EARLY ACTIVATION OF SPLENIC MACROPHAGES BY TUMOR-NECROSIS-FACTOR-ALPHA IS IMPORTANT IN DETERMINING THE OUTCOME OF EXPERIMENTAL HISTOPLASMOSIS IN MICE

Experimental infection of animals with Histoplasma capsulatum caused a massive macrophage infiltration into the spleen and induced the production of tumor necrosis factor alpha (TNF-alpha) locally. The cytokine was also produced in vitro by peritoneal exudate macrophages exposed to a large inoculum of yeast cells. Depletion of the cytokine by injection of polyclonal sheep anti-TNF-alpha antibody was detrimental to sublethally infected mice. Fungous burdens in the spleens of TNF-alpha-depleted mice were higher than they were in the infected control mice at days 2, 7, and 9 after infection, and the antibody-treated animals succumbed to the infection. Histopathological study of spleen sections revealed that splenic macrophages were not able to control proliferation of intracellular yeasts as a result of TNF-alpha depletion. It seems that TNF-alpha plays a role in early activation of splenic macrophages which is important in controlling the outcome of an infection.

Dialysable leukocyte extracts modify the course of experimental paracoccidioidomycosis in the Syrian hamster

The effect of dialysable leukocyte extracts (DLE) obtained from hamsters immunized with Paracoccidioides brasiliensis (immune DLE) and from non-immunized hamsters (non-immune DLE) was studied in hamsters inoculated with P. brasiliensis by the intratesticular route. Treatment with immune or non-immune DLE was started during the third week of infection and was repeated at 7, 11, 15 and 19 weeks. A group of untreated infected animals was used as control. Animals were submitted to the delayed hypersensitivity skin test to P. brasiliensis antigen (PbAg) in vivo and assayed in vitro by the macrophage migration inhibition test in the presence of Phytohemagglutinin (PHA) and PbAg and by immunodiffusion for specific antibody. The animals were sacrificed at 4, 8, 12, 16 and 20 weeks. The morphology and extension of the lesions were studied at the inoculation site, and in lymph nodes, lungs, liver, spleen and kidneys. In contrast to the controls, animals treated with both DLEs maintained a positive cell-mediated immune response throughout the experiment and developed less extensive infection with a significantly lower number of fungi in the lesions. The results suggest that immune and non-immune DLE preparations modified the evolution of experimental paracoccidioidomycosis with equal efficiency. This similarity may be explained by the immunoregulatory activities of both extracts.

Verruciform xanthoma of the oral mucosa. Report of four cases and a review of the literature

We present four new cases of verruciform xanthoma (VX) in the oral mucosa and review the literature. Clinical, histological, and immunohistochemical features of four new cases of VX were analysed together with cases found in a review of the literature. Expression of CD-68 was studied by immunohistochemistry. Only 162 cases were reported in the oral mucosa. Ninety were males (55.5%) and 72 were females (44.5%). Mean age was 44.9 years. The majority of cases occurred in masticatory mucosa (69.7%). Our cases exhibited papillary or verrucous proliferation of squamous epithelium associated with hyperparakeratosis and with numerous foamy cells confined to the lamina propria papillae. Foamy cells were positive to CD-68 antibody, showing a macrophagic nature. VX is a rare benign lesion, and is probably inflammatory. However, its aetiology and pathological mechanisms remain unknown. (C) 2001 Elsevier B.V. Ltd. All rights reserved.

Reduction of erythroid progenitors in protein-energy malnutrition

Protein-energy malnutrition is a syndrome in which anaemia together with multivitamin and mineral deficiency may be present. The pathophysiological mechanisms involved have not, however, yet been completely elucidated. The aim of the present study was to evaluate the pathophysiological processes that occur in this anaemia in animals that were submitted to protein-energy malnutrition, in particular with respect to Fe concentration and the proliferative activity of haemopoietic cells. For this, histological, histochemical, cell culture and immunophenotyping techniques were used. Two-month-old male Swiss mice were submitted to protein-energy malnutrition with a low-protein diet (20g/kg) compared with control diet (400 g/kg). When the experimental group had attained a 20% loss of their original body weight, the animals from both groups received, intravenously, 20IU erythropoietin every other day for 14 d. Malnourished animals showed a decrease in red blood cells, Hb concentration and reticulocytopenia, as well as severe bone marrow and splenic atrophy. The results for serum Fe, total Fe-binding capacity, transferrin and erythropoietin in malnourished animals were no different from those of the control animals. Fe reserves in the spleen, liver and bone marrow were found to be greater in the malnourished animals. The mixed colony-forming unit assays revealed a smaller production of granulocyte-macrophage colony-forming units, erythroid burst-forming units, erythroid colony-forming units and CD45, CD117, CD119 and CD71 expression in the bone marrow and spleen cells of malnourished animals. These findings suggest that, in this protein-energy malnutrition model, anaemia is not caused by Fe deficiency or erythropoietin deficiency, but is a result of ineffective erythropoiesis.

SUPPORT OF PARACOCCIDIOIDES-BRASILIENSIS MULTIPLICATION BY HUMAN MONOCYTES OR MACROPHAGES - INHIBITION BY ACTIVATED PHAGOCYTES

The interaction of human monocytes or monocyte-derived macrophages and yeast-form Paracoccidioides brasiliensis was studied in vitro. Yeast cells were readily ingested by adherent monocytes or macrophages. Multiplication of P. brasiliensis, measured by growth as colony forming units (cfu) on a supplemented medium with good plating efficiency, was greater in monocyte co-cultures compared to the number of cfu obtained from complete tissue-culture medium (CTCM). Multiplication increased with time in macrophage cocultures, e.g., from two-six-fold in 24 h to nine-fold in 72 h. Microscopic observations indicated that ingested yeast cells multiplied inside macrophages. When monocytes were treated with supernate cytokines (CK) from concanavalin-A-stimulated mononuclear cells, then co-cultured with P. brasiliensis, multiplication was significantly inhibited compared with control monocyte co-cultures. Treatment of macrophages-derived from monocytes by culture in vitro for 3 days-for a further 3 days with CK resulted in maximal inhibition of multiplication over the subsequent 72 h. Similarly, when monocyte-derived macrophages (after culture for 7 days) were treated for 3 days with recombinant human gamma-interferon (IFN; 300 U/ml) or CK they restricted multiplication of P. brasiliensis by 65% and 95%, respectively, compared with control macrophages, Antibody to IFN abrogated the effect of IFN or CK treatment. These findings show that ingested P. brasiliensis can multiply in human monocytes or macrophages and that this multiplication can be restricted by activated monocytes or macrophages.

Experimental paracoccidioidomycosis in hamsters (Mesocricetus auratus): Gestational interactions

Paracoccidioides brasiliensis is a dimorphic fungus presenting specific steroid hormone receptors, both in the yeast and mycelial forms and estrogen inhibits the transition from mycelium to yeast. In the acute phase, the disease occurs with equal frequency in both sexes but in adults, females are spared. Placental fungal infection has been reported, but references to fetal infection have not been confirmed. We used 78 Syrian female hamsters divided into 3 groups: GI consisted of 30 infected mated females, GII of 20 infected unmated females and GIII of 28 uninfected mated females. Animals of group I were mated 4 weeks after infection and half of them were submitted to cesarean section on day 15 after successful mating; the other half was maintained and submitted to cesarean section and sacrificed 14 weeks after infection. Half of the animals of group II were sacrificed seven weeks and the other half 14 weeks after infection. Uninfected animals of group III were treated the same as the animals of group I. The animals were infected with strain 18 of P. brasiliensis by the intracardiac route. We evaluated the disease by the volume of granulomas in different organs, number of fungi in liver and spleen and the immunologic responses [ELISA, Double Immunodifusion (DID), Delayed Hypersensitivity Skin Test (DHT) and Macrophage Migration Inhibition (MMI)]. We studied the infection through the gestation by evaluation of the abortions, morphologic and clinic examinations of the fetuses. Our results showed that the infection did not transfer to the fetus through the placenta, but the number of abortions was larger among infected females. The newborns of GI females were smaller, weighed less and showed little vitality. The disease was more severe and disseminated in infected mated females, especially in the second sacrifice 14 weeks after inoculation, when the total volume of granulomas in them (56.3 mm) was much greater than in the infected unmated females (12 mm).

Macrophages and mast cells control the neutrophil migration induced by dentin proteins

Dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), the major dentin proteins, have been shown to induce neutrophil migration through release of IL-1beta, TNF-alpha, MIP-2, and KC. However, the sources of these mediators were not determined. Here, the roles of macrophages and mast cells (MC) in dentin-induced neutrophil accumulation were investigated. Peritoneal MC depletion or the enhancement of macrophage population increased DSP- and DPP-induced neutrophil extravasation. Moreover, supernatants from DSP- and DPP-stimulated macrophages caused neutrophil migration. The release of neutrophil chemotactic factor by macrophages was inhibited by dexamethasone or the supernatant of DSP- treated MC. Consistently, dexamethasone and the MC supernatant inhibited the production of IL-1beta, TNF-alpha, and MIP-2 by macrophages. This inhibitory activity of the DSP- stimulated MC was neutralized by anti-IL-4 and anti-IL-10 antibodies. These results indicate that dentin induces the release of the neutrophil chemotactic substance(s) by macrophages, which are down-modulated by MC-derived IL-4 and IL-10.

SINUS HISTIOCYTOSIS WITH MASSIVE LYMPHADENOPATHY (ROSAI-DORFMAN DISEASE) - CYTOMORPHOLOGIC ANALYSIS ON FINE-NEEDLE ASPIRATES

Cytomorphologic characteristics of smears from fine-needle aspirates (FNA) of three cases of sinus histiocytosis with massive lymphadenopathy (SHML) (Rosai-Dorfman disease) are described. All aspirates showed mature lymphocytes, plasma cells, and large pale histiocytes with well-preserved lymphocytes within the cytoplasm (lymphocytophagocytosis). The cases were confirmed by surgical biopsy, and the immunohistochemical findings suggested a macrophage origin for this lesion. The value of FNA analysis as an aid to diagnosis of SHML is discussed.

PARTICIPATION OF MACROPHAGES IN CHLORAMPHENICOL-POTENTIATED CARRAGEENAN-INDUCED PERITONITIS IN RATS

1. We investigated the possible potentiating effect of chloramphenicol succinate (30 mg/kg, every 12 h for 4 days, ip) on the response of polymorphonuclear neutrophils to carrageenin (150 mug, ip) or dextran (100 mug, ip) in the peritoneal cavity of male Wistar rats (180-230 g; N = 12 in each group).2. Chloramphenicol potentiated the cell migration induced by carrageenin (35%) but not that induced by dextran. Previous macrophage depletion in the peritoneal cavity by washing with sterile saline abolished the cell response, whereas a previous thioglycollate-induced increase in macrophage numbers enhanced the potentiating effect (60%).3. These results suggest that the potentiating effect on polymorphonuclear neutrophil migration induced by chloramphenicol may be related to chemotactic factors released by macrophages.

Modulation of intercellular communication in macrophages: possible interactions between GAP junctions and P2 receptors

Gap junctions are connexin-formed channels that play an important role in intercellular communication in most cell types. In the immune system, specifically in macrophages, the expression of connexins and the establishment of functional gap junctions are still controversial issues. Macrophages express P2X(7) receptors that, once activated by the binding of extracellular ATP, lead to the opening of transmembrane pores permeable to molecules of up to 900 Da. There is evidence suggesting an interplay between gap junctions and P2 receptors in different cell systems. Thus, we used ATP-sensitive and -insensitive J774.G8 macrophage cell lines to investigate this interplay. To study junctional communication in J774-macrophage-like cells, we assessed cell-to-cell communication by microinjecting Lucifer Yellow. Confluent cultures of ATP-sensitive J774 cells (ATP-s cells) are coupled, whereas ATP-insensitive J774 cells (ATP-i cells), derived by overexposing J774 cells to extracellular ATP until they do not display the phenomenon of ATP-induced permeabilization, are essentially uncoupled. Western-blot and reverse-transcription polymerase chain reaction assays revealed that ATP-s and ATP-i cells express connexin43 (Cx43), whereas only ATP-s cells express the P2X(7) receptor. Accordingly, ATP-i cells did not display any detectable ATP-induced current under whole-cell patch-clamp recordings. Using immunofluorescence microscopy, Cx43 reactivity was found at the cell surface and in regions of cell-cell contact of ATP-s cells, whereas, in ATP-i cells, Cx43 immunoreactivity was only present in cytosolic compartments. Using confocal microscopy, it is shown here that, in ATP-s cells as well as in peritoneal macrophages, Cx43 and P2X(7) receptors are co-localized to the membrane of ATP-s cells and peritoneal macrophages.

Low intensity laser therapy (LILT) in vivo acts on the neutrophils recruitment and chemokines/cytokines levels in a model of acute pulmonary inflammation induced by aerosol of lipopolysaccharide from Escherichia coli in rat

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The Paracoccidioides brasiliensis gp70 antigen is encoded by a putative member of the flavoproteins monooxygenase family

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

5-Lipoxygenase Deficiency Impairs Innate and Adaptive Immune Responses during Fungal Infection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Use of In Vivo and In Vitro Systems to Select Leishmania amazonensis Expressing Green Fluorescent Protein

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)