Mucosal leishmaniasis: in situ characterization of the host inflammatory response,before and after treatment

Mucosal leishmaniasis (ML) generally shows progressive tissue destruction, not yet fully elucidated, associated with an intense inflammatory response. To contribute to the understanding of this process and of how treatment interferes with it, we studied several anatomopathological parameters, including those analyzed by immunohistochemistry, such as Leishmania antigens, cells participating in the immune response and cytokine expression. Biopsies were taken from 20 patients with ML before and after treatment. A mixed Th1 and Th2 pattern response occurred inside ML before treatment, persist after treatment. Nevertheless, this mixed response was smaller than in active lesions, with reduced but present numbers of cells expressing TNF-alpha, IFN-gamma and IL-4 and sustained numbers of cells expressing IL-10. We may conclude that specific treatment causes a reduction of inflammatory lesions and disappearance of amastigote forms of Leishmania although the factors related to the pathogenesis of the lesion, such as T CD4+ and T CD8+ lymphocytes and Leishmania antigens, persist in treated lesions. The maintenance of these inflammatory patterns may be due to a specific host-parasite relationship response, strongly indicating the need for continuous surveillance of LM patients at risk of reactivation, despite effective cicatrization after therapy.     

Differential immune regulation of activated T cells between cutaneous and mucosal leishmaniasis as a model for pathogenesis

Cutaneous (CL) and mucosal leishmaniasis (ML) are characterized by a predominant type 1 immune response (IFN-gamma and TNF-alpha production) and strong inflammatory response in the lesions with few parasites. This exacerbated type 1 response is more evident in ML as compared to CL. Our main hypothesis is that a differential immune regulation of T cell activation leads to over reactive T cells in ML. In the present study, we investigated immunological factors that could explain the mechanisms behind it by comparing some immune regulatory mechanisms between ML and CL patients: frequency of cells expressing co-stimulatory molecules, apoptotic markers, T cell activation markers; and ability of neutralizing antibodies to IL-2, IL-12 and IL-15 do down-regulate IFN-gamma production in leishmania antigen-stimulated peripheral blood mononuclear cells (PBMC). Interestingly, in CL anti-IL-2 and anti-IL-15 significantly suppressed antigen-specific IFN-gamma production, while in ML only anti-IL-2 suppressed IFN-gamma production. Finally, higher frequency of CD4+ T cells expressing CD28-, CD69+ and CD62L(low) were observed in ML as compared to CL. These data indicate that an exacerbated type 1 response in ML is differentially regulated and not appropriately down modulated, with increased frequencies of activated effectors T cells, maintaining the persistent inflammatory response and tissue damage observed in ML.     

Signs of an in situ inflammatory reaction in scars of human American tegumentary leishmaniasis

Skin inflammation plays an important role during the healing of American tegumentary leishmaniasis (ATL), the distribution of cells in active lesions may vary according to disease outcome and parasite antigens in ATL scars have already been shown. We evaluated by immunohistochemistry, 18 patients with 1‐ or 3‐year‐old scars and the corresponding active lesions and compared them with healthy skin. Small cell clusters in scars organized as in the active lesions spreaded over the fibrotic tissue were detected, as well as close to vessels and cutaneous glands, despite a reduction in the inflammatory process. Analysis of 1‐year‐old scar tissue showed reduction of NOS2, E‐selectin, Ki67, Bcl‐2 and Fas expression. However, similar percentages of lymphocytes and macrophages were detected when compared to active lesions. Only 3‐year‐old scars showed reduction of CD3⁺, CD4⁺ and CD8⁺T cells, in addition to reduced expression of NOS2, E‐selectin, Ki67 and BCl‐2. These results suggest that the pattern of cellularity of the inflammatory reaction observed in active lesions changes slowly even after clinical healing. Analysis of 3‐year‐old scars showed reduction of the inflammatory reaction as demonstrated by decrease in inflammatory cells and in the expression of cell‐activity markers, suggesting that the host–parasite balance was only established after that period.     

Short report: persistence of tumor necrosis factor-alpha in situ after lesion healing in mucosal leishmaniasis

Mucosal leishmaniasis (ML) is a disease characterized by intense activation of inflammatory cells and extensive tissue destruction. Among the cytokines involved in the immune response to ML, tumor necrosis factor-alpha (TNF-alpha) has attracted strong interest because of its roles in the modulation of the immune response. We studied 20 patients with ML who provided biopsy specimens before treatment and after lesion healing obtained by specific therapy. The biopsy specimens were subjected to immunohistochemical analysis for in situ quantification of cellular and extracellular TNF-alpha. The amount of TNF-alpha was significantly lower in the healed lesions compared with pretreatment biopsy specimens, although TNF-alpha persisted at the tissue level even after lesion healing. This relevant finding demonstrates for the first time an in situ tissue reduction of TNF-alpha after treatment and shows persistence of TNF-alpha in healed lesions may be related to the maintenance of an immunopathologic background for relapses observed in ML.     

Failure of nitric oxide production by macrophages and decrease in CD4+ T cells in oral paracoccidioidomycosis: possible mechanisms that permit local fungal multiplication

Paracoccidioidomycosis is a chronic granulomatous disease that induces a specific inflammatory and immune response. The participation of nitric oxide (NO), a product of the inducible nitric oxide synthase enzyme (iNOS), as an important fungicidal molecule against Paracoccidioides brasiliensis has been demonstrated. In order to further characterize the Oral Paracoccidioidomycosis (OP), we undertook an immunohistochemical study of iNOS+, CD45RO+, CD3+, CD8+, CD20+, CD68+ cells and mast cells. The samples were distributed in groups according to the number of viable fungi per mm2. Our results showed weak immunolabeling for iNOS in the multinucleated giant cells (MNGC) and in most of the mononuclear (MN) cells, and the proportion of iNOS+ MN/MNGC cells in the OP were comparable to Control (clinically healthy oral tissues). Additionally, our analysis revealed a similarity in the number of CD4+ cells between the Control and the OP groups with higher numbers of fungi. These findings suggest that a low expression of iNOS and a decrease in the CD4+ T cells in OP may represent possible mechanisms that permit the local fungal multiplication and maintenance of active oral lesions.     

Tissue eosinophilia and Leishmania mexicana mexicana eosinophil interactions in murine cutaneous leishmaniasis

Summary Outbred albino mice were infected subcutaneously with 10⁶ amastigotes of Leishmania mexicana mexicana and the subsequent lesions were evaluated by light and electron microscopy at various intervals after infection. The animals developed persistent nodules and a spectrum of lesions of variable size which was correlated with the host's ability to control the parasite in the tissue. During the acute phase of the disease the histopathological results showed an accumulation of granulocytes, some mononuclear phagocytes and a predominance of eosinophils as compared to other cell types. In this early acute phase, eosinophils were found in the tissue together with normal and degranulating mast cells. In the granulomatous inflammatory reaction of the chronic phases, there was infiltration of granulocytes parallel to parasite multiplication and the formation of parasitized vacuolated macrophages. The number of eosinophils was consistently greater than neutrophils, regardless of lesion type or number of parasites present in the tissue. During the acute reaction, the granulocytes apparently destroyed many parasites; however, there was an unvaryingly low level of phagocytosis of amastigotes during the chronic stages by both eosinophils and neutrophils. Neutrophils seemed to be more effective than eosinophils in the killing of ingested parasites. A close association between eosinophils and parasitized macrophages was seen in the chronic lesions; thus, eosinophils might contribute to parasite destruction through co-operation with macrophages.     

Amplification of inflammation in emphysema and its association with latent adenoviral infection

This study examines the hypothesis that the cigarette smoke-induced inflammatory process is amplified in severe emphysema and explores the association of this response with latent adenoviral infection. Lung tissue from patients with similar smoking histories and either no (n = 7), mild (n = 7), or severe emphysema (n = 7) was obtained by lung resection. Numbers of polymorphonuclear cells (PMN), macrophages, B cells, CD4, CD8 lymphocytes, and eosinophils present in tissue and airspaces and of epithelial cells expressing adenoviral E1A protein were determined using quantitative techniques. Severe emphysema was associated with an absolute increase in the total number of inflammatory cells in the lung tissue and airspaces. The computed tomography (CT) determined extent of lung destruction was related to the number of cells/m(2) surface area by R(2) values that ranged from 0.858 (CD8 cells) to 0.483 (B cells) in the tissue and 0.630 (CD4 cells) to 0.198 (B cells) in the airspaces. These changes were associated with a 5- to 40-fold increase in the number of alveolar epithelial cells expressing adenoviral E1A protein in mild and severe disease, respectively. We conclude that cigarette smoke-induced lung inflammation is amplified in severe emphysema and that latent expression of the adenoviral E1A protein expressed by alveolar epithelial cells influenced this amplification process.     

Influence of the nutritional status in the clinical and therapeutical evolution in adults and elderly with American Tegumentary Leishmaniasis

The objective of this study is to describe the nutritional status of adult and elderly patients with American Tegumentary Leishmaniasis (ATL). It was conducted a longitudinal study in 68 adult and elderly patients with ATL treating at the Surveillance Leishmaniasis Laboratory at the Evandro Chagas Clinical Research Institute, Oswaldo Cruz Foundation (Fiocruz), from 2009 to 2012. The nutritional assessment included the body mass index (BMI) and serum albumin levels. The clinical evolution (epithelialization and wound healing) was measured up to two years after ATL treatment. Most of the sample was composed of men (71%), adults (73%), with household income of 1–5 minimum wages (79%), and incomplete elementary school (48.5%). The predominant ATL form was cutaneous (72%), and 39% presented comorbidities, the most frequent was hypertension (30.8%). The most prevalent clinical and nutritional events were: recent decrease in food intake (23.9%); nasal obstruction (22.1%); oral ulcer (14.7%), anorexia and dysphagia (13.2% each) and odynophagia (10.3%). The total healing time was 115.00 (IR = 80–230) days for skin lesions, and 120.00 (IR = 104.50–223.50) days for mucous membrane lesions. Low body weight in 10%, and hypoalbuminemia in 12% of the patients have been observed. Low body weight was associated with age, mucosal leishmaniasis (ML), nasal obstruction, recent decrease in food intake and hypoalbuminemia. As for serum albumin depletion, association with the ML, dyspnea, dysphagia, odynophagia, recent decrease in food intake, absence of complete healing of the skin lesions, and increased healing time for mucous membrane lesions, was observed. The ML and their events that affect the alimentary intake have been related to the impairment of the nutritional status. Additionally, serum albumin depletion negatively affected the healing of the lesions, suggesting that a nutritional intervention can increase the effectiveness of the ATL treatment.     

Induction of early atherosclerosis in CBA/J mice by combination of Trypanosoma cruzi infection and a high cholesterol diet

In addition to established factors such as hyperlipidemia, smoking and hypertension, inflammation and infection have recently been implicated as major risk factors for atherosclerotic disease. Proatherogenic effects induced by infection may be related to both systemic inflammation and to direct effects on the vascular wall. We report here that a high fat diet combined with a protozoal infection with known tropism to the heart induced early atherosclerosis and intimal inflammatory infiltrates (CD4+, CD8+ cells and macrophages) in aortas of all (n = 7) CBA/J mice investigated. These mice are normally quite resistant to atherosclerotic development and in the control group (n = 7) receiving only a fatty diet, only one mouse presented a lesion. This lesion was completely devoid of infiltrating CD8+ cells. Parasite-infected mice receiving a normal diet exhibited vasculitis, but no signs of atherosclerosis and control mice receiving normal diet, as expected, exhibited neither signs of vasculitis nor atherosclerosis. Secretion of IL-6, TNF-alpha, and IFN-gamma were demonstrated in all atherosclerotic lesions and IL-6 appeared to be the dominant cytokine, both in the lesions themselves as well as in the intimal-medial junction. There were no traces of parasites present in the artery wall, indicating that atherosclerosis was induced via an indirect route. We conclude that a high fat diet in conjunction with infection and systemic (or localized) inflammation may have a strong proatherogenic effect. Finally, we suggest that CBA/J mice infected with T. cruzi parasites and given a fatty diet could serve as a useful experimental model in the continued analysis of factors contributing to the induction of atherosclerosis.     

Cytokine Profiles Contribute to Understanding the Pathogenic Difference Between Good Syndrome and Oral Lichen Planus: Two Case Reports and Literature Review

We described and analyzed the pathogenic difference between Good syndrome (GS) and oral lichen planus (OLP) in oral mucosa.Good syndrome (GS) is a rare disease characterized by B and T cell immunodeficiency associated with hypogammaglobulinemia and thymoma. GS patients frequently develop oral lichenoid lesions with lymphocytic infiltration beneath the basal layer. Oral lichen planus (OLP) is a chronic inflammatory disease of the oral mucosa characterized by destruction of basal cells by Langerhans cells, macrophages, and T lymphocytes. Although the histological features of the lesions of both diseases are very similar, the pathogenesis of GS in the oral mucosa remains unknown. In this study, we thus investigated the expression of infiltrating lymphocyte subsets (CD3, CD20, CD4, and CD8) and T helper (Th) cytokines including interferon (IFN)-γ (Th1 type), interleukin (IL)-4 (Th2 type), IL-17 (Th17 type), and IL-10 (regulatory T cell type) by immunohistochemistry in buccal mucosa specimens from 2 GS patients compared with 15 OLP patients. All patients showed a predominance of CD3⁺ T cells over CD20⁺ B cells, and CD4⁺ Th cells over CD8⁺ cytotoxic T cells. This polarization was especially prominent in GS. IFN-γ and IL-10 were strongly detected in the infiltrating lymphocytes of all patients. However, IL-4 and IL-17 were detected in OLP patients only.These results suggest that the pathogenesis of GS is different from that of OLP. GS is a unique inflammatory disorder characterized by dysfunction of Th2 and Th17 immune reactions via abnormal T–B cell interaction.     

Relationship between inflammation and joint destruction in early rheumatoid arthritis: a mathematical description

BACKGROUND: The relationship between inflammation and joint destruction in rheumatoid arthritis (RA) has not been unequivocally characterised. Joint destruction may result from the cumulative inflammatory burden over time, modified by an individual constant factor. OBJECTIVE: To test the hypothesis that the relationship between radiological progression and inflammation can mathematically be expressed as: [equation: see text] where Re is a factor that varies from person to person. METHODS: Clinical data and radiographs of 76 patients with early RA receiving different disease modifying antirheumatic drugs were analysed. Radiographs were quantified using the modified Larsen score and the "X-Ray RheumaCoach" software. The cumulative inflammatory burden was estimated by the time integrated 28 joint Disease Activity Score (DAS28), calculated as the area under the curve. RESULTS: 76 patients with early RA who started treatment with methotrexate (n = 20), sulfasalazine (n = 37), or oral gold (n = 19) monotherapy were evaluated. The mean (SEM) DAS28 decreased from 4.6 (0.1) at baseline to 2.3 (0.1) after 2 years. The mean (SEM) DeltaLarsen score from baseline to year 2 was 10.3 (1.5). Correlation between cumulative inflammation and radiographic change was poor. In contrast, when calculating a person's factor Re in year 1 ( Re 1) and year 2 ( Re 2), a strong and significant correlation (r = 0.58, p<0.000001) was seen between Re 1 and Re 2. CONCLUSIONS: Joint destruction is the result of the cumulative burden of inflammation over time, modified by an individual factor Re that remains relatively constant over the first 2 years of observation. The data support a mathematical model that expresses the interrelationship between inflammation and joint destruction.     

AIM2 inflammasome is associated with disease severity in tegumentary leishmaniasis caused by Leishmania (V.) braziliensis

The inflammasome is a multiprotein signalling platform involved in the pathogenesis of various inflammatory skin diseases. Herein, we investigated gene and protein expression of the inflammasome molecules AIM2 and NLRP3 in active lesions from patients with L. (V.) braziliensis‐associated tegumentary leishmaniasis (TL) and correlated these findings with the clinical presentations and responses to therapy. Real‐time PCR assays showed a significantly higher AIM2 gene expression in mucosal leishmaniasis (ML) compared with that in cutaneous leishmaniasis (CL). Additionally, AIM2 mRNA expression was significantly higher in lesions from poor responders than in lesions from good responders. In situ protein quantification analyses revealed greater AIM2 expression in ML lesions than in CL lesions. The percentage of AIM2‐producing cells was higher in poor responders than in good responders. Although not quite significant, IL‐1β+ cells were slightly more prominent in poor responders than in good responders. Similar results were observed when patients were evaluated according to clinical form. GP63 immunostaining was identified in all samples, but no significant variation between mucosal and cutaneous lesions was observed. GP63 could be associated with reduced NLRP3 inflammasome expression in CL and ML patients. Taken together, these data demonstrate that AIM2 is an important component of the inflammasome in TL patients and is directly associated with the severity of lesions.     

Immunohistochemical characterization of the inflammatory infiltrate in placental Chagas' disease: a qualitative and quantitative analysis

Chagas' disease, a systemic illness endemic to some regions of South America, is caused by the protozoan Trypanosoma cruzi. Transplacental infection may occur during any phase and cause fetal death. This study is the first to characterize the inflammatory cells in chagasic villitis by immunohistochemistry. Paraffin sections of 8 placentas with villitis by T. cruzi (4 live births and 4 stillbirths), as well as 8 control placentas without inflammation, were stained with hematoxylin and eosin, monoclonal antibodies for CD45RO, CD20, CD45RO/OPD4, CD8, HNKI, CD15, MAC387, and CD68 proteins, and a polyclonal antibody for S-100 protein. Quantification of positive cells was performed in 3 different high-power fields. In all cases of chagasic villitis, the inflammatory infiltrate was composed mainly of CD68+ macrophages, T lymphocytes, and a few natural killer cells. Among T cells, CD8+ cells outnumbered CD4+ cells in all placentas (CD4+:CD8+ ratios ranged from 0.04 to 0.38). B cells were absent or rare. In stillbirths, villitis was diffuse and severe with numerous T. cruzi, while in live births it was focal with few parasites. Other features that characterized villitis in stillbirths were 1) frequent trophoblastic necrosis, 2) presence of MAC387+ macrophages and CD15+ granulocytes attached to the sites of trophoblastic necrosis, 3) low CD4+: CD8+ ratios in most cases, 4) increased numbers of S-100 positive cells in the villous stroma. In conclusion, CD68+ macrophages and CD8+ T lymphocytes were the major cell population in villitis caused by T. cruzi. However, the pattern of inflammatory reaction differed between stillbirths and live births and was probably related to the number of parasites in the placental villi.     

Inflamed tumor-associated adipose tissue is a depot for macrophages that stimulate tumor growth and angiogenesis

Tumor-associated stroma is typified by a persistent, non-resolving inflammatory response that enhances tumor angiogenesis, growth and metastasis. Inflammation in tumors is instigated by heterotypic interactions between malignant tumor cells, vascular endothelium, fibroblasts, immune and inflammatory cells. We found that tumor-associated adipocytes also contribute to inflammation. We have analyzed peritumoral adipose tissue in a syngeneic mouse melanoma model. Compared to control adipose tissue, adipose tissue juxtaposed to implanted tumors exhibited reduced adipocyte size, extensive fibrosis, increased angiogenesis and a dense macrophage infiltrate. A mouse cytokine protein array revealed up-regulation of inflammatory mediators including IL-6, CXCL1, MCP-1, MIP-2 and TIMP-1 in peritumoral versus counterpart adipose tissues. CD11b(+) macrophages contributed strongly to the inflammatory activity. These macrophages were isolated from peritumoral adipose tissue and found to over-express ARG1, NOS2, CD301, CD163, MCP-1 and VEGF, which are indicative of both M1 and M2 polarization. Tumors implanted at a site distant from subcutaneous, anterior adipose tissue were strongly growth-delayed, had fewer blood vessels and were less populated by CD11b(+) macrophages. In contrast to normal adipose tissue, micro-dissected peritumoral adipose tissue explants launched numerous vascular sprouts when cultured in an ex vivo model. Thus, inflamed tumor-associated adipose tissue fuels the growth of malignant cells by acting as a proximate source for vascular endothelium and activated pro-inflammatory cells, in particular macrophages.     

Plaque inflammation in restenotic coronary lesions of patients with stable or unstable angina

OBJECTIVES: To evaluate immunohistochemically various parameters of inflammation in coronary atherectomy specimens obtained from restenotic culprit lesions of patients presenting with either stable or unstable angina (UA). BACKGROUND: There is no information regarding the relationship between atherosclerotic plaque inflammation and the severity of the coronary syndromes in patients with restenotic coronary lesions. METHODS: A total of 37 patients with either stable angina or UA underwent directional coronary atherectomy for restenotic coronary lesions. Cryostat sections of atherectomy specimen were immunohistochemically stained with monoclonal antibodies CD68 (macrophages [MACs]), CD3 (T-lymphocytes) and alpha-actin (smooth muscle cells [SMCs]). Smooth muscle cell contents and MAC contents were planimetrically quantified as the percentage immunopositive tissue area of the total tissue area. T-lymphocytes were counted at 100-X magnification throughout the entire section and expressed as number of cells per mm2. RESULTS: Restenotic coronary lesions of patients with UA or stable angina showed no significant difference in SMC areas (31.9%+/-16.3% vs. 38.5%+/-18.8%, respectively; p = NS). However, restenotic coronary lesions of patients presenting with unstable angina contained significantly more MACs (24.4%+/-15.1% vs. 10.5%+/-5.8%, p = 0.001) and T-lymphocytes (18.8 cells/mm2+/-15.1 cells/mm2 vs. 8.6 cells/mm2+/-9.8 cells/mm2; p = 0.034) than patients with stable angina. CONCLUSIONS: These results suggested that inflammation appears to affect plaque instability in restenotic coronary lesions resulting in unstable coronary syndromes.     

Increased concentration of soluble CD40 ligand in preeclampsia.

Preeclampsia has been associated with increased platelet activation detected before disease onset. Platelets are involved in hemostasis and also directly initiate an inflammatory response of the vessel wall. Inappropriate activation of platelets may be involved in pathogenesis in preeclampsia by promoting coagulation and thrombosis, and also as a mediator of inflammation. Platelets may release inflammatory mediators such as soluble CD40 ligand. The plasma level of soluble CD40 ligand was investigated during preeclamptic (n =20) and normal pregnancies (n = 20) to emphasize inflammatory response in preeclampsia. The mean soluble CD40 ligand levels were 1.08 +/- 0.43 ng/mL in patients with preeclampsia and 0.76 +/- 0.24 ng/mL in healthy pregnant women, which was statistically significant (P = .01). To clarify whether inflammation may cause inappropriate endothelial cell activation or inappropriate endothelial cell activation may start this inflammatory response, future studies are needed in a larger study population.