Defining quality assurance guidance for effective selection of technical grade zero-valent iron production batch for groundwater remediation using permeable reactive barrier.

Zero-valent iron (ZVI) applied to the remediation of contaminated groundwater (GW) in situ, especially using engineered permeable reactive barriers (PRBs), has been proven to be an effective reactive material. However, many of ZVI brands do not represent tailored reagents specifically regarding destroying pollutants in GW. Thus, their reactivity towards certain contaminants in GW may vary significantly in a wide range even with different production batches of the same ZVI brand. This issue has rarely been known and consequently not addressed to a higher extend so far. Therefore, this study implemented extensive, long-term column experiments followed by short-term batch experiments for chlorinated volatile organic compounds (cVOCs) degradation for developing a semi-empirical test methodology to thoroughly resolve this pivotal issue by achieving an improved quality assurance guidance regarding proper field-scale emplacement of different ZVI brands and their production batches. The results showed that during column experiments perchloroethylene (PCE) led to a significant degradation up to a certain period but sulfate-reducing microorganisms enhanced the dehalogenation and led approximately to 100 % PCE removal. However, the efficacy varied for different ZVI brands, i.e., Gotthart Maier (GM) and Sponge Iron (Responge®). Furthermore, it could be shown that it might even vary among different production batches of the same ZVI brand. It was also observed that evolution of sulfate-reducing microorganisms may improve the efficacy of PCE degradation vastly that occur at different intensities with different ZVI brands and their respective production batches over time. Further, comparing comprehensive long-term column (kobs = 0.0488 1/h) and short-term batch experiments (kobs = 0.07794 1/h) as well as refined kinetic analyses (kobs = 0.0424 1/h) clearly prove that an appropriate guidance protocol for successful full-scale in situ remediation is required for properly select the right ZVI brand and production batch before it is loaded to a PRB in the field.

Knockout mouse models as a resource for the study of rare diseases.

Rare diseases (RDs) are a challenge for medicine due to their heterogeneous clinical manifestations and low prevalence. There is a lack of specific treatments and only a few hundred of the approximately 7,000 RDs have an approved regime. Rapid technological development in genome sequencing enables the mass identification of potential candidates that in their mutated form could trigger diseases but are often not confirmed to be causal. Knockout (KO) mouse models are essential to understand the causality of genes by allowing highly standardized research into the pathogenesis of diseases. The German Mouse Clinic (GMC) is one of the pioneers in mouse research and successfully uses (preclinical) data obtained from single-gene KO mutants for research into monogenic RDs. As part of the International Mouse Phenotyping Consortium (IMPC) and INFRAFRONTIER, the pan-European consortium for modeling human diseases, the GMC expands these preclinical data toward global collaborative approaches with researchers, clinicians, and patient groups.Here, we highlight proprietary genes that when deleted mimic clinical phenotypes associated with known RD targets (Nacc1, Bach2, Klotho alpha). We focus on recognized RD genes with no pre-existing KO mouse models (Kansl1l, Acsf3, Pcdhgb2, Rabgap1, Cox7a2) which highlight novel phenotypes capable of optimizing clinical diagnosis. In addition, we present genes with intriguing phenotypic data (Zdhhc5, Wsb2) that are not presently associated with known human RDs.This report provides comprehensive evidence for genes that when deleted cause differences in the KO mouse across multiple organs, providing a huge translational potential for further understanding monogenic RDs and their clinical spectrum. Genetic KO studies in mice are valuable to further explore the underlying physiological mechanisms and their overall therapeutic potential.

Mouse mutant phenotyping at scale reveals novel genes controlling bone mineral density.

The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease.

Principles and application of LIMS in mouse clinics.

Large-scale systemic mouse phenotyping, as performed by mouse clinics for more than a decade, requires thousands of mice from a multitude of different mutant lines to be bred, individually tracked and subjected to phenotyping procedures according to a standardised schedule. All these efforts are typically organised in overlapping projects, running in parallel. In terms of logistics, data capture, data analysis, result visualisation and reporting, new challenges have emerged from such projects. These challenges could hardly be met with traditional methods such as pen & paper colony management, spreadsheet-based data management and manual data analysis. Hence, different Laboratory Information Management Systems (LIMS) have been developed in mouse clinics to facilitate or even enable mouse and data management in the described order of magnitude. This review shows that general principles of LIMS can be empirically deduced from LIMS used by different mouse clinics, although these have evolved differently. Supported by LIMS descriptions and lessons learned from seven mouse clinics, this review also shows that the unique LIMS environment in a particular facility strongly influences strategic LIMS decisions and LIMS development. As a major conclusion, this review states that there is no universal LIMS for the mouse research domain that fits all requirements. Still, empirically deduced general LIMS principles can serve as a master decision support template, which is provided as a hands-on tool for mouse research facilities looking for a LIMS.

Treatment with granulocyte-macrophage colony-stimulating factor is associated with reduced indoleamine 2,3-dioxygenase activity and kynurenine pathway catabolites in patients with severe sepsis and septic shock.

The immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) controls tryptophan metabolism and is induced by pro-inflammatory stimuli. We investigated whether immunostimulatory treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF) influences IDO activity and tryptophan metabolism in sepsis. Thirty-six patients with severe sepsis/septic shock and sepsis-associated immunosuppression (assessed using monocytic human leukocyte antigen-DR (mHLA-DR) expression) were assessed in a controlled trial of GM-CSF or placebo treatment for 8 days. Using tandem mass spectrometry, levels of tryptophan, kynurenine, kynurenic acid, quinolinic acid, 5-hydroxytryptophan, serotonin, and estimated IDO activity were determined in a blinded fashion over a 9-day interval. At baseline, tryptophan and metabolite levels did not differ between the study groups. Although tryptophan levels were unchanged in both groups over the treatment interval (all p>0.8), IDO activity was markedly reduced after GM-CSF treatment (35.4 +/- 21.0 vs 21.6 +/-9.9 (baseline vs day 9), p = 0.02). IDO activity differed significantly between the 2 groups after therapy (p = 0.03). Metabolites downstream of IDO (kynurenine, quinolinic acid, kynurenic acid) were all induced in sepsis and declined in the GM-CSF group, but not in controls. Serotonin pathway metabolites remained unchanged in both groups (all p>0.15). Moreover, IDO activity correlated with procalcitonin (p< 0.0001, r = 0.56) and mHLA-DR levels (p = 0.005, r = -0.28) in the overall samples group. Thus, GM-CSF therapy is associated with decreased IDO activity and reduced kynurenine pathway catabolites in sepsis. This may be due to an improved antibacterial defence.

Identification of genetic elements in metabolism by high-throughput mouse phenotyping.

Metabolic diseases are a worldwide problem but the underlying genetic factors and their relevance to metabolic disease remain incompletely understood. Genome-wide research is needed to characterize so-far unannotated mammalian metabolic genes. Here, we generate and analyze metabolic phenotypic data of 2016 knockout mouse strains under the aegis of the International Mouse Phenotyping Consortium (IMPC) and find 974 gene knockouts with strong metabolic phenotypes. 429 of those had no previous link to metabolism and 51 genes remain functionally completely unannotated. We compared human orthologues of these uncharacterized genes in five GWAS consortia and indeed 23 candidate genes are associated with metabolic disease. We further identify common regulatory elements in promoters of candidate genes. As each regulatory element is composed of several transcription factor binding sites, our data reveal an extensive metabolic phenotype-associated network of co-regulated genes. Our systematic mouse phenotype analysis thus paves the way for full functional annotation of the genome.

Stress-induced immune conditioning affects the course of experimental peritonitis.

Septic patients show individually different courses of disease that are hard to predict. Little is known about preconditioning influences that may render one person liable to have overwhelming hyperinflammatory response syndrome (systemic inflammatory response syndrome) and another from compensatory anti-inflammatory response syndrome. Here, we show in a murine model that chronic psychological stress before the onset of polymicrobial peritonitis influences the balance between both types of immune response. Chronically stressed mice which had increased lymphocyte apoptosis, severe functional lymphocyte defects, and an anti-inflammatory cytokine bias had a reduced mortality rate during the continuous outflow of gut content in the hyperinflammatory sepsis model of colon ascendens stent peritonitis. In contrast, they had enhanced long-lasting bacterial dissemination in a sepsis model of mild cecal ligation and puncture. Chronic stress therefore is an important preconditioning factor in the individuals' ability to cope with systemic infections after abdominal surgery. It ameliorates lethal shock responses but reduces the capacity to eradicate bacterial infection during mild peritonitis.

Respiratory chlamydial infection based on experimental aerosol challenge of pigs with Chlamydia suis.

An experimental study of aerogeneous challenge in pigs was conducted in order to reveal characteristic features of porcine respiratory chlamydiosis. Eight conventionally raised pigs were exposed to a pathogenic strain of Chlamydia (C.) suis, four controls were mock infected. Besides pathological changes, the acute-phase and humoral immune responses, as well as the dissemination and transmission of the challenge strain was monitored in the course of infection. The data from clinical investigations, LPS-binding protein assay, antibody ELISAs, confocal laser scanning and light microscopy, immunohistochemical staining and PCR provided extensive evidence of the pathogenic potential of C. suis for the porcine respiratory system. This model appears suitable for further pathophysiological and immunological investigations of chlamydial respiratory infections and can also be recommended for studies of Chlamydia-associated infections of the human lung.

Altered hepatic mRNA expression of immune response and apoptosis-associated genes after acute and chronic psychological stress in mice.

Using a combination of transcriptional profiling and Ingenuity Pathway Analysis (IPA, www.ingenuity.com) we investigated acute and chronic psychological stress induced alterations of hepatic gene expression of BALB/c mice. Already after a 2-h single stress session, up-regulation of several LPS and glucocorticoid-sensitive immune response genes and markers related to oxidative stress and apoptotic processes were observed. Support for the existence of oxidative stress was gained by measuring increased protein carbonylation, but no alterations of immune responsiveness or cell death were measured in mice after acute stress compared to the control group. When animals were repeatedly stressed during 4.5-days, we found reduced transcription of antigen presentation molecules, altered mRNA levels of immune cell signaling mediators and persisting high expression of apoptosis-related genes. These alterations were associated with a measurable immune suppression characterized by a reduced ability to clear experimental Salmonella typhimurium infection from the liver and a heightened hepatocyte apoptosis. Moreover, genes associated with anti-oxidative functions and regenerative processes were induced in the hepatic tissue of chronically stressed mice. These findings indicate that modulation of the immune response and of apoptosis-related genes is initiated already during a single acute stress exposure. However, immune suppression will only manifest in repeatedly stressed mice which additionally show induction of protective and liver regenerative genes to prevent further hepatocyte damage.

Chronic alcoholism causes deleterious conditioning of innate immunity.

AIMS: To examine the immune consequences of chronic alcoholism in man, in relation to the known association between alcoholism and raised incidence and severity of infections. METHODS: In 36 alcoholics without liver disease, at the point of commencing withdrawal from alcohol, the following measures of immune competence were measured: the immunophenotypes of cells, acute phase proteins, the endotoxin-neutralizing capacity (ENC) of the serum, titers of anti-lipopolysaccharide (LPS) antibodies, and ex vivo cytokine inducibility in T cells and monocytes (TNFalpha, IL1beta, IL1RA, IL4, IL6, IL8, IL10 and IL12). The results were compared to those from healthy volunteers (day controls). Measures were repeated after 8-13 days of abstinence. RESULTS: LPS-binding protein (LBP) and soluble CD14 (sCD14) were significantly increased in patients' sera at the outset of withdrawal, whereas reduced titers of anti-LPS IgG (P = 0.012) and a reduced ENC (P = 0.001) were measured. Only ENC rapidly returned to normal values after withdrawal therapy. Cytokine induction with phorbol ester showed no significant alterations in patients' T cells. Patients' monocytes, however, responded to LPS stimulation with enhanced IL1beta-, but reduced TNFalpha- and IL12-production (P = 0.004, P = 0.0042 and P = 0.001, respectively). While IL1- and TNFalpha-responses normalized after the withdrawal period, impairment of the IL12 response persisted throughout the observation period of 2 weeks. CONCLUSIONS: Alcoholism results in a prolonged LPS-mediated hypoinflammatory conditioning of the innate but not the adaptive immune system, which is not reversed immediately after withdrawal. This alcohol-induced status of the immune system predisposes to infections and sepsis by blunting initial response to the pathogens.

Concentrations of lipopolysaccharide-binding protein, bactericidal/permeability-increasing protein, soluble CD14 and plasma lipids in relation to endotoxaemia in patients with alcoholic liver disease.

There is increasing evidence that gut leakage in persons with chronic alcohol misuse leads to endotoxaemia, which might contribute to the development of alcoholic hepatitis or cirrhosis. In addition, it was recently shown that the endotoxin-binding capacity of whole blood is reduced in these patients. To analyse this phenomenon, we measured the concentration of functionally important endotoxin-binding plasma components which modify the action of endotoxin. In patients with minimal (n = 10), intermediate (n = 9), and cirrhotic alcoholic liver disease (n = 11), and healthy controls (n = 11), plasma endotoxin was determined in a limulus assay. The concentration of lipoproteins was assessed by measuring apolipoproteins, the other factors were directly measured in immunoassays. In the entire group of alcoholics, endotoxin and the concentration of binding factors that are involved in the action of endotoxin on its target cells (LPS-binding protein and sCD14) were increased. Endotoxin antagonists, such as bactericidal/permeability-increasing protein and high-density lipoprotein, were increased in the pre-cirrhotic stages, whereas a significant reduction of the latter was observed in cirrhosis. Low-density lipoprotein remained unchanged. The elevation of binding factors in the pre-cirrhotic stages of alcoholic liver disease might attenuate the effects of endotoxaemia, whereas in cirrhosis the reduction of high density lipoprotein, to which large quantities of endotoxin bind, may contribute to its pro-inflammatory effects.

Elevated levels of lipopolysaccharide-binding protein and soluble CD14 in plasma in neonatal early-onset sepsis.

No data on lipopolysaccharide-binding protein (LBP) in newborns with sepsis have been available up to now. We therefore determined levels of LBP and soluble CD14 (sCD14) in plasma of healthy and septic neonates in order to evaluate their potential diagnostic role. The study included prospectively collected patient samples of two recently published studies on cytokine expression in neonatal sepsis. Twenty-nine septic patients were enrolled in the present analysis. Samples--either cord blood or peripheral blood--from patients admitted within the first 24 h of life for suspicion of sepsis and cord blood samples of a control group of 40 healthy mature infants delivered spontaneously were analyzed. For seven patients of the septic group, a second sample collected between 24 and 48 h of life was available. Levels of sCD14 and LBP in plasma were determined by an enzyme immunoassay using recombinant CD14 and LBP as standards. LBP and sCD14 were correlated to cytokine plasma levels. In septic neonates, LBP (median, 36.6 versus 7.8 microg/ml; P < 0.001) and sCD14 (median, 0.42 versus 0.28 microg/ml; P < 0.001) levels were highly elevated when compared to those of healthy neonates and strongly correlated to granulocyte colony-stimulating factor (G-CSF), interleukin-1beta (IL-1beta), IL-6, and IL-8 levels. LBP levels in septic neonates analyzed between 24 and 48 h of life even increased when compared to samples obtained at or shortly after delivery (median, 36.6 versus 60 microg/ml; P = 0.038). In summary, levels of LBP in plasma of neonates with early-onset sepsis are significantly elevated; the elevated plasma levels seem to persist for more than 24 h, which could provide the clinician with a prolonged time period to identify the newborn with bacterial sepsis.

Neutrophil influx in response to a peritoneal infection with Salmonella is delayed in lipopolysaccharide-binding protein or CD14-deficient mice.

The induction of an adaptive immune response to a previously unencountered pathogen is a time-consuming process and initially the infection must be held in check by the innate immune system. In the case of an i.p. infection with Salmonella typhimurium, survival requires both CD14 and LPS-binding protein (LBP) which, together with Toll-like receptor 4 and myeloid differentiation protein 2, provide a sensitive means to detect bacterial LPS. In this study, we show that in the first hours after i.p. infection with Salmonella a local inflammatory response is evident and that concomitantly neutrophils flood into the peritoneum. This rapid neutrophil influx is dependent on TNF since it is 1) abolished in TNF KO mice and 2) can be induced by i.p. injection of TNF in uninfected animals. Neutrophil influx is not strictly dependent on the presence of either LBP or CD14. However, in their absence, no local inflammatory response is evident, neutrophil migration is delayed, and the mice succumb to the infection. Using confocal microscopy, we show that the neutrophils which accumulate in CD14 and LBP null mice, albeit with delayed kinetics, are nevertheless fully capable of ingesting the bacteria. We suggest that the short delay in neutrophil influx gives the pathogen a decisive advantage in this infection model.

Cytokine induction by purified lipoteichoic acids from various bacterial species--role of LBP, sCD14, CD14 and failure to induce IL-12 and subsequent IFN-gamma release.

We have recently shown that highly purified lipoteichoic acid (LTA) represents a major immunostimulatory principle of Staphylococcus aureus. In order to test whether this translates to other bacterial species, we extracted and purified LTA from 12 laboratory-grown species. All LTA induced the release of TNF-alpha, IL-1beta, IL-6 and IL-10 in human whole blood. Soluble CD14 (sCD14) inhibited monokine induction by LTA but failed to confer LTA responsiveness for IL-6 and IL-8 release of human umbilical vein endothelial cells (HUVEC). In a competitive LPS-binding protein (LBP) binding assay, the IC(50) of the tested LTA preparations was up to 3,230-fold higher than for LPS. LBP enhanced TNF-alpha release of human peripheral blood mononuclear cells (PBMC) upon LPS but not LTA stimulation. These data demonstrate a differential role for the serum proteins LBP and sCD14 in the recognition of LPS and LTA. Different efficacies of various anti-CD14 antibodies against LPS vs. LTA-induced cytokine release suggest that the recognition sites of CD14 for LPS and LTA are distinct with a partial overlap. While the maximal achievable monokine release in response to LTA was comparable to LPS, all LTA induced significantly less IL-12 and IFN-gamma. IL-12 substitution increased LTA-inducible IFN-gamma release up to 180-fold, suggesting a critical role of poor LTA-inducible IL-12 for IFN-gamma formation. Pretreatment with IFN-gamma rendered galactosamine-sensitized mice sensitive to challenge with LTA. In conclusion, LTA compared to LPS, are weak inducers of IL-12 and subsequent IFN-gamma formation which might explain their lower toxicity in vivo.