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M Campa

Publications and source records attributed to M Campa.

At least 37 records · Page 2Linked to original sources
PubMed1999

In vitro expansion of T-cell-receptor Valpha2.3(+) CD4(+) T lymphocytes in HLA-DR17(3), DQ2(+) individuals upon stimulation with Mycobacterium tuberculosis.

The T-cell receptor (TCR) Valpha/beta gene product expression upon in vitro stimulation with mycobacteria was investigated to assess whether T-cell proliferation was associated with any specific TCR V gene usage. T-cell-enriched populations from peripheral blood of Mycobacterium bovis BCG-vaccinated healthy blood donors were stimulated in vitro with live or killed M. tuberculosis or with a soluble extract thereof. TCR Valpha/beta repertoire analysis of reactive CD4(+) and CD8(+) T cells revealed a selective HLA-DR17(3), DQ2-restricted expansion of Valpha2.3(+) CD4(+) T cells upon stimulation with live M. tuberculosis or its soluble extract. Third-complementarity-determining-region (CDR3) length analysis of the expanded Valpha2.3(+) T cells indicated an oligoclonal pattern with short CDR3 lengths in six of seven HLA-DR17(3), DQ2(+) individuals tested. In addition, Valpha/Vbeta repertoire analysis of T lymphocytes from a DR17(3), DQ2(+) donor before and after BCG vaccination revealed that positivity of skin test reactivity was associated with expansion of Valpha2.3(+) CD4(+) T lymphocytes with preferential use of a short CDR3 peak length after in vitro stimulation. Separation of M. tuberculosis soluble extract by fast protein liquid chromatography (FPLC) purification indicated that fractions corresponding to molecular masses of 60 to 70 and 15 to 25 kDa were particularly effective in eliciting Valpha2.3(+) CD4(+) T-cell expansion.

BCG Vaccine↗
PubMed1999

Periodontal tissue disposition of azithromycin in patients affected by chronic inflammatory periodontal diseases.

BACKGROUND: The recognition that periodontal diseases are associated with specific pathogens has led to interest in the use of antibacterial drugs for inhibition of these microorganisms. On these bases, the present study was aimed at evaluating the tissue distribution of the new macrolide antibiotic azithromycin in patients subjected to oral surgery for chronic inflammatory diseases of both marginal and periapical periodontium. METHODS: Thirty-two patients were treated with azithromycin 500 mg/day orally for 3 consecutive days, and drug concentrations in plasma, saliva, normal gingiva, and pathological periodontal tissues were evaluated. For this purpose, samples of blood, saliva, normal gingiva, granulation tissue, and radicular granuloma or cyst wall (from dentigerous cyst) were collected during oral surgery or 0.5, 2.5, 4.5, and 6.5 days after the end of pharmacological treatment; then, azithromycin levels were measured by a microbiological plate assay, using Micrococcus luteus NCTC 8440 as the indicator organism. RESULTS: The concentrations of azithromycin in plasma, saliva, normal gingiva, and pathological tissues reached the highest values 12 hours after the last dose (0.37+/-0.05 mg/l, 2.12+/-0.30 mg/l, 6.30+/-0.68 mg/kg, and 11.60+/-1.50 mg/kg, respectively) and then declined gradually. Consistent levels of the drug in normal gingiva and pathological tissues could be detected, however, up to 6.5 days, indicating that azithromycin was retained in target tissues for a long time after the end of treatment. Moreover, azithromycin levels in both normal gingiva and pathological tissues exceeded the minimum inhibitory concentrations of most pathogens involved in the pathophysiology of chronic inflammatory periodontal diseases. Notably, azithromycin levels in pathological tissues were significantly higher than those in normal gingiva 0.5, 2.5, and 4.5 days after the last dose. CONCLUSIONS: The present results indicate a marked penetration of azithromycin into both normal and pathological periodontal tissues, suggesting that azithromycin represents a promising option in both adjunctive and prophylactic treatments of chronic inflammatory periodontal diseases.

Adolescent↗
PubMed1998

Analysis of the Mycobacterium bovis hsp60 promoter activity in recombinant Mycobacterium avium.

A clinical isolate of Mycobacterium avium was transformed with a new shuttle plasmid containing the Escherichia coli beta-galactosidase reporter gene under the control of the Mycobacterium bovis bacillus Calmette-Guérin (BCG) hsp60 promoter. beta-Galactosidase activity was assayed spectrophotometrically in bacterial homogenates of the recombinant strain (M. avium::lacZ) and used for quantification of the hsp60 promoter strength in different conditions of extra- and intracellular growth. Very low levels of beta-galactosidase were recorded during the exponential phase of in vitro growth, while they increased progressively during the late exponential and stationary phases. A significant increase in enzyme activity was also induced in exponentially growing cells by shifting the incubation temperature from 37 to 45 degrees C, but not from 37 to 42 degrees C nor from 30 to 42 degrees C. No induction of the promoter was observed by adding hydrogen peroxide to the cultures. Finally, beta-galactosidase levels were quantified during growth of M. avium::lacZ in murine macrophages. Soon after phagocytosis and, to a lesser extent at 1, 5 and 7 days after infection, increased levels of bacterial beta-galactosidase were observed indicating an increment in transcriptional activity of hsp60 promoter both at early phases of infection and during the course of intracellular growth.

Animals↗
PubMed1998

Identification and molecular cloning of a novel secretion antigen from Mycobacterium tuberculosis and Mycobacterium bovis BCG.

A novel protein called SA-5K was identified in Mycobacterium bovis BCG (BCG) short-term culture filtrates (CFs) by means of a recently described monoclonal antibody (mAb), L8D8. This protein had an apparent molecular mass (MM) of 5 kDa, as judged by Western blotting after sodium dodecyl sulphate-polyacrylamide gel electrophoresis in reducing conditions, and did not seem to contain any sugar or lipid substituents. In the present work, SA-5K was purified from BCG CFs by affinity chromatography. A protein that could be detected in Western blot but not by standard protein staining techniques was obtained. When SA-5K was subjected to aminoterminal sequencing, the 10 amino acids (aa) found matched the first 10-aa sequence deduced from an open reading frame (ORF) of M. tuberculosis. The ORF encodes a polypeptide, likely to include a signal for secretion, with an estimated MM of 8.3 kDa after signal peptide cleavage. The secretory nature of SA-5K was confirmed by the fact that it could only be detected in CFs, but not in other BCG subcellular fractions. After size exclusion chromatography, reactivity with mAb L8D8 was found to peak in the 45-50- and 14-16-kDa fractions. The latter MM was close to that estimated from the ORF of M. tuberculosis, implying that the 5-kDa antigen detected initially by Western blot in reducing conditions was a portion of SA-5K released after reduction of a disulphide bridge. The presence of the gene for SA-5K in BCG and its identity were confirmed by PCR (polymerase chain reaction) with specific primers and restriction analysis: the PCR product was slightly shorter in BCG than in M. tuberculosis. The gene coding for SA-5K was cloned by PCR from BCG and M. tuberculosis DNA and was expressed in Escherichia coli.

Amino Acid Sequence↗
PubMed1998

Gammadelta+ and CD4+ alphabeta+ human T cell subset responses upon stimulation with various Mycobacterium tuberculosis soluble extracts.

By using a flow cytometric technique which allows direct identification of proliferating cells within mixed cell populations, we have previously described that soluble extracts obtained from Mycobacterium tuberculosis or M. avium represent strong stimuli for human gammadelta+ T cells. In the present study, we demonstrate that the protocol used for the preparation of M. tuberculosis soluble extracts may have an impact on their gammadelta+ T cell stimulatory capacity. In agreement with our previous data, soluble extracts prepared from bacteria killed at 85 degrees C and directly disrupted by prolonged sonication (TBe), elicited a strong proliferation of gammadelta+ T cells after 6-7 days of stimulation. In contrast, when soluble extracts were obtained from bacteria autoclaved (121 degrees C, 25 min) and then washed by centrifugation, a predominant proportion of CD4+ alphabeta+ T cells was achieved in the responding population. The stimulatory activity for gammadelta+ T cells was recovered in the supernatant of the autoclaved bacteria, indicating that autoclaving of M. tuberculosis bacilli releases an antigen(s) into the supernatant which stimulates human gammadelta+ T cells. While protease digestion of TBe only partially reduced its stimulatory capacity on gammadelta+ T cells, the stimulatory component(s) released into the supernatant after autoclavation of bacilli was found to be sensitive to protease digestion. Interestingly, in contrast to the preponderant proportion of gammadelta+ T cells induced in the responding population by unfractionated TBe, when the extract was fractionated by fast performance liquid chromatography (FPLC), most of the fractions exhibited a strong stimulatory capacity on CD4+ alphabeta+ T cells only. The gammadelta+ T cell stimulatory activity was confined to the low molecular weight range FPLC fractions. Such results may suggest a possible regulatory role of gammadelta+ T cells on CD4+ alphabeta+ T cells.

Antigen Presentation↗
PubMed1998

Synergic interactions of macrolides and proton-pump inhibitors against Helicobacter pylori: a comparative in-vitro study.

Thirty-eight clinical strains of Helicobacter pylori were isolated from patients with chronic gastritis and gastroduodenal ulceration, and their susceptibility to macrolide antibiotics (roxithromycin, flurithromycin, azithromycin, erythromycin) in combination with proton-pump inhibitors (lansoprazole and omeprazole) and bismuth subcitrate was assayed. Chequerboard titration was used to analyse the results of antimicrobial interactions and showed that the activity of macrolides was enhanced by combining them with lansoprazole, omeprazole or, to a lesser extent, bismuth subcitrate. While the interactions between erythromycin and the proton-pump inhibitors or bismuth subcitrate were always additive, the combinations of roxithromycin-lansoprazole, flurithromycin-omeprazole and azithromycin-lansoprazole acted synergically on 82%, 60% and 60% of H. pylori strains, respectively. These results may, in part, account for the enhanced clinical efficacy of macrolides administered with proton-pump inhibitors in the treatment of H. pylori-associated diseases.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1998

Gastric mucosal distribution and clinical efficacy of azithromycin in patients with Helicobacter pylori related gastritis.

The gastric mucosal distribution of azithromycin, the prototype of a new class of macrolide antibiotics named azalides, was studied in patients with duodenal ulcer and Helicobacter pylori-related gastritis. The time course of ulcer healing, H. pylori infection, and gastritis activity was also evaluated. Twenty patients (median age 50 years) received the following treatment for 1 month: three cycles of azithromycin (500 mg/day for 3 consecutive days) on days 1-3, 11-13 and 21-23 plus omeprazole (40 mg/day) for 30 consecutive days. Endoscopic biopsy specimens of gastric mucosa and blood samples were collected on days 0, 4, 7, 10, 20 and 30. An additional follow-up endoscopy was carried out on day 60. H. pylori infection was determined by both histology and rapid urease test. Azithromycin concentrations in both plasma and gastric mucosa were measured by a microbiological plate assay, using Micrococcus luteus NCTC 8440 as the reference organism. Azithromycin concentrations in plasma ranged between 0.17 mg/L (95% CI: 0.08-0.26; n = 5) and 0.32 mg/L (95% CI: 0.21-0.43; n = 5) throughout the treatment period. In addition, azithromycin concentrations in gastric mucosa were significantly higher than plasma concentrations at all times examined and ranged from 18.5 mg/kg (95% CI: 15-20; n = 20) to 24.6 mg/kg (95% CI: 16.8-32.4; n = 5), Indicating that the drug was highly retained in the target tissue. Accordingly, the ratio of azithromycin mucosal level to plasma concentration varied between 77.9 (95% CI: 56.5-99.3; n = 5) and 112.7 (95% CI: 100.2-125.2; n = 5). At the end of treatment (day 30) H. pylori was no longer detected in 16 of 20 patients (80%), and this finding was consistent with a marked decrease in the grading of gastritis activity. At the follow-up endoscopy (day 60) the infection was eradicated in only four patients (20%). These data indicate a favourable distribution of azithromycin into gastric mucosa of patients with H. pylori infection and suggest that this new macrolide antibiotic represents a valuable option for treatment regimens against H. pylori. However, the low eradication rate achieved with azithromycin plus omeprazole is a source of concern and requires further investigation.

Adult↗
PubMed1998

Characterization of antigens recognized by new monoclonal antibodies raised against culture filtrate proteins of Mycobacterium bovis bacillus Calmette-Guérin.

Effective protection against Mycobacterium tuberculosis may be achieved in experimental animals by immunization with proteins secreted by tuberculous bacilli in the extracellular milieu during growth. In this study, monoclonal antibodies were raised against Mycobacterium bovis bacillus Calmette-Guérin (BCG) culture filtrate proteins or live BCG, in an attempt to identify novel mycobacterial secretion antigens: the localization of the antigens recognized by the monoclonal antibodies within the mycobacterial cell was studied and interspecies reactivity was also investigated. The monoclonal antibodies obtained recognized proteins of molecular mass ranging from 5 to 82 kDa, with a prevailing frequency in the 30 kDa region. Three of the monoclonal antibodies recognized proteins present only in culture filtrates, one reacted with a cytoplasmic antigen, while the remaining antibodies recognized components which were mainly associated with the cell wall and the cytoplasmic membrane. The chemical nature and possible identity of the antigens was checked. Three monoclonal antibodies are likely to react with novel mycobacterial antigens of 5, 42 and 82 kDa, respectively.

Antibodies, Bacterial↗
PubMed1997

Comparative analysis of subcellular distribution of protein antigens in Mycobacterium bovis bacillus Calmette-Guérin.

The distribution of protein antigens in purified subcellular fractions of Mycobacterium bovis bacillus Calmette-Guérin (BCG) was comparatively analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with specific monoclonal antibodies and polyclonal sera. The 19- and 38-kDa lipoproteins were mainly detected in the cell wall and cell membrane enriched fractions, and they were extracted from the former by Triton X-114 and Nonidet P-40. The 65-kDa heat-shock protein (hsp) was present in the cytoplasmic fraction and only trace amounts were found in the crude cell wall preparation. In contrast, the 14-kDa hsp was highly represented in the cell wall fraction, besides being present in cytoplasmic fraction. Both superoxide dismutase (SOD) and antigen 85 complex (Ag 85) were abundantly released in culture medium, and to a lower extent, they were present in the cell wall fraction; SOD was present in comparable amounts also in the cytoplasmic fraction, while Ag 85 was far less represented in the same. Sera from mice immunized with culture filtrate (CF) proteins of BCG recognized several antigens in CFs, which were not detectable in cell wall, cell membrane, and cytoplasmic fractions, indicating that CF proteins include secreted antigens which have not yet been identified.

Animals↗
PubMed1997

Periodontal tissue disposition of azithromycin.

The tissue penetration of azithromycin, the prototype of a new class of macrolide antibiotics named azalides, was studied in patients undergoing surgery for third-molar removal. Drug concentrations in plasma, saliva, and periodontal tissues were evaluated in 28 patients treated with azithromycin 500 mg/day per os for 3 consecutive days. Samples of blood, saliva, gingiva, and alveolar bone were collected during oral surgery, 12 hours, and 2.5, 4.5, and 6.5 days after the last dosing, and the azithromycin concentration was measured microbiologically by using Micrococcus luteus NCTC 8440 as the reference organism. The highest concentrations of azithromycin were observed 12 hours after the last dose in plasma, saliva, gingiva, and bone (0.33 +/- 0.04 mg/l, 2.14 +/- 0.30 mg/l, 6.47 +/- 0.57 mg/kg, and 1.86 +/- 0.15 mg/kg, respectively) and then declined gradually. However, consistent levels of the drug in saliva and periodontal tissues could be detected up to 6.5 days, indicating that azithromycin was retained in target tissues and fluids for a long time after the end of treatment. Among the samples examined, the highest concentration of azithromycin was found in the gingiva at each time studied. Moreover, the ratios of salivary or periodontal tissue levels versus plasma concentrations remained nearly unmodified from 12 hours up to 6.5 days. Overall, these results indicate a favorable disposition of azithromycin into saliva and periodontal tissues and suggest that this macrolide antibiotic represents a valuable option in the pharmacologic treatment of odontogenic infections.

Adult↗
PubMed1996

Proliferation of distinct human T cell subsets in response to live, killed or soluble extracts of Mycobacterium tuberculosis and Myco. avium.

The proliferative responses of distinct cell subsets from healthy, bacille Calmette-Guérin (BCG)-vaccinated blood donors were assessed after in vitro stimulation with live or UV-killed Mycobacterium tuberculosis and Myco. avium or with soluble extracts obtained from either mycobacterial species. Proliferation of cell subsets was evaluated by flow cytometric determination of 5-bromo-2'-deoxy-uridine incorporation into DNA and simultaneous identification of surface phenotypic markers. In the presence of monocytes, the response to whole (live or killed) bacteria was characterized by a predominant proliferation of CD4+ alphabeta+ T cells and, to a lesser extent, of CD8+ alphabeta+ T cells. Proliferation of CD8+ alphabeta+ T cells was primarily elicited by live rather than killed bacilli (P < 0.05). Conversely, when soluble bacterial extracts were used as stimulators, a preferential proliferation of gammadelta+ T cells, expressing predominantly Vgamma9+ and Vdelta2+ T cell receptor chains, was recorded. Moreover, when monocyte-depleted cell populations were directly cultured with live bacteria, a marked proportion of CD3- CD16+ (natural killer (NK)) cells was detected among the responding cells. Although both alphabeta, gammadelta and NK cells have been previously shown to react with mycobacteria in vitro, their relative contributions to the response have been difficult to assess. Using a flow cytometric technique which allows direct identification of proliferating cells within complex cell populations, our study demonstrates significant differences in the ability of various mycobacterial antigen preparations to elicit proliferation of distinct cell subsets.

Antigens, Bacterial↗
PubMed1995

Molecular typing of Candida albicans in oral candidiasis: karyotype epidemiology with human immunodeficiency virus-seropositive patients in comparison with that with healthy carriers.

Candida albicans organisms isolated from the oral cavities of healthy carriers (26 individuals) and compromised hosts (40 human immunodeficiency virus [HIV]-seropositive patients, all showing symptomatic oral candidiasis) were compared by resolving chromosome-sized DNA molecules into electrophoretic karyotypes. Seven- to 10-band electrophoretic patterns were obtained, with significant and reproducible differences in the distributions of the DNA bands. Seven distinct classes were identified and were designated type a (8 bands), type b (8 bands), type c (7 bands), type d (9 bands), type x (10 bands), type y (10 bands), and type z (9 bands). Four of these (types a to d) were the most representative within all of the isolated strains (95.5%), and the other three (types x to z) were observed only once in three HIV-seropositive individuals (4.5%). Only types b and c were isolated from healthy carriers, with the percentage of their isolation being 61.5 and 38.5%, respectively, while all the described karyotypes were isolated from HIV-seropositive patients, with type b being the most frequent (45%); this was followed by types c (25%), a (15%), and d (7.5%). The prevalence of type b and c karyotypes in HIV-infected individuals, as well as in healthy carriers, suggests that commensal strains in the oral cavities of healthy individuals may become the prevalent agents of subsequent oral candidiasis in compromised hosts. However, replacement of the original, commensal strain, if there is one, cannot be excluded in a compromised host, although strain replacement may be more reasonably hypothesized for types a and d, since only these types were isolated at a relative high percentage from the oral lesions of HIV-infected individuals.

AIDS-Related Opportunistic Infections↗
PubMed1993

BCG-activated NK cells regulate the antibody response to SRBC and restore immune reactivity to PPD in BCG-infected mice.

C57B1/6 mice show a significant increase in the number of natural killer (NK) cells in the peritoneal cavity, four days after intraperitoneal infection with Mycobacterium bovis strain BCG. Cell transfer experiments demonstrated that BCG-induced NK cells are able to depress the induction of antibody response to an unrelated antigen (i.e., sheep red blood cells) in recipient mice. The involvement of macrophages, B and T cells in the phenomenon was ruled out by using different purification steps. In addition, BCG-induced NK cells were shown to be able of partially restoring the DTH response to PPD in recipient mice that were anergic to the latter antigen as a consequence of intravenous infection with large doses of BCG.

Animals↗