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

Freer, Giulia; Florio, Walter; Casa, B Dalla; Bottai, Daria; Batoni, Giovanna; Maisetta, Giuseppantonio; Senesi, Sonia; Campa, Mario

doi:10.1016/s0923-2508(98)80302-1

Cited by 13 publications

(23 citation statements)

References 25 publications

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“…Anti-feline B7.1 (B7.1.66) was a kind gift from Wayne Tompkins, North Carolina State University (25). The negative control for all unlabeled immunoglobulin G1 (IgG1) isotype antibodies was monoclonal antibody L8D8 (6). All unlabeled monoclonal antibodies were mouse IgG1.…”

Section: Methodsmentioning

confidence: 99%

Generation of Feline Dendritic Cells Derived from Peripheral Blood Monocytes for In Vivo Use

Freer

Matteucci

Mazzetti

et al. 2005

Clin Vaccine Immunol

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Dendritic cells (DCs) are professional antigen-presenting cells that can prime T cells and polarize the cellular immune response. Because Th1-type immune responses have been connected to success in combating viral infection, a promising therapeutic application of DCs would be their differentiation in vitro and injection back into the host to boost an immune response in infected animals. This study was aimed both at developing a protocol to cultivate feline DCs in the absence of exogenous proteins for their use in vivo and at investigating what might be the most appropriate stimulus to induce their maturation in vitro and finding correlates of maturation. We generated DCs from peripheral blood monocytes in the presence of feline interleukin-4 and granulocyte-macrophage colony stimulating factor, and after 5 days their maturation was induced with either lipopolysaccharide, human recombinant tumor necrosis factor alpha, poly(I:C), or activated feline platelets. After 48 h, their CD14, CD1a, major histocompatibility complex class II, and B7.1 surface expression was analyzed in parallel with their ability to uptake antigen or prime a mixed leukocyte reaction. The results presented show that feline DCs cultured in autologous plasma differentiate and are able to mature in the presence of stimuli similar to the ones currently used for other species. The present work sets the grounds for future use of DCs obtained by the protocol described for in vivo vaccination and immunotherapy of feline immunodeficiency virus-infected cats.

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Section: Methodsmentioning

confidence: 99%

Generation of Feline Dendritic Cells Derived from Peripheral Blood Monocytes for In Vivo Use

Freer

Matteucci

Mazzetti

et al. 2005

Clin Vaccine Immunol

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“…CFs were prepared from 12-day-old cultures of BCG as described previously (10). MAb WB8A11-reacting Ag was purified by immunoaffinity chromatography from CFs of BCG.…”

Section: Methodsmentioning

confidence: 99%

“…MAb WB8A11-reacting Ag was purified by immunoaffinity chromatography from CFs of BCG. To this end, ascitic fluid containing MAb WB8A11 was added to Sepharose-protein A at 2 mg/ml of gel slurry and was covalently bound to Sepharose-protein A by use of dimethyl pimelidate as described previously (10). CFs of BCG were added to the gel slurry at 0.5 mg/ml in phosphate-buffered saline (PBS), and the mixture was incubated for 6 h at 4°C with gentle agitation.…”

Section: Methodsmentioning

confidence: 99%

“…The icd2 gene was obtained by PCR amplification from genomic DNA of BCG with a high-fidelity DNA polymerase with proofreading activity (Expand High Fidelity PCR system; Boehringer Mannheim, Mannheim, Germany). Genomic DNA of BCG was obtained as described previously (10). The upstream primer was UPPER24 (5Ј-GCCTTGGACAGCCTCCAGCGCTGC-3Ј), and the downstream primer was LOWER25 (5Ј-ATGAGCGCCGAACAGCCGACCATCA-3Ј).…”

Section: Methodsmentioning

confidence: 99%

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Identification, Molecular Cloning, and Evaluation of Potential Use of Isocitrate Dehydrogenase II of Mycobacterium bovis BCG in Serodiagnosis of Tuberculosis

Florio

Bottai

Batoni

et al. 2002

Clin Vaccine Immunol

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Diagnosis of tuberculosis is time-consuming and requires infrastructures which are often not available in countries with high incidences of the disease. In the present study, an 82-kDa protein antigen was isolated by affinity chromatography and was identified by peptide mass fingerprinting as isocitrate dehydrogenase II, which is encoded by the icd2 gene of Mycobacterium bovis BCG. The icd2 gene of BCG was cloned by PCR, and the product of recombinant gene expression was purified and analyzed by two-dimensional polyacrylamide gel electrophoresis. The recombinant protein, named rICD2, was tested for its recognition by immunoglobulin G (IgG) antibodies from the sera of 16 patients with tuberculosis (TB) and 23 healthy individuals by Western blotting. The results showed that rICD2 is recognized by IgG antibodies from the sera of all TB patients tested at serum dilutions of >1:640. At a serum dilution of 1:1,280, the sensitivity was 50% and the specificity was 86.9%. These results indicate that rICD2 might represent a candidate for use in a new assay for the serodiagnosis of TB.Tuberculosis (TB) remains a major cause of death and disabilities in developing countries, where over 90% of global cases occur, and is now also a cause for growing concern in industrialized countries, where the incidence of the disease has also increased (6). Diagnosis of TB in developing countries mainly relies on examination of chest X rays and/or examination of smears under a microscope for detection of acid-fast bacilli. However, only about 50% of the patients with pulmonary TB are smear positive, and chest X rays can detect advanced pulmonary TB only after extensive damage of lung tissues has already occurred (22). At present, the most reliable method for diagnosis of TB is still isolation of organisms by culture and biochemical identification of the tubercle bacilli, but because of the slow growth rate of Mycobacterium tuberculosis, results are not seen until several weeks after specimen collection. The time to detection might be shortened to 1 week by the use of radiometric systems and nucleic acid probes, but these techniques are still too expensive for use by laboratories in developing countries. A simple and inexpensive diagnostic assay might be of great benefit for global control of the disease (9). Considerable efforts have been made to evaluate assays based on specific recognition of selected mycobacterial antigens (Ags) in vitro by serum antibodies (Abs) (16,17,25,34) or the release of gamma interferon by peripheral blood mononuclear cells stimulated with M. tuberculosis-specific Ags (33, 34). In the latter case, the use of cocktails of immunodominant Ags of M. tuberculosis has been indicated to increase the sensitivity of the assay significantly without affecting the specificity of the assay (33), and the same strategy has been suggested for use in the diagnosis of TB based on detection of specific Ab responses (13,14). As the pattern of Ag recognition by patient Abs may be influenced by the stage of the disease (15, 28) and b...

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“…By means of a recently described monoclonal antibody (mAb) (L8D8) [5] we had previously identi¢ed a new protein (SA-5K) with an apparent molecular mass of 5 kDa, secreted in culture ¢ltrates by M. tuberculosis, Mycobacterium bovis bacillus Calmette-Guërin (BCG) and few other mycobacterial species [6]. No signi¢cant homology with other proteins in the databases was found and the physiologic role of SA-5K for mycobacterial cells is unknown.…”

Section: Introductionmentioning

confidence: 99%

Involvement of the Mycobacterium tuberculosis secreted antigen SA-5K in intracellular survival of recombinant Mycobacterium smegmatis

Batoni

2001

FEMS Microbiology Letters

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A new protein (SA-5K) secreted in culture filtrates by Mycobacterium bovis, Mycobacterium tuberculosis, and few other mycobacterial species was previously identified and purified in our laboratory. In order to evaluate the putative role of SA-5K during intracellular mycobacterial growth, in the present study the SA-5K gene was cloned and expressed in Mycobacterium smegmatis, a rapid growing nonpathogenic mycobacterium which does not contain the gene for the protein.

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

Cited by 13 publications

References 25 publications

Generation of Feline Dendritic Cells Derived from Peripheral Blood Monocytes for In Vivo Use

Generation of Feline Dendritic Cells Derived from Peripheral Blood Monocytes for In Vivo Use

Identification, Molecular Cloning, and Evaluation of Potential Use of Isocitrate Dehydrogenase II of Mycobacterium bovis BCG in Serodiagnosis of Tuberculosis

Involvement of the Mycobacterium tuberculosis secreted antigen SA-5K in intracellular survival of recombinant Mycobacterium smegmatis

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