Commentary: Revisiting nanoparticle-assay interference: There's plenty of room at the bottom for misinterpretation.

Engineered nanomaterials (ENMs) are a diverse class of materials whose distinct properties make them desirable in a multitude of applications. The proliferation of nanotoxicology research has improved our understanding of ENM toxicity, but an under appreciation for their potential to interfere with biochemical assays has hampered progress in the field. The physicochemical properties of ENMs can promote their interaction with membranes or biomacromolecules (e.g. proteins, genomic material). This can influence the activity of enzymes used as biomarkers or as reagents in biochemical assay protocols, bind indicator dyes in cytotoxicity tests, and/or interfere with the cellular mechanisms controlling the uptake of such dyes. The spectral characteristics of some ENMs can cause interference with common assay chromophores, fluorophores, and radioisotope scintillation cocktails. Finally, the inherent chemical reactivity of some ENMs can short circuit assay mechanisms by directly oxidizing or reducing indicator dyes. These processes affect data quality and may lead to significant misinterpretations regarding ENM safety. We provide an overview of some ENM properties that facilitate assay interference, examples of interference and the erroneous conclusions that may result from it, and a number of general and specific recommendations for validating cellular and biochemical assay protocols in nanotoxicology studies.

Towards an integrative approach to understanding the role of chemerin in human health and disease.

Chemerin is an adipocyte-secreted protein with autocrine/paracrine roles on adipose development and function as well as endocrine roles in metabolism and immunity. Following prochemerin secretion, protease-mediated generation of chemerin isoforms with a range of biological activities is a key regulatory mechanism controlling local, context-specific chemerin bioactivity. Together, experimental and clinical data indicate that localized and/or circulating chemerin expression and activation are elevated in numerous metabolic and inflammatory diseases including psoriasis, obesity, type 2 diabetes, metabolic syndrome and cardiovascular disease. These elevations are positively correlated with deleterious changes in glucose, lipid, and cytokine homeostasis, and may serve as a link between obesity, inflammation and other metabolic disorders. This review highlights the current state of knowledge regarding chemerin expression, processing, biological function and relevance to human disease, particularly with respect to adipose tissue development, inflammation, glucose homeostasis and cardiovascular disease. Furthermore, it discusses study variability, deficiencies in current measurement, and questions concerning chemerin function in disease, with a special emphasis on techniques and tools used to properly assess chemerin biology. An integration of basic and clinical research is key to understanding how chemerin influences disease pathobiology, and whether modulation of chemerin levels and/or activity may serve as a potential method to prevent and treat metabolic diseases.

Gastric B-cell mucosa-associated lymphoid tissue (MALT) lymphoma in an animal model of 'Helicobacter heilmannii' infection.

While Helicobacter pylori is accepted as the dominant human gastric bacterial pathogen, a small percentage of human infections have been associated with another organism, commonly referred to as 'Helicobacter heilmannii', which is more prevalent in a range of animal species. This latter bacterium has been seen in association with the full spectrum of human gastric diseases including gastritis, peptic ulceration, and gastric carcinomas, including gastric B-cell mucosa-associated lymphoid tissue (MALT) lymphoma. This study describes an analysis of the pathogenic potential of a number of 'H heilmannii' isolates in an animal model of gastric MALT lymphoma. BALB/c mice were infected with ten different 'H heilmannii' isolates originating from both human and animal hosts. The animals were examined at various time points for up to 28 months after infection. The infected animals initially developed a chronic inflammatory response within 6 months. This histological response increased in severity with the length of infection, with the development of overt lymphoma in some animals 18 months after infection. MALT lymphomas were detected in up to 25% of the infected animals. The prevalence of lymphoma was dependent on the length of infection and the origin of the infecting isolates. A range of other histological features accompanied the lymphocytic infiltration, including invaginations of the gastric epithelium and associated hyperplastic tissue, mucus metaplasia, and a small number of diffuse large B-cell lymphomas. The ability to manipulate experientially the presence of the bacterium in the animal model will allow further studies examining the role of antigen drive in the development of Helicobacter-associated MALT lymphoma.

Gastritis associated with Helicobacter-like organisms in baboons.

Subclinical gastritis was observed in 10 of 10 baboons (Papio spp.) from a toxicity study in a research facility. The lesions were similar in xenobiotic-treated and control animals, suggesting a spontaneous rather than chemical-induced disease. Histologic examination revealed lymphoplasmacytic gastritis in the antral mucosa. The fundic mucosa contained minor, scattered aggregates of lymphocytes and plasma cells. A Warthin-Starry silver stain and ultrastructural examination revealed numerous spiral-shaped bacteria morphologically resembling Helicobacter pylori in antral glands and numerous spiral-shaped bacteria morphologically consistent with H. heilmannii-like organisms in fundic glands. Polymerase chain reaction assay of paraffin-embedded antral and fundic tissue using primers for the urease gene and 16S ribosomal ribonucleic acid gene amplified deoxyribonucleic acid fragments with a high degree of sequence homology for H. pylori and H. heilmannii. This is the first report of gastritis associated with Helicobacter-like organisms in baboons.

Helicobacter ganmani sp. nov., a urease-negative anaerobe isolated from the intestines of laboratory mice.

Spiral bacteria were isolated from the intestines of laboratory mice during a study examining the presence of Helicobacter species and other spiral organisms naturally infecting mice maintained at four different animal facilities in Sydney, Australia. One group of 17 isolates, cultured from mice from three of the four facilities, were found to be helicobacters but did not fall within any of the 18 currently recognized species. These isolates were unusual in that they only grew anaerobically at 37 degrees C and were incapable of growth under microaerobic conditions. Like Helicobacter rodentium, isolates possessed single, bipolar, unsheathed flagella and were urease-negative. They were positive for oxidase and reduced nitrate to nitrite but did not hydrolyse hippurate or indoxyl acetate, grew on charcoal agar and were resistant to cephalothin. 16S rDNA sequences from four strains were determined and found to be identical to one another. H. rodentium was the most closely related species in terms of 16S rDNA sequence similarity (98.2%). Numerical analysis of whole-cell proteins by SDS-PAGE for nine isolates was carried out with a comparison to all known Helicobacter species, including newly determined profiles from three H. rodentium strains. The new isolates were clearly differentiated from H. rodentium and other Helicobacter spp. On the basis of this data, including genetic, biochemical and protein analysis, it is proposed that these isolates belong to Helicobacter ganmani sp. nov. (type strain CMRI H02T = CCUG 43526T = CIP 106846T).

Helicobacter-induced expression of Bcl-X(L) in B lymphocytes in the mouse model: a possible step in the development of gastric mucosa-associated lymphoid tissue (MALT) lymphoma.

Primary gastric mucosa-associated lymphoid tissue (MALT) lymphoma may develop from chronic infection with Helicobacter sp. in the mouse model. The mechanisms of pathogenesis remain unclear. Regulation of B-cell proliferation and death are important features to investigate. Proteins encoded by bcl-2 family genes, e.g., Bcl-X(L), regulate apoptosis; and alterations in the expression of these genes can contribute to the development of cancer. Our aim was to determine the role of Bcl-X(L) in B lymphocytes in the development of gastric MALT lymphoma associated with Helicobacter infection using the BALB/c mouse model. We analyzed 37 animals with Helicobacter-associated MALT (n = 25), low-grade MALT lymphoma (n = 10) and high-grade lymphoma (n = 2), investigating the in vivo distribution of Bcl-X(L) in B cells/B-lymphoma cells using immunohistochemical analysis. In vitro cultivation of B cells/B-lymphoma cells was employed to perform RT-PCR analysis of Bcl-X(L) mRNA expression after cell stimulation with Helicobacter antigen. We found significant Bcl-X(L) protein expression in B lymphocytes within MALT and low-grade MALT lymphoma, whereas there was no and minimal expression, respectively, of Bcl-X(L) in the 2 high-grade MALT lymphoma cases. Expression of bcl-X(L) mRNA in B lymphocytes was up-regulated in vitro upon Helicobacter-antigen stimulation and associated with prolonged cell survival. These findings support the hypothesis that Bcl-X(L) plays a role in the pathogenesis of B-cell MALT lymphoma by providing cell-survival signals and by triggering the acquisition of MALT.

MALToma-like lesions in the murine gastric mucosa after long-term infection with Helicobacter felis. A mouse model of Helicobacter pylori-induced gastric lymphoma.

The long-term consequences of helicobacter infection were observed in an established murine model of human helicobacter infection. Stomachs of specific pathogen-free BALB/c mice infected with Helicobacter felis were examined for inflammation with particular reference to lymphoid cell proliferation and lymphoepithelial lesions. There was little evidence of an inflammatory response in animals sacrificed up to 19 months after infection. In contrast, from 22 months, 38% of infected animals had lymphoid follicles, whereas no lymphoid follicles were found in noninfected control animals. Lymphoepithelial lesions were observed in 25% of infected mice compared with none in controls. Immunostaining confirmed the B-cell nature of the lymphoid infiltrate. The morphology of these lesions closely resemble those seen in human gastric MALToma. This animal model would provide an opportunity to study the pathogenesis of lymphoproliferative disease.

The importance of local acid production in the distribution of Helicobacter felis in the mouse stomach.

BACKGROUND/AIMS: Helicobacter felis colonizes the gastric mucosa of rodents. Preliminary studies showed differences in the distribution of the organism in different parts of the stomach that seemed related to the secretory capacity of the mucosa. The aim of this study was to determine the localization of H. felis in the mouse stomach and to investigate the influence of acid-suppressive agents. METHODS: Specific-pathogen-free BALB/c mice were infected with H. felis. Colonization was assessed in longitudinal sections of gastric tissue from animals untreated or treated with omeprazole or ranitidine. RESULTS: In untreated H. felis-infected animals, the preferred ecological niche was the antrum and cardia equivalent. The density of colonization correlated with the number of parietal cells per gland. Partial acid suppression with ranitidine produced a slight increase in the colonization of the body but was restricted to the upper portions of the gastric gland. Omeprazole treatment produced a greater colonization of the body with bacteria traversing the entire gland. Some reduction in antral colonization occurred. CONCLUSIONS: These results are consistent with the hypothesis that local acid output is a crucial determinant in the distribution of Helicobacter species in the stomach. Differences in local acid output may explain the different patterns of Helicobacter pylori-induced gastric pathology.

Animal and public health implications of gastric colonization of cats by Helicobacter-like organisms.

The bacterial genus Helicobacter contains a number of species which colonize the gastric mucosa of mammals. Natural and/or experimental gastric pathology has been correlated with colonization in humans and a wide variety of animal species. Historical reports in the literature suggest that a high percentage of cats are colonized by large, spiral, gastric helicobacter-like organisms (GHLOs). One of these bacteria (Helicobacter felis) has been isolated on artificial media and has experimentally caused gastritis in gnotobiotic dogs. This study surveyed the prevalence of helicobacter colonization in random-source cats by using the urease assay. Histologic examination was performed to determine the degree of associated pathology present. GHLOs associated with chronic gastritis were present in 70% of the juvenile and 97% of the adult cats studied. Although further study is needed to determine specifically what role GHLOs play in feline gastrointestinal disease, these results indicate that helicobacter colonization should be considered in the pathogenesis of feline gastroenteropathy. Furthermore, the high prevalence of feline infection is interesting because cats have recently been implicated as a potential reservoir for human infection by helicobacter-like organisms.

Helicobacter muridarum sp. nov., a microaerophilic helical bacterium with a novel ultrastructure isolated from the intestinal mucosa of rodents.

Helical organisms with novel ultrastructural characteristics were isolated from the intestinal mucosa of rats and mice. These bacteria were characterized by the presence of 9 to 11 periplasmic fibers which appeared as concentric helical ridges on the surface of each cell. The cells were motile with a rapid corkscrewlike motion and had bipolar tufts of 10 to 14 sheathed flagella. The bacteria were microaerophilic, nutritionally fastidious, and physiologically similar to Helicobacter species and Wolinella succinogenes but could be differentiated from these organisms by their unique cellular ultrastructure. Using 16S rRNA sequencing, we found that strain ST1T (T = type strain) was related to previously described Helicobacter species, "Flexispira rappini," and W. succinogenes. The closest relatives of strain ST1T were Helicobacter mustelae and "F. rappini" (average similarity value, 96%). On the basis of phylogenetic data, strain ST1T (= ATCC 49282T) represents a new species of the genus Helicobacter, for which we propose the name Helicobacter muridarum.

Phylogeny of Helicobacter felis sp. nov., Helicobacter mustelae, and related bacteria.

Strain CS1T (T = type strain) is a gram-negative, microaerophilic, urease-positive, spiral-shaped bacterium that was isolated from the gastric mucosa of a cat. Additional strains which possessed biochemical and ultrastructural characteristics similar to those of strain CS1T were isolated from the gastric mucosa of cats and dogs. The guanine-plus-cytosine content of the DNA of strain CS1T was 42.5 mol%. The 16S rRNA sequences of strain CS1T, strain DS3 (a spiral-shaped isolate from a dog), and Helicobacter mustelae were determined by direct RNA sequencing, using a modified Sanger method. These sequences were compared with the 16S rRNA sequences of Helicobacter pylori, "Flexispira rappini," Wolinella succinogenes, and 11 species of campylobacters. A dendrogram was constructed based upon sequence similarities. Strains CS1T and DS3 were very closely related (level of similarity, 99.3%). Two major phylogenetic groups were formed; one group consisted of strains CS1T and DS3, H. mustelae, H. pylori, "F. rappini," and W. succinogenes, and the other group contained the true campylobacters. The average level of similarity between members of these two groups was 84.9%. Within the first group, strains CS1T and DS3, H. pylori, and H. mustelae formed a cluster of organisms with an interspecies similarity level of 94.5%. The phylogenetic positions of W. succinogenes and "F. rappini" were just outside this cluster. On the basis of the results of this study, we believe that strains CS1T (= ATCC 49179T) and DS3 represent a new species of the genus Helicobacter, for which we propose the name Helicobacter felis.

Mucus colonization as a determinant of pathogenicity in intestinal infection by Campylobacter jejuni: a mouse cecal model.

Human isolates of the intestinal pathogen Campylobacter jejuni have been shown to colonize mucus on the outer surface and deep within the intestinal crypts of gnotobiotic or germfree mice. The cecal crypts are preferentially colonized. A model of mucus colonization by C. jejuni in the mouse cecum has been developed, using antibiotic- and magnesium sulfate-treated specific-pathogen-free animals. These spiral-shaped bacteria colonize the mucus in a similar manner to the normal spiral-shaped microbiota. No evidence of adhesion to the intestinal surface was found with a wide variety of microscopic techniques. The campylobacters were seen to be highly motile in living preparations of gut tissue and rapidly tracked along intestinal mucus. Just as many of the normal spiral-shaped bacteria of intestinal surfaces can achieve close association with the epithelium through mucus association and do not adhere to the surface, C. jejuni colonizes the intestinal mucosa via mucus colonization. Thus, a major determinant of pathogenicity in intestinal infection with C. jejuni is proposed to be an ability to colonize intestinal mucus. The possession of specific adhesins is unlikely to be a significant determinant of pathogenicity. Better understanding of the mechanism of mucus association and the properties of the bacterium that are responsible will provide a basis for the rational selection of preventative measures. The model of mucus association in adult antibiotic-treated mice provides an opportunity for colonization studies with variant organisms and immunization studies.