Transcriptomes from German shepherd dogs reveal differences in immune activity between atopic dermatitis affected and control skin.

Canine atopic dermatitis (CAD) is an inflammatory and pruritic allergic skin disease with both genetic and environmental risk factors described. We performed mRNA sequencing of non-lesional axillary skin biopsies from nine German shepherd dogs. Obtained RNA sequences were mapped to the dog genome (CanFam3.1) and a high-quality skin transcriptome was generated with 23,510 expressed gene transcripts. Differentially expressed genes (DEGs) were defined by comparing three controls to five treated CAD cases. Using a leave-one-out analysis, we identified seven DEGs: five known to encode proteins with functions related to an activated immune system (CD209, CLEC4G, LOC102156842 (lipopolysaccharide-binding protein-like), LOC480601 (regakine-1-like), LOC479668 (haptoglobin-like)), one (OBP) encoding an odorant-binding protein potentially connected to rhinitis, and the last (LOC607095) encoding a novel long non-coding RNA. Furthermore, high mRNA expression of inflammatory genes was found in axillary skin from an untreated mild CAD case compared with healthy skin. In conclusion, we define genes with different expression patterns in CAD case skin helping us understand post-treatment atopic skin. Further studies in larger sample sets are warranted to confirm and to transfer these results into clinical practice.

Small molecule PGC-1α1 protein stabilizers induce adipocyte Ucp1 expression and uncoupled mitochondrial respiration.

OBJECTIVE: The peroxisome proliferator-activated receptor-γ coactivator-1α1 (PGC-1α1) regulates genes involved in energy metabolism. Increasing adipose tissue energy expenditure through PGC-1α1 activation is potentially beneficial for systemic metabolism. Pharmacological PGC-1α1 activators could be valuable tools in the fight against obesity and metabolic disease. Finding such compounds has been challenging partly because PGC-1α1 is a transcriptional coactivator with no known ligand-binding properties. While, PGC-1α1 activation is regulated by several mechanisms, protein stabilization is a crucial limiting step due to its short half-life under unstimulated conditions. METHODS: We designed a cell-based high-throughput screening system to identify PGC-1α1 protein stabilizers. Positive hits were tested for their ability to induce endogenous PGC-1α1 protein accumulation and activate target gene expression in brown adipocytes. Select compounds were analyzed for their effects on global gene expression and cellular respiration in adipocytes. RESULTS: Among 7,040 compounds screened, we highlight four small molecules with high activity as measured by: PGC-1α1 protein accumulation, target gene expression, and uncoupled mitochondrial respiration in brown adipocytes. CONCLUSIONS: We identify compounds that induce PGC-1α1 protein accumulation and show that this increases uncoupled respiration in brown adipocytes. This screening platform establishes the foundation for a new class of therapeutics with potential use in obesity and associated disorders.

Outbreak of wound botulism in people who inject drugs, Norway, October to November 2013.

In October and November 2013, four cases of wound botulism were confirmed in people who inject drugs (PWID) in Norway. Two additional cases are suspected. Because of the international distribution pathways for heroin ­ the likely source of the outbreak ­ healthcare workers and public health authorities in other countries should remain vigilant for wound botulism in PWID. This outbreak serves as a reminder that countries should ensure access to botulinum antitoxin in case of outbreak situations.

Escherichia coli clonal group A causing bacteraemia of urinary tract origin.

Escherichia coli clonal group A (CgA) causes disease in humans. This is the first study investigating the prevalence of CgA among E. coli from non-urine, extraintestinal infections in a northern European country. E. coli blood (n = 196) and paired urine (n = 195) isolates from the same patients with bacteraemia of urinary tract origin were analysed. The isolates were collected from January 2003 through May 2005 at four hospitals in Copenhagen, Denmark. Pulsed-field gel electrophoresis (PFGE) patterns, antimicrobial resistance and patient characteristics were determined for all CgA isolates; presence of virulence-associated genes (VAGs) and serotypes were determined for the blood CgA isolates. Thirty blood isolates (15%) belonged to CgA. CgA blood isolates were associated with female patients and sulfamethoxazole-trimethoprim resistance and they harboured a distinctive VAG profile. The blood and urine isolates from each pair were found to be related in 26 of 27 CgA blood/urine pairs, confirming a urinary tract origin of infection. Furthermore, a relationship between the PFGE patterns of CgA blood/urine isolates and CgA isolates from UTI patients in general practice and a CgA isolate from a community-dwelling human reported previously, was found, suggesting a community origin of CgA. The finding of CgA strains in 15% of the E. coli bloodstream infections with a urinary tract origin in Denmark suggests that CgA constitutes an important clonal lineage among extraintestinal pathogenic E. coli. A reservoir of this pathogenic E. coli group in the community causing not only UTI but also more severe infections such as bacteraemia has implications for public health.

An abbreviated MLVA identifies Escherichia coli ST131 as the major extended-spectrum ß-lactamase-producing lineage in the Copenhagen area.

Rapid bacterial typing is a valuable and necessary tool in the prevention and detection of outbreaks. The purpose of this study was to adapt a multilocus variable number of tandem repeats analysis (MLVA) for analysis on a benchtop capillary electrophoresis instrument and compare the modified assay with multilocus sequence typing (MLST) for typing cefpodoxime-resistant Escherichia coli (E. coli). Further, we identified the causative resistance mechanisms and epidemiological type of infection for isolates producing extended-spectrum ß-lactamases (ESBLs). A collection of E. coli resistant to cefpodoxime was typed by MLST and a modified MLVA assay using a benchtop capillary electrophoresis instrument. Resistance mechanisms were identified by polymerase chain reaction (PCR) and sequencing. Patient history was examined to establish the epidemiological type of infection for ESBL-producing E. coli. MLVA yielded typing results homologous with MLST and it correctly identified E. coli sequence type (ST) 131 that was accounting for 45 % of all ESBL-producing isolates in the sample collection. The majority (76.7 %) of ESBL-producing isolates was healthcare-related and only 23.3 % of the ESBL-producing isolates were community-onset infections (COI), regardless of the ST. Patients with COI were significantly more often of female gender and younger age compared to healthcare-associated infections (HCAI) and hospital-onset infections (HOI). In conclusion, the modified MLVA is a useful tool for the rapid typing of E. coli and it identified ST131 as the predominating ESBL-producing lineage in Copenhagen. Healthcare-related infections were the predominant infection setting of ESBL-producing E. coli and the demographic characteristics differed between patients with COI and healthcare-related infections.

CBG Santiago: a novel CBG mutation.

CONTEXT: Corticosteroid-binding globulin (CBG; SERPIN A6) gene mutations are rare; only four mutations have been described, often in association with fatigue and chronic pain, albeit with incomplete penetrance. PATIENT: We report a kindred with a novel SERPINA6 mutation. The proband, a 9-yr-old male, had excessive postexertional fatigue, weakness, and migraine. MAIN OUTCOME MEASURES AND RESULTS: Investigations revealed low morning and ACTH-stimulated peak cortisol levels. SERPIN A6 sequencing detected a novel exon 2 single base deletion (c.13delC) leading to a frameshift generating a stop codon within the signal peptide coding region (p.Leu5CysfsX26) and 50% reduced CBG levels in heterozygotes. The patient's father and two sisters share the mutation. Symptom expression within the family may have been modified by a polymorphic CBG allele (c.735G>T). Exogenous hydrocortisone had no effect on the fatigue. CONCLUSION: This report documents the fifth CBG gene mutation in humans and the second causing major effects on CBG levels. Individuals with low CBG levels may be misdiagnosed as having secondary hypocortisolism. The association with fatigue and idiopathic pain is again noted and may relate to altered stress system function. Variability of the phenotype may relate to other genetic variations of the CBG gene or environmental factors.

Environmental meticillin-resistant Staphylococcus aureus (MRSA) disinfection using dry-mist-generated hydrogen peroxide.

Meticillin-resistant Staphylococcus aureus (MRSA) is a major problem in hospitals worldwide. Hand hygiene is recognised as crucial in limiting the spread of MRSA but less is known about the role of MRSA reservoirs in the inanimate hospital environment. We evaluated the effect of hydrogen peroxide vapour diffused by Sterinis((R)) against MRSA in two experimental hospital settings and in two field trials. Dipslides were used for MRSA detection and quantification before and after using the Sterinis disinfection process. In the first experimental hospital setting, four epidemic MRSA strains were placed at five locations and left for one week. All strains survived the week but not the disinfection process. In field trial one 14 upholstered chairs from a department with many MRSA positive patients were left for one month in a closed room prior to disinfection. MRSA was found on the upholstery of four of the 14 chairs. Three chairs became MRSA negative immediately after the disinfection, the fourth 24h later. The second field trial was in the private home of a MRSA positive family of four individuals. One location was found MRSA positive, remaining so after the Sterinis cycles. We found Sterinis to be effective against MRSA in the experimental hospital setting and upholstered chairs, but not in the private home of heavily colonised MRSA patients.

European surveillance study on antimicrobial susceptibility of Gram-positive anaerobic cocci.

Gram-positive anaerobic cocci (GPAC) are a heterogeneous group of microorganisms frequently isolated from local and systemic infections. In this study, the antimicrobial susceptibilities of clinical strains isolated in 10 European countries were investigated. After identification of 299 GPAC to species level, the minimum inhibitory concentrations of penicillin, imipenem, clindamycin, metronidazole, vancomycin and linezolid were determined by the agar dilution method according to the Clinical and Laboratory Standards Institute. The majority of isolates were identified as Finegoldia magna and Parvimonas micra (formerly Peptostreptococcus micros), isolated from skin and soft tissue infections. All isolates were susceptible to imipenem, metronidazole, vancomycin and linezolid. Twenty-one isolates (7%) were resistant to penicillin (n=13) and/or to clindamycin (n=12). Four isolates were resistant to both agents. The majority of resistant isolates were identified as F. magna and originated from blood, abscesses and soft tissue infections.

Diagnosis of pulmonary infection with Toxoplasma gondii in immunocompromised HIV-positive patients by real-time PCR.

The aim of the study presented here was to evaluate the use of PCR for improving the diagnosis of Toxoplasma gondii infection in immunocompromised hosts. Three hundred thirty-two bronchoalveolar lavage (BAL) fluid samples were analyzed by real-time PCR targeting a 529 bp element of T. gondii. In positive samples, the genotype of the parasite was determined by sequence analysis of the GRA6 gene. Positive results were achieved for 2% (7/332) of the samples tested. Genotyping was possible in two samples and revealed GRA6 type II T. gondii. PCR for detecting T. gondii in BAL samples should be performed in all immunosuppressed HIV-positive patients with symptoms of a systemic infection of unknown etiology. Trimethoprim-sulfamethoxazole prophylaxis does not exclude concomitant infection with T. gondii.

The rationale behind a vaccine based on multiple HIV antigens.

The viral diversity of HIV-1 is likely to require a vaccine strategy that induces broad cellular and humoral anti-HIV-1 immunity. Our strategy is based on multiple HIV-1 DNA immunogens together with adjuvant recombinant granulocyte-macrophage stimulating factor. This article describes pre-clinical and clinical work preceding the initiation of clinical HIV-1 phase I/II trials.

Stable suppression of MDR1 gene expression and function by RNAi in Caco-2 cells.

Vector-based RNAi was used to establish a stable Caco-2 cell line with a persistent knockdown of multidrug resistant gene 1 (MDR1) and P-glycoprotein (P-gp). Several positive clones were collected, many of which showed significantly reduced levels of MDR1 mRNA and P-gp compared to wt Caco-2 cells. Selected clones were sub-cultivated for six passages and real-time PCR showed that MDR1 expression remained significantly reduced (up to 96%) over this period of time. RNAi-MDR1 clones frozen long term also kept their low MDR1 expression levels when re-cultured. Permeability studies were performed across RNAi-MDR1 clone cell monolayers, and the efflux of cyclosporine A, digoxin, vinblastine, and vincristine showed 58%, 61%, 91%, and 78% decrease in active transport, respectively, compared to wt Caco-2 cells. This stably modified Caco-2 cell line provides a novel tool for studies on MDR1 and other ABC transporter protein gene cellular functions.

Service screening with mammography. Long-term effects on breast cancer mortality in the county of Gävleborg, Sweden.

In the current study the long-term effects of a pilot service screening programme in the Swedish county of Gävleborg were studied. Women aged 40-64 years in 13 sub-areas were followed from start of screening between 1974 and 1979. Two control groups were used for comparison; the neighbouring counties and all of Sweden. A reference period prior to the study period was used to facilitate an adjustment for possible differences in baseline breast cancer mortality. Only deaths from breast cancer diagnosed after the first invitation to screening were analysed. Two outcome measures were used for breast cancer mortality; the underlying cause of death and excess mortality. Using the neighbouring counties as a control group, the relative risk, after 22 years of follow-up, of 10 years of screening was estimated at 0.84 (95% CI 0.71-1.00) using excess mortality. Due to lead time bias the relative risk was overestimated by 4%. Hence, a significant 20% reduction of breast cancer excess mortality was found.

Antisecretory factor counteracts secretory diarrhoea of endocrine origin.

BACKGROUND: Dietary induction of antisecretory factor (AF) can reduce diarrhoea in patients with inflammatory bowel disease. Patients with neuroendocrine tumours may suffer from diarrhoea with a prominent secretory component. We studied if AF-therapy could affect this type of diarrhoea. METHODS: Six patients with the midgut carcinoid syndrome and two with metastasizing medullary thyroid carcinoma (MTC) participated. Effects of intake of AF, in the form of AF-rich egg powder (AF-egg), and induction of endogenous AF-activity by intake of specially processed cereals (SPCs) were studied. In an initial open part of the study all patients received AF-egg for 4 weeks, followed by a double-blind crossover period with SPC and control cereals (CCs) for 6 weeks each. Daily number of bowel movements at the end of each treatment period was registered. RESULTS: Treatment with AF-egg resulted in a decrease of bowel movements in seven patients (P<0.01). Registrations of bowel movements from both SPC and CC diet periods were obtained from five patients. The daily number of bowel movements was lower during the SPC-period compared to the period with CC (P<0.05). All patients had low levels of AF-activity in serum at baseline. During treatment with AF-egg, the mean level increased slightly. AF-activity was higher (P<0.05) after SPC compared to the CC diet. CONCLUSIONS: In a group of patients with endocrine diarrhoea, AF-activity could be induced, and AF-therapy reduced the number of bowel movements.

Comparison of three small chamber test methods for the measurement of VOC emission rates from paint.

The aim of this study was to demonstrate a correlation between the measurement of emission rates of volatile organic compounds (VOCs) in three different climate chambers. In order to achieve this aim, the early state of the emission process in the three chambers was investigated and the effects of some important factors on the emission rates from paint were determined. The paper presents results of measurements in three different climate chambers. For the study, a 1-m3 chamber, a field and laboratory emission cell (FLEC), and a chamber for laboratory investigation of materials pollution and air quality (CLIMPAQ) were used. The airflow and surface area were selected so that the area-specific ventilation rates were identical in the three chambers. Temperature and relative humidity were identical during all the measurements. The paint examined was a solvent-based alkyd paint intended for indoor, which use contained between 30 and 60% of white spirit in wet condition. The paint was applied to electropolished and cleaned stainless steel plates. After application, the test material was stored for 14 days for drying in a well-ventilated conditioning room before the measurements were made. After 2 weeks storage, the most pronounced emissions were pentanal, hexanal, octanal, and decanol. The period before the emission rate stabilized differed for the three chambers studied. However, all chambers gave similar emission rates within the overall uncertainty used in these experiments.

Zinc deficiency is of limited clinical importance in the elderly (>69 y) of Oslo.

BACKGROUND: Zinc is an important trace element, and deficiency can cause disease and impairment of several physiological functions. OBJECTIVE: To examine s-zinc concentration in a large elderly population (347 subjects), and correlate it to standard biochemical markers, nutritional core indicators, and anamnestic data. DESIGN: A randomized population survey, studying two groups of elderly; one living at home and the other recently admitted to hospital. RESULTS: Serum zinc concentration was (Mean SD) in the home group (11.6 1.8 micromoles /L), and in the hospital group (11,5 2.5 micromoles /L). S-zinc was below 8 micromol/L in the hospital group in 22 of 250 patients and in 4 out of 97 of the home group. There was no significant difference in prevalence of zinc deficiency in hospital versus the home living group. Low s-zinc was significantly correlated to diarrhea, but to no other marker used in this study. CONCLUSION: Zinc deficiency is most probably of limited clinical importance in the elderly of Oslo, and there is no biochemical or nutritional marker that in addition to s-zinc can aid in the diagnosis.

Evaluation of a new commercial assay for diagnosis of pulmonary and nonpulmonary tuberculosis.

A new commercial assay for the diagnosis of tuberculosis, the BDProbeTec ET Direct Detection assay (Becton Dickinson, USA), was evaluated using 351 respiratory and 372 nonrespiratory specimens. The results were compared to detection of Mycobacterium tuberculosis complex (MTC) by conventional culture. Among the 351 respiratory specimens, MTC bacteria were identified in 150, of which 85 were positive by both microscopy and the assay. Sixty-five specimens culture positive for MTC were microscopy negative; of these, 39 were positive in the assay. All 26 specimens culture positive for nontuberculous mycobacteria (NTM) were negative by the assay. Of 175 specimens culture negative for MTC, 3 were falsely positive by the assay and 1 yielded inhibition. The overall sensitivity and specificity values were 82.7% and 98.5%, respectively. The sensitivity for microscopy-positive and -negative respiratory specimens was 100% and 60%, respectively. After correction for discrepancies, the specificity was 99% compared with notification data. The BDProbeTec ET assay detected 66 of 67 microscopy-positive and 50 of 125 microscopy-negative nonrespiratory specimens. The result for one specimen was inconclusive. All nine specimens containing NTM were negative by the assay. Of 171 specimens culture negative for MTC, 6 were falsely positive by the assay. The overall sensitivity and specificity values obtained with nonrespiratory specimens were 60.7% and 96.7%, respectively. After examining discrepancies by reviewing the patients' histories, the specificity was 98.9%. The sensitivity was 98.5% in microscopy-positive specimens and 40.3% in microscopy-negative specimens. The overall inhibition rate was 0.3%. The BDProbeTec ET assay is a fast, effective, and user-friendly system that can be used for rapid detection of MTC bacteria in respiratory and microscopy-positive nonrespiratory specimens as an important supplement to smear and culture.

Development of posterior capsule opacification in the rabbit.

PURPOSE: The aim of the present study was to characterize the development of after-cataract in the rabbit by measuring its wet weight, protein, DNA and glycosaminoglycan (GAG) contents and using Scheimpflug and slitlamp analysis. Further, aqueous humor (AqH) leukocytes, protein and lens epithelial cell proliferation activity were determined. METHODS: AqH was collected and capsular bags including after-cataract were dissected free on days 0, 1, 7, 14, 28 and 56 after cataract surgery. The wet weights were determined and the contents of DNA, protein and GAG in the capsular bags including after-cataract were analyzed. AqH was analyzed for leukocytes, protein and proliferative activity. In another set of experiments, rabbit eyes were analyzed by the Scheimpflug technique and slitlamp examination on days 0, 1, 7, 14, 28 and 56 after cataract surgery. The wet weight of the capsular bag with the after-cataract was also determined. RESULTS: An increase was found in the wet weight (480%) and the contents of protein (221%), DNA (945%) and GAG (336%) of the capsular bags including after-cataract during the experimental period. In the AqH, all 3 variables measured, leukocytes, protein and proliferative activity, reached their highest levels on day 1 after surgery. In the second set of experiments, the wet weight of the capsular bag including after-cataract increased by 391% during the 56-day experimental period. Posterior capsule opacification (PCO), as measured by Scheimpflug analysis, increased from 0.8 to 81.7% and the scores of Elschnig's pearls as well as fibrosis, analyzed by slitlamp, increased from 0.0 to 2.8 and 3.0, respectively. CONCLUSIONS: This study shows that the same components that are reported to be important in human PCO are also components of PCO in the rabbit. Thus, the rabbit model seems to accurately reflect human PCO development, and because PCO develops much faster in rabbits that would make the rabbit model suitable for studies to elucidate human PCO development.

Effect of oral washes on the diagnosis of Pneumocystis carinii pneumonia with a low parasite burden and on detection of organisms in subclinical infections.

This study was designed to assess the efficacy of using oral washes (OWs) to diagnose Pneumocystis carinii pneumonia (PCP) in patients with a low parasite burden and to detect cases of subclinical infection. A total of 104 paired induced sputum (IS) samples and OWs from 104 HIV-seropositive patients and 32 OWs from immunocompetent healthy controls were studied. All of the control samples were negative. Fifty-two IS specimens were positive for Pneumocystis carinii, and 26 of these cases were also detected in the OWs using conventional stain or polymerase chain reaction. Twenty-four of the PCP cases had a high or a moderate parasite load and 28 had a low parasite load; among them, Pneumocystis carinii was detected in the OWs of 15 and 11 cases, respectively. Fifteen of the 104 IS samples studied belonged to patients who were asymptomatic carriers or who had a subclinical infection, and Pneumocystis carinii was detected in the OWs of 4 of these cases. The parasite was not detected in 37 IS samples and in 74 OWs. The results of this study indicate that in patients with a low pulmonary parasite burden, the number of organisms reaching the oral cavity is insufficient for reliable detection in OWs. Thus, OWs are less useful than IS samples for detecting Pneumocystis carinii in cases of pneumonia in which a low parasite burden and/or subclinical infection are present.

Correlation of gene expression of ten drug efflux proteins of the ATP-binding cassette transporter family in normal human jejunum and in human intestinal epithelial Caco-2 cell monolayers.

This investigation describes the expression and interindividual variability in transcript levels of multiple drug efflux systems in the human jejunum and compares the expression profiles in these cells with that of the commonly used Caco-2 cell drug absorption model. Transcript levels of ten-drug efflux proteins of the ATP-binding cassette (ABC) transporter family [MDR1, MDR3, ABCB5, MRP1-6, and breast cancer resistance protein (BCRP)], lung resistance-related protein (LRP), and CYP3A4 were determined using quantitative polymerase chain reaction in jejunal biopsies from 13 healthy human subjects and in Caco-2 cells. All genes except ABCB5 were expressed, and transcript levels varied between individuals only by a factor of 2 to 3. Surprisingly, BCRP and MRP2 transcripts were more abundant in jejunum than MDR1 transcripts. Jejunal transcript levels of the different ABC transporters spanned a range of three log units with the rank order: BCRP approximately MRP2 > MDR1 approximately MRP3 approximately MRP6 approximately MRP5 approximately MRP1 > MRP4 > MDR3. Furthermore, transcript levels of 9 of 10 ABC transporters correlated well between jejunum and Caco-2 cells (r2 = 0.90). However, BCRP exhibited a 100-fold lower transcript level in Caco-2 cells compared with jejunum. Thus, the expression of a number of efflux protein transcripts in jejunum are equal to, or even higher than, that of MDR1, suggesting that the roles of these proteins (in particular BCRP and MRP2) in intestinal drug efflux have been underestimated. Also, we tentatively conclude that the Caco-2 cell line is a useful model of jejunal drug efflux, if the low expression of BCRP is taken into account.