Molecular Analysis of Sarcoidosis Tissues for<i>Mycobacterium</i>Species DNA

Culture of bronchoalveolar lavage fluid (BALF) is the gold standard for detection of pathogens in the lower airways in cystic fibrosis (CF). However, current culture results do not explain all clinical observations in CF, including negative culture results during pulmonary exacerbation and inflammation in the absence of pathogens. We hypothesize that organisms not routinely identified by culture occur in the CF airway and may contribute to disease. To test this hypothesis we used a culture-independent molecular approach, based on use of rRNA sequence analysis, to assess the bacterial composition of BALF from children with CF and disease controls (DC). Specimens from 42 subjects (28 CF) were examined, and Ϸ6,600 total clones were screened to identify 121 species of bacteria. In general, a single rRNA type dominated clone libraries from CF specimens, but not DC. Thirteen CF subjects contained bacteria that are not routinely assessed by culture. In four CF subjects, candidate pathogens were identified and include the anaerobe Prevotella denticola, a Lysobacter sp., and members of the Rickettsiales. The presumptive pathogens Tropheryma whipplei and Granulicatella elegans were identified in cases from the DC group. The presence of unexpected bacteria in CF may explain inflammation without documented pathogens and consequent failure to respond to standard treatment. These results show that molecular techniques provide a broader perspective on airway bacteria than do routine clinical cultures and thus can identify targets for further clinical evaluation. pulmonary microbiology ͉ ribosomal RNA C ystic fibrosis (CF), the most common autosomal lethal disease in Caucasians, is caused by mutation in the CF transmembrane conductance regulator gene, which results in a generalized exocrinopathy (1). Thickened secretions caused by improper regulation of airway surface liquid contribute to the accumulation of mucus in the airway and defective mucociliary clearance (2). Retained mucus plugs provide a niche for bacterial colonization and persistence. Most morbidity and mortality associated with CF is attributed to microbial infections in the airway and the persistent inflammatory response (3).A small number of pathogens are recognized traditionally in CF airway disease. These include Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, and the Burkholderia cepacia complex (4-7). Other bacteria, including Stenotrophomonas maltophilia and Achromobacter xylosoxidans, also are associated with CF, but there is controversy as to their roles as pathogens (8)(9)(10). This view of bacteria associated with CF is based on culture, however, which may not recover or identify all bacteria present in a specimen. Although known pathogens clearly are important in airway disease, a more comprehensive picture of the bacterial community in the CF airway potentially could lead to improved insight about the disease and thus better treatment. For instance, ''normal'' microbiota that are commonly present, but not considered pathogenic, ...

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“…In cases where no rDNA PCR product was evident, human ␤-actin controls were performed as in ref. 27.…”

Section: Methodsmentioning

confidence: 99%

Molecular identification of bacteria in bronchoalveolar lavage fluid from children with cystic fibrosis

Harris

Groote

Sagel³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

350

342

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“…In Japanese patients with sarcoidosis, quantitative differences in Propionibacterium acnes DNA among sarcoidosis and control subjects were detected (1). Among American patients with sarcoidosis, the presence of genetically distinct mycobacterial nucleic acids in sarcoidosis granulomas was reported (2). Song and colleagues (3) used matrix-assisted laser desorption/ionization time of flight mass spectrometry to demonstrate the presence of mycobacterial catalase-peroxidase (katG) proteins in sarcoidosis granulomas.…”

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confidence: 99%

Development of a Sarcoidosis Murine Lung Granuloma Model UsingMycobacteriumSuperoxide Dismutase A Peptide

Swaisgood¹,

Oswald-Richter²,

Moeller³

et al. 2011

Am J Respir Cell Mol Biol

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Sarcoidosis is characterized by noncaseating granulomas containing CD4 1 T cells with a Th1 immunophenotype. Although the causative antigens remain unknown, independent studies noted molecular and immunologic evidence of mycobacterial virulence factors in sarcoidosis specimens. A major limiting factor in discovering new insights into the pathogenesis of sarcoidosis is the lack of an animal model. Using a distinct superoxide dismutase A peptide (sodA) associated with sarcoidosis granulomas, we developed a pulmonary model of sarcoidosis granulomatous inflammation. Mice were sensitized by a subcutaneous injection of sodA, incorporated in incomplete Freund's adjuvant (IFA). Control subjects consisted of mice with no sensitization (ConNS), sensitized with IFA only (ConIFA), or with Schistosoma mansoni eggs. Fourteen days later, sensitized mice were challenged by tail-vein injection of naked beads, covalently coupled to sodA peptides or to schistosome egg antigens (SEA). Histologic analysis revealed hilar lymphadenopathy and noncaseating granulomas in the lungs of sodA-treated or SEA-treated mice. Flow cytometry of bronchoalveolar lavage (BAL) demonstrated CD4 1 T-cell responses against sodA peptide in the sodA-sensitized mice only. Cytometric bead analysis revealed significant differences in IL-2 and IFN-g secretion in the BAL fluid of sodA-treated mice, compared with mice that received SEA or naked beads (P 5 0.008, Wilcoxon rank sum test). ConNS and ConIFA mice demonstrated no significant formation of granuloma, and no Th1 immunophenotype. The use of microbial peptides distinct for sarcoidosis reveals a histologic and immunologic profile in the murine model that correlates well with those profiles noted in human sarcoidosis, providing the framework to investigate the molecular basis for the progression or resolution of sarcoidosis.

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“…Correspondence and requests for reprints should be addressed to David R. Moller control tissues (4,5). The completed ACCESS (A Case Control Etiologic Study of Sarcoidosis) study provides data that support possible disease associations with select microbially rich environments (6).…”

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confidence: 99%

State of the Art. Potential Etiologic Agents in Sarcoidosis

Möller

2007

Proceedings of the American Thoracic Society

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The etiology of sarcoidosis remains uncertain. The hallmark of sarcoidosis is the epithelioid granuloma, which serves as a necessary starting point for considering disease etiology. Any etiologic agent of sarcoidosis must also explain the typical clinical behaviors and characteristic immunopathologic features of the disease. One clinical observation that serves as a bridge to the etiology of sarcoidosis is the Kveim reaction. In this reaction, local epithelioid granulomas develop several weeks after the intradermal injection of homogenates of sarcoidosis tissue. Our group capitalized on the known properties of the Kveim reagent to search for candidate pathogenic tissue antigens in sarcoidosis without other a priori hypotheses regarding possible microbial or autoimmune etiologies. Using a limited proteomics approach based on the physicochemical properties of Kveim reagent, we detected a limited number of poorly soluble antigenic proteins in sarcoidosis tissues by protein immunoblotting, using sarcoidosis sera. Matrix-associated laser desorption/ ionization-time of flight mass spectrometry identified one of these antigens to be the Mycobacterium tuberculosis catalase-peroxidase protein (mKatG). We found IgG responses to recombinant mKatG in more than 50% of patients with sarcoidosis but rarely in purified protein derivative (PPD)-negative control subjects. These findings support the conclusion that mKatG is a tissue antigen and target of the adaptive immune response in sarcoidosis, providing further evidence of a mycobacterial etiology in a subset of sarcoidosis. More generally, the approach used in these studies might be employed to discover and validate other candidate pathogenic antigens in sarcoidosis or other granulomatous disorders.

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Molecular Analysis of Sarcoidosis Tissues forMycobacteriumSpecies DNA

Cited by 119 publications

References 30 publications

Molecular identification of bacteria in bronchoalveolar lavage fluid from children with cystic fibrosis

Molecular identification of bacteria in bronchoalveolar lavage fluid from children with cystic fibrosis

Development of a Sarcoidosis Murine Lung Granuloma Model UsingMycobacteriumSuperoxide Dismutase A Peptide

State of the Art. Potential Etiologic Agents in Sarcoidosis

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