Profiling circulating microRNAs in the serum of pregnant and non-pregnant pigs reveals a plethora of reproductive status-dependent microRNAs.

Circulating, non-coding RNAs, such as microRNAs (miRNAs) have been proposed to be powerful pathophysiological indicators of pregnancy in animals and humans. Since their discovery, it is known that miRNAs can take part in numerous biological processes, including cell proliferation and differentiation during early embryonic development and establishment of pregnancy. Our recent studies have indicated that maternal blood can carry miRNAs reported previously at the embryo-maternal interface in pigs. To expand the scope of our research, we tested the hypothesis that miRNAs previously identified in conceptuses, trophoblasts, endometrium and uterine lumen-derived extracellular vesicles (EVs) collected before Day 20 of pregnancy can show reproductive status-dependent profiles in the serum of cyclic and pregnant crossbred pigs. Custom-designed TaqMan arrays, multiplex real-time reverse transcription (RT)-PCR and real-time RT-PCR allowed us to identify a number of reproductive status-dependent miRNAs in serum samples collected from pigs during the estrous cycle or pregnancy (Days 16 and 20). We found that serum samples were enriched with miRNAs involved in processes important during the estrous cycle and early pregnancy, e.g. cell sensitivity and viability, angiogenesis, embryonic cell proliferation and differentiation. Further validation revealed different abundance of ssc-miR-143-3p and ssc-miR-125b in pregnant and non-pregnant animals and correlation of ssc-miR-125b levels with litter size. In addition, analyzed serum samples contained both EVs and Argonaute2 proteins, which are known to be involved in miRNA transportation and intercellular communication. In summary, we identified several circulating miRNAs that differ in abundance between cyclic and pregnant animals and could serve as potential indicators of reproductive status in pigs during breeding management.

EuroGuiDerm Guideline on the systemic treatment of Psoriasis vulgaris - Part 2: specific clinical and comorbid situations.

This evidence- and consensus-based guideline on the treatment of psoriasis vulgaris was developed following the EuroGuiDerm Guideline and Consensus Statement Development Manual. The second part of the guideline provides guidance for specific clinical and comorbid situations such as treating psoriasis vulgaris patient with concomitant psoriatic arthritis, concomitant inflammatory bowel disease, a history of malignancies or a history of depression or suicidal ideation. It further holds recommendations for concomitant diabetes, viral hepatitis, disease affecting the heart or the kidneys as well as concomitant neurological disease. Advice on how to screen for tuberculosis and recommendations on how to manage patients with a positive tuberculosis test result are given. It further covers treatment for pregnant women or patients with a wish for a child in the near future. Information on vaccination, immunogenicity and systemic treatment during the COVID-19 pandemic is also provided.

Claudin-3 - a new intestinal integrity marker in patients with psoriasis: association with disease severity.

BACKGROUND: Gut dysbiosis and increased intestinal permeability play a significant role in the pathogenesis of psoriasis and its comorbidities. Claudin-3 is a key component of tight junctions, which may serve as marker of gut barrier integrity. OBJECTIVES: The aim of the study was to investigate circulating plasma claudin-3 in patients with psoriasis and to evaluate clinical and metabolic factors, which determine its concentration. METHODS: This cross-sectional study included 60 patients with psoriasis (39 men and 21 women, mean age: 45.6 ± 12.1 years) and 30 healthy controls (18 men and 12 women, mean age: 46.3 ± 15.5 years) age, sex and body mass index-matched. Plasma claudin-3 concentration was measured using an enzyme-linked immunosorbent assay. RESULTS: Plasma claudin-3 concentration was significantly higher in patients with psoriasis in comparison with healthy control [median (interquartile range), 50.7 ng/mL (47.3-54.2) vs. 43.3 ng/mL (42.3-44.2), P < 0.001]. Patients who achieved ΔPASI90 response after 16 weeks of treatment showed tendency to decrease in circulating claudin-3 plasma concentration. Positive correlations between claudin-3 concentration and the PASI score (r = 0.828; P < 0.001) as well as claudin-3 and neutrophil-to-lymphocyte ratio (r = 0.847; P < 0.001) were found. A multivariable linear regression analysis confirmed association of claudin-3 with the PASI score (P < 0.001), neutrophil-to-lymphocyte ratio (P < 0.01) and active smoking (P < 0.05). CONCLUSION: Claudin-3, a biomarker for gut permeability, is increased in psoriasis and correlates with disease severity and smoking. Further investigations are needed to determine whether reinforcing intestinal barrier may be a new therapeutic target in psoriasis.

Predominance of Comorbidities in the Detriment of Daily Activity in Sarcoidosis Patients.

Sarcoidosis may affect lung function, working ability, overall mobility, and daily activity. In the present study we performed an analysis of clinical settings in patients with sarcoidosis to disentangle its influence on daily physical activity (PA). PA assessment (number of steps per day, daily energy expenditure) was performed by accelerometry during consecutive 7 days after discharge from hospital. Thirty patients with sarcoidosis, aged 46.4 ± 10.5, were enrolled in the study. Clinical data (age, gender, steroid consumption, weight, and comorbidities), lung function tests (forced expiratory volume in one second - FEV1, forced vital capacity - FVC, and lung diffusion for carbon monoxide - DLCO), mobility (6-minute walk test - 6 MWT) and physical performance (oxygen consumption at anaerobic threshold - VO2/AT) were estimated. The mean daily PA (5214 ± 2699 steps/day) and VO2max (22.3 ± 7.0 ml/kg/min) were lower when referenced to the age-group predicted values. A significant greater daily PA was observed in sarcoidosis patients without comorbidities compared with those having more than two comorbidities (p = 0.046). No association was found between steroid use, lung function, and 6MWT. Daily PA was associated with patients aerobic efficacy and VO2max (r = 0.38, p < 0.04). The findings demonstrate a significant influence of comorbidities on sarcoidosis patients' exercise tolerance and daily PA. Special treatment considerations, including the potential impact of comorbidities, may help optimize exercise regimes, link physical activity with health, and prevent sarcoidosis complications.

Physical Functioning and Symptoms of Chronic Fatigue in Sarcoidosis Patients.

Scientific reports underscore the importance of measuring the health-related quality of life in sarcoidosis patients. The present study seeks to define how sarcoidosis patients' quality of life, daily physical activity, and physical performance are related to each other. Seventeen patients (mean age 46.8 ± 8.8 years) suffering from sarcoidosis completed the following questionnaires: the fatigue assessment scale (FAS), the quality of life scale (SF-36 questionnaire), and the Borg dyspnea scale. Physical activity (PA) was assessed using accelerometry. Respiratory function, consisting of forced expiratory volume in one second (FEV1), forced vital capacity (FVC), forced expiratory volume in one second as a percentage of vital capacity (FEV1/%FVC), and diffusing capacity of the lungs for carbon monoxide (DLCO), were assessed. In addition, performance in 6-min walk test (MWT), aerobic capacity assessed from maximal oxygen uptake (VO2max), and the metabolic equivalent of task (MET) were evaluated. We found that daily PA (4566 ± 2378 steps/day) and VO2max (21.8 ± 5.9 ml/kg/min) were lower in sarcoidosis patients than the known predicted values in healthy age-matched individuals. There were significant inverse associations between the FAS score and 6MWT (r = -0.62; p < 0.01), and between SF-36 score and 6MWT (r = -0.55; p < 0.03). In contrast, SF-36 scores associated with fatigue and dyspnea scores (r = 0.72; p < 0.001 and r = 0.85; p < 0.001). These findings imply that sarcoidosis patients are less active compared with healthy subjects. The FAS and SF-36 scales seem to be effective tools for assessing the severity of fatigue in sarcoidosis patients.

CYP2D6 genotype is not associated with survival in breast cancer patients treated with tamoxifen: results from a population-based study.

PURPOSE: A number of studies have tested the hypothesis that breast cancer patients with low-activity CYP2D6 genotypes achieve inferior benefit from tamoxifen treatment, putatively due to lack of metabolic activation to endoxifen. Studies have provided conflicting data, and meta-analyses suggest a small but significant increase in cancer recurrence, necessitating additional studies to allow for accurate effect assessment. We conducted a retrospective pharmacogenomic analysis of a prospectively collected community-based cohort of patients with estrogen receptor-positive breast cancer to test for associations between low-activity CYP2D6 genotype and disease outcome in 500 patients treated with adjuvant tamoxifen monotherapy and 500 who did not receive any systemic adjuvant therapy. METHODS: Tumor-derived DNA was genotyped for common, functionally consequential CYP2D6 polymorphisms (*2, *3, *4, *6, *10, *41, and copy number variants) and assigned a CYP2D6 activity score (AS) ranging from none (0) to full (2). Patients with poor metabolizer (AS = 0) phenotype were compared to patients with AS > 0 and in secondary analyses AS was analyzed quantitatively. Clinical outcome of interest was recurrence free survival (RFS) and analyses using long-rank test were adjusted for relevant clinical covariates (nodal status, tumor size, etc.). RESULTS: CYP2D6 AS was not associated with RFS in tamoxifen treated patients in univariate analyses (p > 0.2). In adjusted analyses, increasing AS was associated with inferior RFS (Hazard ratio 1.43, 95% confidence interval 1.00-2.04, p = 0.05). In patients that did not receive tamoxifen treatment, increasing CYP2D6 AS, and AS > 0, were associated with superior RFS (each p = 0.0015). CONCLUSIONS: This population-based study does not support the hypothesis that patients with diminished CYP2D6 activity achieve inferior tamoxifen benefit. These contradictory findings suggest that the association between CYP2D6 genotype and tamoxifen treatment efficacy is null or near null, and unlikely to be useful in clinical practice.

Polymerase chain reaction melting profiles of Candida glabrata clinical isolates in solid organ transplant recipients in comparison to the other group of surgical patients.

The aim of the retrospective study were to estimate the prevalence of Candida glabrata in liver and kidney transplant recipients compared to patients with short bowel syndrome receiving chronic total parenteral nutrition and relevance of the polymerase chain reaction melting profile (PCR MP) method for Candida glabrata strains differentiation. C. glabrata clinical strains isolated from patients were identified by using standard mycological procedures. The analysis of genetic relatedness of the isolated strains was conducted using the PCR MP method. The prevalence of C. glabrata comprised 29% of all episodes of fungal colonization and infection in solid organ transplant recipients, and 54% of those in hospitalized patients receiving long-term total parenteral nutrition. Among 78 isolates obtained from 55 solid organ transplant recipients and 2 organ donors, 44 different C. glabrata PCR MP fingerprints were observed. Forty-seven organ recipients and one organ donor carried unique C. glabrata strains. Among 37 isolates obtained from 31 patients receiving long-term TPN, 8 different PCR MP profiles of C. glabrata strains were observed. Two patients carried unique C. glabrata strains. Most of the C. glabrata colonization and infections in solid-organ transplant recipients were caused by endogenic strains. Most of the C. glabrata colonization and infections in hospitalized patients receiving long-term total parenteral nutrition could result by patient-to-patient transmission. The results showed that the PCR MP technique is a good discriminatory method for genotyping for C. glabrata strains.

Acinetobacter baumannii multidrug-resistant strain occurrence in liver recipients with reference to other high-risk groups.

INTRODUCTION: The increasing clinical significance of Acinetobacter baumannii species is due to its ability to survive in hospital environments, its species-specific multidrug resistance, and its ability to instantly develop various drug-resistance mechanisms through antibiotic pressure. MATERIALS AND METHODS: We identified 16 A baumannii strains isolated from patients presenting postoperative infections in 2010. A baumannii isolates were obtained from clinical specimens by standard microbiologic methods. As previously described, we performed polymerase chain reaction (PCR) analysis for carbapenemase-encoding genes (VIM, IMP, SPM, OXA23, OXA24, OXA51, OXA58) in Acinetobacter spp. RESULTS: The double-disk synergy test phenotypic method did not detect any A baumannii strains producing metallo-beta-lactamaus cultured from swabs from all the patient groups. No products of PCR amplification with specific starters for VIM, IMP, and SPM (Sao Paulo metallo-ß-lactamase) genes were found. All analyzed strains were colistin-sensitive. Among five strains from liver recipients, one was imipenem- and meropenem-resistant. Four among six strains isolated from cancer patients were resistant to imipenem and/or meropenem; 1/5 were imipenem-and meropenem-resistant; 1, meropenem-resistant and imipenem-sensitive; 1, meropenem- and imipenem-resistant; and 1 with intermediate resistance to both meropenem and imipenem among swabs cultured from patients with postoperative complication after bone fracture. Fifteen among 16 analyzed A baumannii strains had an OXA51 gene. Two among five A baumannii strains isolated in liver recipients had only an OXA51 gene; one, OXA51 and OXA24 genes; one, OXA51 and OXA23 genes.

High-efficiency genotype analysis from formalin-fixed, paraffin-embedded tumor tissues.

Single-nucleotide polymorphisms (SNPs) can be assayed using DNA isolated from archival formalin-fixed, paraffin-embedded (FFPE) samples, making retrospective pharmacogenetic studies possible. In this study, we describe methods that significantly increase the number of SNP determinations possible using FFPE samples. Quantifying the amount of DNA amenable to PCR (amplification-quality DNA, AQ-DNA) allows a significant reduction in the amount of sample required for Taqman-based SNP assays. Optimizing AQ-DNA input increases PCR amplification efficiency and SNP determination accuracy. DNA was extracted from 39 FFPE tumor sections and matched tumor and stromal cores, which were of the type used to generate tissue microarrays. Sections and tumor cores yielded sufficient AQ-DNA for more than 1000 SNP determinations. Seven SNPs were assessed following individual assay optimization for minimal AQ-DNA. Genotypes from tumor cores for single SNPs were 92.3-100% concordant with those obtained from sections. Using these methods, the number of SNP genotypes that can be determined from single FFPE samples is greatly increased expanding the genetic association studies possible from limited archival specimens. The use of tumor cores is of particular importance as the harvesting of tumor cores has minimal impact on the utility of the donor blocks for other purposes.

Efavirenz directly modulates the oestrogen receptor and induces breast cancer cell growth.

OBJECTIVES: Efavirenz-based HIV therapy is associated with breast hypertrophy and gynaecomastia. Here, we tested the hypothesis that efavirenz induces gynaecomastia through direct binding and modulation of the oestrogen receptor (ER). METHODS: To determine the effect of efavirenz on growth, the oestrogen-dependent, ER-positive breast cancer cell lines MCF-7, T47D and ZR-75-1 were treated with efavirenz under oestrogen-free conditions in the presence or absence of the anti-oestrogen ICI 182,780. Cells treated with 17ß-oestradiol in the absence or presence of ICI 182,780 served as positive and negative controls, respectively. Cellular growth was assayed using the crystal violet staining method and an in vitro receptor binding assay was used to measure the ER binding affinity of efavirenz. RESULTS: Efavirenz induced growth in MCF-7 cells with an estimated effective concentration for half-maximal growth (EC(50)) of 15.7 µM. This growth was reversed by ICI 182,780. Further, efavirenz binds directly to the ER [inhibitory concentration for half maximal binding (IC(50)) of ∼52 µM] at a roughly 1000-fold higher concentration than observed with 17ß-oestradiol. CONCLUSIONS: Our data suggest that efavirenz-induced gynaecomastia may be caused, at least in part, by drug-induced ER activation in breast tissues.

A virtual source model of electron contamination of a therapeutic photon beam.

The most efficient way of generating particles for Monte Carlo (MC) dose calculation is through a virtual source model (VSM) of the linear accelerator head. We have previously developed a VSM based on three sources: a primary photon source, a secondary photon source and an electron contamination source (Sikora et al 2007). In this work, we present an improvement of the electron contamination source. The VSM of contamination electrons (eVSM) is derived from a full MC simulation of the accelerator head with the BEAMnrc MC system. It comprises a Gaussian source located at the base of the flattening filter. The eVSM models two effects: an energy-dependent source diameter and an angular dependence of the particle fluence. The air scatter of the contamination electrons is approximated by energetic properties of the eVSM so that explicit in-air transport is not required during MC simulation of the dose distributions in the patient. The calculations of electron dose distributions were compared between the eVSM and the full MC simulation. Good agreement was achieved for various rectangular field sizes as well as for complex conformal segment shapes for the contamination electrons of 6 and 15 MV beams. The 3D dose evaluation of the surface dose in a CT-based patient geometry shows high accuracy (2%/2 mm) of the eVSM for both energies. The model has one tunable parameter, the mean energy of the spectrum at the patient surface. High accuracy and efficiency of particle generation make the eVSM a valuable virtual source of contamination electrons for MC treatment planning systems.

Cytochrome P450 2D6 activity predicts discontinuation of tamoxifen therapy in breast cancer patients.

The selective estrogen receptor modulator tamoxifen is routinely used for treatment and prevention of estrogen-receptor-positive breast cancer. Studies of tamoxifen adherence suggest that over half of patients discontinue treatment before the recommended 5 years. We hypothesized that polymorphisms in CYP2D6, the enzyme responsible for tamoxifen activation, predict for tamoxifen discontinuation. Tamoxifen-treated women (n=297) were genotyped for CYP2D6 variants and assigned a 'score' based on predicted allele activities from 0 (no activity) to 2 (high activity). Correlation between CYP2D6 score and discontinuation rates at 4 months was tested. We observed a strong nonlinear correlation between higher CYP2D6 score and increased rates of discontinuation (r(2)=0.935, P=0.018). These data suggest that presence of active CYP2D6 alleles may predict for higher likelihood of tamoxifen discontinuation. Therefore, patients who may be most likely to benefit from tamoxifen may paradoxically be most likely to discontinue treatment prematurely.

Trichosporon asahii as a prospective pathogen in solid organ transplant recipients.

Trichosporon spp. is not an important factor of mycotic infections in immunocompetent patients. It may be a cause of invasive mycoses with a high mortality rate in patients undergoing solid organ transplantation. We have analysed the antifungal agents' susceptibility of Trichosporon asahii and its frequency of occurrence as a prospective etiological agent of infections in liver, kidney and simultaneous pancreas-kidney transplant recipients. Clinical specimens (urine, blood, peritoneal fluid and swabs) were obtained from patients hospitalised in the Institute of Transplantation Medicine, Department of General and Transplantation Surgery, Medical University of Warsaw in 2005 and 2006. Microbiological tests were performed in Mycological Laboratory, Department of Microbiology, Medical University of Warsaw. A total of 475 strains of yeast-like fungi were isolated from clinical specimens taken from 263 liver, kidney and simultaneous pancreas-kidney transplant recipients and from 26 organ donors. Trichosporon asahii was found in 26 clinical samples taken from 18 patients and one organ donor. Positive cultures were obtained from 22 urine samples, one stoma fluid, one wound swab, one tracheal aspirate and one ejaculate. Isolates of Trichosporon asahii were found in 6% of total positive mycological cultures in the solid organ transplant recipients. Among cultured strains, 11 isolates were resistant to fluconazole, four to itraconazole and three of them demonstrated resistance to amphotericin B.

A virtual photon source model of an Elekta linear accelerator with integrated mini MLC for Monte Carlo based IMRT dose calculation.

A dedicated, efficient Monte Carlo (MC) accelerator head model for intensity modulated stereotactic radiosurgery treatment planning is needed to afford a highly accurate simulation of tiny IMRT fields. A virtual source model (VSM) of a mini multi-leaf collimator (MLC) (the Elekta Beam Modulator (EBM)) is presented, allowing efficient generation of particles even for small fields. The VSM of the EBM is based on a previously published virtual photon energy fluence model (VEF) (Fippel et al 2003 Med. Phys. 30 301) commissioned with large field measurements in air and in water. The original commissioning procedure of the VEF, based on large field measurements only, leads to inaccuracies for small fields. In order to improve the VSM, it was necessary to change the VEF model by developing (1) a method to determine the primary photon source diameter, relevant for output factor calculations, (2) a model of the influence of the flattening filter on the secondary photon spectrum and (3) a more realistic primary photon spectrum. The VSM model is used to generate the source phase space data above the mini-MLC. Later the particles are transmitted through the mini-MLC by a passive filter function which significantly speeds up the time of generation of the phase space data after the mini-MLC, used for calculation of the dose distribution in the patient. The improved VSM model was commissioned for 6 and 15 MV beams. The results of MC simulation are in very good agreement with measurements. Less than 2% of local difference between the MC simulation and the diamond detector measurement of the output factors in water was achieved. The X, Y and Z profiles measured in water with an ion chamber (V = 0.125 cm(3)) and a diamond detector were used to validate the models. An overall agreement of 2%/2 mm for high dose regions and 3%/2 mm in low dose regions between measurement and MC simulation for field sizes from 0.8 x 0.8 cm(2) to 16 x 21 cm(2) was achieved. An IMRT plan film verification was performed for two cases: 6 MV head&neck and 15 MV prostate. The simulation is in agreement with film measurements within 2%/2 mm in the high dose regions (> or = 0.1 Gy = 5% D(max)) and 5%/2 mm in low dose regions (<0.1 Gy).

DNA released from rejecting organs is an indicator of the degree of graft cellular damage.

Warm and cold ischemia as well as rejection of the transplanted organ or tissue cause destructive changes in the graft parenchyma. Fragments of disintegrated cellular organelles are phagocytized and digested by recipient scavenger cells in lymph nodes, spleen, and liver. Some fragments engulfed by dendritic cells are processed including donor DNA present in the ingested cellular debris. The question arises as to whether the DNA from the disintegrated cells may be used as a measure of graft damage. In this study we provide evidence that both syngeneic and allogeneic organ transplantation followed by "seeding" of donor DNA from graft cells is internalized in recipient macrophages and dendritic cells in lymphoid organs. The kinetics of accumulation of donor DNA in recipient tissues reflected the degree of ischemic and immune graft damage. Immunosuppression with cyclosporine or tacrolimus did not significantly attenuate the DNA release. Measurements of the concentration of donor DNA gives insight into the kinetics of allograft rejection. Real-time polymerase chain reaction for donor DNA in recipient serum and blood leukocytes that have engulfed donor cell debris may be useful for clinical diagnostic application.

Compound action potential input-output decruitment. Effect of topically applied antiseptics.

The ototoxic effect of povidone-iodine antiseptics topically applied to the chinchilla round window was examined with particular emphasis on the action potential (AP) input-output function at 2 and 4 kHz. A group of chinchillas exhibited a marked elevation of AP threshold at 8 and 12 kHz, with only a slight threshold elevation at 2 and 4 kHz. A distinct decruitment (less than normal growth of response with increasing sound intensity) of the AP input-output function was, however, found at the lower frequencies. There are implications of an ototoxically induced high-frequency hearing loss on speech frequencies.