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.

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.

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.

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.

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.

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.

Purification and characterization of a major human Pneumocystis carinii surface antigen.

Previous studies of Pneumocystis carinii have identified the major surface antigen of rat and human isolates as proteins of 116,000 and 95,000 mol wt, respectively, that are antigenically not identical. In this study both rat and human P. carinii proteins were purified by solubilization with zymolyase followed by molecular sieve and ion exchange chromatography. The native proteins had an apparent mol wt of 290,000 or greater, based on molecular sieve studies as well as cross-linking studies. Both proteins were glycoproteins; treatment with endoglycosidase H resulted in a 9% decrease in mol wt. The carbohydrate composition of the rat P. carinii glycoprotein was distinct from the human isolate; glucose, mannose, galactose, and glucosamine occurred in approximately equimolar ratios in the human P. carinii protein, whereas glucose and mannose were the predominant sugars of the rat P. carinii protein. To evaluate humoral immune responses to the human P. carinii protein, an enzyme-linked immunosorbent assay using purified protein was developed. Some, but not all, patients who subsequently developed P. carinii pneumonia demonstrated a serum antibody response to the surface antigen. Nearly all subjects without a history of P. carinii pneumonia had no detectable antibodies. Purified P. carinii proteins will greatly facilitate the investigation of host-P. carinii interactions.

Efficiency of single-view mammography: rate of interval cancer cases.

A nonselected population of women screened by single-view mammography was followed up to 2 years after screening, and breast carcinoma cases diagnosed were registered; 28,350 women were followed for 1 year. At screening, 132 invasive carcinoma cases were detected, and during the first year after screening, 10 cases were detected. According to an efficiency index, which takes into consideration both detections at screening and interval cases, the 1-year efficiency was 93%. The efficiency of single-view mammography for breast cancer screening was found to be satisfactory.

Population screening for breast cancer by single-view mammography in a geographic region in Sweden.

Single-view mammography was used for the screening of a total population of women living in a defined geographic region in Sweden; 37,640 women were invited and 31,074 participated. The average participation rate was 82.6%, and in the age group 40-69 years it was 91.7%. The rate of referrals from screening to clinical examination was 1.2%. Of 209 surgical biopsies performed, 130 primary mammary carcinomas were detected, 91% of which had no clinically detectable metastatic nodes. Because of the few false-positive cases, the low rate of benign biopsy specimens, the high rate of early detected carcinomas, and the low costs, screening by single-view mammography is considered a satisfactory method, provided the positioning of the oblique view is correct and the image quality is high.

Single oblique-view mammography for periodic screening for breast cancer in women.

A population, originally 6,845 women, 40 years of age and over, was screened for breast cancer by single oblique-view mammography in 1974, 1977, and 1980. Of the 5,789 women remaining in their parish in 1980, 94% had attended the screening at least once. Of 111 breast cancers diagnosed in the 6.25-year study period, 78% were detected at screening, 15% as interval cancers, and 7% among nonresponders. Sixty-three percent of the women had tumors in clinical stage I, and 59% had tumors less than or equal to 10 mm. The predictive value for biopsy recommendations by mammography was 79%. This method was considered very suitable for screening. In the older age groups there was probably some overdiagnosis of cancers that would never have become symptomatic.

Effects of dietary treatment with clofibrate, nafenopin or WY-14.643 on mitochondria and DNA in mouse liver.

Male C57bl/6 mice were administered clofibrate (0.5%, w/w), nafenopin (0.125%, w/w) or WY-14.643 (0.125%, w/w) in their diet for 4 days. Assay of eight mitochondrial marker enzymes, -i.e., malate and glutamate dehydrogenases (matrix markers), cytochrome oxidase and cytochromes c + c1 and a (inner membrane), adenylate kinase (intermembrane space) and monoamine oxidase and microsomal glutathione transferase (outer membrane)--and morphometric analysis of electron micrographs was used to examine hepatic mitochondria after treatment with these peroxisome proliferators. A moderate increase in the number of hepatic mitochondrial profiles, with a simultaneous decrease in the average size of these organelles, was observed. The total mitochondrial volume is apparently unchanged during this process. An important experimental consequence of the apparent decrease in mitochondrial size is the redistribution of a large portion of the total hepatic mitochondria from the 'nuclear' to the mitochondrial fraction. A similar effect was seen with rats.

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.

Clinical correlation of variations in the internal transcribed spacer regions of rRNA genes in Pneumocystis carinii f.sp. hominis.

OBJECTIVES: To analyse the importance of sequence variations in the internal transcribed spacer (ITS) regions 1 and 2 of the nuclear rRNA operon in AIDS patients with Pneumocystis carinii pneumonia (PCP). DESIGN AND METHODS: ITS 1 and 2 genotypes were determined in 162 bronchoalveolar lavage samples from 130 patients participating in a prospective cohort study of PCP. RESULTS: A total of 49 different ITS genotypes were detected. ITS genotype was not associated with the clinical severity or outcome of PCP. In 37 of 162 (23%) samples infection with two or more genotypes was observed. A genotype switch was detected in six of 10 patients (60%) with recurrent episodes of PCP. However, genotype changes were also seen in 10 of 19 patients (53%) who had repeated bronchoscopies within the same episode of PCP. The same ITS type was observed twice in 13 (46%) of the 28 patients with repeat bronchoscopies during single or recurrent episodes of pneumonia, but in only 14 of 81 (17%) randomly selected pairs (P < 0.01). CONCLUSION: Although the detection of ITS genotypes is not a random event, changes in genotype can be detected in a single episode of disease, with 23% of PCP patients being infected with more than one P. carinii genotype, thus complicating the use of this locus as a genetic marker to separate new infection from the reactivation of latent infection. ITS genotypes are not associated with the clinical severity of PCP.

Growth hormone (GH) therapy in GH-deficient adults influences the response to a dietary load of cholesterol and saturated fat in terms of cholesterol synthesis, but not serum low density lipoprotein cholesterol levels.

An increased dietary load of cholesterol (ch) and saturated fat increases serum low density lipoprotein ch (LDL-ch) levels. GH therapy in GH-deficient adults decreases serum LDL-ch levels. In the rat, GH is important for resistance to dietary cholesterol in terms of serum cholesterol levels. The aim of this study was to investigate the influence of GH on the effects of an increase in the intake of cholesterol and saturated fat on serum lipoproteins and markers for cholesterol synthesis in man. Six GH-deficient adults were given an isocaloric diet enriched in cholesterol and saturated fat for 17 days with and without GH therapy (1-1.5 U/day). Serum cholesterol, LDL-ch, apolipoprotein B (apoB), and apoA1 levels increased during the diet period with GH therapy and tended to increase during the diet period without GH. However, GH therapy did not influence the dietary effect on serum cholesterol, LDL-ch, apoA1, or apoB levels. Serum levels of triglycerides, very low density lipoprotein ch, high density lipoprotein ch, and apoE were not affected by diet or GH therapy. GH therapy increased serum lipoprotein(a) levels, but did not affect the response to diet. The serum total delta7-lathosterol/cholesterol ratio increased less during the diet period with GH therapy than during the diet period without GH. Serum 7alpha-hydroxy-4-cholesten-3-one levels tended to increase during both diet periods, but were not influenced by GH treatment. Serum plant sterol levels did not change. These results indicate that GH counteracts an increase in cholesterol synthesis induced by a high fat diet without affecting bile acid synthesis or sterol absorption. GH therapy did not have any major influence on the dietary effects on serum lipoprotein levels.

Is the cytosolic catalase induced by peroxisome proliferators in mouse liver on its way to the peroxisomes?

Dietary treatment of male C57B1/6 mice with clofibrate, nafenopin or WY-14.643 resulted in a modest (at most 2-fold) increase in the total catalase activity in the whole homogenate and mitochondrial fraction prepared from the livers of these animals. On the other hand, the catalase activity recovered in the cytosolic fraction was increased 12- to 18-fold, i.e. 30-35% of the total catalase activity in the hepatic homogenate was present in the high-speed supernatant fraction after treatment with these peroxisome proliferators. A study of the time course of the changes in peroxisomal and cytosolic catalase activities demonstrated that the peroxisomal activity both increased upon initiation of exposure and decreased after termination of treatment several days after the increase and decrease, respectively, in the corresponding cytosolic activity. This finding suggests that the cytosolic catalase may be on its way to incorporation into peroxisomes.

Antiviral effects of 3'-fluorothymidine and 3'-azidothymidine in cynomolgus monkeys infected with simian immunodeficiency virus.

An acute infection with simian immunodeficiency virus (SIVSM) in cynomolgus monkeys was used to evaluate the antiviral effects of 3'-fluorothymidine (FLT) and 3'-azidothymidine [zidovudine (ZDV)]. Neither compound prevented the infection despite dosing prior to virus inoculation. FLT was about ten times more potent than ZDV in delaying the appearance of SIVSM antigen in the monkeys. The serum half-life of FLT was longer than that of ZDV and ZDV was bound to plasma proteins to about 60% while FLT was virtually unbound. It is proposed that the in vivo difference in potency between ZDV and FLT could, at least partly, be explained as the combined effects of a longer plasma half-life and a higher free concentration of FLT and possibly a higher intracellular concentration of the triphosphate of FLT.

Experimental infection of cynomolgus monkeys (Macaca fascicularis) with simian immunodeficiency virus (SIVsm).

Five healthy cynomolgus monkeys were inoculated intravenously with simian immunodeficiency virus (SIVsm) propagated in human lymphocytes. All five animals became infected. Virus was recovered from blood mononuclear cells and viral antigen was detected in serum 12 days postinoculation (PI) in all inoculated animals. Virus was also isolated in all five animals tested 74 to 226 days PI. Antibodies to different structural proteins of SIV and HIV-2 were demonstrated by ELISA, Western blot, and radioimmunoprecipitation assay from day 31 PI concomitantly with a reduction of viral proteins in the serum. Reappearance of antigen accompanied by a fall in antibody to gag products (p26) was observed in two monkeys 69 days PI. All SIV-infected monkeys showed a pronounced decrease in CD4+ lymphocytes demonstrable already 12 days PI. They also developed persistent lymphadenopathy. Thus, infection of cynomolgus monkeys with SIVsm mimics events in human immunodeficiency virus infection in humans but the course of evolution of pathogenic events in the monkey is markedly compressed. This experimental model will be useful for evaluation of HIV vaccines and antiviral testing.

Effects of dietary treatment with 11 dicarboxylic acids, diethylcarboxylic esters and fatty acids on peroxisomal fatty acid beta-oxidation, epoxide hydrolases and lauric acid omega-hydroxylation in mouse liver.

C57B1/6 male mice were exposed through their diet to 11 dicarboxylic acids, carboxylic acids and diethyldicarboxylesters for 10 days. For the diacids and diethylesters this treatment resulted in a chain length-dependent induction of lauryl-CoA oxidase and cyanide-insensitive palmitoyl-CoA oxidation activities. A chain length of 12 carbon atoms or more seemed to be necessary for induction of these two activities. In addition, the same chain length dependence was observed for induction of lauric acid omega + omega-1 hydroxylase activity and increase in the protein content of the mitochondrial fraction. Treatment with two "natural" fatty acids, i.e. lauric and palmitic acid gave no effect at all on these various parameters. In no case was induction of cytosolic and mitochondrial epoxide hydrolase activities observed. Instead, a slight decrease in these activities was observed after administration of diacids with a chain length of 4-8 carbon atoms, whereas microsomal epoxide hydrolase activity was concurrently induced.

Induction of cytosolic and microsomal epoxide hydrolases and proliferation of peroxisomes and mitochondria in mouse liver after dietary exposure to p-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid.

The effects of dietary exposure to 0.125% (w/w) p-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid or 2,4,5-trichlorophenoxyacetic acid on the content of peroxisomes and levels of certain xenobiotic-metabolizing enzymes in mouse liver have been investigated. In agreement with the literature on rat liver 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid were found to cause extensive proliferation of peroxisomes (as judged by the total levels of "mitochondrial" protein, carnitine acetyltransferase, cyanide-insensitive palmitoyl-CoA oxidation and catalase) in mouse liver. On the other hand, exposure to p-chlorophenoxyacetic acid did not significantly affect any of these parameters. As with certain other peroxisome proliferators, 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid increased total cytochrome oxidase activity as well. In addition, dietary exposure to 2,4-dichlorophenoxyacetic acid and to 2,4,5-trichlorophenoxyacetic acid resulted in increases in the activities of cytosolic and microsomal epoxide hydrolases in mouse liver and generally less pronounced increases in the total cytosolic glutathione transferase activity and microsomal content of cytochrome P-450. In the case of cytochrome P-450, this process can be said to be a true induction (i.e. the amount of enzyme protein is increased), because the assay procedure for cytochrome P-450 measures holoenzyme amount. Immunoquantitation demonstrated that this was also the case for the changes in cytosolic epoxide hydrolase. The dramatic differences in proliferation of peroxisomes and induction of xenobiotic-metabolizing enzymes seen here with compounds differing relatively little in structure may indicate that a receptor mechanism of some kind is involved.

Effects of perfluoro fatty acids on xenobiotic-metabolizing enzymes, enzymes which detoxify reactive forms of oxygen and lipid peroxidation in mouse liver.

Male mice were exposed via their diet to perfluoro fatty acids of various chain-lengths (2-10 carbon atoms) at different doses (0.02 and 0.1% weight) and for different periods of time (2-10 days). Thereafter, we monitored effects on liver and body weights and a number of hepatic parameters, including mitochondrial protein content, microsomal contents of cytochromes P450 and b5, NADPH-cytochrome P450 reductase activity [measured as NADPH-cytochrome c reductase (EC 1.6.2.3)], microsomal and cytosolic epoxide hydrolase (EC 3.3.2.3) activities, cytosolic DT-diaphorase (EC 1.6.99.2), glutathione transferase (EC 2.5.1.18), glutathione peroxidase (EC 1.11.1.9) and superoxide dismutase (EC 1.15.1.1) activities, and levels of thiobarbituric acid-reactive material (as an indicator of lipid peroxidation) in the mitochondrial subfraction. The most dramatic changes observed were a 5-9-fold increase in mitochondrial protein, a 3-6-fold increase in the microsomal content of cytochrome P450, a 3-10-fold increase in cytosolic DT-diaphorase activity, an approximately 2-fold increase in cytosolic epoxide hydrolase activity and as much as a 60% decrease in the level of thiobarbituric acid-reactive compounds in the mitochondrial fraction. Smaller increases in microsomal epoxide hydrolase activity and decreases in cytosolic glutathione peroxidase activity were also observed. Of the perfluoro fatty acids tested, perfluorooctanoic acid caused the largest changes in the parameters examined here. Dietary exposure of mice to a 0.02% dose of this substance for 10 days results in a maximal or near-maximal effect in most cases.