Acute Effects of Glucocorticoids on Serum Markers of Osteoclasts, Osteoblasts, and Osteocytes

The aim of this study was to investigate the acute effects of oral glucocorticoids in doses used in clinical practice on biochemical indices of the function of osteoclasts, osteoblasts, and osteocytes. In 17 adult patients suffering from various medical pathologies requiring systemic steroid therapy that were never before treated with glucocorticoids, glucocorticoid treatment was initiated (mean prednisolone equivalent dose of 23.1 ± 12.7 mg/day, range 10–50). Fasting morning serum concentrations of osteocalcin (OC), amino-terminal propeptide of type I procollagen (PINP), type 1 collagen cross-linked C-telopeptide (βCTX), soluble receptor activator of nuclear factor kappaB ligand (sRANKL), osteoprotegerin (OPG), sclerostin, Dickkopf-1 (Dkk-1), and high-sensitivity C-reactive protein (hsCRP) were measured at baseline and on three consecutive days. Significant reductions in serum OC, PINP, OPG, sclerostin, and hsCRP were observed during 96 h of glucocorticoid administration, while serum βCTX showed a significant percentual increase. A significant positive correlation was found between serum concentrations of Dkk-1 and βCTX after 96 h of treatment with glucocorticoids. A significant drop in serum sclerostin, OPG, and OC observed in this study may reflect the rapid glucocorticoid-induced apoptosis of osteocytes.     

Rapid Detection and Identification of Mucormycetes in Bronchoalveolar Lavage Samples from Immunocompromised Patients with Pulmonary Infiltrates by Use of High-Resolution Melt Analysis

Rapid differential diagnostics of pulmonary infiltrates suspected of invasive fungal disease in an immunocompromised host and early initiation of effective antifungal therapy are crucial for patient outcomes. There are no serological tests available to detect mucormycetes; therefore, PCR-based methods are highly suitable. We validated our previously published PCR followed by high-resolution melt analysis (PCR/HRMA) to detect Rhizopus spp., Rhizomucor pusillus, Lichtheimia corymbifera, and Mucor spp. in bronchoalveolar lavage (BAL) samples from immunocompromised patients who were at risk of invasive fungal disease. All PCR/HRMA-positive samples were retested using novel real-time quantitative PCR (RQ PCR) assays specific to the species identified. In total, between January 2009 and December 2012 we analyzed 99 BAL samples from 86 patients with pulmonary abnormalities using PCR/HRMA. Ninety (91%) BAL samples were negative, and 9 (9%) samples were positive. The sensitivity and specificity of PCR/HRMA were 100% and 93%, respectively. By combining the positive results of PCR/HRMA with positive RQ PCR results, the specificity was raised to 98%. PCR/HRMA, due to its high negative predictive value (99%), represents a fast and reliable tool for routine BAL sample screening for the differential diagnosis of pulmonary infiltrates in immunocompromised patients for the four most clinically important mucormycetes.     

Interleukin-10 modulation of alloreactivity and graft-versus-host reactions

BACKGROUND: The biological properties of interleukin (IL)-10 in tolerance induction and inhibition of alloreactivity have suggested a therapeutic use of this cytokine as an additional or alternative prophylaxis for graft-versus-host disease (GvHD). However, the effects of exogenous IL-10 on GvHD are mainly studied in animal models, and the results remain conflicting. This study aims to demonstrate, for the first time, whether the addition of exogenous IL-10 can reduce the severity of graft-versus-host reactions (GvHR) in humans. METHODS: The regulatory role of exogenous IL-10 in GvHR was investigated using an in vitro human skin explant model. The effects of IL-10 on skin GvHR were tested in parallel with allo-antigen induced T-cell proliferation, cytolytic reactivity, and cytokine production. RESULTS: In the presence of IL-10, the mixed lymphocyte reaction (MLR) primed responder cells showed significantly lower proliferative and cytolytic responses compared with the responder cells from the control MLR carried out in the absence of IL-10. The responder cells from IL-10 containing MLR induced significantly less severe skin GvHR and displayed a significantly reduced T-cell activation and cytokine production. A significant correlation was observed between the levels of TNF-alpha production and the sensitivity to IL-10 modulation of GvHR. CONCLUSIONS: The addition of exogenous IL-10 strongly inhibited the broad alloreactivity initiated by primary MLR and significantly reduced the overall severity of skin GvHR induced by MLR primed responder cells. Responder cells producing high TNF-alpha following allogeneic stimulation appeared to be less sensitive to IL-10 modulation of GvHR.     

The use of auto-titrating continuous positive airway pressure for treatment of adult obstructive sleep apnea. An American Academy of Sleep Medicine review

This paper reviews the efficacy of auto-titrating continuous positive airway pressure (APAP) for treatment of obstructive sleep apnea. It is based on a review of 30 articles published in peer review journals conducted by a task force appointed by the American Academy of Sleep Medicine to develop practice parameters for use of APAP devices for treatment of obstructive sleep apnea (OSA). The data indicate that APAP can be used to treat many patients with OSA (auto-adjusting) or to identify an effective optimal fixed level of continuous positive airway pressure (CPAP) for treatment (auto-titration). Patients with significant congestive heart failure, chronic obstructive pulmonary disease (COPD), or significant amounts of central apnea were excluded from many treatment trials and there is insufficient evidence that APAP can be used to treat these patients. Many clinical trials have been performed in patients already on CPAP or with the initial APAP night in a laboratory setting. At this time only a few studies have evaluated initial titration with APAP in CPAP-na?ve patients in an unattended setting. Further studies of APAP in this circumstance are needed. No studies have systematically compared the efficacy of one APAP technology with another. Devices using different technology may not give the same results in a given patient. Devices solely dependent on vibration may not work in non-snorers or patient who have undergone upper-airway surgery. High mask or mouth leaks may prevent adequate titration in devices monitoring snoring, flow, or impedance (forced oscillation technique). Review of the raw data to identify periods of high leak was performed in several of the APAP titration studies, to identify a pressure for fixed CPAP treatment or to determine if the titration was adequate. There is conflicting evidence for and against the premise that treatment with APAP increases acceptance and adherence compared to fixed CPAP. In studies demonstrating an increase in adherence with APAP, there was similar improvement in measures of daytime sleepiness as with fixed CPAP treatment. Further studies are needed to determine if APAP can increase acceptance or adherence with positive pressure treatment in patients with OSA.     

Virtual ambulatory care. Computer simulation applications

Computer simulation modeling has evolved during the past twenty years into an effective tool for analyzing and planning ambulatory care facilities. This article explains the use of this tool in three case-study, ambulatory care settings--a GI lab, holding beds for a cardiac catheterization laboratory, and in emergency services. These examples also illustrate the use of three software packages currently available: MedModel, Simul8, and WITNESS.     

The Dynamic Assessment and Referral System for Substance Abuse (DARSSA): development, functionality, and end-user satisfaction

The Dynamic Assessment and Referral System for Substance Abuse (DARSSA) conducts a computerized substance abuse assessment; prints personalized summary reports that include tailored substance abuse treatment referral lists; and, for individuals who provide authorization, automatically faxes their contact information to a "best match" substance abuse treatment provider (dynamic referral). After piloting the program and resolving problems that were noted, we enrolled a sample of 85 medical patients. The DARSSA identified 48 (56%) participants who were risky substance users, many of whom had not been identified during their routine medical assessment. Mean satisfaction scores for all domains ranged between "Good" to "Excellent" across patients, nurses, doctors, and substance abuse treatment providers. The median completion time was 13min. Of the 48 risky substance using participants, 20 (42%) chose to receive a dynamic referral. The DARSSA provides a user-friendly, desirable service for patients and providers. It has the potential to improve identification of substance abuse in medical settings and to provide referrals that would not routinely be provided. Future studies are planned to establish its efficacy at promoting treatment initiation and abstinence.     

Human papillomavirus (HPV) profiles of vulvar lesions: possible implications for the classification of vulvar squamous cell carcinoma precursors and for the efficacy of prophylactic HPV vaccination

The term vulvar intraepithelial neoplasia (VIN) introduced in 1986 incorporates 3 grades of usual VIN (u-VIN I-III) and the differentiated VIN (d-VIN). Although u-VIN is etiologically associated with the human papillomavirus (HPV) infection, d-VIN represents an alternative HPV negative pathway of vulvar carcinogenesis. In 2004, the u-VIN I category was abandoned and u-VIN II and III were merged. Further, an alternative Bethesda-like terminology scheme presenting the term vulvar intraepithelial lesion was proposed recently. To analyze the impact of HPV profiles of vulvar precancerous lesions for their classification and to assess the presumable efficacy of the prophylactic HPV vaccination, 269 vulvar excisions representing lichen sclerosus, lichen simplex chronicus, condylomata acuminata, d-VIN, all grades of u-VIN and squamous cell carcinomas were subjected to the HPV typing by use of GP5+/6+ polymerase chain reaction and reverse line blot hybridization. The results showed different HPV profiles, and also differing frequency of multiple-type HPV infection and the age structure in patients with u-VIN II and III. The biologic heterogeneity within the u-VIN II category was also demonstrated. u-VIN I was distinguished as a rare disorder associated with high-risk HPV infection. We conclude that the original VIN terminology proposed in 1986 seems to be appropriate for the classification of vulvar squamous dysplastic lesions. The spectrum of HPV types found in vulvar squamous cell carcinomas indicates that the efficacy of HPV vaccination in preventing vulvar cancer might be diminished in the studied population, because the recently developed prophylactic vaccines are targeted against a limited number of HPV types.     

Rapid Detection and Identification of Mucormycetes from Culture and Tissue Samples by Use of High-Resolution Melt Analysis

We present a method for rapid and simple detection of clinically relevant mucormycetes of the Mucorales order in cultures and clinical samples. This seminested real-time PCR uses mucormycete-specific primers and is followed by species identification using high-resolution melt (HRM) analysis. The method is highly suitable for routine clinical diagnostics.Invasive infections caused by mucormycetes started to occur more frequently in the last decade and are connected with rapid progression and high mortality rates. Early diagnostics and targeted treatment are crucial. Most mucormycosis cases (over 90%) are caused by Rhizopus spp., followed by Mucor spp., Lichtheimia spp., Rhizomucor pusillus, and, rarely, some other species (2, 9, 11, 16).Definitive diagnosis of mucormycosis is usually made after histopathological proof of mucormycete-like hyphae in involved tissue; the causative agent can be determined only by culture (13). So far, no serological test is available and radiological methods are nonspecific.Molecular detection of mucormycetes is complicated by several factors, and we still do not have any standard protocol. Few methods for the detection of mucormycetes have been published, and only some have been evaluated using clinical samples (1, 5, 10, 14, 15, 17) or samples from animal models (6, 7).The aim of this study was to develop a rapid and sensitive technique for the detection and identification of clinically important mucormycetes. We adopted primers from a qualitative method previously published by Bialek et al. (1) that is specific for members of the order Mucorales targeting 18S ribosomal DNA (rDNA). We modified it to seminested real-time PCR with EvaGreen dye, followed by species distinction by high-resolution melt (HRM) analysis. HRM analysis uses amplification of DNA in the presence of intercalation dye. Fluorescence is measured during a controlled melting of PCR product that results in a melt curve that depends mainly on GC content, length, and sequence of the PCR product. This simple method can be used for genotyping or mutation scanning without the need for time-consuming sequencing (4, 12).DNA was isolated from 50 μl of fungal culture (inoculum was prepared by covering sporulating colonies with approximately 2 ml of sterile 0.85% saline) or a piece of fresh tissue (2 by 1 mm) using the ZR fungal/bacterial DNA kit (Zymo Research). Tissue samples were incubated in lysis buffer overnight, and cultures were immediately processed according to the manufacturer''s protocol. Disruption was extended to 15 min (Disruptor Genie; Scientific Industries). DNA from formalin-fixed, paraffin wax-embedded (FFPE) tissue samples was isolated from 2 or 3 scrolls (5 to 10 μm each) of paraffin block using a DNeasy blood and tissue kit (Qiagen). Paraffin was dissolved in 1 ml of xylene, and then the tissue was washed two times using 1 ml of 96% ethanol and incubated in 180 μl of ATL buffer (Qiagen) and 20 μl of proteinase K (600 mAU/ml solution, where one mAU represents the activity of proteinase K that releases folin-positive amino acids and peptides corresponding to 1 μmol of tyrosine per min) at 55°C overnight and then at 90°C for 1 h. The next steps were done in accordance with the manufacturer''s protocol. DNA isolation from clinical samples was done in a biological safety cabinet. An aliquot of sterile water was processed with each set of samples as a control of potential contamination during the isolation process.Five microliters of DNA was amplified in 25 μl of amplification mixture that contained a 0.2 μM concentration each of primers ZM1 and ZM2 (1), 120 μM deoxynucleoside triphosphates (dNTPs; Roche, Germany), 2.5 mM MgCl₂, 1× GeneAmp PCR Gold buffer, and 1.5 U AmpliTaq Gold DNA polymerase (Applied Biosystems). The cycling conditions were 10 min at 95°C, 16 cycles of 30 s at 94°C, 30 s at 50°C, and 60 s at 72°C, and 7 min at 72°C. One microliter of PCR product from the external round was then amplified in duplicate using Rotorgene 6000 (Corbett Research, Australia). Twenty-five microliters of the amplification mixture contained a 0.4 μM concentration each of primers ZM1 and ZM3 (1), 12.5 μl of SensiMix HRM, and 1 μl of EvaGreen (both from a SensiMix HRM kit; Quantace, United Kingdom). The cycling conditions were 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, 20 s at 60°C, and 30 s at 72°C (acquired on the green channel), followed by HRM analysis (ramp from 74°C to 79.5°C, rising by 0.1°C each cycle, acquired on the HRM channel). Rotorgene 6000 series software (version 1.7) was used for analysis of the results. All positive results were confirmed by sequencing of the PCR product. DNA was purified using a QIAquick PCR purification kit (Qiagen, Germany) and sequenced using a BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems) on an ABI Prism 310 genetic analyzer (Applied Biosystems). Sequences were analyzed using the BLAST alignment program of the GenBank database.We used DNA extracted from five mucormycete cultures diluted in Tris-EDTA (TE) buffer as positive controls in every run. A DNA isolation control (sterile water processed with clinical samples) and a negative control of PCR (sterile water) were added to each run as well.In this study, we tested 31 fungal isolates, comprising 10 mucormycete isolates and 21 isolates from other filamentous fungal groups (Department of Clinical Microbiology, University Hospital Brno and Czech Collection of Microorganisms, Czech Republic). All mucormycete isolates were correctly identified. The melting temperatures (T_m) for each species were as follows: for Rhizopus microsporus, 76.46°C; for Rhizopus oryzae, 76.59°C; for Mucor racemosus, 76.78°C; for Mucor circinelloides, 76.98°C; for Rhizomucor pusillus, 77.87°C; and for Lichtheimia corymbifera, 78.56°C. Representative HRM curves for six different mucormycetes are shown in Fig. ​Fig.1.1. All HRM analysis results were confirmed by sequencing. None of the nonmucormycete fungi were positively tested. The results are summarized in Table ​Table11.Open in a separate windowFIG. 1.Representative result of high-resolution melt (HRM) analysis. Shown are HRM curves for six mucormycete isolates (black curves) and one negative and one positive tissue sample (gray curves).

TABLE 1.

List of fungal isolates used in this study and results of HRM analysis^a

Neurotoxic protein expression reveals connections between the circadian clock and mating behavior in Drosophila

To investigate the functions of circadian neurons, we added two strategies to the standard Drosophila behavioral genetics repertoire. The first was to express a polyglutamine-expanded neurotoxic protein (MJDtr78Q; MJD, Machado-Joseph disease) in the major timeless (tim)-expressing cells of the adult brain. These Tim-MJD flies were viable, in contrast to the use of cell-death gene expression for tim neuron inactivation. Moreover, they were more arrhythmic than flies expressing other neurotoxins and had low but detectable tim mRNA levels. The second extended standard microarray technology from fly heads to dissected fly brains. By combining the two approaches, we identified a population of Tim-MJD-affected mRNAs. Some had been previously identified as sex-specific and relevant to courtship, including mRNAs localized to brain-proximal fat-body tissue and brain courtship centers. Finally, we found a decrease in the number of neurons that expressed male-specific forms of the fruitless protein in the laterodorsal region of the brain. The decrease was not a consequence of toxic protein expression within these specialized cells but a likely effect of communication with neighboring TIM-expressing neurons. The data suggest a functional interaction between adjacent circadian and mating circuits within the fly brain, as well as an interaction between circadian circuits and brain-proximal fat body.