Werner Nicklas

Receptor for advanced glycation end products (RAGE) regulates sepsis but not the adaptive immune response

While the initiation of the adaptive and innate immune response is well understood, less is known about cellular mechanisms propagating inflammation. The receptor for advanced glycation end products (RAGE), a transmembrane receptor of the immunoglobulin superfamily, leads to perpetuated cell activation. Using novel animal models with defective or tissue-specific RAGE expression, we show that in these animal models RAGE does not play a role in the adaptive immune response. However, deletion of RAGE provides protection from the lethal effects of septic shock caused by cecal ligation and puncture. Such protection is reversed by reconstitution of RAGE in endothelial and hematopoietic cells. These results indicate that the innate immune response is controlled by pattern-recognition receptors not only at the initiating steps but also at the phase of perpetuation.

Implications of infectious agents on results of animal experiments: Report of the Working Group on Hygiene of the Gesellschaft für Versuchstierkunde-Society for Laboratory Animal Science (GV-SOLAS)

Report of the Working Group on Hygiene of the Gesellschaft für Versuchstierkunde–Society for Laboratory Animal Science (GV-SOLAS) GV-SOLAS Working Group on Hygiene: Werner Nicklas (Chairman), Felix R. Homberger, Brunhilde Illgen-Wilcke, Karin Jacobi, Volker Kraft, Ivo Kunstyr, Michael Mähler, Herbert Meyer & Gabi Pohlmeyer-Esch

Molecular characterization of pneumococcal isolates from pets and laboratory animals.

Background Between 1986 and 2008 Streptococcus pneumoniae was isolated from 41 pets/zoo animals (guinea pigs (n = 17), cats (n = 12), horses (n = 4), dogs (n = 3), dolphins (n = 2), rat (n = 2), gorilla (n = 1)) treated in medical veterinary laboratories and zoos, and 44 laboratory animals (mastomys (multimammate mice; n = 32), mice (n = 6), rats (n = 4), guinea pigs (n = 2)) during routine health monitoring in an animal facility. S. pneumoniae was isolated from nose, lung and respiratory tract, eye, ear and other sites. Methodology/Principal Findings Carriage of the same isolate of S. pneumoniae over a period of up to 22 weeks was shown for four mastomys. Forty-one animals showed disease symptoms. Pneumococcal isolates were characterized by optochin sensitivity, bile solubility, DNA hybridization, pneumolysin PCR, serotyping and multilocus sequence typing. Eighteen of the 32 mastomys isolates (56%) were optochin resistant, all other isolates were optochin susceptible. All mastomys isolates were serotype 14, all guinea pig isolates serotype 19F, all horse isolates serotype 3. Rats had serotypes 14 or 19A, mice 33A or 33F. Dolphins had serotype 23F, the gorilla serotype 14. Cats and dogs had many different serotypes. Four isolates were resistant to macrolides, three isolates also to clindamycin and tetracyclin. Mastomys isolates were sequence type (ST) 15 (serotype 14), an ST/serotype combination commonly found in human isolates. Cats, dogs, pet rats, gorilla and dolphins showed various human ST/serotype combinations. Lab rats and lab mice showed single locus variants (SLV) of human STs, in human ST/serotype combinations. All guinea pig isolates showed the same completely new combination of known alleles. The horse isolates showed an unknown allele combination and three new alleles. Conclusions/Significance The isolates found in mastomys, mice, rats, cats, dogs, gorilla and dolphins are most likely identical to human pneumococcal isolates. Isolates from guinea pigs and horses appear to be specialized clones for these animals. Our data redraw attention to the fact that pneumococci are not strictly human pathogens. Pet animals that live in close contact to humans, especially children, can be infected by human isolates and also carriage of even resistant isolates is a realistic possibility.

FELASA guidelines for the accreditation of health monitoring programs and testing laboratories involved in health monitoring

Monitoring the health of research animals is an essential part of the research process and helps to ensure that experiments yield reliable and reproducible results. The Federation of European Laboratory Animal Science Associations (FELASA) is one organization that accredits and evaluates health monitoring programs and laboratories involved with health monitoring. In this article, the authors (who are members of the FELASA working group Accreditation Board for Health Monitoring) describe the guidelines of the FELASA health monitoring accreditation process. The ultimate goal of this accreditation program is to make health monitoring reports more thorough and reliable, thereby increasing the standardization of health monitoring of laboratory animals.

Induction of Interleukin-6 in Murine Bone Marrow-Derived Macrophages Stimulated by the Mycoplasma Arthritidis Mitogen Mas

Cell-free supernatant of cultures from Mycoplasma arthritidis (MAS) functions as an extremely potent T-cell mitogen for human and murine lymphocytes. The T-cell response is dependent on the presence of accessory cells, presenting the intact E2 molecule on the cell surface. Until now, pure MAS protein has not been available. We developed a new multi-step method for MAS purification. The main steps in this protocol are ammonium sulfate precipitation, anion exchange and hydroxyapatite chromatography followed by gel filtration. With this efficient protocol we obtained fractions of extremely potent mitogenic properties, the purification rate was about 5 x 10(5). Although this protease-sensitive mitogenic activity was highly enriched, we failed to detect the protein by sensitive staining methods of SDS-PAGE. In previous studies, we showed that MAS induces the synthesis of interferon gamma in human and murine lymphocyte cultures. Here we demonstrate that MAS induces interleukin-6 (IL-6) in murine bone-marrow derived macrophage cultures. Since IL-6 is also induced by endotoxin, we used C3H/HeJ mice, which are known to be LPS-nonresponders, in all our studies.

Streptobacillus felis sp. nov., isolated from a cat with pneumonia, and emended descriptions of the genus Streptobacillus and of Streptobacillus moniliformis

A pleomorphic, Gram-stain-negative, rod-shaped, indole-, oxidase- and catalase-negative, non-spore-forming, non-motile bacterium (strain 131000547(T)) was isolated from the lungs of a cat with pneumonia. On the basis of 16S rRNA gene sequence analyses the strain was assigned to the genus Streptobacillus with 97.6% sequence similarity to the type strain of Streptobacillus moniliformis and 94.6% to that of Streptobacillus hongkongensis. The clear differentiation of strain 131000547(T) from Streptobacillus moniliformis and Streptobacillus hongkongensis was also supported by gyrB, groEL, and recA nucleotide and amino acid sequence analysis. DNA-DNA hybridization demonstrated ≤ 19.9% (reciprocal 28.7%) DNA-DNA relatedness between strain 131000547(T) and Streptobacillus moniliformis DSM 12112(T). Physiological data confirmed the allocation of strain 131000547(T) to the family Leptotrichiaceae. Strain 131000547(T) has a unique profile of enzyme activities allowing differentiation from the most closely related species. Within the genus Streptobacillus, isolate 131000547(T) could also unambiguously be separated from Streptobacillus moniliformis and Streptobacillus hongkongensis by both matrix-assisted laser desorption ionization time-of-flight mass spectrometry and Fourier transform-infrared spectroscopy. On the basis of these data, a novel species of the genus Streptobacillus, Streptobacillus felis sp. nov., is proposed with the type strain 131000547(T) ( = DSM 29248(T) = CCUG 66203(T) = CCM 8542(T)). Emended descriptions of the genus Streptobacillus and of Streptobacillus moniliformis are also given.

Phenotypic and Genotypic Characteristics of Members of the Genus Streptobacillus

The genus Streptobacillus (S.) remained monotypic for almost 90 years until two new species were recently described. The type species, S. moniliformis, is one of the two etiological agents of rat bite fever, an under-diagnosed, worldwide occurring zoonosis. In a polyphasic approach field isolates and reference strains of S. moniliformis, S. hongkongensis, S. felis as well as divergent isolates were characterized by comparison of molecular data (n = 29) and from the majority also by their physiological as well as proteomic properties (n = 22). Based on growth-independent physiological profiling using VITEK2-compact, API ZYM and the Micronaut system fastidious growth-related difficulties could be overcome and streptobacilli could definitively be typed despite generally few differences. While differing in their isolation sites and dates, S. moniliformis isolates were found to possess almost identical spectra in matrix-assisted laser desorption ionization—time of flight mass spectrometry and Fourier transform infrared spectroscopy. Spectroscopic methods facilitated differentiation of S. moniliformis, S. hongkongensis and S. felis as well as one divergent isolate. Sequencing of 16S rRNA gene as well as functional genes groEL, recA and gyrB revealed only little intraspecific variability, but generally proved suitable for interspecies discrimination between all three taxa and two groups of divergent isolates.

Streptobacillus ratti sp. nov., isolated from a black rat (Rattus rattus)

An indole-, oxidase- and catalase-negative, non-motile bacterium, strain OGS16T, was isolated from an oral swab of a feral black rat (Rattus rattus) in 2007 in Japan. It stained Gram-negative and had pleomorphic, rod-shaped, non-spore-forming cells. Based on 16S rRNA gene sequence analyses, strain OGS16T was assigned to the genus Streptobacillus, with 16S rRNA gene sequence similarities of 99.3, 99.0, 98.6 and 95.5% to the type strains of Streptobacillus moniliformis, Streptobacillus notomytis, Streptobacillus felis and Streptobacillus hongkongensis, respectively. Strain OGS16T could also be differentiated clearly from other species of the genus Streptobacillus by rpoB, groEL and recA nucleotide and deduced amino acid sequence analysis. DNA-DNA relatedness as obtained by average nucleotide identity was 89.10% between strain OGS16T and Streptobacillus moniliformis DSM 12112T. Chemotaxonomic and physiological data for strain OGS16T were congruent with results for other closely related members of the family Leptotrichiaceae, represented by highly similar enzyme profiles and fatty acid patterns. MALDI-TOF MS analysis also proved suitable in discriminating strain OGS16T unequivocally from all currently described taxa of the genus Streptobacillus. On the basis of these data, we propose the novel species Streptobacillus ratti sp. nov., with the type strain OGS16T (=JCM 31098T=DSM 101843T). The G+C content of the DNA of the type strain is 25.9 mol% and the genome size is 1.50 Mbp.

Use of ciprofloxacin and bm-cyclin in mycoplasma decontamination

Dear Editor: The contamination of cultured cell lines and hybridomas with mycoplasmas remains a formidable problem in medical and biological research. The insidious infection has been shown to mediate a wide variety of adverse effects on the growth and functional activity of cultured cell lines and hybridomas regardless of their origin (Shoenfeld and Schwartz, 1985). Thus, it is very important to undertake the necessary precautionary measures to prevent any cell line from being contaminated with mycoplasmas. It is usually recommended that all contaminated cultures in a laboratory are immediately autoclaved to prevent the infection from spreading and to use only mycoplasma-free cultures. However, as some cell lines and hybridomas are irreplaceable, an effective eradication program is necessary. Methods of elimination have included passage in athymic mice (van Diggelen et al., t977), growth of cells in rabbit or guinea pig serum (Nair, 1985), use of nucleic acid analogues (Marcus et al., 1980), antibiotic treatment (Schmidt and Erfle, 1984), supplementation of culture media with specific antisera against mycoplasmas (Jeansson and Brorson, 1985) and many other techniques. However, none of these techniques produced satisfactory and/or consistent results. Recently, the use of the fluoroquinolone antibiotic ciprofloxacin (CF) has been reported to be successfully used in decontaminating mycoplasma-infected cell cultures (Mowles, 1988; Schmitt et at., 1988; Gignac et at., 1991). Ciprofloxacin acts by inhibiting the enzyme DNA gyrase (topoisomerase II) which is essential for the supercoiling of bacterial DNA (Dabbs et at., 1987). As mycoplasma screening and subsequent decontamination is becoming a routine procedure in cell culture laboratories and many new products are marketed specially for these purposes, selecting the simplest and the most effective mycoplasma decontamination product may pose as a problem, especially to the novice in this field. In this study, we compared the established efficacy of ciprofloxacin with the commercially marketed mycoplasma elimination product BM-Cyclin (BC) in decontaminating 3 cultured cell lines and 6 hybridomas which were chronically infected with Mycoplasma hyorhinis and Acholeplasma laidlawii. The effectiveness of these 2 treatment programs was monitored using different mycoplasma detection methods viz., DNA stain (Hoechst 33258), microbiological culture and mycoplasma species-specific ELISA. The results of this comparative study are summarized in Table 1. Mycoplasma contaminated cell cultures were effectively decontaminated within 2 weeks of ciprofloxacin treatment with no apparent cytotoxicity or re-emergence. The treatment was continued for an additional week to ensure that all contaminating mycoplasma had been eradicated and to prevent selection of ciprofloxacin resistant strains. Both the microbiological culture and ELISA techniques further confirmed the successful decontamination of the cells with ciprofloxacin. In contrast, BM-Cyclin which is commercially marketed specially for mycoplasma decontamination was found to be very cytotoxic to all our cell lines and there was also a case of treatment failure. These findings were in contradiction to the reported efficiency of BM-Cyclin in mycoplasma decontamination (Branch and Guilbert, 1986; Ravaoarinoro and Lecomte, 1988). The cytotoxic effect was already observed within 1 week of treatment. As the manufacturer recommends at least 3 weeks of treatment for effective decontamination, the treatment was continued for another 2 weeks. By the third week, even though all cell lines appeared decontaminated, the cytotoxicity of the BM-Cyclin drug combinations resulted in extensive cell mortality and poor growth. Thus, although the treatment was discontinued after the third week, there were no living ceils by Day 42. Since all our cell lines appeared very sensitive to BM-Cyclin, lower concentrations of the drug combinations in BM-Cyclin have to be tried on a trial-and-error basis as recommended by the manufacturer. As we observed that ciprofloxacin treatment was very effective and very much simpler than the sequential treatment of BM Cyclin and a recent study also reported that BM-Cyclin was effective for only some cells and not for all cell lines (Kotani et at., 1991), we did not further attempt to decontaminate our cell cultures with lower concentrations of BM Cyclin. However, it must be pointed out that all the 9 cell lines examined in this study may be unusually very sensitive to BM-Cyclin and that other mycoplasmainfected cell cultures may respond differently with respect to the relative effectiveness of mycoplasma decontamination compared to the cytotoxicity of ciprofloxacin vs. BM-Cyclin. The 3 different mycoplasma detection methods viz., DNA stain, microbiological culture and ELISA revealed similar results except in one case. The treatment failure of BJAB ceils with BM-Cyclin was noticed when A. laidlawii was cultured from these cells even after 3 weeks of treatment. Although both the DNA stain technique and ELISA indicated successful decontamination, the microbiological culture technique which is 100× more sensitive than the DNA stain technique (Stanbridge, 1981) and ELISA (detection limit for A. laidlawii is 104-105 CFU/ml), indicated a persistent mycoplasmal infection in BM-Cyclin treated B JAB cells. In conclusion, ciprofloxacin proved to be a better choice of antibiotic in the mycoplasma eradication program of our contaminated cell lines because of its simplicity in use, effectiveness in the elimination of mycoplasmas, lack of re-emergence of infection and its lack of cytotoxicity. In contrast, we found BM-Cyclin to be very cytotoxic to all the 9 cell lines examined in this study. Furthermore, we found the sequential and cyclic use of 2 different antibiotic combinations in BM-Cyclin and the necessity to empirically determine the optimal concentrations of these 2 antibiotics for treating BM-Cyclin sensitive cell lines both time-consuming and cumbersome.

Phylogenetic and comparative genomics of the family Leptotrichiaceae and introduction of a novel fingerprinting MLVA for Streptobacillus moniliformis

BackgroundThe Leptotrichiaceae are a family of fairly unnoticed bacteria containing both microbiota on mucous membranes as well as significant pathogens such as Streptobacillus moniliformis, the causative organism of streptobacillary rat bite fever. Comprehensive genomic studies in members of this family have so far not been carried out. We aimed to analyze 47 genomes from 20 different member species to illuminate phylogenetic aspects, as well as genomic and discriminatory properties.ResultsOur data provide a novel and reliable basis of support for previously established phylogeny from this group and give a deeper insight into characteristics of genome structure and gene functions. Full genome analyses revealed that most S. moniliformis strains under study form a heterogeneous population without any significant clustering. Analysis of infra-species variability for this highly pathogenic rat bite fever organism led to the detection of three specific variable number tandem analysis loci with high discriminatory power.ConclusionsThis highly useful and economical tool can be directly employed in clinical samples without laborious prior cultivation. Our and prospective case-specific data can now easily be compared by using a newly established MLVA database in order to gain a better insight into the epidemiology of this presumably under-reported zoonosis.