Experimental paracoccidioidomycosis in the Syrian hamster

Male hamsters (105) received intratesticular injection of suspension of a live yeast phase culture ofParacoccidioides brasiliensis and were sacrificed weekly during 20 weeks. Humoral immunity was studied by the agar-gel immunodiffusion (ID) and indirect immunofluorescence (IF) tests. Cell-mediated immunity was determined by the macrophage migration inhibition test in the presence of phytohemagglutinin (PHA) andParacoccidioides brasiliensis soluble antigen (PbAg). The morphology of the lesions was studied in the inoculation site, lymph nodes, lung, liver, spleen and kidneys.Disseminated paracoccidioidomycosis was observed in 100% of the animals after the first week. The lesions were initially made up of fungi surrounded by polymorphonuclear neutrophils and macrophages. Up to the 10th week the majority of the lesions appeared as compact confluent ephitelioid granulomas containing rare large fungi, some showing signs of degeneration. At this time, the specific antibody titers and the cellular immune response to PHA and PbAg were highest.From the 11th week on the granulomas became less compact, edematous with the epithelioid cells loosely arranged. This change was accompanied by an increase in the number of fungi showing reproductive activity and was associated with renal amyloidosis and progressive decline of cellular immune response both to PHA and PbAg. Contrariwise the titers of circulating antibodies were maintained.In the present model, disseminated paracoccidioidomycosis of the hamster was associated with depression of cellular immunity, change in the pattern of the granuloma, intense fungi proliferation and amyloidosis.     

Electron microscopic cytochemical analysis of hepatic granuloma induced by Cryptococcus neoformans

The hepatic granulomas in experimental cryptococcosis were analyzed by peroxidase (PO) cytochemistry. Cryptococcus neoformans was inoculated intravenously into rats (group A), and some rats were administrated with dextran sulphate to suppress Kupffer cell functions before inoculation (group B). All rats were sacrificed 7 days after inoculation. The livers were examined PO cytochemically. In addition, the liver, spleen, lungs, kidneys and brain were also examined histopathologically. The hepatic granulomas consisted of the following four type cells; exudate macrophages (type I), PO-negative macrophages (type II), Kupffer cells (type III), and other inflammatory cells (type IV) such as neutrophils and lymphocytes. The percentages of the granulomacomposing cells in group A were 10.3% (type I), 27.3% (type II), 52.9% (type III) and 9.5% (type IV), respectively. In contrast in group B, type II cells outnumbered type III cells by a ratio of 53. In group B, necrosis and hemorrhage were observed in the granuloma. The lesions in the lungs changed from granulomatous to cystic ones after suppression of the Kupffer cell functions. These results suggest that resident macrophages such as Kupffer cells may play an important role in the formation of cryptococcal lesions.     

Measurement of the immunoperoxidase staining of macrophages within liver granulomata of mice infected with Mycobacterium tuberculosis.

A quantitative image analysis technique developed for the measurement of the extent of macrophage activation and epithelioid cell differentiation was performed on mice infected experimentally with Mycobacterium tuberculosis. The granulomatous inflammatory response within the liver reached a peak at day 23 and declined by day 33. Animals of strain B10.BR (H-2k) showed an increased granuloma fraction as compared to Balb/k (H-2k) mice, thus confirming the influence of non-H2 genes in the control of granuloma formation in mice. Using a monoclonal antibody against CD11b/CD18 (Mac1;CR3), we observed two subpopulations of macrophages within the granulomata. The small, darkly staining cells at the periphery of granulomata appear to be newly recruited macrophages. Larger, paler staining cells toward the center of granulomata represent activated and mature epithelioid macrophages. Using a semiautomated image analyzer (Quantimet 970), we measured the relative numbers of these macrophage subpopulations. There were more activated macrophages (epithelioid cells) associated with the increased granuloma fraction in the B10.BR mice than in the Balb/k. However, similar numbers of newly recruited peripheral macrophages were found in both Balb/k and B10.BR strains. This technique has shown qualitative as well as quantitative differences in the granulomatous inflammatory response in this murine model of tuberculosis in strains of mice with quite different antibody repertoires to mycobacterial antigens.     

Presence of Schistosoma mansoni antigens in liver, spleen and kidney of infected mice: a sequential study

In the present study the kinetics of the uptake and deposition of Schistosoma mansoni antigens in liver, spleen and kidney of S. mansoni infected Swiss mice have been investigated in relation to duration of infection and infection dose (50, 100, 200 cercariae). The presence of antigen was studied with a direct immunofluorescence reaction on frozen sections of the organs, using a number of fluorescein isothiocyanate (FITC)-labeled antisera produced against various antigen preparations isolated from different life-cycle stages of the parasite. The presence of antigen was demonstrable with two of the antisera, directed against the circulating anodic antigen (CAA) and against total soluble egg antigen (SEA). CAA was demonstrable from 1 week post infection (p.i.) onwards in Kupffer cells in the liver, from 2-3 weeks p.i. onwards in macrophages in the marginal zones in the spleen and from 3 weeks onwards in kidney glomeruli. Immunofluorescence reactions on CAA in kidney glomeruli, however, were only weak positive until 12 weeks p.i., whereafter strong positive reactions were found. SEA was demonstrable from 5 weeks p.i. onwards in Kupffer cells in the liver and from 4 weeks p.i. onwards in macrophages of the spleen. In contrast to CAA, SEA was not detectable in kidney glomeruli.     

Classification of lesions and comparison of immunohistochemical and acid fast staining in diagnosis of naturally occurring paratuberculosis in goats

Lesions associated with paratuberculosis and degrees of positivity by Ziehl-Neelsen (ZN) and Avidin–Biotin Complex peroxidase (ABC) techniques in 26 adult goats grossly or clinically suspected to paratuberculosis were studied. Of these, 17 (65.4%) had microscopic lesions associated with paratuberculosis which were classified into three categories. Diffuse multibacillary lesions (30.8%) characterized by a diffuse granulomatous enteritis with epithelioid macrophages infiltration as main inflammatory cells into lamina propria that arranged mosaic-like appearance, and filled with numerous acid fast bacilli in ZN and highly positive in ABC. Diffuse lymphocytic lesions (11.5%) composed of lymphocytes as main inflammatory infiltrate, with some epithelioid macrophages or giant cells containing few if any mycobacteria. Diffuse mixed lesions (23%) characterized by a diffuse granulomatous enteritis with infiltration of mixture of large numbers of lymphocytes and epithelioid macrophages with varying degrees of positivity in ZN and ABC. In all types of lesions, other lesions such as granulomatous lymphangitis and arteritis, lymphangiectasis, thrombi composed of degenerated epithelioid macrophages were observed. Caseous necrosis, calcification and fibrosis in mesenteric lymph nodes commonly, especially in diffuse lymphocytic form, were observed. In conclusion, the present study showed three categories of lesions in which diffuse multibacillary form was the commonest type. Both ABC and ZN methods can detect (ABC with more positivity) almost all the goats with diffuse multibacillary and mixed lesions; more cases with lymphocytic forms were positive by ABC than by ZN. ABC method seems to be an efficient diagnostic adjunct to conventional ZN staining for diagnosis of paratuberculosis in goats. The importance of sampling the ileum (especially ileocecal valve), jejunum and mesenteric lymph nodes to find microscopic lesions of paratuberculosis in goats is emphasized.     

Human macrophage maturation and heterogeneity: Restricted expression of late differentiation antigens in situ

Summary Terminal maturation of human macrophages is an important step for creation of cell diversity amongst site-specific subpopulations and their functional competence in situ. As monocytes undergo differentiation in vitro, they start to express lineage-restricted antigens specific for differentiation stages beyond the blood monocyte level as detected by monoclonal antibodies of the MAX series. We have analyzed the expression of MAX.1, MAX.2, MAX.3 and MAX.11 on exudate-type macrophages from pleural and peritoneal cavity and the alveolar space, as well as on resident and activated tissue macrophages in cryostat sections of spleen, lymph node, tonsil, liver, gut mucosa, skin, placenta, kidney and bone. It was found that free macrophages in serous cavities expressed MAX antigens in a heterogenous pattern, whereas none of the organ-specific tissue macrophages subsets did so (with the exception being the weak label of MAX.2 on Kupffer cells). Only during allograft rejection were infiltrating macrophages found to express MAX antigens but not at sites of nonspecific inflammation or granuloma formation. However, Cyclosporin A treatment seems to suppress the induction of MAX antigen expression on intragraft macrophages. In addition, freshly harvested MAX-negative exudate macrophages converted to the complete Max⁺ phenotype on further cultivation. Isolated Kupffer cells were able only to express the MAX.2 antigen in culture but still did not react with the MAX.1 and MAX.3 monoclonal antibodies. Some MAX antigens are co-expressed on glomerular mesangial cells, dendritic reticulum cells and placental cells (MAX.1/. 11) as well as on capillary endothelium within tissues of active immune response (MAX.2). These results add to the knowledge of the phenotypic heterogeneity within the macrophage system as a result of site-specific influences and modulation during a cell-mediated immune response. They also give evidence for a major difference between free exudate-type macrophages and resident tissue macrophages.This work has been supported by Deutsche Forschungsgemeinschaft (AN111) and Boehringer Ingelheim Fonds Stiftung für Grundlagenforschung, Stuttgart, FRGReinhard Andreesen is a recipient of a Heisenberg Award from the Deutsche Forschungsgemeinschaft     

Experimental pulmonary paracoccidioidomycosis in mice: Morphology and correlation of lesions with humoral and cellular immune response

The present paper describes a murine model for pulmonary paracoccidioidomycosis injecting 6×10⁵ yeast forms ofParacoccidioides brasiliensis (Pb) by the direct intratracheal route. The sequential histopathology of lung and dissemination lesions together with humoral (immunodiffusion test) and cellular immune response (footpad test and macrophage inhibition factor assay — MIF assay) were investigated since the 1^st to the 360^th day after infection. All infected animal showed pulmonary Pbmycosis up to Day 30; onwards the lesions subsided being found only in one mouse at Day 360. Dissemination lesions were observed in paratracheal and cervical lymph nodes in 9 out of 68 infected animals. Histologically early lesions were rich in polymorphonuclear cells and evolved to a macrophage desquamative pneumonitis at Day 15 and to typical epithelioid granulomata from Day 30 up to Day 360. Specific precipitating antibodies were first detected 15 days after infection, peaked from Day 30 to 60 and were not observed at Day 360. Significant cell-mediated immunity to Pb was noted at Day 15 with the peak reaction at Day 60 and 90.The intratracheal route represents a highly effective way of infecting mouse with Pb. This experimental pulmonary Pbmycosis is a granulomatous inflammation which courses with specific humoral and cellular immune response. It may be a good tool for further investigation in the pathogenesis and natural history of the disease.     

Localisation of parasite antigens and inflammatory responses in experimental opisthorchiasis

The time course localisation of parasite antigens and related host pathology were studied in hamsters infected with 100 metacercariae of Opisthorchis viverrini for up to 6 months. Parasite antigens, as detected by immunofluorescence and/or immunoperoxidase-staining, were first observed in the flukes and the biliary epithelium of the intrahepatic and extrahepatic bile ducts as early as day 3 p.i. Antigens increased as the parasite matured, both in tissues in direct contact with the flukes and those surrounding the infection. Opisthorchis antigens were also observed in the first order bile ducts (small bile ducts) of the liver, which are not normally inhabited by flukes. In addition, they were found in damaged liver cells, Kupffer cells, macrophages, and within epithelioid and giant cells in the egg granuloma. The presence of the antigens was associated with heavy inflammatory cell infiltration, particularly with mononuclear cells. The results strongly support the role of fluke-associated antigens and local parasite-specific immune responses in the pathogenesis of opisthorchiasis.     

Histochemical localization of autometallographically detectable mercury in tissues of the immune system from mice exposed to mercuric chloride

Summary The distribution of mercury in the spleen, liver, lymph nodes, thymus and bone marrow was studied by autometallography in mice exposed to mercuric chloride intraperitoneally. Application of immunofluorescence histochemistry and an autometallographic silver amplification method was employed to the same tissue section. Mercury was not only detected in macrophages marked by the antibody M1/70 but also in macrophage-like cells, which were either autofluorescent or devoid of fluorescent signals. These two cell types were identified as macrophages at the electron microscopical level. Autometallographically stained macrophages were observed in the spleen, lymph nodes, thymus and in Kupffer cells of the liver. Furthermore, mercury was observed in endothelial cells. No obvious pathological disturbances were observed at light and electron microscopical level. At the subcellular level mercury was localized in lysosomes of macrophages and endothelial cells.     

A monoclonal antibody against an antigen present on mouse macrophages and absent from monocytes

Summary A monoclonal antibody (BM8) raised in the rat against cultured mouse bone marrow-derived macrophages reacted only with macrophages and not with granulocytes, mast cells, platelets, lymphocytes, fibroblasts, and endothelial cells. BM8 did not detect blood monocytes. In cultured bone-marrow cells, expression of BM8-antigen was found on a few macrophages after one day of culture and reached its maximum level with 80% positive macrophages after 7–10 days of culture. The antibody BM8 belonged to the IgG_2a subclass, was non-cytotoxic and directed against a 125 kD membrane antigen. In cryostat sections of normal mouse tissues (spleen, lymph node, thymus, liver, skin) BM8 detected tissue-fixed macrophages and Langerhans cells in the skin. In spleen and lymph node, BM8 reacted with macrophages in the red pulp and in the medullary cords, respectively, but not with heavily phagocytosing marginal-zone macrophages, as revealed by in-vivo phagocytosis of colloidal carbon. In granulomata induced by complete Freund's adjuvant, BM8 detected inflammatory macrophages but not epithelioid cells. Thus, antibody BM8 detected a differentiation antigen expressed only on mature, tissue-fixed macrophages.     

The murine Kupffer cell. I. Characterization of the cell serving accessory function in antigen-specific T cell proliferation.

Murine Kupffer cells, the tissue macrophages of the liver, were isolated by collagenase digestion, differential sedimentation over Metrizamide, and glass adherence. The resultant cell population was more than 86% phagocytic, and 95% of cells stained positively for alpha-naphthyl butyrate esterase activity. The cells also had cell surface receptors for complement (C) and the Fc portion of IgG. In addition, a large proportion of Kupffer cells was shown to bear Ia antigens: about half of the cells bore I-A subregion-encoded antigens and about half bore I-BJE or I-EC subregion-encoded antigens. Kupffer cell populations were capable of reconstituting antigen-stimulated proliferative responses of antigen-primed, macrophage-depleted, lymph node T cells. The ability to reconstitute proliferation was enriched in the adherent population and was resistant to radiation and treatment with an anti-Thy antiserum and C. We conclude that isolated murine Kupffer cells bear the Ia phenotype of accessory cells that function in antigen presentation and that Kupffer cells can participate in the induction of antigen-specific immune responses. These data suggest that Kupffer cells may play a role in modulating responses to enterically derived antigens.     

Immunocytochemical localization of cytokines and inducible nitric oxide synthase (iNOS) in oral mucosa and lymph nodes of patients with paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is a deep mycosis caused by Paracoccidioides brasiliensis, with high incidence in Brazil. In order to examine the immune response in lesional tissue from patients with PCM, we analyzed cytokines as well as the phenotype of the cell infiltrate. Paraffin-embedded tissue from the oral mucosa of eight patients with the localized adult form (AF) of PCM and from the lymph nodes of 10 patients with the juvenile form (JF) of PCM was analyzed by immunohistochemistry to detect tumor necrosis factor-alpha (TNF-alpha), inducible nitric oxide synthase (iNOS), transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10). Most of the inflammatory cells in the lymph nodes were CD68+ (macrophages, epithelioid and giant cells), while a mixed infiltrate with macrophages, plasma cells and neutrophils was detected in the oral mucosa. TNF-alpha as well as iNOS expression was similar in lymph nodes and oral mucosa, whereas TGF-beta and IL-10 were observed in a larger number of macrophages, epithelioid and giant cells in the lymph nodes, where numerous yeast cells were visualized. The higher expression of anti-inflammatory cytokines (IL-10 and TGF-beta) in lesions of patients with the JF of PCM (lymph nodes) may represent a mechanism by which the fungus evades the host immune response, contributing to a more severe and disseminated form of the disease.     

Evidence for a migratory capability of rat Kupffer cells to portal tracts and hepatic lymph nodes

The present study concerns the migratory ability of Kupffer cells in the rat. Phagocytic cells were labeled with colloidal carbon or gold, these markers being administered intravenously either into a tail vein, which resulted in generalized reticuloendothelial uptake, or in low dose into the portal vein, which produced uptake by Kupffer cells alone. Cells containing marker were observed in the portal tracts and in hepatic lymph nodes from 1 to 3 days after injection into the portal vein. The direct movement of single marker particles to the portal tracts could be excluded. Since injection of marker into the portal vein labeled Kupffer cells exclusively, whereas blood cells, splenic and bone marrow macrophages remained unlabeled, the labeled cells in the portal tracts and hepatic lymph nodes appeared to be former Kupffer cells migrating which had migrated to these sites.     

Micro-CT based experimental liver imaging using a nanoparticulate contrast agent: a longitudinal study in mice

Background

Micro-CT imaging of liver disease in mice relies on high soft tissue contrast to detect small lesions like liver metastases. Purpose of this study was to characterize the localization and time course of contrast enhancement of a nanoparticular alkaline earth metal-based contrast agent (VISCOVER ExiTron nano) developed for small animal liver CT imaging.

Methodology

ExiTron nano 6000 and ExiTron nano 12000, formulated for liver/spleen imaging and angiography, respectively, were intravenously injected in C57BL/6J-mice. The distribution and time course of contrast enhancement were analysed by repeated micro-CT up to 6 months. Finally, mice developing liver metastases after intrasplenic injection of colon carcinoma cells underwent longitudinal micro-CT imaging after a single injection of ExiTron nano.

Principal Findings

After a single injection of ExiTron nano the contrast of liver and spleen peaked after 4–8 hours, lasted up to several months and was tolerated well by all mice. In addition, strong contrast enhancement of abdominal and mediastinal lymph nodes and the adrenal glands was observed. Within the first two hours after injection, particularly ExiTron nano 12000 provided pronounced contrast for imaging of vascular structures. ExiTron nano facilitated detection of liver metastases and provided sufficient contrast for longitudinal observation of tumor development over weeks.

Conclusions

The nanoparticulate contrast agents ExiTron nano 6000 and 12000 provide strong contrast of the liver, spleen, lymph nodes and adrenal glands up to weeks, hereby allowing longitudinal monitoring of pathological processes of these organs in small animals, with ExiTron nano 12000 being particularly optimized for angiography due to its very high initial vessel contrast.     

Immune response regulation by lymphocyte products. Potentiator and suppressor factors detected during responses to concanavalin A, E. coli lipopolysaccharide and dinitrophenylated bovine serum albumin.

The efferent lymph from immunostimulated popliteal lymph nodes of sheep was analysed for modulatory effects on the in vitro response of peripheral blood lymphocytes to phytohemagglutinin. Both efferent lymph and the supernates prepared from lymph cells exhibited potentiating activity when collected during the first 6 hours of responses to Concanavalin A and dinitrophenylated bovine serum albumin.² Cells collected on the third day of responses to these antigens and to E. coli lipopolysaccharide produced supernates that almost totally suppressed the in vitro response. The possible contribution made by these cells and cell products to the regulation of the immune response in vivo is discussed.     

Dendritic cells take up and present antigens from viable and apoptotic polymorphonuclear leukocytes

Dendritic cells (DC) are endowed with the ability to cross-present antigens from other cell types to cognate T cells. DC are poised to meet polymorphonuclear leukocytes (PMNs) as a result of being co-attracted by interleukin-8 (IL-8), for instance as produced by tumor cells or infected tissue. Human monocyte-derived and mouse bone marrow-derived DC can readily internalize viable or UV-irradiated PMNs. Such internalization was abrogated at 4°C and partly inhibited by anti-CD18 mAb. In mice, DC which had internalized PMNs containing electroporated ovalbumin (OVA) protein, were able to cross-present the antigen to CD8 (OT-1) and CD4 (OT-2) TCR-transgenic T cells. Moreover, in humans, tumor cell debris is internalized by PMNs and the tumor-cell material can be subsequently taken up from the immunomagnetically re-isolated PMNs by DC. Importantly, if human neutrophils had endocytosed bacteria, they were able to trigger the maturation program of the DC. Moreover, when mouse PMNs with E. coli in their interior are co-injected in the foot pad with DC, many DC loaded with fluorescent material from the PMNs reach draining lymph nodes. Using CT26 (H-2(d)) mouse tumor cells, it was observed that if tumor cells are intracellularly loaded with OVA protein and UV-irradiated, they become phagocytic prey of H-2(d) PMNs. If such PMNs, that cannot present antigens to OT-1 T cells, are immunomagnetically re-isolated and phagocytosed by H-2(b) DC, such DC productively cross-present OVA antigen determinants to OT-1 T cells. Cross-presentation to adoptively transferred OT-1 lymphocytes at draining lymph nodes also take place when OVA-loaded PMNs (H-2(d)) are coinjected in the footpad of mice with autologous DC (H-2(b)). In summary, our results indicate that antigens phagocytosed by short-lived PMNs can be in turn internalized and productively cross-presented by DC.     

Early detection of Leishmania (Leishmania) chagasi in draining lymph node after subcutaneous inoculation in hamster

To study the route of migration of Leishmania (Leishmania) chagasi to the viscera from the subcutaneous inoculation site, ultrastructural studies were carried out. These studies on popliteal draining lymph nodes of hamsters inoculated with 10⁷ promastigotes of Leishmania (Leishmania) chagasi in the hind footpad were carried out. Parasites were detected in lymph nodes just 2 h after inoculation. Parasites were either preserved or degenerated in macrophages. In 24 h signs of binary division of the parasites were found in macrophages. This fact suggests that the parasites migrate from skin to the lymph node early in the infection within phagocytes and proliferate in lymph nodes before dissemination to the viscera.     

Tissue-specific expression of LeY antigen in high endothelial venules of human lymphoid tissues

In this study, we demonstrated that the anti-Le^Yantibody (BM-1) especially reacted with high endothelial venules (HEVs) in peripheral lymph nodes of blood group O individuals. The Le^Yexpression on HEVs showed a unique tissue-specific pattern, i.e., a large amount of the Le^Yexpression in peripheral lymph nodes and no or small amounts in mesenteric lymph node. Statistical analysis showed that there was the significant difference between the percentage of Le^Y-positive HEVs in peripheral lymph nodes and mesenteric lymph nodes. No expression of Le^Ywas observed in vessels of Payer's patch, thymus, spleen and other non-lymphoid organs. In blood group A or B individuals, the reactivity between HEVs and anti-Le^Yantibody increased after enzyme digestion with -N-acetylgalactosaminidase or -galactosidase. These findings show that the expression of difucosylated blood group ABH antigens are especially expressed on HEVs in peripheral lymph nodes. Furthermore, the tissue-specific pattern suggests that these antigens may be related to intercellular adhesion between lymphocytes and HEVs.     

Intravesical BCG administration in the guinea pig. A histomorphological study

Intravesical BCG administration is used as an adjuvant therapy after transurethral resection for superficial bladder cancer in man. The mechanisms of its antitumor activity are not known. The aim of this study was to characterize the histomorphological changes in various organs of the guinea pig after intravesical BCG administration. The BCG preparation used was BCG-RIVM, a Dutch BCG preparation. Instillations were performed in previously undamaged bladders weekly for 6 consecutive weeks and lasted 30 min or 1 h. Different doses were used ranging from 10(3) culturable particles (c.p.) to 5 x 10(7) c.p. of BCG. After 6 weeks, the animals were killed and postmortem examination was performed. The bladder wall, retroperitoneal lymph nodes, spleen, liver, lungs and distant lymph nodes were examined histologically. The BCG therapy, with a dose of 10(6) culturable particles and higher, induced an inflammatory reaction consisting of mononuclear infiltrates in the subepithelial tissue of the bladder wall. In approximately 50% of the animals investigated, the infiltrates were accompanied by non-caseating granulomatous lesions indicated by the presence of epithelioid cells. In general, the epithelial layer of the bladder showed no visible alterations. Similarly, a granulomatous inflammatory reaction was observed in the first retroperitoneal (iliac) lymph nodes draining the bladder. Granulomatous lesions were occasionally also present in liver and lung. In three of the 29 animals investigated, lesions were present both in liver and lungs, and in two of these three animals a granulomatous reaction was observed in the spleen and distant lymph nodes indicating a generalized inflammatory response induced by BCG.(ABSTRACT TRUNCATED AT 250 WORDS)