Gero Hilken

Role of intestinal microbiota in transformation of bismuth and other metals and metalloids into volatile methyl and hydride derivatives in humans and mice.

ABSTRACT The present study shows that feces samples of 14 human volunteers and isolated gut segments of mice (small intestine, cecum, and large intestine) are able to transform metals and metalloids into volatile derivatives ex situ during anaerobic incubation at 37°C and neutral pH. Human feces and the gut of mice exhibit highly productive mechanisms for the formation of the toxic volatile derivative trimethylbismuth [(CH3)3Bi] at rather low concentrations of bismuth (0.2 to 1 μmol kg−1 [dry weight]). An increase of bismuth up to 2 to 14 mmol kg−1 (dry weight) upon a single (human volunteers) or continuous (mouse study) administration of colloidal bismuth subcitrate resulted in an average increase of the derivatization rate from approximately 4 pmol h−1 kg−1 (dry weight) to 2,100 pmol h−1 kg−1 (dry weight) in human feces samples and from approximately 5 pmol h−1 kg−1 (dry weight) to 120 pmol h−1 kg−1 (dry weight) in mouse gut samples, respectively. The upshift of the bismuth content also led to an increase of derivatives of other elements (such as arsenic, antimony, and lead in human feces or tellurium and lead in the murine large intestine). The assumption that the gut microbiota plays a dominant role for these transformation processes, as indicated by the production of volatile derivatives of various elements in feces samples, is supported by the observation that the gut segments of germfree mice are unable to transform administered bismuth to (CH3)3Bi.

Growth of Xenopus laevis under different laboratory rearing conditions.

Since the European frogs (Rana spp.) have fallen under the German endangered species regulation, Xenopus laevis (South African Clawed Frog) is being used increasingly in animal research and education. Optimal growth rates and homogeneity of groups have not necessarily been attained as little statistical analysis of growth data has been available. Following metamorphosis, an as yet not understood variability of growth is exhibited by X. laevis. In this study the effect of environmental factors on this variability was determined. Feeding, population density, background colouring, water temperature, the availability of hiding places, water level and water care were each examined separately. Development of body weight and body length were recorded. A definite correlation between the feeding programme, population density, cover and water care on the one hand and growth on the other were seen. Of lesser importance were water temperature, water level and background colouring. The observed variability of growth is assumed to also be of ethological origin.

Woodchuck Gamma Interferon Upregulates Major Histocompatibility Complex Class I Transcription but Is Unable To Deplete Woodchuck Hepatitis Virus Replication Intermediates and RNAs in Persistently Infected Woodchuck Primary Hepatocytes

ABSTRACT Gamma interferon (IFN-γ) is an important mediator with multiple functions in the host defense against viral infection. IFN-γ, in concert with tumor necrosis factor alpha (TNF-α), leads to a remarkable reduction of intrahepatic replication intermediates and specific mRNAs of hepatitis B virus (HBV) by a noncytolytic mechanism in the transgenic mouse model. Thus, it is rational to evaluate the potential value of IFN-γ for the treatment of chronic HBV infection. In the present study, we expressed recombinant woodchuck IFN-γ (wIFN-γ) in Escherichia coli and mammalian cells. wIFN-γ protected woodchuck cells against infection of murine encephalomyocarditis virus in a species-specific manner. It upregulated the mRNA level of the woodchuck major histocompatibility complex class I (MHC-I) heavy chain in permanent woodchuck WH12/6 cells and regulated differentially the gene expression. However, the level of the replication intermediates and specific RNAs of woodchuck hepatitis virus (WHV) in persistently WHV-infected primary woodchuck hepatocytes did not change despite a treatment with 1,000 U of wIFN-γ per ml or with a combination of wIFN-γ and woodchuck TNF-α. Rather, hepatocytes derived from chronic carriers had an elevated level of the MHC-I heavy-chain mRNAs, most probably due to the exposure to inflammatory cytokines in vivo. Treatment with high doses of wIFN-γ led to an abnormal cell morphology and loss of hepatocytes. Thus, wIFN-γ regulates the gene expression in woodchuck hepatocytes but could not deplete WHV replication intermediates and mRNAs in persistently infected hepatocytes. The cellular response to wIFN-γ may be changed in hepatocytes from chronically WHV-infected woodchucks. It should be clarified in the future whether the continuous exposure of hepatocytes to inflammatory cytokines or the presence of viral proteins leads to changes of the cellular response to wIFN-γ.

Immunization with the Gene Expressing Woodchuck Hepatitis Virus Nucleocapsid Protein Fused to Cytotoxic-T-Lymphocyte-Associated Antigen 4 Leads to Enhanced Specific Immune Responses in Mice and Woodchucks

ABSTRACT A number of options are available to modify and improve DNA vaccines. An interesting approach to improve DNA vaccines is to fuse bioactive domains, like cytotoxic-T-lymphocyte-associated protein 4 (CTLA-4), to an antigen. Such fusion antigens are expressed in vivo and directed to immune cells by the specific bioactive domain and therefore possess great potential to induce and modulate antigen-specific immune responses. In the present study, we tested this new approach for immunomodulation against hepadnavirus infection in the woodchuck model. Plasmids expressing the nucleocapsid protein (WHcAg) and e antigen (WHeAg) of woodchuck hepatitis virus (WHV) alone or in fusion to the extracellular domain of woodchuck CTLA-4 and CD28 were constructed. Immunizations of mice with plasmids expressing WHcAg or WHeAg led to a specific immunoglobulin G2a (IgG2a)-dominant antibody response. In contrast, fusions of WHcAg to CTLA-4 and CD28 induced a specific antibody response with comparable levels of IgG1 and IgG2a. Furthermore, the specific IgG1 response to WHcAg/WHeAg developed immediately after a single immunization with the CTLA-4-WHcAg fusion. Woodchucks were immunized with plasmids expressing WHeAg or the CTLA-4-WHcAg fusion and subsequently challenged with WHV. CTLA-4-WHcAg showed an improved efficacy in induction of protective immune responses to WHV. In particular, the anti-WHsAg antibody response developed earlier after challenge in woodchucks that received immunizations with CTLA-4-WHcAg, consistent with the hypothesis that anti-WHs response is dependent on a Th cell response to WHcAg. In conclusion, the use of fusion genes represents a generally applicable strategy to improve DNA vaccination.

Polyethylene Particle–Induced Bone Resorption in α-Calcitonin Gene–Related Peptide–Deficient Mice†

This study investigates the impact of α‐CGRP on bone metabolism after implantation of polyethylene particles. α‐CGRP knockout mice showed less osteolysis compared with wildtype mice. The local neurogenic microenvironment might be a crucial factor in particle‐induced osteolysis.

RANKL-associated suppression of particle-induced osteolysis in an aged model of Calcitonin and α-CGRP deficiency.

An aging population with higher bone turnover intensifies the need for joint replacement surgery. However, particle-induced osteolysis (PIO) remains a major cause of early implant loosening. Differences in bone remodeling between young and aged Calcitonin (CT)- and α-CGRP (Calcitonin gene-related peptide)-deficient mice (Calca(-/-)) might modify our previous findings regarding CT/α-CGRP in PIO. This may have important implications for PIO in an aging population. Four groups of twelve-month-old wild-type and Calca(-/-) mice underwent either SHAM surgery with and without CT, or polyethylene-particle implantation with related treatment. Morphometric changes were detected using μ-CT, histomorphometric analysis and by counting TRAP(+) cells (osteoclast-staining). Bone remodeling was assessed using serum and urinary markers. There was no osteolysis in aged particle-treated Calca(-/-) animals and the effect of CT on PIO was reduced compared to wild-type mice. However, there were significantly higher numbers of TRAP(+) cells in Calca(-/-) animals, and bone remodeling markers revealed a significant increase in OPG/OCN and a significant reduction in RANKL compared to aged wild-type mice. CT/α-CGRP modulates bone cell activity in PIO in aged mice in a way that is distinct from young animals. This may have implications for the treatment of PIO in the periprosthetic surface of joint replacements in an aging population.

Calcitonin substitution in calcitonin deficiency reduces particle-induced osteolysis

BackgroundPeriprosthetic osteolysis is a major cause of aseptic loosening in joint arthroplasty. This study investigates the impact of CT (calcitonin) deficiency and CT substitution under in-vivo circumstances on particle-induced osteolysis in Calca -/- mice.MethodsWe used the murine calvarial osteolysis model based on ultra-high molecular weight polyethylene (UHMWPE) particles in 10 C57BL/6J wild-type (WT) mice and twenty Calca -/- mice. The mice were divided into six groups: WT without UHMWPE particles (Group 1), WT with UHMWPE particles (Group 2), Calca -/- mice without UHMWPE particles (Group 3), Calca -/- mice with UHMWPE particles (Group 4), Calca -/- mice without UHMWPE particles and calcitonin substitution (Group 5), and Calca -/- mice with UHMWPE particle implantation and calcitonin substitution (Group 6). Analytes were extracted from serum and urine. Bone resorption was measured by bone histomorphometry. The number of osteoclasts was determined by counting the tartrate-resistant acid phosphatase (TRACP) + cells.ResultsBone resorption was significantly increased in Calca -/- mice compared with their corresponding WT. The eroded surface in Calca -/- mice with particle implantation was reduced by 20.6% after CT substitution. Osteoclast numbers were significantly increased in Calca -/- mice after particle implantation. Serum OPG (osteoprotegerin) increased significantly after CT substitution.ConclusionsAs anticipated, Calca -/- mice show extensive osteolysis compared with wild-type mice, and CT substitution reduces particle-induced osteolysis.

Infectious microorganisms in mice (Mus musculus) purchased from commercial pet shops in Germany

In this study, we investigated the prevalence of infectious microorganisms (viruses, bacteria, fungi and eukaryotic parasites) in mice from different pet shops in Germany; such animals may compromise the hygienic integrity of laboratory animal vivaria if private pet holders act as unintended vectors of infections carried by them. House mice sold as pets or feed specimens were purchased from different pet shops and tested for a comprehensive panel of unwanted microorganisms. We found a number of microorganisms in these pet shop mice, the most prevalent of which were Helicobacter species (92.9%), mouse parvovirus (89.3%), mouse hepatitis virus (82.7%), Pasteurella pneumotropica (71.4%) and Syphacia species (57.1 %). Several microorganisms (e.g. mouse parvovirus, Theilers murine encephalomyelitis virus, pneumonia virus of mice, Encephalitozoon cuniculi, Clostridium piliforme) had considerably higher prevalences than those reported in similar studies on wild mice from North America, Europe or Australia. Our study shows that direct contact with pet shop mice may constitute a risk for laboratory animal vivaria if hygienic precautions are not taken. However, even relatively simple precautions seem effective enough to hold the risk at bay.

Replication of Naturally Occurring Woodchuck Hepatitis Virus Deletion Mutants in Primary Hepatocyte Cultures and after Transmission to Naive Woodchucks

ABSTRACT Woodchuck hepatitis virus (WHV) mutants with core internal deletions (CID) occur naturally in chronically WHV-infected woodchucks, as do hepatitis B virus mutants in humans. We studied the replication of WHV deletion mutants in primary woodchuck hepatocyte cultures and in vivo after transmission to naive woodchucks. By screening 14 wild-caught, chronically WHV-infected woodchucks, two woodchucks, WH69 and WH70, were found to harbor WHV CID mutants. Consistent with previous results, WHV CID mutants from both animals had deletions of variable lengths (90 to 135 bp) within the middle of the WHV core gene. In woodchuck WH69, WHV CID mutants represented a predominant fraction of the viral population in sera, normal liver tissues, and to a lesser extent, in liver tumor tissues. In primary hepatocytes of WH69, the replication of wild-type WHV and CID mutants was maintained at least for 7 days. Although WHV CID mutants were predominant in fractions of cellular WHV replicative intermediates, mutant covalently closed circular DNAs (cccDNAs) appeared to be a small part of cccDNA-enriched fractions. Analysis of cccDNA-enriched fractions from liver tissues of other woodchucks confirmed that mutant cccDNA represents only a small fraction of the total cccDNA pool. Four naive woodchucks were inoculated with sera from woodchuck WH69 or WH70 containing WHV CID mutants. All four woodchucks developed viremia after 3 to 4 weeks postinoculation (p.i.). They developed anti-WHV core antigen (WHcAg) antibody, lymphoproliferative response to WHcAg, and anti-WHV surface antigen. Only wild-type WHV, but no CID mutant, was found in sera from these woodchucks. The WHV CID mutant was also not identified in liver tissue from one woodchuck sacrificed in week 7 p.i. Three remaining woodchucks cleared WHV. Thus, the presence of WHV CID mutants in the inocula did not significantly change the course of acute self-limiting WHV infection. Our results indicate that the replication of WHV CID mutants might require some specific selective conditions. Further investigations on WHV CID mutants will allow us to have more insight into hepadnavirus replication.

Biodistribution of 10B for Boron Neutron Capture Therapy (BNCT) in a Mouse Model after Injection of Sodium Mercaptoundecahydro-closo-dodecaborate and l-para-Boronophenylalanine

Abstract In boron neutron capture therapy, the absorbed dose from the 10B(n,α)7Li reaction depends on the 10B concentration and 10B distribution in the irradiated volume. Thus compounds used in BNCT should have tumor-specific uptake and low accumulation in normal tissues. This study compares in a mouse model the 10B uptake in different organs as delivered by l-para-boronophenylalanine (BPA, 700 mg/kg body weight, i.p.) and/or sodium mercaptoundecahydro-closo-dodecaborate (BSH, 200 mg/kg body weight, i.p). After BSH injection, the 10B concentration was high in kidneys (20 ± 12 µg/g) and liver (20 ± 12 µg/g) but was low in brain (1.0 ± 0.8 µg/g) and muscle (1.9 ± 1.2 µg/g). After BPA injection, the 10B concentration was high in kidneys (38 ± 25 µg/g) and spleen (17 ± 8 µg/g) but low in brain (5 ± 3 µg/g). After combined BPA and BSH injection, the effect on the absolute 10B concentration was additive in all organs. The ratio of the 10B concentrations in tissues and blood differed significantly for the two compounds depending on the compound combination, which implies a different uptake profile for normal organs.