Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity.

The genome of Mycobacterium tuberculosis H37Rv contains three contiguous genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa phospholipase C (PLC) genes. Expression of mycobacterial PLC-a and PLC-b in E. coli and M. smegmatis has been reported, whereas expression of the native proteins in M. tuberculosis H37Rv has not been demonstrated. The objective of the present study was to demonstrate that native PLC-a is expressed in M. tuberculosis H37Rv. Sera from mice immunized with recombinant PLC-a expressed in E. coli were used in immunoblots to evaluate PLC-a expression. The immune serum recognized a 49-kDa protein in immunoblots against M. tuberculosis extracts. No bands were visible in M. tuberculosis culture supernatants or extracts from M. avium, M. bovis and M. smegmatis. A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of beta-galactosidase activity. beta-Galactosidase activity was detected in M. smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages. The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive. In conclusion, expression of non-secreted native PLC-a was demonstrated in M. tuberculosis.

Functional differences between two morphologically distinct cell subpopulations within a human colorectal carcinoma cell line.

The LISP-I human colorectal adenocarcinoma cell line was isolated from a hepatic metastasis at the Ludwig Institute, São Paulo, SP, Brazil. The objective of the present study was to isolate morphologically different subpopulations within the LISP-I cell line, and characterize some of their behavioral aspects such as adhesion to and migration towards extracellular matrix components, expression of intercellular adhesion molecules and tumorigenicity in vitro. Once isolated, the subpopulations were submitted to adhesion and migration assays on laminin and fibronectin (crucial proteins to invasion and metastasis), as well as to anchorage-independent growth. Two morphologically different subpopulations were isolated: LISP-A10 and LISP-E11. LISP-A10 presents a differentiated epithelial pattern, and LISP-E11 is fibroblastoid, suggesting a poorly differentiated pattern. LISP-A10 expressed the two intercellular adhesion molecules tested, carcinoembryonic antigen (CEA) and desmoglein, while LISP-E11 expressed only low amounts of CEA. On the other hand, adhesion to laminin and fibronectin as well as migration towards these extracellular matrix proteins were higher in LISP-E11, as expected from its poorly differentiated phenotype. Both subpopulations showed anchorage-independent growth on a semi-solid substrate. These results raise the possibility that the heterogeneity found in the LISP-I cell line, which might have contributed to its ability to metastasize, was due to at least two different subpopulations herein identified.

Participation of glycosylation sites in the binding of Staphylococcus aureus to laminin.

1. Microbial pathogenicity is in many instances associated with the ability to adhere to host surfaces or to extracellular matrix components. 2. Laminin is a major glycoprotein of basement membranes which can promote specific bacterial adhesion. Staphylococcus aureus is a pathogenic bacterium which presents a laminin receptor of about 50-kDa molecular mass (Lopes JD, Reis M & Brentani RR (1985). Science, 229: 275-277). 3. Adhesion inhibition assays of [125iodine]-labeled bacteria to laminin demonstrate that the receptor binding site is contained in the pepsin-derived (P1) laminin fragment. 4. Cell adhesion to laminin is unaffected by periodate oxidation of sugars on the surface of bacteria or by removal of divalent cations by ethylenediaminetetraacetic acid (EDTA). In contrast, bacterial adhesion is reduced when laminin is deglycosylated with N-glycosidase F or when bacteria are submitted to controlled trypsin digestion. 5. Laminin binding to the S. aureus 50-kDa band in immunoblotting assays has confirmed all of these results obtained in cell adhesion experiments.

Evaluation of an anti-carcinoembryonic monoclonal antibody suitable for immunoscintigraphy.

An anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb 6D1. 1) was evaluated in vitro and in vivo to determine its suitability as a tracer for immunoscintigraphy of colorectal carcinomas. Determination of mAb affinity for CEA showed a constant of association of 0.63 +/- 0.11 x 10(9) M-1. Binding of technetium-99m (99mTc)-6D1.1, labeled by a direct method, to human cultured lineages was highly specific. Binding to only CEA-positive LS-174T cells resulted in a saturable curve inhibited by pre-incubation with unlabeled mAb. No binding at all was observed for the human lineages MeWo (melanoma) or ZR75-30 (breast carcinoma), neither of them expressing CEA cells. Intravenous injection of 99mTc-6D1.1 into nude mice xenografted with human LS-174T tumors resulted in planar images of excellent quality. Localization of an irrelevant mAb labeled with either 99mTc or iodine-125 was never observed in tumor masses. Biodistribution studies on excised tumoral tissue showed retention of 28.48% of the injected dose per gram of LS-174T tumor. The tumor-to-blood ratio was 3.46. The same analysis performed on the other three human xenografted tumors studied demonstrated that only the CEA-producing HT-29 (colorectal adenocarcinoma) retained 99mTc-6D1.1 while the other two (ZR75-30 and MeWo) did not. These data demonstrate that this mAb is an adequate tool for targeting CEA-expressing tumors in experimental models.

The immunodominant surface antigen of Plasmodium gallinaceum is present in both the salivary gland and oocyst sporozoites.

1. Monoclonal antibodies (MAbs) against surface antigens of Plasmodium gallinaceum sporozoites, an avian malaria parasite, were produced using spleen cells from mice immunized with sporozoites from mosquito salivary glands (SGS) or from midguts containing oocysts (OoS). 2. All of the 15 MAbs tested (11 anti-SGS and 4 anti-OoS) reacted with SGS and OoS by indirect immunofluorescence and circumsporozoite precipitation reactions. Fourteen of these MAbs (11 anti-SGS and 3 anti-OoS) produced a Western blot (WB) pattern identical to that produced with serum from mice hyperimmunized with viable intact sporozoites. 3. All MAbs and the immune sera recognized only two polypeptide bands of approximate molecular weight 76 and 64 kDa. 4. No difference in the WB pattern was observed when 9- or 12-day SGS or OoS extracts were used as antigens in WB. This antigenic similarity was confirmed when the total protein extracts were visualized on silver-stained SDS-PAGE gel.

Evolutionary conservation of laminin-binding proteins.

1. The virulence of pathogens and metastatic capacity of cancer cells seems to correlate with the ability to adhere to cells and/or to basement membrane components. A key feature of this mechanism is the expression of specific receptors for the basement membrane protein laminin. Three different receptors have been already described in cells phylogenetically very distant, such as human white blood cells, Trichomonas vaginalis and Staphylococcus aureus, all recognizing laminin with the same range of affinity. 2. We have shown that laminin, which is also found in the circulation, enhances phagocytosis of S. aureus by macrophages in a species-specific fashion. Also, monoclonal antibodies (MAb) raised against the bacterial receptor inhibit the phagocytic enhancement mediated by laminin and recognize laminin-binding proteins in unicellular parasites and mammalian cells. The same Mab 1.H12 elutes a 52-kDa protein from bacterial extracts and a 67-kDa band from cancer cell extracts. Since the MAb is a monospecific reagent, results with 1.H12 strongly suggest an evolutionary conservation of the binding site of phylogenetically different laminin receptors.

Surface antigen detected by a Schistosoma mansoni monoclonal antibody in worm extracts and kidney deposits of infected mice and hamsters.

Four monoclonal antibodies (MAbs) derived from a Schistosoma mansoni-infected mouse reacted against the tegument or the cell layer of the digestive tract of the adult worm. They also showed similar patterns of immunofluorescence staining when schistosomula were used as antigens. Two of the MAbs (4A10 and 4D3) recognized immune complexes deposited in the kidneys of infected mice and hamsters as detected by indirect and direct immunofluorescence reactions. When adsorbed to polystyrene beads, both MAbs allowed the quantitative detection of antigen by an enzyme immunoassay. 125I-labeled 4A10 binding to live schistosomula and corresponding inhibition assay ruled out the possibility that this binding could be through its Fc fragment. The same MAb detected an antigen migrating as an 80-kilodalton protein by Western blot analysis of soluble worm antigen preparation after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Use of MAb 4A10 may help to elucidate mechanisms of renal pathology and also be of value in the development of immunodiagnostic assays.

Susceptibility of colorectal-carcinoma cells to natural-killer-mediated lysis: relationship to CEA expression and degree of differentiation.

This study addresses the relevance of colorectal-carcinoma-cell (CRC) CEA expression and degree of differentiation in natural-killer(NK)-mediated lysis susceptibility. A 51Cr-release cytotoxicity assay performed with 5 human CRC lines demonstrated that CRC CEA expression was related to resistance to NK lysis. Moreover, the addition of anti-CEA Fab fragments to the assay led to a significant increase of lysability of high-CEA-producing and NK-resistant cells (LS 174-T), whereas purified CEA drastically reduced lysis of low-CEA-producing and NK-susceptible cells (LISP-I) in a dose-dependent manner. These results strongly suggest that CEA plays a causal role in CRC resistance to NK lysis. Nevertheless, our data did not demonstrate CEA binding to effector cell surface, suggesting that CEA expression can protect CRC, possibly by preventing NK-tumor-cell adhesion to occur. Our results also show that CRC susceptibility to NK lysis was related to a less differentiated phenotype. HCT-8, which are poorly differentiated and low-CEA-producing cells, were cultured in vitro in the presence of the differentiation agent sodium butyrate. Treated cells became less susceptible to NK lysis as they progressed towards a more differentiated phenotype. However, CEA production was not altered upon differentiation. Our study thus demonstrates that both features, CEA expression and degree of cellular differentiation, may individually influence CRC susceptibility to NK lysis.

Postexercise facilitation of motor evoked potentials elicited by ipsilateral voluntary contraction.

Motor evoked potentials (MEPs) to transcranial magnetic stimulation (TMS) increase in amplitude when obtained immediately after a period of exercise of the target muscle (postexercise facilitation). We studied postexercise facilitation of MEPs to TMS after periods of voluntary activation of either the ipsilateral or contralateral primary motor cortex (simple finger movements) or supplementary motor area (complex finger movements). Postexercise facilitation of the first dorsal interosseous MEPs occurred ipsilaterally even after simple, unilateral finger movements of the dominant hand. The findings are taken to suggest transcallosal transfer of excitability from the dominant to nondominant cerebral hemisphere, perhaps related to mechanisms involved in bimanual motor coordination.

Monoclonal antibodies to Staphylococcus aureus laminin-binding proteins cross-react with mammalian cells.

We and others have previously shown that some microorganisms, including bacteria, express on their surfaces receptors that specifically recognize extracellular matrix proteins, such as laminin, fibronectin, or both. The ability of microorganisms to adhere and to invade might depend on the existence of receptors which could, thus, be correlated with pathogenicity. In the present paper, we report the isolation of five stable cell lines that were producers of monoclonal antibodies to Staphylococcus aureus laminin receptors. One of these antibodies, which was of the immunoglobulin M isotype, blocked the binding of laminin to bacteria before and after fixation and recognized the putative 52-kilodalton laminin-binding protein in whole bacterial extracts. Also, purified receptor was isolated by immunoaffinity chromatography and shown to bind laminin. Furthermore, the same antibodies bound the 67-kilodalton putative receptor from mouse melanoma cells and gave positive immunofluorescence reactions against mammalian tumor cells. These data strongly suggest either the evolutionary conservation of at least some sequences in both procaryotic and eucaryotic laminin-binding proteins or convergent evolution and positive selection of epitopes cross-reacting with laminin. Some of these antibodies to the procaryotic protein could therefore become useful markers for the expression of laminin receptors by cancer cells.

Variable region structure and staphylococcal protein A binding specificity of a mouse monoclonal IgM anti-laminin-receptor antibody.

Staphylococcal protein A is a cell wall-attached polypeptide that acts as a B-lymphocyte superantigen. This activation correlates with specific VH gene segment usage in the B-cell receptor. B-cell receptor assembled from members of the VH3 family in humans, or S107 family in mice, has an intrinsic affinity for protein A. Human VH3-derived antibodies bind to domain D of protein A. We have characterized a mouse IgM monoclonal antibody that binds protein A. The sequencing of the variable region suggests an almost germline-encoded VH derived from S107 family and a V kappa 8-derived VL. The binding specificity of the monoclonal antibody was tested with various recombinant constructions derived from protein A. We show that, unlike human VH3-derived antibody, mouse S107-derived immunoglobulin binds to the B domain of the bacterial superantigen.

Antibodies directed to antigens secreted by murine epithelioid macrophages modulate BCG-induced granulomata.

The authors have previously shown that epithelioid cells isolated from mice secrete a factor, called macrophage deactivating factor (MDF), that promptly deactivates superoxide release by activated macrophages and neutrophils. In this paper some biological properties of a polyclonal rat antiserum directed to MDF and other substances secreted by these cells are described. The immunoglobulin fraction of this antiserum reacted, by immunocytochemical methods, with epitopes in the cell membrane of macrophages adherent to coverslips subcutaneously implanted for 14 days; but not for 5 days. It also reacted with antigens within and outside cells in BCG-induced granulomas. This antiserum blocked completely the macrophage deactivating activity of epithelioid cell culture supernatants. Anti-IL-10 monoclonal antibody, did not block MDF activity. The administration of the immunoglobulin fraction from immunized rats to C(5) deficient mice bearing BCG-induced granulomatas in the footpad, significantly reduced the size of the lesions. A marked necrosis of inflammatory cells and mononuclear cells phagocyting debris of necrotic cells were observed in these lesions.

Induction of an immune response through the idiotypic network with monoclonal anti-idiotype antibodies in the carcinoembryonic antigen system.

Anti-idiotype antibodies can mimic the conformational epitopes of the original antigen and act as antigen substitutes for vaccination and/or serological purposes. To investigate this possibility concerning the tumor marker carcinoembryonic antigen (CEA), BALB/c mice were immunized with the previously described anti-CEA monoclonal antibody (MAb) 5.D11 (AB1). After cell fusion, 15 stable cloned cell lines secreting anti-Ids (AB2) were obtained. Selected MAbs gave various degrees of inhibition (up to 100%) of the binding of 125I-labeled CEA to MAb 5.D11. Absence of reactivity of anti-Id MAbs with normal mouse IgG was first demonstrated by the fact that anti-Id MAbs were not absorbed by passage through a mouse IgG column, and second because they bound specifically to non-reduced MAb 5.D11 on Western blots. Anti-5.D11 MAbs did not inhibit binding to CEA of MAb 10.B9, another anti-CEA antibody obtained in the same fusion as 5.D11, or that of several anti-CEA MAbs reported in an international workshop, with the exception of two other anti-CEA MAbs, both directed against the GOLD IV epitope. When applied to an Id-anti-Id competitive radioimmunoassay, a sensitivity of 2 ng/ml of CEA was obtained, which is sufficient for monitoring circulating CEA in carcinoma patients. To verify that the anti-Id MAbs have the potential to be used as CEA vaccines, syngeneic BALB/c mice were immunized with these MAbs (AB2). Sera from immunized mice were demonstrated to contain AB3 antibodies recognizing the original antigen, CEA, both in enzyme immunoassay and by immunoperoxidase staining of human colon carcinoma. These results open the perspective of vaccination against colorectal carcinoma through the use of anti-idiotype antibodies as antigen substitutes.

Circadian latency variability of sympathetic skin responses.

Sympathetic skin responses (SSRs) have been increasingly used as tests for autonomic function in the clinical setting. In spite of the known circadian rhythmicity of sympathetic function, however, normative studies have not addressed the possibility of circadian variability of SSR parameters. Ten normal volunteers (7 men, 3 women, aged 19 to 43) had SSR testing performed in the morning, at noon, and in the early evening, and response latencies and amplitudes were compared for the different day periods. Although amplitude values varied in a random fashion, regardless of the time of testing, there was a statistically significant variability in response latencies, which were, on the average, approximately 150 ms shorter in the morning trials, as compared to the early evening ones. This difference was statistically significant (ANOVA, p < 0.01). We propose that circadian variability of SSR latencies should be taken into account in normative studies of SSR parameters.

Localization by immunoelectron microscopy of Schistosoma mansoni antigens in the glomerulus of the hamster (Mesocricetus auratus) kidney.

Adult worm antigen (AWA) and soluble egg antigen (SEA) were localized ultrastructurally by immunoelectron microscopy using two monoclonal antibodies in the glomeruli of hamsters infected with Schistosoma mansoni cercariae or injected with S. mansoni eggs. AWA was detected in all cercaria-infected groups from the 30th day on and was present mainly in cytoplasm of mesangial cells, mesangial matrix, and glomerular basement membrane, either as isolated gold particles or in small electron-dense deposits of probable immune origin. AWA was encountered also on the inner side of the glomerular basal membrane, close to endothelial cells, and in the foot processes of the glomerular epithelial cells. SEA was detected at similar sites, apparently in lesser amounts, in uninfected hamsters inoculated with S. mansoni eggs into the jugular vein. Schistosomal antigens are apparently processed mainly bymesangial cells which are considered to be critical in the pathogenesis of S. mansoni associated glomerulopathy. Mesangioproliferative glomerulonephritis, immunoglobulin (IgG and IgM), and C3 deposits were observed in hamsters in which AWA and SEA were visualized. During early phases of the infection and in hamsters in which granulomatous pneumonitis was induced by S. mansoni eggs, glomeruli were unchanged or showed a slight mesangial proliferation. Our findings suggests that egg antigens also contribute to the pathogenesis of experimental glomerulopathy in the hamster.

Praziquantel in the treatment of the hepatosplenic form of Schistosomiasis mansoni.

UNLABELLED: 40 patients with the hepatosplenic form of schistosomiasis mansoni were selected for this study. Among 29 patients submitted to oral endoscopy there were 28 in whom oesophageal varices were demonstrated and 20 out of 21 patients who presented gastrointestinal haemorrhage had been operated on. All patients received a single oral dose of 40 mg/kg b.w. of 2-cyclohexylcarbonyl-1,2,3,6,7,11b-hexahydro-4H-pyrazino[2,1-a]isoquinolin-4-one (praziquantel, EMBAY 8440, Biltricide). Unwanted side effects were recorded when spontaneously reported. These were rather frequent but generally mild and disappeared within hours without additional medication. Comprehensive laboratory tests as well as ECG and EEG dit not show any significant changes. 27 patients were parasitologically followed up for 3-6 months; 26 of them were egg-negative. In the 18 patients who could be followed up for more than 6 months, the cure rate was 94.4%. CONCLUSION: praziquantel can be safely given also to patients with hepatosplenic complications.