Prevalent bacterial species and novel phylotypes in advanced noma lesions.

The purpose of this study was to determine the bacterial diversity in advanced noma lesions using culture-independent molecular methods. 16S ribosomal DNA bacterial genes from DNA isolated from advanced noma lesions of four Nigerian children were PCR amplified with universally conserved primers and spirochetal selective primers and cloned into Escherichia coli. Partial 16S rRNA sequences of approximately 500 bases from 212 cloned inserts were used initially to determine species identity or closest relatives by comparison with sequences of known species or phylotypes. Nearly complete sequences of approximately 1,500 bases were obtained for most of the potentially novel species. A total of 67 bacterial species or phylotypes were detected, 25 of which have not yet been grown in vitro. Nineteen of the species or phylotypes, including Propionibacterium acnes, Staphylococcus spp., and the opportunistic pathogens Stenotrophomonas maltophilia and Ochrobactrum anthropi were detected in more than one subject. Other known species that were detected included Achromobacter spp., Afipia spp., Brevundimonas diminuta, Capnocytophaga spp., Cardiobacterium sp., Eikenella corrodens, Fusobacterium spp., Gemella haemoylsans, and Neisseria spp. Phylotypes that were unique to noma infections included those in the genera Eubacterium, Flavobacterium, Kocuria, Microbacterium, and Porphyromonas and the related Streptococcus salivarius and genera Sphingomonas and TREPONEMA: Since advanced noma lesions are infections open to the environment, it was not surprising to detect species not commonly associated with the oral cavity, e.g., from soil. Several species previously implicated as putative pathogens of noma, such as spirochetes and Fusobacterium spp., were detected in at least one subject. However, due to the limited number of available noma subjects, it was not possible at this time to associate specific species with the disease.

A novel lacrimal gland autoantigen in the NOD mouse model of Sjögren's syndrome.

Recent research has demonstrated a crucial role for autoantibodies in the pathogenesis of Sjögren's syndrome (SS)-like disease in the non-obese diabetic (NOD) mouse, but it remains to be determined which antibody species among all those present are directly related to the various aspects of pathology. To identify autoantigens in the NOD mouse system, we have taken the approach of using immunoglobulin (Ig)G purified from sera of NOD mice exhibiting SS-like symptoms to screen cDNA expression libraries derived from exocrine gland mRNA. Here we report the identification of a novel autoantigen, designated LGP10, expressed in lacrimal and submandibular glands. Autoantibodies to this protein are prominent in the sera of NOD mice starting at 11-12 weeks of age, but not in control nonautoimmune mice. LGP10 has no known function, but bears similarities to various other proteins produced by epithelia. Interestingly, all of these similar proteins have been linked to immunosuppression and/or steroid binding, both processes that could have a significant impact on pathological features of SS.

Bacterial diversity in human subgingival plaque.

The purpose of this study was to determine the bacterial diversity in the human subgingival plaque by using culture-independent molecular methods as part of an ongoing effort to obtain full 16S rRNA sequences for all cultivable and not-yet-cultivated species of human oral bacteria. Subgingival plaque was analyzed from healthy subjects and subjects with refractory periodontitis, adult periodontitis, human immunodeficiency virus periodontitis, and acute necrotizing ulcerative gingivitis. 16S ribosomal DNA (rDNA) bacterial genes from DNA isolated from subgingival plaque samples were PCR amplified with all-bacterial or selective primers and cloned into Escherichia coli. The sequences of cloned 16S rDNA inserts were used to determine species identity or closest relatives by comparison with sequences of known species. A total of 2,522 clones were analyzed. Nearly complete sequences of approximately 1,500 bases were obtained for putative new species. About 60% of the clones fell into 132 known species, 70 of which were identified from multiple subjects. About 40% of the clones were novel phylotypes. Of the 215 novel phylotypes, 75 were identified from multiple subjects. Known putative periodontal pathogens such as Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola were identified from multiple subjects, but typically as a minor component of the plaque as seen in cultivable studies. Several phylotypes fell into two recently described phyla previously associated with extreme natural environments, for which there are no cultivable species. A number of species or phylotypes were found only in subjects with disease, and a few were found only in healthy subjects. The organisms identified only from diseased sites deserve further study as potential pathogens. Based on the sequence data in this study, the predominant subgingival microbial community consisted of 347 species or phylotypes that fall into 9 bacterial phyla. Based on the 347 species seen in our sample of 2,522 clones, we estimate that there are 68 additional unseen species, for a total estimate of 415 species in the subgingival plaque. When organisms found on other oral surfaces such as the cheek, tongue, and teeth are added to this number, the best estimate of the total species diversity in the oral cavity is approximately 500 species, as previously proposed.

Leukocytes infiltrating the submandibular glands of NOD mice express E-cadherin.

Sjögren's syndrome [SS] is typified by infiltration of mononuclear cells [MNC] into the salivary and lacrimal glands, although the biological role of these infiltrating cells remains unclear. We report here that E-cadherin, which mediates cell-cell adhesion and regulates differentiation, proliferation, and apoptosis of epithelial cells, is expressed ectopically by MNC in the salivary glands in the NOD mouse model of SS. Flow cytometric analysis of CD45(+)cells from NOD submandibular glands revealed that over 90% express E-cadherin. More detailed phenotypic analyses demonstrated that E-cadherin expression is high (>90%) among mature T cells (CD3(+)), B cells (CD19(+)), NK cells (DX5(+)), and monocyte/macrophages (CD11b(+)) within the infiltrates. Expression of other surface antigens, such as CD90 and CD117, above expected values suggests the presence of immature leukocytes, possibly of the T cell lineage, within the foci. We also present evidence that E-cadherin-expressing T cells in the glands do not exhibit normal proliferative responses to immobilized anti-CD3 antibody. While infiltrating MNC are not likely to be the direct cause of salivary hypofunction, the expression of E-cadherin by these cells may have implications for the progression of disease.

The diversity of periodontal spirochetes by 16S rRNA analysis.

The purpose of this study was to examine the diversity of spirochetes in the subgingival pocket of multiple subjects with a range of periodontal conditions, including two healthy, one adult periodontitis, three acute necrotizing ulcerative gingivitis, eight refractory periodontitis, and one human immunodeficiency virus (HIV) periodontitis. The 16S rRNA genes of spirochetes in plaque were amplified by polymerase chain reaction using spirochete selective primers. Over 500 clones were sequenced and subjected to phylogenetic analysis. The sequences clustered into the 10 known cultivated Treponema species and into 47 as-yet-uncultivated Treponema species. Most of these Treponema species were identified from multiple clones and subjects. The human periodontal pocket harbors a highly diverse treponeme population. Of the cultivated species, Treponema denticola, Treponema maltophilum and Treponema sp. Smibert-3 were most commonly encountered in diseased subjects but rarely in healthy subjects. Molecular tools based on the sequence data from this study will allow determination of the prevalence of cultivable and uncultivable treponemes in oral diseases.