Analysis of the sex-specific variability of blood parameters in C3H inbred mice by using data from a long-term, high-throughput project.

Mice are important models for biomedical research by providing the possibility of standardizing genetic background and environmental conditions, which both affect phenotypic variability. Use of both sexes in experiments is strongly recommended because of possible differences in the outcome. However, sex-specific phenotypic variability is discussed with regard to putative consequences on the group size which is necessary for achieving valid and reproducible results. Here, we retrospectively analyzed the sex-specific variability of 25 blood parameters of C3H inbred mice in two different mouse facilities withinthe long-term, high-throughput Munich ENU mouse mutagenesis project. Using the 95 % data range, data of4,780-20,706 mice per parameter were analyzed and resulted in ratios of the coefficient of variation (= female CV / (female CV + male CV)) from 0.44 to 0.58 for the 25 parameters, with an overall mean of 0.51 in both facilities. Together with data analyses of three additional, smaller studies with 72-247 animals per parameter examined and various genetic backgrounds (inbred strains, F1 hybrids) included, hints for reproducible sex-specific variability were observed for particular parameters. Thus, the overall analysis comprising all 25 clinical chemical and hematological parameters of the standardized, long-term analysis of a high number of group housed, young adult, twelve-week-old C3H inbred mice showed no evidence for substantial sex-specific variability. The results may provide a basis for the examination of sex-specific variability in particular blood parameters.

Brown Marmorated Stink Bug (Hemiptera: Pentatomidae) Infestations in Tree Borders and Subsequent Patterns of Abundance in Soybean Fields.

The invasive brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), is an important pest of soybean (Glycine max L. Merr.) in the Mid-Atlantic United States. In order to assess the influence of nonmanaged wooded borders on H. halys infestation patterns in soybean, 12 soybean fields in Orange and Madison Counties, VA, were sampled each week from July to October in 2013 or 2014 for H. halys. At each location, five 2-min visual counts of H. halys life stages were made on tree of heaven (Ailanthus altissima Mill.) and other favorable host trees along a wooded border, on the adjacent soybean edge, 15 m into the soybean field, and 30 m into the field. Seasonal data showed a clear trend at all locations of H. halys densities building up on A. altissima-dominated wooded borders in July, then, gradually moving into adjacent soybean field edges later in the summer. Halyomorpha halys did not move far from the invading field edge, with approximately half as many bugs being present at 15 m into the field and very few being detected 30 m into the field. These results have implications for continued monitoring and management using field border sprays, particularly on edges adjacent to woods.

Comparison of Two Sampling Methods for Assessing Halyomorpha halys (Hemiptera: Pentatomidae) Numbers in Soybean Fields.

Sampling soybean fields for the brown marmorated stink bug, Halyomorpha halys Stål (Hemiptera: Pentatomidae), can be challenging. Both adults and nymphs have a "startle response" and drop to the ground with even the slightest disturbance. This behavior could reduce the effectiveness of the traditional sweep net and ground cloth sampling methods. In 2013 and 2014, in Virginia, Delaware, and Maryland, we evaluated a visual plant inspection method that consisted of counting the number of brown marmorated stink bug nymphs and adults seen on soybean plants in a 2-min inspection period while walking carefully between two rows. After a 30-min interval, which allowed the stink bugs to reposition in the canopy, the area was resampled using 15 sweeps with a 38-cm-diameter sweep net. In total, 76 soybean fields and 2,042 paired comparisons were used to determine a strong linear relationship between sampling methods (y = 0.984x + 0.4359, R2 = 0.6934, where y = brown marmorated stink bugs/2-min visual count and x = brown marmorated stink bugs/15 sweeps). An average visual count of 5.4 brown marmorated stink bugs in 2 min was estimated as being equivalent to the current economic threshold of 5 stink bugs per 15 sweeps. Visual inspection appears to be an effective method for assessing brown marmorated stink bug populations in soybeans.

Bioavailability of insulin detemir and human insulin at the level of peripheral interstitial fluid in humans, assessed by open-flow microperfusion.

AIMS: To find an explanation for the lower potency of insulin detemir observed in humans compared with unmodified human insulin by investigating insulin detemir and human insulin concentrations directly at the level of peripheral insulin-sensitive tissues in humans in vivo. METHODS: Euglycaemic-hyperinsulinaemic clamp experiments were performed in healthy volunteers. Human insulin was administered i.v. at 6 pmol/kg/min and insulin detemir at 60 pmol/kg/min, achieving a comparable steady-state pharmacodynamic action. In addition, insulin detemir was doubled to 120 pmol/kg/min. Minimally invasive open-flow microperfusion (OFM) sampling methodology was combined with inulin calibration to quantify human insulin and insulin detemir in the interstitial fluid (ISF) of subcutaneous adipose and skeletal muscle tissue. RESULTS: The human insulin concentration in the ISF was ∼115 pmol/l or ∼30% of the serum concentration, whereas the insulin detemir concentration in the ISF was ∼680 pmol/l or ∼2% of the serum concentration. The molar insulin detemir interstitial concentration was five to six times higher than the human insulin interstitial concentration and metabolic clearance of insulin detemir from serum was substantially reduced compared with human insulin. CONCLUSIONS: OFM proved useful for target tissue measurements of human insulin and the analogue insulin detemir. Our tissue data confirm a highly effective retention of insulin detemir in the vascular compartment. The higher insulin detemir relative to human insulin tissue concentrations at comparable pharmacodynamics, however, indicate that the lower potency of insulin detemir in humans is attributable to a reduced effect in peripheral insulin-sensitive tissues and is consistent with the reduced in vitro receptor affinity.

[Cutaneous sarcomas: update on selected fibrohistiocytic and myofibroblastic tumors]. / Kutane Sarkome: update ausgewählter Entitäten fibrohistiozytärer und myofibrozytärer Morphologie.

BACKGROUND: Malignant fibrohistiocytic tumors are a heterogeneous group of mesenchymal neoplasms that may occur in the skin and subcutaneous tissues. DIAGNOSIS: Diagnosis of these tumors may be difficult, as they are rare, and a wide morphological diversity of types and subtypes has been described. In this update, relevant aspects of selected entities like dermatofibrosarcoma protuberans, desmoid tumor, atypical fibroxanthoma, pleomorphic dermal sarcoma, and myxofibrosarcoma are discussed according to the WHO classification of 2013. The typical clinical feature of these tumors is their mostly asymptomatic appearance. For diagnosis, the histologic workup is therefore the key feature; herein immunohistochemistry as well as molecular diagnostics become increasingly important. THERAPY: The primary treatment for locally resectable tumors is complete surgical removal; chemotherapy, radiation, and targeted therapies with kinase inhibitors are available for inoperable and metastatic disease.

Plantar erythrodysesthesia caused by antiretroviral treatment: a case report and review of the literature.

Palmoplantar erythrodysesthesia is an uncommon localised cutaneous reaction to certain chemotherapeutic agents and characterized by painful palmoplantar erythema and dysesthesia. To the best of our knowledge, we report the first case of plantar erythrodysesthesia in a 40-year-old male patient receiving an antiretroviral combination therapy for HIV.

Clinical applicability of dOFM devices for dermal sampling.

BACKGROUND: Sampling the dermal interstitial fluid (ISF) allows the pharmacokinetics and pharmacodynamics of dermatological drugs to be studied directly at their site of action. Dermal open-flow microperfusion (dOFM) is a recently developed technique that can provide minimally invasive, continuous, membrane-free (thus unfiltered) access to the dermal ISF. Herein, we evaluate the clinical applicability and reliability of novel wearable dOFM devices in a clinical setting. METHODS: Physicians inserted 141 membrane-free dOFM probes into the dermis of 17 healthy and psoriatic volunteers and sampled dermal ISF for 25 h by using wearable push-pull pumps. The tolerability, applicability, reproducibility, and reliability of multiple insertions and 25 h continuous sampling was assessed by pain scoring, physician feedback, ultrasound probe depth measurements, and 25 h-drift and variability of the sodium relative recovery. RESULTS: Insertion pain was moderate and decreased with each additional probe. Probe insertion was precise, although slightly deeper in lesional skin. The wearable push-pull pump enabled uninterrupted ISF sampling over 25 h with low variability. The relative recovery was drift-free and highly reproducible. CONCLUSION: dOFM sampling devices are tolerable and reliable for prolonged continuous dermal sampling in a multiprobe clinical setting. These devices should enable the study of a wide range of drugs and their biomarkers in the skin.

Enhanced oxidative stress and endocrine pancreas alterations are linked to a novel glucokinase missense mutation in ENU-derived Munich Gck(D217V) mutants.

In the large-scale Munich N-ethyl-N-nitrosourea (ENU) mouse mutagenesis project murine models recapitulating human diseases were generated. In one strain, a novel missense mutation (D217V) in the glucokinase (Gck) gene was identified, resulting in decreased glucokinase activity. Heterozygous mutants display mild hyperglycaemia, disturbed glucose tolerance, and decreased glucose-induced insulin secretion. In contrast, homozygous mutants exhibit severe but not survival affecting hyperglycaemia, mild growth retardation, diminished oxidative capacity, and increased abundance of CHOP protein in the islets. Furthermore, the total islet and ß-cell volumes and the total volume of isolated ß-cells are significantly decreased in adult homozygous mutants, whereas in neonatal mice, ß-cell mass is not yet significantly decreased and islet neogenesis is unaltered. Therefore, reduced total islet and ß-cell volumes of adult homozygous mutants might predominantly emerge from disturbed postnatal islet neogenesis. Thus, we identified a novel Gck mutation in mice, with relevance in humans, leading to glycaemic disease.

Multiple basal cell carcinomas after long-term exposure to hydrazine: case report and review of the literature.

Hydrazine (N(2)H(4)) is a clear, inorganic colourless liquid. It is known to be a skin sensitizer, a corrosive agent and it causes dermatitis on contact. Hydrazine is employed in chemical plants, used as a corrosion inhibitor for feed waters and may be added to rocket fuels. The authors report the case of a 68-year-old man with multiple basal cell carcinomas (BCCs) covering his arms and face. The patient worked in a steam power plant with extensive exposure to hydrazine for a period of over 10 years. The present case report strongly suggests that there may be a correlation between the long-term exposure to hydrazine and an increased risk for multiple BCCs.

Phenotypic and pathomorphological characteristics of a novel mutant mouse model for maturity-onset diabetes of the young type 2 (MODY 2).

Several mutant mouse models for human diseases such as diabetes mellitus have been generated in the large-scale Munich ENU (N-ethyl-N-nitrosourea) mouse mutagenesis project. The aim of this study was to identify the causal mutation of one of these strains and to characterize the resulting diabetic phenotype. Mutants exhibit a T to G transversion mutation at nt 629 in the glucokinase (Gck) gene, leading to an amino acid exchange from methionine to arginine at position 210. Adult Munich Gck(M210R) mutant mice demonstrated a significant reduction of hepatic glucokinase enzyme activity but equal glucokinase mRNA and protein abundances. While homozygous mutant mice exhibited growth retardation and died soon after birth in consequence of severe hyperglycemia, heterozygous mutant mice displayed only slightly elevated blood glucose levels, present from birth, with development of disturbed glucose tolerance and glucose-induced insulin secretion. Additionally, insulin sensitivity and fasting serum insulin levels were slightly reduced in male mutant mice from an age of 90 days onward. While beta-cell mass was unaltered in neonate heterozygous and homozygous mutant mice, the total islet and beta-cell volumes and the total volume of isolated beta-cells were significantly decreased in 210-day-old male, but not female heterozygous mutant mice despite undetectable apoptosis. These findings indicate that reduced total islet and beta-cell volumes of male mutants might emerge from disturbed postnatal islet neogenesis. Considering the lack of knowledge about the pathomorphology of maturity-onset diabetes of the young type 2 (MODY 2), this glucokinase mutant model of reduced total islet and total beta-cell volume provides the opportunity to elucidate the impact of a defective glucokinase on development and maintenance of beta-cell mass and its relevance in MODY 2 patients.

Polymorphic microsatellite markers in the outbred CFW and ICR stocks for the generation of speed congenic mice on C57BL/6 background.

Reliable definition of the phenotype of particular alleles is carried out in the genetic background of inbred strains. Appearance of mutations in outbred mice therefore requires the generation of congenic mice. The aim of this study was the establishment of a list of polymorphic microsatellite markers which can be used in a polymerase chain reaction (PCR)-based marker-assisted selection protocol (MASP) to allow the use of the two common outbred stocks, CFW and ICR, as donor animals for the fast generation of congenic C57BL/6 mice. The selection of informative microsatellite markers was carried out to provide a simple evaluation of the PCR products by conventional agarose gel electrophoresis. Outbred mice from three suppliers were examined. In total, 153 microsatellite loci were analysed. Here we present 76 and 70 microsatellite markers polymorphic for the outbred ICR and CFW stocks compared to C57BL/6. At least three microsatellite loci per chromosome were chosen as informative markers for the autosomal genome, giving rise to a maximum marker distance of 58 cM. Thus, additional individual markers have to be selected for the respective outbred mouse which is chosen as a donor animal.

A novel leptin receptor variant with a conservative amino acid substitution (I359 V) in body weight selected and unselected mouse lines.

Recently published results revealed linkage of obesity traits to a chromosomal region near the leptin receptor gene (Lepr) locus in high body weight selected big and fat DU6i mice. The purpose of this study was the search for variants of the Lepr gene in selected and unselected mouse lines as a candidate for different body composition traits. The complete Lepr cDNA sequence was analysed in DU6i mice. In addition, body weight, abdominal fat weight and serum leptin levels were measured in 42 day old, male mice of the strains DU6i, DUKs, Him : OF1 and DBA/2. Sequence comparison to the published wild type sequence revealed three silent mutations and an amino acid exchange (I359 V) in the C2 domain of the putative leptin binding site in the line DU6i. Compared to the high body weight selected mice also unselected lean control DUKs mice and Him : OF1 mice harbour the amino acid substitution, although they show significantly lower values for body weight, abdominal fat weight and serum leptin levels. Therefore, we assume that the mutation in the C2 domain of Lepr alone might not result in impaired leptin binding or signaling.

Porcine 11beta-hydroxysteroid dehydrogenase type 2 isoform: complete coding sequence and polymorphisms.

11Beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) is involved in the regulation of the peripheral glucocorticoid concentrations. Due to the central role of glucocorticoids in protein turnover, 11beta-HSD2 is a candidate gene for optimising production traits in livestock. In addition, mutant 11beta-HSD2 animals may be used as models for human disorders. Here, we present the complete porcine 11beta-HSD2 coding sequence, the RT-PCR strategy for the examination of the coding sequence and the polymorphisms found in the pig.

Partial leptin receptor gene deletion in transgenic mice prevents expression of the membrane-bound isoforms except for Ob-Rc.

Examination of random insertional mutations in transgenic animals harbouring an abnormal phenotype contributes to the discovery of new genes and/or the understanding of already known genes. Here we describe a transgenic mouse line showing early-onset obesity as consequence of the transgene insertion. Molecular genetic analysis revealed a partial deletion of the leptin receptor (Lepr, Ob-R) gene including the coding sequences downstream of exon 17'. This defect prevents the expression of all described membrane-bound isoforms of Ob-R except for isoform Ob-Rc in the homozygous transgenic animals. Thus, this mouse model might be useful for the investigation of the function of the short Ob-R isoforms.

Contrasting obesity phenotypes uncovered by partial leptin receptor gene deletion in transgenic mice.

Non-insulin-dependent diabetes mellitus (type 2 diabetes) is known to be a polygenic and polyfactorial disorder. Here we describe the long-term examination of a transgenic mouse line showing the disruption of the leptin receptor (Lepr, Ob-R) gene caused by transgene insertion. The absence of the expression of the long isoform Ob-Rb uncovered a strong variation of the obesity and diabetes phenotype in the homozygous mutant mice of the outbred strain used. One part of the homozygous mice developed severe persistent early-onset obesity, whereas the other part developed cachexia after having shown initial obesity in the examination period up to 26 weeks p.p. The leptin-receptor-defective mice of this line might serve as a model for the investigation of genes modulating the development and mode of expression of diabetes.

Comparison of MB/BacT and BACTEC 460 TB systems for recovery of mycobacteria in a routine diagnostic laboratory.

MB/BacT, BACTEC 460 TB, and Löwenstein-Jensen (LJ) medium were evaluated in parallel for recovery of mycobacteria from 3,700 continuous clinical specimens in a routine laboratory. Mycobacteria were identified from 123 (3.3%) specimens. The recovery rates for all mycobacteria by the different systems were 91.0, 73.0, and 53.6% for BACTEC 460 TB, MB/BacT, and LJ medium, respectively. The recovery rates for Mycobacterium tuberculosis complex were 97.1, 80. 2, and 67.6%, respectively. The lack of sensitivity of the MB/BacT system was more pronounced with smear-negative specimens and resulted in a failure to detect three patients with infectious tuberculosis.

Chimeric pigs following blastocyst injection of transgenic porcine primordial germ cells.

Porcine primordial germ cell (PGC) derived cell lines of WAPhGH-transgenic pigs have been established that were able to contribute to chimeras. PGCs were isolated from day 25 to 28 genital ridges of more than 30 individual transgenic fetuses in order to have an easy to follow marker gene. To support undifferentiated growth, cell lines were derived and stable maintained on STO no. 8 feeder cells, a murine embryonic fibroblast cell line expressing recombinant, membrane-bound porcine stem cell factor (SCF). Fifteen lines proliferated in an undifferentiated state up to passage 13; two lines were maintained for more than 23 passages. Cell staining experiments for differentiation markers in several cell lines, indicated the presence of pluripotent cells in prolonged cultures. Further characterization using karyotyping revealed a normal, euploid set of chromosomes in cells of passages 15 and higher. Pluripotency of freshly isolated, short-term (up to 24 hr before injection) and long-term cultured, frozen/thawed cells was tested by injection into day 6 recipient blastocysts to give rise to chimeric piglets. The injected embryos (n = 209) were endoscopically transferred into the uterine horns of 11 recipient gilts. Tissue analysis from 49 fetuses and eighteen liveborn piglets for PGC contribution in chimeras was carried out using PCR analysis for the presence of the marker transgene. Thirty-two fetuses showed detectable chimerism in up to five out of 12 tissues analyzed. Skin samples from eight piglets were positive for the transgene, four of them displayed coat colour chimerism.

Stable long-term germ-line transmission of transgene integration sites in mice.

Transgenic mice provide a valuable tool in all fields of basic and applied biological and medical research. In this study, we describe the fate of integrated transgenes in the mammalian host genome over a large number of generations. The stability of the germ-line transmission of integrated tyrosinase transgene copies was monitored up to generation F20 in a large number of individuals from seven transgenic mouse lines. Phenotypic and molecular genetic analysis of the offspring both within the different lines and in cross-breeding experiments revealed the high stability of the transgene integration sites in mice. Only very few individuals were affected by a transgene copy loss. These results indicate that, once homozygous transgenic lines are established, breeding programs can be continued to a high number of generations without further stringent molecular genetic analysis.