In vitro isolation and cultivation of Cowdria ruminantium under serum-free culture conditions

Imagined halitosis is poorly documented in the psychiatric literature. It is perhaps best thought of as a symptom rather than as a specific syndrome (a collection of symptoms that co-vary together). Many of the cases with imagined halitosis described in the literature resemble the psychiatric syndrome of social phobia.     

Cloning and partial characterization of the Cr32 gene of Cowdria ruminantium

Cowdria organisms were purified by density gradient centrifugation. The DNA was used to construct expression libraries. The immunodominant Cr32 protein was purified and its N-terminal amino acid sequence was determined. The expression libraries were screened with Cr32-specific monoclonal antibodies, but did not yield Cr32-positive clones. Therefore a part of the Cr32-gene was amplified using primers derived from the N-terminal and an internal amino acid sequence. This DNA was used as a probe to detect the genomic DNA fragment encoding the Cr32 protein. This fragment was cloned, using genomic DNA of the Senegal strain of Cowdria ruminantium. A part of the gene comprising two third of its total length has been expressed in vector pGEX2T. This expression product is recognized by Cr32-specific monoclonal antibodies.     

Demonstration of a carrier state for Cowdria ruminantium in wild ruminants from Africa

We recently conducted a study of the behavioral effects of combined cocaine and ethanol in genetically defined mice. Male and female C57BL/6 (B6) and DBA/2 (D2) were tested in an automated activity monitor on 2 consecutive days. On day 1, all animals received an IP injection of sterile saline and were placed into the activity monitor for 30 min. Behaviors measured were total distance traveled, stereotypy, nosepokes, and wall-seeking. On day 2, all animals were tested again for 15 min following injection of one of the following: saline, 10% v/v ethanol at 2.0 g kg(-1) or 2.0 g kg(-1) ethanol plus 5, 15, or 30 mg kg(-1) cocaine. Cocaine alone at the same doses was injected into separate groups of animals. For the B6 strain, the overall effect of ethanol was to reduce cocaine-induced locomotor stimulation; no consistent effect of ethanol on cocaine-induced locomotion was observed in D2 mice. Cocaine-induced inhibition of nosepokes in both strains and sexes was partially reversed by ethanol. Ethanol also partially reversed cocaine-elevated stereotypy in both strains and both sexes. In B6 mice, cocaine-increased wall seeking tended to be reversed by coadministration of ethanol, whereas no consistent pattern was observed in the D2s. Results from this study suggest that the several measures affected by cocaine (locomotor activity, stereotypy, exploration, thigmotaxis) were, in turn, differentially affected by concurrent treatment with ethanol. Furthermore, our results point to genetic-based differences in ethanol's effects on cocaine-related behaviors. We address the implications for combined ethanol and cocaine use in humans.     

The inactivated Cowdria ruminantium vaccine for heartwater protects against heterologous strains and against laboratory and field tick challenge

We previously described that an inactivated vaccine against heartwater prepared from Cowdria ruminantium (Crystal Springs strain) organisms and administered in complete Freund's adjuvant (cFA) protected sheep against homologous needle challenge. Further studies, described herein, demonstrated that this vaccine protected 100% of sheep against death on challenge with laboratory-infected ticks and with field ticks in a heartwater endemic farm, whereas a mortality rate of 44% and 62%, respectively, was recorded in the control sheep. Subsequently, the Mbizi strain of C. ruminantium was incorporated into the vaccine because of its wider cross-protective capacity, and trial data suggested that protection may be achieved against challenge with diverse geographical strains using this strain. The efficacy of five adjuvants with acceptable safety was compared with that of cFA. Against a homologous intravenous challenge, highest survival rates were observed in sheep vaccinated with inactivated C. ruminantium in either cFA, Montanide ISA 50 or Quil A. The vaccine prepared in Montanide ISA 50 protected six of seven sheep against natural challenge from field ticks on a farm in Zimbabwe where heartwater is endemic, whereas six of seven control sheep died (P = 0.029). These data support optimization of the vaccine prepared in Montanide ISA 50, followed by evaluation of its efficacy in all target domestic ruminant species and in other geographical regions where heartwater constrains livestock production.     

On the masking of signals on immunoblots by cellular proteins

Functional magnetic resonance imaging (fMRI) is capable of detecting task-induced blood oxygenation changes using susceptibility sensitive pulse sequences such as gradient-recalled echo-planar imaging (EPI). The local signal increases seen in the time course are believed to be due to an increase in oxygen delivery that is incommensurate with oxygen demands. To help isolate the sources of functional signal changes, the authors have incorporated various forms of diffusion weighting into EPI pulse sequences to characterize the apparent mobility of the functionally modulated protons. Results suggest that the majority of the functional signal at 1.5 T arises from protons that have apparent diffusion coefficients that are approximately four or five times higher than that of brain tissue. This implies that significant functional signal sources are either protons within the vascular space or protons from the perivascular space that is occupied by cerebrospinal fluid.     

Comparison of chagasic and non-chagasic myocardiopathies by ELISA and immunoblotting with antigens of Trypanosoma cruzi and Trypanosoma rangeli

Trypanosoma cruzi associated myocardiopathy, or Chagas disease, continues to be a serious problem in Venezuela, for which there is neither a vaccine nor a cure. In order to learn more about the humoral immune response to trypanosomal antigens, and to try to identify dominant antigens, we used ELISA and immunoblotting to study the reactivity of sera from patients with chagasic and non-chagasic myocardiopathies, against surface and secreted proteins from T. cruzi and T. rangeli. Both species are found in the same insect vector, but only T. cruzi is thought to be pathogenic in vertebrates. The ELISA results fell into three patterns: (1) high reactivity values with both T. cruzi and T. rangeli surface and secreted proteins; (2) high values to T. cruzi but low values with T. rangeli; and (3) high values to T. rangeli and low values with T. cruzi. This finding that some chagasic sera react more strongly against T. rangeli than against T. cruzi is intriguing, and warrants further investigation. When chagasic sera were tested on Western blots of total extracts of T. cruzi and T. rangeli, the pattern of reactive bands was similar against both parasites, but no two sera showed an identical pattern. Furthermore, there was no correlation between a particular immunoblotting pattern and either the antibody titer, or the severity of the disease. Several T. cruzi and T. rangeli antigens were recognized by sera from healthy controls as well as from patients with other tropical diseases endemic in Venezuela. Overall, our results suggest that the humoral immune response to trypanosomal antigens is complex, and no single antigen may be the determining factor in the pathogenesis of chagasic myocardiopathy.     

Use of immunoplot analysis for the identification of immunodominant non-variant antigens of Trypanosoma brucei rhodesiense

BACKGROUND: Common elements in many different types of amyloid may have important roles in amyloidogenesis. The proteinaceous tissue deposits have a common appearance in polarized light and other similar features. The present investigation describes for the first time the relation between beta 2-microglobulin (beta 2-M)-type amyloidosis and colocalized materials, as demonstrated using specific antibodies and hyaluronan-binding protein. EXPERIMENTAL DESIGN: Amyloid-rich carpal tunnel synovium was obtained surgically from 28 patients who were being treated by maintenance hemodialysis. Serial sections were examined using a hyaluronan (hyaluronic acid)-binding protein and antibodies against heparan sulfate-glycosaminoglycan, chondroitin sulfate-proteoglycan, dermatan sulfate-proteoglycan, alpha 1-antichymotrypsin, alpha 1-antitrypsin, inter-alpha-trypsin inhibitor, haptoglobin, and ubiquitin. RESULTS: Accumulation of hyaluronan was of three types, namely, localization around beta 2-M deposits, colocalization with deposition of beta 2-M itself and localization at a small distance from beta 2-M deposits. Immunostaining for heparan sulfate glycosaminoglycan was demonstrated at the sites of beta 2-M plaques. Chondroitin sulfate-proteoglycan did not show specific patterns of immunostaining, resembling hyaluronan rather than heparan sulfate. The other materials tested, alpha 1-antichymotrypsin, alpha 1-antitrypsin, inter-alpha-trypsin, haptoglobin and ubiquitin, were not immunostained at sites of beta 2-M plaques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed that the molecular weight of heparan sulfate-glycosaminoglycan was 16,000. CONCLUSIONS: These results suggest that HS has an important role in hemodialysis-associated amyloidosis as it does in other types of amyloidosis. Moreover, accumulation of hyaluronan may be an indication of inflammation of the carpal synovium.     

Detection of cross-reactive antigens shared by Fusarium oxysporum and Glycine max by indirect ELISA and their cellular location in root tissues

Pathogenicity test of Fusarium oxysporum on ten cultivars of soybean revealed Soymax and Punjab-1 to be most resistant while JS-2 and UPSM-19 were most susceptible. Antigens were prepared from the roots of all the ten varieties of soybean and the mycelium of F. oxysporum. Polyclonal antisera were raised against the mycelial suspension of F. oxysporum and the root antigen of the susceptible cultivar UPSM-19. Cross reactive antigens shared by the host and the pathogen were detected first by immunodiffusion. The immunoglobulin fraction of the antiserum was purified by ammonium sulfate precipitation and DEAE-Sephadex column chromatography. The immunoglobulin fractions were used for detection of cross-reactive antigens by enzyme-linked immunosorbent assay. In enzyme-linked immunosorbent assay, antigens of susceptible cultivars showed higher absorbance values when tested against the purified anti-F. oxysporum antiserum. Antiserum produced against UPSM-19 showed cross-reactivity with the antigens of other cultivars. Indirect staining of antibodies using fluorescein isothiocyanate indicated that in cross-sections of roots of susceptible cultivar (UPSM-19) cross-reactive antigens were concentrated around xylem elements, endodermis and epidermal cells, while in the resistant variety, fluorescence was concentrated mainly around epidermal cells and distributed in the cortical tissues. CRAs were also present in microconidia, macroconidia and chlamydospores of the fungus.     

The possibilities for improving the serological diagnosis of active tuberculosis by using new mycobacterial antigens and immunoblot and ELISA technics

The study concerns 264 cases among which: 119 active lung tb. eliminating and 11 cases not-eliminating M. Tuberculosis; 17 cases of extrarespiratory tb. confirmed by bacteriology and/or by anatomopathology; 18 cases of bone-joint non-tb disease; 38 cases of chronic lung disease other than tb; 61 healthy persons (controls). Sera from these cases were collected before treatment and submitted concomitantly to two different methods: (1) Mycodot test (immunoblot) with lipoarabinomannan (LAM) as antigen, on nitrocellulose discs (Dynagen, Cambridge, MA, USA); (2) ELISA test with antigen 60 (A60) (ANDA-Biologicals, Strasbourg, France) and with antigen I.C. (Cantacuzino Institute, Bucharest). The results were estimated on terms of sensitivity and specificity. As for sensitivity the results show 74-90%. the highest values were reached in ELISA with A60 IgA. The specificity of the Mycodot test was highest: 90-100% in the two successive experiments. The active tb diagnosis discrimination capacity of the studied methods allows the following classification: 1. Mycodot test with LAM antigen 2. ELISA with A60-Ig G complex 3. ELISA with I.C. antigen The Mycodot test is more advantageous being more rapid and more simple to perform.     

Characterization and comparison of merozoite antigens of different Babesia canis isolates by serological and immunological investigations

Merozoites of four Babesia canis isolates from Hungary, France, Africa, and Egypt were purified. Antigens were compared in an enzyme-linked immunosorbent assay (ELISA) and by immunoblotting. In the ELISA, antigen from the highly pathogenic isolate from Hungary showed the highest sensitivity for homologous and heterologous immune sera. This was confirmed by immunoblotting. Protein bands of the Hungarian isolate were strongly recognized by all B. canis immune sera, whereas the antigens from the other isolates showed only weak reactions with homologous and heterologous immune sera. Significant was a protein band of about 12 kDa appearing in all pathogenic isolates from Hungary, France, and South Africa but not in the apathogenic Egyptian isolate. This protein band may determine the virulence. For serological tests, the B. canis isolate from Hungary seems to be the one most suitable for detection of even mild infections.     

Shaping the repertoire of cytotoxic T-lymphocyte responses: explanation for the immunodominance effect whereby cytotoxic T lymphocytes specific for immunodominant antigens prevent recognition of nondominant antigens

The immunodominance effect, whereby the presence of immunodominant epitopes prevents recognition of nondominant determinants presented on the same antigen-presenting cell (APC) considerably restricts the repertoire of cytotoxic T lymphocyte (CTL) responses. To elucidate the molecular basis of the immunodominance effect, we compared the interactions of a dominant (B6(dom1)) and a nondominant epitope (H-Y) with their restricting class I molecule (H2-Db), and their ability to trigger cognate CTLs. We found that B6(dom1)/Db complexes behaved as optimal T-cell receptor (TCR) ligands and triggered a more rapid in vivo expansion of cognate CTLs than H-Y/Db complexes. The superiority of the dominant epitope was explained by its high cell surface density (1,012 copies/cell for B6(dom1) v 10 copies/cell for H-Y) and its optimal affinity for cognate TCRs. Based on these results, we conclude that dominant class I-associated epitopes are those that have optimal ability to trigger TCR signals in CTLs. We propose that the rapid expansion of CTLs specific for dominant antigens should enable them to compete more successfully than other CTLs for occupancy of the APC surface.     

HA-1 and the SMCY-derived peptide FIDSYICQV (H-Y) are immunodominant minor histocompatibility antigens after bone marrow transplantation

BACKGROUND: Allogeneic bone marrow donors can be incompatible at different levels. Even HLA-identical pairs will be still incompatible for numerous minor histocompatibility antigens (mHag). Nevertheless, some incompatibilities are found to be associated with an increased risk of graft-versus-host disease (GVHD), which could be related to the way the immune system recognizes these antigens. METHODS: We determined the specificity of cytotoxic T-cell clones isolated during acute GVHD or during bone marrow graft rejection in patients (n=14) transplanted with marrow from donors who were histoincompatible for different minor and/or major histocompatibility antigens. RESULTS: We found a clear hierarchy among the different types of histoincompatibilities. In three combinations mismatched for a class I allele, all 27 clones isolated during GVHD were specific for the incompatible HLA molecule. In the 11 class I-identical combinations, 14 different mHags were recognized. The mHag HA-1, known to have a significant impact on the development of GVHD, was recognized in the two HA-1-incompatible combinations. In one of these combinations, which was sex mismatched, all 56 clones analyzed were directed against HA-1, demonstrating the dominance of this mHag. In the four HA-1-compatible, sex-mismatched combinations, the anti-H-Y response was directed against one immunodominant epitope rather than against multiple Y-chromosome-encoded epitopes. All male specific cytotoxic T lymphocytes (n=15) recognized the same high-performance liquid chromatography-purified peptide fraction presented by T2 cells. Moreover, all cytotoxic T lymphocytes tested (n=6) were specific for the SMCY-derived peptide FIDSYICQV, originally described as being the H-Y epitope recognized in the context of HLA-A*0201. CONCLUSIONS: Some histocompatibility antigens are recognized in an immunodominant fashion and will therefore be recognized in the majority of mismatched combinations. Only for such antigens, correlations between mismatches and the occurrence of GVHD or graft rejections will be found.     

Characterization of seven low incidence blood group antigens carried by erythrocyte band 3 protein

Recent studies have demonstrated that band 3 carries antigens of the Diego blood group system and have elucidated the molecular basis of several previously unassigned low incidence and high incidence antigens. Because the available serological data suggested that band 3 may carry additional low incidence blood group antigens, we screened band 3 genomic DNA encoding the membrane domain of band 3 for single-strand conformational polymorphisms. We found that the putative first ectoplasmic loop of band 3 carries blood group antigen ELO, 432 Arg-->Trp; the third putative loop harbors antigens Vga (Van Vugt), 555 Tyr-->His, BOW 561 Pro-->Ser, Wu (Wulfsberg), 565 Gly-->Ala, and Bpa (Bishop), 569 Asn-->Lys; and the putative fourth ectoplasmic loop carries antigens Hga (Hughes), 656 Arg-->Cys, and Moa (Moen), 656 Arg-->His. We studied erythrocytes from carriers of five of these blood group antigens. We found similar levels of reticulocyte mRNA corresponding to the two band 3 gene alleles, normal content and glycosylation of band 3 in the red blood cell membrane, and normal band 3-mediated sulfate influx into red blood cells, suggesting that the mutations do not have major effect on band 3 structure and function. In addition to elucidating the molecular basis of seven low incidence blood group antigens, these results help to create a more accurate structural model of band 3.     

Characterization of two corneal epithelium-derived antigens associated with vasculitis

PURPOSE: In a previous investigation into corneal autoimmunity, it was demonstrated that a putative autoantigen, a protein of 66 kDa, present in bovine corneal epithelium, binds circulating autoantibodies in approximately 60% of patients with Wegener's granulomatosis (WG). The aim of the present study was to characterize and identify the 66-kDa protein. METHODS: A purification protocol was established for the 66-kDa protein using standard chromatography techniques. During the purification procedure it became clear that the 66-kDa protein detected in patients' sera was in fact two proteins, both running at 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, that eluted in different fractions on DE-52 chromatography columns. These two proteins have been labeled bovine corneal epithelial antigen-A and -B (BCEA-A and BCEA-B). Further investigations of antibody binding have demonstrated that patients' sera bind to either one or the other of these proteins with no cross-reactivity between them. Separated BCEA-A and BCEA-B protein extracts were immunoblotted with 27 WG patients' sera, 10 Churg-Strauss syndrome (CSS) patients' sera, 31 rheumatoid arthritis (RA) patients' sera, and 40 healthy control subjects' sera from the blood bank. RESULTS: Forty-six percent of WG patients' sera had antibodies to one of the 66-kDa antigens, whereas none of the healthy control subjects' sera had 66-kDa antibodies (P < 10(-5)). In the WG group, 31% were positive to BCEA-A (versus controls, P = 0.0023), and 15% were positive to BCEA-B. WG patients with peripheral ulcerative keratitis (PUK) had a significant association with anti-BCEA-A antibodies when compared with healthy control subjects (50%, P < 10(-6)). However, in the RA group with no eye disease there was an association with BCEA-A (25%, P = 0.011) but not in the RA group with PUK. The frequency of anti-BCEA-B antibodies was significantly increased in patients with CSS (60%, P < 10(-7)). CONCLUSIONS: In summary, it has been shown that vasculitis patients have antibodies to two 66-kDa corneal antigens and that autoantibodies to these antigens are mutually exclusive. It has also been shown that antibodies to BCEA-B are associated with CSS, whereas BCEA-A antibodies are associated with WG and RA.     

Characterization of three new apparently related high frequency antigens

1. To study the relative contributions of luminal nutrition, bile and pancreatic secretions and hormonal factors in intestinal adaptation, lactation hyperphagia was chosen as a model for increased luminal nutrition, either alone (intestinal transection control group) or in combination with (i) exclusion of bile and pancreatic secretions from the jejunum (by transposition of the jejunum above the Ampulla of Vater) or (ii) exclusion of bile, pancreatic secretions and exogenous luminal nutrition from the jejunum (proximal Thiry-Vella by-pass group). 2. The results confirm that in lactation there is mucosal hyperplasia with increases in villus height and crypt depth, and in small-bowel wet and defatted dry-tissue weights per unit length of intestine. 3. There are corresponding changes in absorptive function with increased glucose and water absorption per unit length of intestine. 4. These structural and functional adaptive changes are proportionately greater in ileum than in jejunum. 5. The exclusion of exogenous luminal nutrition, bile and pancreatic secretions from the jejunum did not diminish the degree of intestinal mucosal hyperplasia and functional adaptation seen in lactation. 6. Diversion to the ileum of greater than normal amounts of bile, pancreatic secretions and luminal nutrition did not further increase the degree of mucosal hyperplasia and enhanced absorption seen in the lactating intestinal transection control group. 7. Unlike other models of intestinal adaptation, the changes in small-bowel mucosal structure and function seen in lactation are probably due to hormonal factors.     

Simultaneous analysis of cyclin and oncogene expression using multiple monoclonal antibody immunoblots

Cell dysfunction or dysregulation in cancer generally results from complex gene interactions, numerous cellular events and environmental influences which modify gene expression or post-translational protein modifications. Genetic analysis in itself cannot always predict or diagnose multigenic diseases. The major technical difficulty is thus to detect, identify and measure simultaneously the expression of several genes and the post-translational modifications of their products. In order to progress to this direction, this paper describes a simple immunoblot method using several monoclonal anti-bodies to simultaneously analyze oncogene expression and cell cycle specific checkpoints in patient solid biopsies and transformed cell lines. One mg of normal human liver biopsy and HEPG2 (hepatoblastoma-derived cell line) protein samples have been separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were stained with amido black, scanned and tested separately with the nine monoclonal antibodies p53, c-myc, PCNA, MEK1, pan-ras, Cip1, Cdc2, Kip1, and TCTP. The nine antibodies of interest were then combined to form a mixture, and simultaneously used as the primary antibodies. This antibody mixture simultaneously detected the nine proteins of interest on both samples and it demonstrated the extensive expression changes and the presence of various isoforms most likely due to post-translational modifications of gene products.