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Cultured peritoneal macrophages from intact (control) and BCG-infected (experiment) male BALB/c mice were studied 90 days after infection. Polarization of macrophages by M1 (expression of GM-CSF, IFN γ , and CD16/32) and M2 (expression of bFGF and CD36) differentiation pathways was studied with consideration for their the nuclearity class. Mononuclear cells predominated (90% and higher) in macrophage cultures of both groups and presumably, were presented by mainly epithelioid cells. The results indicated polarization of mononuclear and multinuclear macrophages in the M2 direction under conditions of BCG granulomatosis and a higher initial M2 polarization of binuclear macrophages. In control cultures, the ratio of M2 to M1 macrophages was 0.57, in experimental cultures this ratio was 1.6. It seems that long persistence of Mycobacterium tuberculosis in macrophages served as a factor stimulating the plastic processes and transformation of macrophages into epithelioid cells that form the "core" of granulomas and their enlargement upon incorporation of macrophages.

Transforming growth factor beta (TGF-β) has been implicated in the pathogenesis of several diseases including infection with intracellular pathogens such as the Mycobacterium avium complex. Infection of macrophages with M. avium induces TGF-β production and neutralization of this cytokine has been associated with decreased intracellular bacterial growth. We have previously demonstrated that epithelioid cell surrogates (ECs) derived from primary murine peritoneal macrophages through a process of differentiation induced by IL-4 overlap several features of epithelioid cells found in granulomas. In contrast to undifferentiated macrophages, ECs produce larger amounts of TGF-β and inhibit the intracellular growth of M. avium. Here we asked whether the levels of TGF-β produced by ECs are sufficient to induce a self-sustaining autocrine TGF-β signaling controlling mycobacterial replication in infected-cells. We showed that while exogenous addition of increased concentration of TGF-β to infected-macrophages counteracted M. avium replication, pharmacological blockage of TGF-β receptor kinase activity with SB-431542 augmented bacterial load in infected-ECs. Moreover, the levels of TGF-β produced by ECs correlated with high and sustained levels of ERK1/2 activity. Inhibition of ERK1/2 activity with U0126 increased M. avium replication in infected-cells, suggesting that modulation of intracellular bacterial growth is dependent on the activation of ERK1/2. Interestingly, blockage of TGF-β receptor kinase activity with SB-431542 in infected-ECs inhibited ERK1/2 activity, enhanced intracellular M. avium burden and these effects were followed by a severe decrease in TGF-β production. In summary, our findings indicate that the amplitude of TGF-β signaling coordinates the strength and duration of ERK1/2 activity that is determinant for the control of intracellular mycobacterial growth.

Background: The diagnosis of tuberculoid leprosy is often difficult on hematoxylin and eosin (H&E) due to the absence of demonstrable nerve destruction. This study evaluates the utility of S-100 staining in identifying nerve fragmentation and differentiation of tuberculoid leprosy from other cutaneous granulomatous diseases., Methods: Fifty cases of leprosy including 38 borderline tuberculoid (BT), two tuberculoid (TT), and 10 indeterminate leprosy (IL) were studied. Eleven controls of non-lepromatous cutaneous granulomatous lesions were included. S-100 was used for identifying the following dermal nerve patterns: infiltrated (A), fragmented (B), absent (C), and intact (D) nerves., Results: On H&E, only 18/38 (47.4%) BT cases and 1/2 (50%) TT cases revealed neural inflammation. On S-100 staining of BT cases, 28/38 (73.7%) showed pattern B followed by patterns C and A in 8/38 (21.1%) and 2/38 (5.3%) cases, respectively. Both the TT cases showed pattern B. Only intact nerves (D) were seen in all the control cases. S-100 identified nerve damage in 4/10 (40%) IL cases. The patterns A, B, and C had sensitivity, specificity, and positive and negative predictive values of 100% in diagnosing tuberculoid (BT + TT) leprosy., Conclusions: S-100 is superior to H&E in identifying nerve fragmentation (p < 0.01). It also aids the differential diagnosis of tuberculoid leprosy.

Our group has previously described that murine peritoneal macrophages treated in vitro for 7 days with recombinant interleukin-4 (rIL-4) acquire morphological and functional characteristics of epithelioid cells (ECs) found in granulomatous lesions. Although EC function has not been clarified so far, it has been suggested that these cells could present antigens and control multiplication of mycobacteria. These aspects have been addressed here using in vitro EC surrogates. Using immunocytochemistry and immunofluorescence methods, we have observed an increased expression of CD11b, CD54, CD86 and CD40 molecules on rIL-4-treated macrophages when compared to untreated ones. Cytokine-treated cells were less phagocytic for latex beads (P<0.03) and more pinocytic for dextran particles than untreated macrophages. T-cell lymphoproliferation assays using ovalbumin (OVA) and Mycobacterium avium as antigens showed that both cultured macrophages were equally efficient as antigen presenting cells (APCs). However, M. avium antigens were better presented in vivo by EC surrogates (P<0.01). Both macrophage cultures were similarly infected by M. avium. However, while the infection level was maintained in the cytokine-treated population, untreated macrophages showed a progressive increase in the number of bacilli/cell with time (P<0.01) and a reduction of about 65% in cell population. After 96 h of M. avium infection, untreated cells secreted higher amounts of tumor necrosis factor-alpha (P<0.005) while rIL-4-treated macrophages showed higher, although not significant, transforming growth factor-beta production. Also, EC surrogates produced less nitric oxide than control macrophages (P<0.05). Hence, EC surrogates restrain M. avium growth and act as APCs in vitro and in vivo.

Following reports that a VacA+cag+ toxigenic but not a VacA-cag- non-toxigenic Helicobacter pylori strain induced homotypic phagosome fusion in murine macrophages, we addressed that phenomenon in human cells. Mononuclear phagocytes and epitheloid cells were challenged with H. pylori strains of different vacA and cag genotypes and with VacA- and Cag- isogenic mutants, and chased in the absence or presence of signal transduction modulators. Electron microscopy revealed that, in monocytes: (i) homotypic phagosome fusion was frequently induced by all live H. pylori strains investigated but not by exogenous VacA; (ii) phagosomes containing bacteria fused, but not those containing latex beads; (iii) fusion resulted in communal compartments resembling giant multivesicular bodies; and (iv) formation of these compartments was blocked by inhibiting the host cell regulators PI 3-kinase, phospholipase C and p42 MAP kinase. Whereas some internalized bacteria remained viable 1 h after uptake, none survived a 24 h period. In contrast to monocytes, infected epitheloid cells rarely developed communal compartments. In combination, these results demonstrate that, in human monocytes, the H. pylori-induced homotypic phagosome fusion depends on neither the vacuolating cytotoxin VacA nor the cag pathogenicity island of H. pylori and does not result in prolonged intracellular survival.

A case in which malignant lymphoma occurred in association with a tuberculosis focus in a 70-year-old man is reported. Surrounding the epithelioid cell granulomas with caseous necrosis was a dense and diffuse monotonous infiltration of atypical lymphoid cells. Acid-fast bacilli were found in the granulomas and pulmonary tuberculosis was diagnosed. The infiltrating atypical lymphoid cells occasionally invaded the respiratory epithelium producing lymphoepithelial lesions. Immunohistochemically, the lymphoid cells were positive for CD20, and clonal rearrangement of the immunoglobulin heavy chain gene was demonstrated by polymerase chain reaction (PCR). We diagnosed the lesion as a pulmonary malignant lymphoma of bronchus-associated lymphoid tissue (BALT) occurring in the background of tuberculosis. This is the first reported case of pulmonary BALT lymphoma coexistent with pulmonary tuberculosis.

The aim was to evaluate the cellular immune response in atypical tuberculosis and granulomatous inflammation consistent with tuberculosis (TBC), negative histochemically for acid-fast bacilli and analysed by PCR for Mycobacterium tuberculosis (MT) detection in paraffin-embedded tissue. Thirty six samples of differently localized atypical tuberculous lesions and granulomatous tuberculoid lesions negative for acid fast bacilli and 4 positive cases on Ziehl-Nielsen stain were analysed by PCR for MT detection and were tested immunohistochemically (IHC) for the cellular immune response in the granulomas and perigranulomatous tissue. The samples selected were: 7 pulmonary and 33 extrapulmonary specimens, especially lymph nodes. Histologically, the atypical tuberculous lesions contained supurative necrosis, defective granulomas and cellular polymorphism. The epithelioid cells showed frequent mitoses. The immunoprofile of cells was polymorphous. L26 positive small lymphocytes were found in nodular lymphoid aggregates surrounding granulomas. A significantly increased number of positive UCHL1 cells were found in 33 out of the 40 analysed cases, with a larger percentage of CD4 positive T cells (81.8% of cases). CD44 was positive in multinucleated giant cells (17.5% of cases), epithelioid cells (60% of cases) and lymphocytes (30% of cases). CD68 was localized in multinucleated giant cells and epithelioid cells, in a 4%, respectively 62.5% of cases. The PCR was performed in all 40 cases; the tissue samples were heterogeneous (lung, lymph nodes, lever, nasopharynx, etc.) and needed a good quality extraction of DNA. Performing a control PCR for Beta Globin tested the extraction; a good result was obtained in 31 cases (77.5%); from these, 19 cases had amplification for IS 6110. The cellular immune response in the atypical tuberculous lesions was similar in cases with and without acid-fast bacilli, but positive for PCR. In the most cases with negative PCR reaction, it was due to a deficient fixation of the material. The T lymphocytes were numerous in all types of tuberculous granulomas, with the prominence of CD4 positive subtype. The immunoprofile of the epithelioid cells, positive for CD44 and CD68, presenting frequently mitoses suggests an activate state in a possible relationship to the T-cell-mediated immune response in tuberculosis.

Between 1989 and 1996, fine-needle aspiration biopsy was performed in 22 patients with palpable colonic masses. In all of these patients colonoscopic examination was either not possible or could not be carried out successfully. The results of aspiration biopsy were confirmed by surgery and histopathological examination of the tissue. Aspiration biopsy correctly diagnosed all except one case. It identified all cases of colonic cancer. One patient with colonic tuberculosis was also diagnosed correctly. However, in another patient with colonic tuberculosis, aspiration biopsy showed only nonspecific changes in the form of inflammatory and epithelial cells. There were no false-positive results or complications from the procedure. It is concluded that fine-needle aspiration biopsy is a simple, rapid, and accurate method of diagnosing palpable colonic masses in patients in whom a colonoscopy is not possible or cannot be performed satisfactorily.

Some believe that asteroid bodies (AB) in sporotrichosis are nonspecific and are equivalent to the AB of sarcoidosis and other granulomatous diseases. We studied 25 skin biopsy specimens of sporotrichosis in which AB were demonstrated, ten of them with Sporothrix-positive culture. Immunohistochemistry was performed in paraffin-embedded biopsy specimens using an anti-Sporothrix antibody. The same procedures were done with seven biopsy specimens of lobomycosis, which contained AB within giant cells. These did not react with the anti-Sporothrix antibody, and by electron microscopy they displayed filamentous and myelin figures similar to the AB of sarcoidosis. In sporotrichosis, the AB are extracellular eosinophilic structures, 15-35 microm in diameter, and located within abscesses. One to three are found in a section. They consist of a central yeast, surrounded by eosinophilic spicules. The yeast stains with the anti-Sporothrix antibody, while the spicules do not. Therefore, AB in sporotrichosis are specific for disease. Visualization of the spicules alone can lead to the demonstration of the AB in adjacent sections, and thus is a useful clue in the diagnosis of sporotrichosis. Sporotrichotic AB must not be confused with the intracellular AB seen in giant cells of granulomatous reactions, which are filamentous and myelin figures that contain lipid.

Fifty-nine cases of tuberculous cervical lymphadenitis were analysed histologically. Characteristic epithelioid cell granulomas were seen in all the cases with central areas of caseation necrosis in 96.6% (57/59) of these cases. The diagnosis of tuberculosis was further established by the demonstration of acid-fast bacilli (AFB) in the tissue sections in 29 cases. These AFB, although occasional, were found more frequently within the epithelioid cells as compared with other zones of the granuloma. There was no significant association between necrosis and bacillary content. We conclude that light microscopical assessment is still a useful screening method to diagnose tuberculosis in cases of cervical lymphadenopathy.