40.3 ± 5.7% in healing lesions) and T-cell ratios favor CD8+ cells in both lesion types. The percentage of cells expressing class II antigen (HLA-DR+) in active lesions (95 ± 7.1%) was significantly higher (P < 0.005) from the healing lesions (42.7 ± 12.7%). Moreover, the expression of the activation molecules CD25 (@ 16%), the receptor for interleukin-2, suggests that many T cells are primed and proliferating in active lesions. Distinct histopathological patterns were observed in lesions at biopsy, but healing lesions contained more organized epithelioid granulomas and activated macrophages, followed by fibrotic substitution. The progression and resolution of skin lesions appears to be very similar to that observed in humans, confirming the potential for this to be used as a viable model to study the immune response in human cutaneous leishmaniasis.

Histopathology of Leishmania major infection: revisiting L. major histopathology in the ear dermis infection model

We describe the relationship between lesion outcome and histopathological hallmarks in susceptible (BALB/c) and resistant (C57BL/6 and IL-4-deficient BALB/c) mouse strains over the course of a 12-week-infection with Leishmania major in the ear. The infiltration of mononuclear cells and polymorphonuclear cells occurred within 6 h and mononuclear cells predominated one week post-infection. Permissive intracellular growth of the pathogen was associated with non-healing lesions. In contrast, tissue damage and clearance of the parasite was observed in healing lesions and was associated with inducible nitric oxide synthase expression. The identification of the structural components of tissue reaction to the parasite in this study furthers our understanding of subjacent immune effector mechanisms.

The role of dentritic cells in leishmania infection : use of novel DC lines for the development of monoclonal antibodies

Les cellules dendritiques (DCs) sont des cellules multifonctionnelles qui font le lien entre le sytème immunitaire inné et adaptatif chez les mammifères. Il existe plusieurs sous-types de DCs basés sur leurs fonctions et l'endroit où elles se situent dans le corps. Dans le cadre de cette thèse, nous avons étudié le rôle de ces cellules face à une infection parasitaire. La Leishmania est un parasite causant une maladie appelée Leishmaniose, maladie endémique de l'Afrique, de l'Asie et de certaines régions de l'Amérique du Sud. Certaines espèces causent des lésions cutanées, alors que d'autres causent des lésions dans les muqueuses ou dans les organes internes. Le système immunitaire répond en générant une réponse inflammatoire qui élimine l'infection. Lors d'une réponse non-inflammatoire (de type cytokines, chemokines), cela va amener à une persistance du parasite sur le long terme. Les DC s'activant en présence du parasite dans la peau, vont le transporter vers un ganglion. A cet endroit, se trouvent différents sous-types de DC qui ont la particularité de présenter l'antigène (spécifique à la Leishmaniose) aux lymphocytes T, ce qui va alors amener à une réponse immunitaire puissante contre le parasite. Nous avons comparé différentes espèces de Leishmaniose dans leur façon d'activer les DC et différents modèles de souris ont été utilisé dans ce but-là. Les souris du type C57BL/6 sont connues pour être résistantes à L. major et sensibles à L. mexicana, alors qu'au contraire, les souris Balb/c sont connues pour être sensibles à ces deux espèces. En utilisant des parasites fluorescents transgéniques, nous avons comparé ces deux espèces de parasites (L. major et L. mexicana) en recherchant quelles cellules elles sont capables d'infecter in-vivo dans un modèle murin. Le rôle général des DC dans une infection à L. major a déjà été décrit. Dans notre étude, nous avons étudié le besoin en DC CD8a+ dans les ganglions afin d'engendrer une réponse face à une infection à L. major. Les souris qui n'ont pas ce sous-type de DC sont beaucoup plus sensibles à l'infection : elles ont des marqueurs inflammatoires plus bas et des lésions plus grandes. Nous avons également remarqué que les DC CD8a+ jouent un rôle crucial dans une phase plus avancée de l'infection. Dans notre laboratoire, nous avons la chance d'avoir une source illimitée de DCs de sous-type CD8a+ provenant d'une souris génétiquement modifiée par nos soin. Grâce à cela, nous avons utilisé ces cellules CD8a+ pour immuniser des rats afin de produire des anticorps monoclonaux ayant des propriétés spécifiques comme l'identification de protéines uniques présentes à la surface des DC et qui ensuite, modulent une réponse immunitaire in-vivo. Nous sommes actuellement en phase de caractérisation de plus de 750 hybridomes générés dans notre laboratoire. - Les cellules dendritiques (DCs) constituent le lien entre le système inné et adaptatif de la réponse immunitaire, car elles sont capables de présenter l'antigène, de donner la co- stimulation et de relâcher des cytokines et chimokines. Au cours de cette thèse, nous avons exploré différentes familles de DC lors d'infections parasitaires, telles que la Leishmaniose, parasite intracellulaire qui infecte les mammifères. La plupart des lésions cutanées résistantes sont caractérisées par une réponse pro-inflammatoire générée par l'IL-12. A l'inverse, pour la forme non résistante, la réponse est générée par l'IL-4 et l'IL-10, dans les modèles murins vulnérables. L'infection avec Lmajor a été caractérisée chez la souris C57BL/6 (Thl) et chez la souris Balb/c (Th2). Chez la souris C57BL/6 la lésion guérit, alors que chez la souris Balb/c, la lésion est au contraire non-cicatrisante. Nous avons comparé l'activation causée dans l'ensemble des DC par différentes espéces de Leishmania, et plus spécifiquement dans les DC CD8a+ présentes dans les ganglions lymphatiques et leur rôle dans la vulnérabilité à L. major. Ces cellules sont spécialisées dans la présentation croisée d'antigènes exogènes par le CMH-I et le haut taux de production d'IL-12 après activation. En utilisant des DC dérivées de moelle osseuse, nous avons constaté que L. guyanensis V+ (transportant un retrovirus) était le plus efficace pour l'activation des DC in-vitro comparé à L. major, L. mexicana et L. guyanensis (V-). Toutefois, in-vivo, les souris infectées avec L. major ont vu la taille de leur ganglions lymphatiques drainants augmentée, 3-6 semaines après l'infection dans les deux espèces de souris (les C57BL/6 résistantes et les Balb/c sensibles). En utilisant un parasite fluorescent transgénique, nous avons trouvé que les souris C57BL/6 sensibles à Lmexicana ont un nombre plus important de cellules Β infectées et un plus petit nombre de DC dérivées des monocytes inflammatoires, comparé au souris infectées avec L. major. Les conséquences de ces observations sont encore à l'étude. Des souris déficientes en CD8ct+DC et CD103+ sont plus sensibles à L. major que les souris WT: leurs lésions sont plus grandes et la charge parasitaire est plus importante. Nous avons généré une chimère de moelles osseuse CD11-DTR et Batf3-/- en mélangeant les moelles de ces deux souris, afin de déterminer le temps après infection où le manque de DC's CD8a+ contribue le plus à l'augmentation de la vulnérabilité chez la souris KO. Ces souris produisent plus d'IgG1 et IgE, font une réponse Th2 plus forte et Thl moins forte. Nous avons constaté que les souris déficientes en DC CD8a+ au début de la réponse immunitaire adaptive (trois semaines après injection) maintiennent un haut taux de lésions de grande taille, semblable à celui des souris chez qui les cellules ont été déplétées avant l'injection. Cela indique que les DC CD8a+ sont nécessaires pour l'efficacité de l'immunité dans la phase chronique de l'infection à L. major. Parallèlement à cela, nous avons aussi commencé une génération d'anticorps monoclonaux dirigés contre les DC CD8a+ activés en utilisant des souches établies dans notre laboratoire. En partant d'une librairie de 763 hybridomes, nous avons identifié plusieurs clones dignes d'intérêt avec une capacité fonctionnelle à moduler la prolifération et la sécrétion de cytokines des cellules T, ainsi que les molécules de co-stimulation présentes à la surface des DC activées elle-même. - Dendritic cells (DCs) are the bridge between the innate and the adaptive arms of the immune systems. They are professional antigen presentation cells and have important cytokine/chemokine release functions. In this dissertation we have focussed on the study of the different subsets of DCs in parasitic infection immunity. Leishmania are intra-cellular parasites of many different species that infect mammals. Most cutaneous lesions that are self- healing are characterized with a pro-inflammatory response with IL-12 while high levels of cytokines such as IL-4 and IL-10 characterized in susceptible mouse models. In mice L. major infection has been well characterized in C57BL/6 mice (Thl) that form healing lesions while Balb/c mice (Th2) form non-healing lesions. This thesis is focussed on comparing DC activation at large by different strains of Leishmania and more specifically, dLN resident CD8a+ DCs and their role in L. major susceptibility. This subset is specialized in cross- presentation of exogenous antigens in the MHC-I pathway and produce high levels of EL-12. Using bone marrow derived DCs we found that L. guyanensis V+ (carrying a retro-virus) was the most efficient at activating DCs in-vitro. In-vivo however L. major infected mice had the largest dLNs 3-6 weeks after infection in both genetically resistant C57BL/6 and susceptible Balb/c mice. Using transgenic fluorescent parasites, we found that C57BL/6 mice which are susceptible to L. mexicana had more number of infected Β cells and fewer number of infected inflammatory monocyte derived DCs in contrast to L. major infection. Using mice deficient in CD8a+ DCs, we found that these mice were more susceptible to L. major than their WT counterparts. They made larger lesions, had higher parasite burdens, higher levels of Th2 indicating immunolgloblins as measured by higher serie IgE levels and lower CD4+ IFNy+ cells. A mixed bone marrow chimera system of CDllc-DTR and Batf3~'~ was generated to determine the time point at which the lack of CD8a+ DCs most contributes to the increased susceptibility in KO mice. We found that mice depleted of CD8a+ DCs at the advent of the adaptive response (3 weeks after infection) maintained the significantly higher lesion size similar to mice whose cells were depleted from the onset of infection. This indicates that CD8a+ DCs are required for effective immunity in the chronic phase of L. major infection. We also began the generation of a valuable tool of monoclonal antibodies against activated CD8a+ DCs using our in-house DC line. From a library of 763 hybridomas we have identified several interesting clones with a functional ability to modulate Τ cell proliferation and cytokine secretion as well as down-modulating co-stimulatory molecules on activated DC cells themselves.

Implication of TNF family members in the immune response to the infection with the intracellular protozoan parasite "Leishmania major"

Suite à une infection avec le protozoaire Leishmania major (L. major), les souris sensibles de souche BALB/c développent des lésions progressives associées à une maturation des cellules CD4+ TH2 sécrétant de l'IL-4. A l'inverse, les souris résistantes de souche C57BL/6 guérissent à terme, sous l'influence de l'expansion des cellules CD4+ TH1 produisant de l'IFNy qui a un effet synergique avec le TNF ("tumor necrosis factor") sur l'activation des macrophages et leur fonction leishmanicide. Lors de notre étude nous avons montré que des souris C57BL/6 doublement déficientes en TNF et FasL ("Fas ligand") infectées par L. major ne guérissaient ni leur lésions ni ne contrôlaient la réplication de parasites malgré une réponse de type TH1. Bien que l'activité de synthétase inductible de l'oxyde nitrique ("iNOs") soit comparable chez les souris doublement ou simplement déficientes, seules celles déficientes en FasL ont démontré une incapacité à contrôler la réplication parasitaire. De surcroît il est apparu que le FasL a un effet synergique avec l'IFNy. L'adjonction de FasL à une culture cellulaire de macrophages stimulés par l'IFNy conduit à une activation de ces cellules. Celle-ci est démontrée par l'augmentation de la production de TNF et de NO par les macrophages ainsi que par l'élimination des parasites intracellulaires par ces mêmes cellules. Alors que le FasL et l'IFNy semblent essentiels au contrôle de la réplication des pathogènes intracellulaires, la contribution de TNF s'oriente davantage vers le contrôle de l'inflammation. L'activation macrophagique via Fas précède la mort cellulaire qui survient quelques jours plus tard. Cette mort cellulaire programmée était indépendante de la cascade enzymatique des caspases, au vu de l'absence d'effet de l'inhibiteur non-spécifique ZVAD-fmk des caspases. Ces résultats suggèrent que l'interaction Fas-FasL agit comme une costimulation nécessaire à une activation efficace des macrophages, la mort cellulaire survenant consécutivement à l'activation des macrophages.¦-¦Upon infection with the protozoan parasite Leishmania major (L. major), susceptible BALB/c mice develop non healing lesions associated with the maturation of CD4+ TH2 cells secreting IL-4. In contrast, resistant C57BL/6 mice are able to heal their lesions, because of CD4+ TH1 cell expansion and production of high levels of IFNy, which synergizes with tumour necrosis factor (TNF) in activating macrophages to their microbicidal state. In our study we showed that C57BL/6 mice lacking both TNF and Fas ligand (FasL) infected with L. major neither resolved their lesions nor controlled L. major replication despite a strong TH1 response. Although comparable inducible nitric oxide synthase (iNOs) was measured in single or double deficient mice, only mice deficient in FasL failed to control the parasite replication. Moreover FasL synergized with IFNy for the induction of leishmanicidal activity within macrophages infected with L. major in vitro. Addition of FasL to IFNy stimulated macrophages led to their activation, as reflected by the secretion of tumour necrosis factor and nitrite oxide, as well as the induction of their microbicidal activity, resulting in the killing of intracellular L. major. While FasL along with IFNy and iNOs appeared to be essential for the complete control of intracellular pathogen replication, the contribution of TNF appeared more important in controlling the inflammation on the site of infection. Macrophage activation via Fas pathway preceded cell death, which occurred a few days after Fas mediated activation. This program cell death was independent of caspase enzymatic activities as revealed by the lack of effect of ZVAD-fmk, a pan-caspase inhibitor. These results suggested that the Fas-FasL pathway, as part of the classical activation pathway of the macrophages, is essential in the stimulation of macrophage leading to a microbicidal state and to AICD, and may thus contribute to the pathogenesis of L. major infection.

An essential role for transmembrane TNF in the resolution of the inflammatory lesion induced by Leishmania major infection.

TNF is an essential player in infections with Leishmania major, contributing to the control of the inflammatory lesion and, to a lesser degree, to parasite killing. However, the relative contribution of the soluble and transmembrane forms of TNF in these processes is unknown. To investigate the role of transmembrane TNF (mTNF) in the control of L. major infections, mTNF-knock-in (mTNF(Delta/Delta)) mice, which express functional mTNF but do not release soluble TNF, were infected with L. major, and the development of the inflammatory lesion and the immune response was compared to that occurring in L. major-infected TNF(-/-) and wild-type mice. mTNF(Delta/Delta) mice controlled the infection and resolved their inflammatory lesion as well as wild-type mice, a process associated with the early clearance of neutrophils at the site of parasite infection. In contrast, L. major-infected TNF(-/-) mice developed non-healing lesions, characterized by an elevated presence of neutrophils at the site of infection and partial control of parasite number within the lesions. Altogether, the results presented here demonstrate that mTNF, in absence of soluble TNF, is sufficient to control infection due to L. major, enabling the regulation of inflammation, and the optimal killing of Leishmania parasites at the site of infection.

Healable supramolecular polymers

This mini-review details the recent development of self-healing and mendable polymeric materials which take advantage of the reversible characteristics of non-covalent interactions during their physical recovery process. Supramolecular polymer systems which undergo spontaneous (autonomous) healing, as well as those which require external stimuli to initiate the healing process (healable/mendable), are introduced and discussed. Supramolecular polymers offer key advantages over alternative approaches, as these materials can typically withstand multiple healing cycles without substantial loss of performance, as a consequence of the highly directional and fully reversible non-covalent interactions present within the polymer matrix.

An essential role for transmembrane TNF in the resolution of the inflammatory lesion induced by Leishmania major infection

TNF is an essential player in infections with Leishmania major, contributing to the control of the inflammatory lesion and, to a lesser degree, to parasite killing. However, the relative contribution of the soluble and transmembrane forms of TNF in these processes is unknown. To investigate the role of transmembrane TNF (mTNF) in the control of L. major infections, mTNF-knock-in (mTNF(Delta/Delta)) mice, which express functional mTNF but do not release soluble TNF, were infected with L. major, and the development of the inflammatory lesion and the immune response was compared to that occurring in L. major-infected TNF(-/-) and wild-type mice. mTNF(Delta/Delta) mice controlled the infection and resolved their inflammatory lesion as well as wild-type mice, a process associated with the early clearance of neutrophils at the site of parasite infection. In contrast, L. major-infected TNF(-/-) mice developed non-healing lesions, characterized by an elevated presence of neutrophils at the site of infection and partial control of parasite number within the lesions. Altogether, the results presented here demonstrate that mTNF, in absence of soluble TNF, is sufficient to control infection due to L. major, enabling the regulation of inflammation, and the optimal killing of Leishmania parasites at the site of infection.

Spontaneous healing of Mycobacterium ulcerans lesions in the guinea pig model

Buruli Ulcer (BU) is a necrotizing skin disease caused by Mycobacterium ulcerans infection. BU is characterized by a wide range of clinical forms, including non-ulcerative cutaneous lesions that can evolve into severe ulcers if left untreated. Nevertheless, spontaneous healing has been reported to occur, although knowledge on this process is scarce both in naturally infected humans and experimental models of infection. Animal models are useful since they mimic different spectrums of human BU disease and have the potential to elucidate the pathogenic/protective pathway(s) involved in disease/healing. In this time-lapsed study, we characterized the guinea pig, an animal model of resistance to M. ulcerans, focusing on the macroscopic, microbiological and histological evolution throughout the entire experimental infectious process. Subcutaneous infection of guinea pigs with a virulent strain of M. ulcerans led to early localized swelling, which evolved into small well defined ulcers. These macroscopic observations correlated with the presence of necrosis, acute inflammatory infiltrate and an abundant bacterial load. By the end of the infectious process when ulcerative lesions healed, M. ulcerans viability decreased and the subcutaneous tissue organization returned to its normal state after a process of continuous healing characterized by tissue granulation and reepethelialization. In conclusion, we show that the experimental M. ulcerans infection of the guinea pig mimics the process of spontaneous healing described in BU patients, displaying the potential to uncover correlates of protection against BU, which can ultimately contribute to the development of new prophylactic and therapeutic strategies.

ChronOS Inject in Children with Benign Bone Lesions – Does It Increase the Healing Rate?

Benign bone lesions in children although rare, can result in a pathological fracture. Although their etiology and pathogenesis are not yet entirely clear, the phenomenon of spontaneous healing is well known. Nevertheless, some benign bone lesions are unlikely to heal spontaneously due to the patient's age or high risk of fracture and deformity due to the lesion's location or size. The following study presents our results after treatment of these bone cysts with chronOS Inject.

S-nitrosoglutathione (gsno) Is Cytotoxic To Intracellular Amastigotes And Promotes Healing Of Topically Treated Leishmania Major Or Leishmania Braziliensis Skin Lesions-authors' Response.

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Influence of apical foramen widening and sealer on the healing of chronic periapical lesions induced in dogs' teeth

Objective. The aim of this study was to evaluate the influence of apical foramen widening on the healing of chronic periapical lesions in dogs' teeth after root canal filling with Sealer 26 or Endomethasone.Study design. Forty root canals of dogs' teeth were used. After pulp extirpation, the canals were exposed to the oral cavity for 180 days for induction of periapical lesions, and then instrumented up to a size 55 K-file at the apical cemental barrier. In 20 roots, the cemental canal was penetrated and widened up to a size 25 K-file; in the other 20 roots, the cemental canal was preserved (no apical foramen widening). All canals received a calcium hydroxide intracanal dressing for 21 days and were filled with gutta-percha and 1 of the 2 sealers: group 1: Sealer 26/apical foramen widening; group 2: Sealer 26/no apical foramen widening; group 3: Endomethasone/apical foramen widening; group 4: Endomethasone/no apical foramen widening. The animals were killed after 180 days, and serial histologic sections from the roots were prepared for histomorphologic analysis. Scores were assigned according to preestablished histomorphologic parameters and analyzed statistically by Kruskal-Wallis and Mann-Whitney U tests.Results. Regarding new cementum formation, repair of cementum and bone resorption areas, presence of microorganisms, inflammatory cell infiltrate and periodontal ligament conditions, significantly better periapical healing was obtained when foramen widening was done and Sealer 26 was used.Conclusion. Apical foramen widening and calcium hydroxide-containing sealer were more favorable to the healing of chronic periapical lesions. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109:932-940)

Age-related histopathological lesions in the Mongolian gerbil ventral prostate as a good model for studies of spontaneous hormone-related disorders

The Meriones unguiculatus (Mongolian) gerbil has demonstrated significant prostatic responses to hormonal treatments, and to drugs against human prostatic hyperplasia Spontaneous neoplasia develops in the older animals. Thirty gerbils (age 18 months) were divided into non-affected and prostatic lesion bearers and the prostate lesions were evaluated morphologically, immunohistochemically and quantitatively. The most frequent changes were in epithelial sites and, namely prostatic intraepithelial neoplasias, microinvasive carcinomas and adenocarcinomas. In the stromal compartment, cellular hyperplasia, when verified, was always associated with the sites of anomalous epithelium. Additionally, larger deposition of collagen fibrils, generating stromal fibrosis, was found in all the old gerbils analysed. The quantitative analysis showed that prostatic tissue proportions differed in altered areas, being specific for each lesion type. Isolated nuclear and nucleolar parameters were not effective in diagnosing the malign potential of lesions. However, the cellular proliferation and death indexes indicated larger cellular turnover in invasive lesions such as carcinomas. With these analyses, it could be verified that old gerbils present high propensity to develop spontaneous prostate changes and this may aid in a better understanding of the biological behaviour of human prostate cancer.