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AIM: This paper presents the findings of a staff survey conducted at a 350-bed acute care facility located on the periphery of Toronto, Canada. BACKGROUND: Toronto's severe acute respiratory syndrome (SARS) crisis resulted in trauma-like effects at hospitals hardest hit by the disease. A systematic examination of the impact on staff working in hospitals that saw relatively few cases, while maintaining the precautions associated with elevated alert levels, has not been undertaken. METHODS: A questionnaire was distributed for 1 month commencing 17 April 2003 and 300 completed responses were obtained (approximately one in six staff members). The data collected included demographic and occupational information, in addition to perceptions of SARS' impact on patient care, factors contributing to adverse impacts on patient care, working conditions, decision-making, communication and relations, sources of support, and the impact on workers' lives outside work. Items for these sections were developed by a multi-disciplinary team of health care workers and hospital administrators. RESULTS: In the absence of pre-SARS normative data for the survey, demographic and occupational variables were used to look for patterns of differences between relevant subgroups of respondents. Statistically significant differences were found for gender (73.9% women), nurses (24.7%) vs. others, doctors (20.3%) vs. others, older (40 years or older, 60.0%) vs. younger persons, emergency or intensive care unit workers (8.0%) vs. others, and those employed fewer years at the hospital (less than 5 years: 46.2%) vs. five or more years. These differences varied across the following domains: factors adversely affecting patients, communications, support, working conditions, decision-making and, to a lesser extent, impact on life outside work. While all groups found SARS stressful, nurses reported a greater impact on morale and job satisfaction. Nurses relied more on peer support than doctors, felt less informed and less involved in decision-making than doctors felt, and were more likely to report that infection control procedures were not strict enough. CONCLUSIONS: The between-group differences and the pattern of these differences clearly illustrate the polarizing and stressful impact SARS had at a hospital with only a small number of probable or suspect cases. The clear differences between groups defined by demographics, professions and clinical roles suggest a subtle and pervasive secondary impact of the SARS outbreak, with repercussions health care facilities must contend with while maintaining increased levels of vigilance in the wake of SARS. [ABSTRACT FROM AUTHOR]

We investigated the basic characteristics of a new murine cytomegalovirus (MCMV) vector platform. Using BAC technology, we engineered replication-competent recombinant MCMVs with deletions of up to 26% of the wild-type genome. To this end, we targeted five gene blocks (m01-m17, m106-m109, m129-m141, m144-m158, and m159-m170). BACs featuring deletions from 18% to 26% of the wild-type genome exhibited delayed virus reconstitution, while smaller deletions (up to 16%) demonstrated reconstitution kinetics similar to those of the wild type. Utilizing an innovative methodology, we introduced large genomic DNA segments, up to 35 kbp, along with reporter genes into a newly designed vector with a potential cloning capacity of 46 kbp (Q4). Surprisingly, the insertion of diverse foreign DNAs alleviated the delayed plaque formation phenotype of Q4, and these large inserts remained stable through serial in vitro passages. With reporter-gene-expressing recombinant MCMVs, we successfully transduced not only mouse cell lines but also non-rodent mammalian cells, including those of human, monkey, bovine, and bat origin. Remarkably, even non-mammalian cell lines derived from chickens exhibited successful transduction.

Importance: The influenza A virus genome consists of eight distinct viral RNAs (vRNAs) that are typically packaged into a single virion as an octameric complex. How this genome complex is assembled and incorporated into the virion is poorly understood, but previous research suggests a coordinative role for packaging signals present in all vRNAs. Here, we show that disruption of two packaging signals in a model H7N7 influenza A virus results in a mixture of virions with unusual vRNA content, including empty virions, virions with one to four vRNAs, and virions with octameric complexes composed of vRNA duplicates. Our results suggest that (i) the assembly of error-free octameric complexes proceeds through a series of defined vRNA sub-complexes and (ii) virions can bud without incorporating complete octameric complexes., Competing Interests: The authors declare no conflict of interest.

The homogeneity of the genetically modified single-cells is a necessity for many applications such as cell line development, gene therapy, and tissue engineering and in particular for regenerative medical applications. The lack of tools to effectively isolate and characterize CRISPR/Cas9 engineered cells is considered as a significant bottleneck in these applications. Especially the incompatibility of protein detection technologies to confirm protein expression changes without a preconditional large-scale clonal expansion creates a gridlock in many applications. To ameliorate the characterization of engineered cells, we propose an improved workflow, including single-cell printing/isolation technology based on fluorescent properties with high yield, a genomic edit screen (Surveyor assay), mRNA RT-PCR assessing altered gene expression, and a versatile protein detection tool called emulsion-coupling to deliver a high-content, unified single-cell workflow. The workflow was exemplified by engineering and functionally validating RANKL knockout immortalized mesenchymal stem cells showing bone formation capacity of these cells. The resulting workflow is economical, without the requirement of large-scale clonal expansions of the cells with overall cloning efficiency above 30% of CRISPR/Cas9 edited cells. Nevertheless, as the single-cell clones are comprehensively characterized at an early, highly parallel phase of the development of cells including DNA, RNA, and protein levels, the workflow delivers a higher number of successfully edited cells for further characterization, lowering the chance of late failures in the development process., Competing Interests: NO authors have competing interests.

Cervical cancer is a common malignant tumor worldwide ranking fourth in incidence and mortality among females, which was reduced significantly by cytology screening and human papilloma virus (HPV) DNA testing. The specificity of cytology is high; however, the sensitivity is low, in contrast to the HPV DNA testing. Despite the success of these measures, new biomarkers are still considered to aim increasing sensitivity and specificity of screening and diagnosis. Significant alterations in microRNA (miRNA) expression have been detected in several cancers with variable consistency. To investigate the stratification role of miRNAs between normal epithelium and cervical intraepithelial neoplasia (CIN2-3), we screened the expression of 667 miRNAs to identify significant markers (n = 10), out of them 9 miRNAs were applied in the study (miR-20b, -24, -26a, -29b, -99a, -100, -147, -212, -515-3p) along with RNU48 and U6 as the references. To benchmark the miRNAs, 22 paired (tumor-free and tumor tissue pairs) laser microdissection-obtained cervical formalin fixed, paraffin embedded tissue samples were assayed. The expression of miR-20b was 2.4 times higher in CIN2-3 samples as compared to normal tissues (p < 0.0001). In the HPV16-positive subsets of the samples (n = 13), miR-20b showed 2.9-times elevation (p < 0.001), whereas miR-515 was 1.15-times downregulated (p < 0.05) in CIN2-3 as compared to normal tissue. These results suggest the potential value of miR-20b as a statification biomarker in order to differentiate neoplastic and non-tumorous cases.

Several biomarkers are in use to improve the sensitivity and specificity of cervical cancer screening. Previously, increased expression of tight junction protein claudin-1 (CLDN1) was detected in premalignant and malignant cervical lesions and applied for cytology screening. To improve the specificity, a double immunoreaction with CLDN1/Ki67 was developed in the recent study. Parallel p16/Ki67 (CINtec® PLUS) and CLDN1/Ki67 dual-stained cytology and histology were performed and compared. p16/Ki67 immunoreaction showed positivity in 317 out of 1596 smears with negativity in 1072 and unacceptable reactions in 207 samples. CLDN1/Ki67 dual staining was positive in 200 of 1358 samples, negative in 962, whereas 196 smears could not be evaluated due to technical reasons. Considering the high-grade squamous intraepithelial lesion cytology as gold standard, sensitivity of CLDN1/Ki67 reaction was 76%, specificity was 85.67%, while for p16/Ki67 sensitivity was 74% and specificity was 81.38%. Comparison of CLDN1/Ki67 and p16/Ki67 dual stainings showed the results of the two tests not to be significantly different. Analysing histological slides from 63 cases, the results of the two tests agreed perfectly. As conclusion the sensitivity and specificity proved to be similar using p16/Ki67 and CLDN1/Ki67 double immunoreactions both on LBC samples and on histological slides.

Zoonotic influenza A viruses of avian origin can cause severe disease in individuals, or even global pandemics, and thus pose a threat to human populations. Waterfowl and shorebirds are believed to be the reservoir for all influenza A viruses, but this has recently been challenged by the identification of novel influenza A viruses in bats 1,2 . The major bat influenza A virus envelope glycoprotein, haemagglutinin, does not bind the canonical influenza A virus receptor, sialic acid or any other glycan 1,3,4 , despite its high sequence and structural homology with conventional haemagglutinins. This functionally uncharacterized plasticity of the bat influenza A virus haemagglutinin means the tropism and zoonotic potential of these viruses has not been fully determined. Here we show, using transcriptomic profiling of susceptible versus non-susceptible cells in combination with genome-wide CRISPR-Cas9 screening, that the major histocompatibility complex class II (MHC-II) human leukocyte antigen DR isotype (HLA-DR) is an essential entry determinant for bat influenza A viruses. Genetic ablation of the HLA-DR α-chain rendered cells resistant to infection by bat influenza A virus, whereas ectopic expression of the HLA-DR complex in non-susceptible cells conferred susceptibility. Expression of MHC-II from different bat species, pigs, mice or chickens also conferred susceptibility to infection. Notably, the infection of mice with bat influenza A virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant. Collectively, our data identify MHC-II as a crucial entry mediator for bat influenza A viruses in multiple species, which permits a broad vertebrate tropism.

In the era of primary vaccination against HPV and at the beginning of the low prevalence of cervical lesions, introduction of screening methods that can distinguish between low- and high-grade lesions is necessary in order to maintain the positive predictive value of screening. This case-control study included 562 women who attended cervical screening or were referred for colposcopy and 140 disease free controls, confirmed by histology and/or cytology. The cases were stratified by age. Using routine exfoliated liquid based cytological samples RT-PCR measurements of biomarker genes, high-risk HPV testing and liquid based cytology were performed and used to evaluate different testing protocols including sets of genes/tests with different test cut-offs for the diagnostic panels. Three new panels of cellular biomarkers for improved triage of hrHPV positive women (diagnostic panel) and for prognostic assessment of CIN lesions were proposed. The diagnostic panel (PIK3AP1, TP63 and DSG3) has the potential to distinguish cytologically normal hrHPV+ women from hrHPV+ women with CIN2+. The prognostic gene panels (KRT78, MUC5AC, BPIFB1 and CXCL13, TP63, DSG3) have the ability to differentiate hrHPV+ CIN1 and carcinoma cases. The diagnostic triage panel showed good likelihood ratios for all age groups. The panel showed age-unrelated performance and even better diagnostic value under age 30, a unique feature among the established cervical triage tests. The prognostic gene-panels demonstrated good discriminatory power and oncogenic, anti-oncogenic grouping of genes. The study highlights the potential for the gene expression panels to be used for diagnostic triage and lesion prognostics in cervical cancer screening.

The ongoing Triage and Risk Assessment of Cervical Precancer by Epigenetic Biomarker (TRACE) prospective, multicenter study aimed to provide a clinical evaluation of the CONFIDENCE™ assay, which comprises a human papillomavirus (HPV) DNA and a human epigenetic biomarker test. Between 2013 and 2015 over 6,000 women aged 18 or older were recruited in Hungary. Liquid-based cytology (LBC), high-risk HPV (hrHPV) DNA detection and single target host gene methylation test of the promoter sequence of the POU4F3 gene by quantitative methylation-specific polymerase chain reaction (PCR) were performed from the same liquid-based cytology sample. The current analysis is focused on the baseline cross-sectional clinical results of 5,384 LBC samples collected from subjects aged 25 years or older. The performance of the CONFIDENCE HPV™ test was found to be comparable to the cobas® HPV test with good agreement. When applying the CONFIDENCE Marker™ test alone in hrHPV positives, it showed significantly higher sensitivity with matching specificity compared to LBC-based triage. For CIN3+ histological endpoint in the age group of 25-65 and 30-65, the methylation test of POU4F3 achieved relative sensitivities of 1.74 (95% CI: 1.25-2.33) and 1.64 (95% CI: 1.08-2.27), respectively, after verification bias adjustment. On the basis of our findings, POU4F3 methylation as a triage test of hrHPV positives appears to be a noteworthy method. We can reasonably assume that its quantitative nature offers the potential for a more objective and discriminative risk assessment tool in the prevention and diagnostics of high-grade cervical intraepithelial neoplasia (CIN) lesions and cervical cancer., (© 2016 UICC.)

Background: In the 1990s, azithromycin became the drug of choice for many infectious diseases but emerging resistance to the drug has only been reported in the last decade. In the last 5 years, the National Neisseria gonorrhoeae Reference Laboratory of Hungary (NNGRLH) has also observed an increased number of N. gonorrhoeae strains resistant to azithromycin. The aim of this study was to determine the most frequent sequence types (ST) of N. gonorrhoeae related to elevated levels of azithromycin MIC (minimal inhibitory concentration). Previously and currently isolated azithromycin-resistant strains have been investigated for the existence of molecular relationship., Methods: Maldi-Tof technic was applied for the identification of the strains isolated from outpatients attending the reference laboratory. Testing antibiotic susceptibility of azithromycin, cefixime, ceftriaxone, tetracycline, spectinomycin and ciprofloxacin was carried out for all the identified strains, using MIC strip test Liofilchem(®). N. gonorrhoeae multiantigen sequence typing (NG-MAST) was performed exclusively on azithromycin-resistant isolates. A phylogenetic tree was drawn using MEGA6 (Molecular Evolutionary Genetics Analysis Version 6.0) Neighbour-Joining method., Results: Out of 192 N. gonorrhoeae isolates, 30.0 % (58/192) proved resistant to azithromycin (MIC > 0.5 mg/L). Of the azithromycin-resistant isolates, ST1407, ST4995 and ST11064 were the most prevalent. Based on the phylogenetic analysis, the latter two STs are closely related., Conclusions: In contrast to West-European countries, in our region, resistance to azithromycin has increased up to 30 % in the last 5 years, so the recommendation of the European Guideline -500 mg of ceftriaxone combined with 2 g of azithromycin as first choice therapy against N. gonorrhoeae- should be seriously considered in case of Hungary.

Several immunochemistry tests are used for triaging human papilloma virus (HPV) and cytology positive cases in cervical cancer screening and as an adjunct test to diagnose cervical cancer. Claudin-1 (CLDN1) protein is a major component of the tight junction, shown to have altered expression in cervical cancer. In this study, value of CLDN1 was analysed as a screening and triage immunochemistry test compared to cytology and HPV testing. A population of 352 women attending colposcopic referral visits resulting in cervical conisation and a second population of 150 women attending routine gynaecological visits with negative cervical cytology were enrolled in a multi-centre clinical study in Hungary. Cytology and HPV (Genoid Full Spectrum HPVAmplification and Detection System) testing were carried out along with immunocytochemistry for CLDN1, and as a reference, using CINtec p16 Cytology Kit. Three different evaluation protocols were used which assessed immunostaining characteristics with or without cytological readings. High correlation observable between p16INK4a and CLDN1 established CLDN1 as a competing marker in cervical cancer. Concordance of CLDN1 immunostaining of cervical intraepithelial neoplasia 2 and above (CIN2+) positives was 84.0 % (73.8–89.3); concordance of CIN2+ negatives was 69.0 % (59.6–75.8). In conclusion, CLDN1 has similar diagnostic potential as p16INK4a, our results established it as a histological and cytological biomarker with the potential to improve the clinical performance of cervical cytology and histology.

Background: Entrustable professional activities (EPAs) are routine tasks considered essential to a professional practice. An EPA can serve as a performance-based outcome that a clinical supervisor would progressively entrust a learner to perform., Objective: Our aim was to identify, develop, and validate a set of EPAs for the palliative medicine discipline., Methods: The design was a sequential qualitative and quantitative mixed methods study. A working group was convened to develop a set of EPAs. Focus groups and surveys were used for validation purposes. Palliative medicine educators and content experts from across Canada participated in both the working group as well as the focus groups. Attendees of the 2014 Canadian Society of Palliative Care Physicians (CSPCP) annual conference completed surveys. A questionnaire was used to collect survey participant sociodemographic, clinical, and academic information along with ratings of the importance of the EPAs individually and collectively. Cronbach's alpha examined internal consistency of the set of EPAs., Results: Focus group participants strongly endorsed the 12 EPAs. Virtually all survey participants rated the individual EPAs as being "fairly/very important" (range 94% to 100%). Of the participants, 97% agreed that residents able to perform the set of EPAs would be practicing palliative medicine and 87% indicated strong agreement that this collective set of EPAs captures activities that all palliative medicine physicians must be able to perform. A Cronbach's alpha of 0.841 confirmed good internal consistency., Conclusions: Near uniform agreement from a national group of palliative medicine physicians provides strong validation for the set of 12 EPAs.

Data discussed in recent reviews demonstrated that dysregulation of microRNA (miRNA) expression profiles occurs during cervical carcinogenesis and characteristic up- or downregulation of certain miRNAs might be used as biomarkers. The majority of altered miRNAs, however were found to be inconsistent upon comparison with cancerous and normal cervical epithelia in the discussed studies due to several reasons. The results obtained in this present review suggest the need for further investigations on miRNAs on larger sample sizes in order to indicate sensitivity and specificity by means of well defined, "unified" methods. In addition, obtaining further data on the clinical course and outcome of patients in comparison to the dysregulation of miRNA expression profile could turn miRNAs into prognostic and/or progression markers. Inhibition of overexpressed miRNAs, as suggested by some authors, might even serve as target for cancer therapy.

Cervical screening programmes are moving towards HPV testing as part of the screening process and as a triage for colposcopy. Three HPV detection methods were evaluated using cervical cytology specimens from colposcopy patients. PreservCyt™ liquid based cytology specimens from 241 women attending colposcopy clinics with greater than 2 persistently abnormal smears were recruited through the Coombe Women and Infants University Hospital, Dublin. HPV DNA was detected by Hybrid Capture (HC2) for 13 high-risk HPV types, Full-Spectrum HPV (FS-HPV) for 49 high and low-risk types and Molecular Beacon Real-Time HPV assay (MBRT-HPV) for 16 high and low-risk types. HPV genotyping was performed using Linear Array HPV Assay (LA-HPV). HPV was detected in 83.3% (195/234), 91.9% (217/236) and 80.1% (169/211) of cytology specimens by HC2, FS-HPV and MBRT-HPV, HPV DNA detection assays. The sensitivity of the assays for the detection of high-risk HPV in cytology specimens that had a Cervical Intraepithelial Neoplasia Grade 2+ result by histology were, 98%, 97% and 94% for HC2, FS-HPV and MBRT-HPV assays with positive predictive values of 94.1%, 94.1% and 97.3%. The most common HPV genotypes were HPV 16, 31, 33, 58, 42, 61 and 53, and the most common high-risk HPV genotypes were HPV 16, 31, 33, 58, 18, 45, 59, 51, 56 and 39, with detection of multiple infections in 57.7% of all cases. FS-HPV and MBRT-HPV are highly sensitive and have a similarly high PPV as the HC2 assay for detection of HPV in patients with Cervical Intraepithelial Neoplasia Grade 2+ disease. HPV genotyping of women with persistent abnormalities is warranted prior to the introduction of HPV DNA testing in a colposcopy setting., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Recently, a PCR-derived method for serotyping Streptococcus pneumoniae has been devised to substitute the conventional antiserum phenotypic method. The method initially used a multiplex PCR reaction, dividing the isolates into 6 different groups based on the detected PCR gel pattern. In order to optimise and refine this crucial step, the Taguchi technique was employed, which can evaluate the individual effect of six parameters (in this case: primers, MgCl2, nucleotide mix, polymerase and buffer), with only 18 experiments; varying the parameter levels in an orthogonal matrix which suppresses the interactions between them. With this method, clear and sharp bands were observed in 5 experiments out of the 18, while the PCR did not work reliably in the remaining cases. In addition, the PCR-based technique could be rendered more economic by the 10-fold lowering of the quantities of two primers. The modified reaction yielded identical results to those obtained with the original method. Furthermore, we have designed serotype-specific primers for 11 new serotypes. The most important ones are those that can distinguish the very closely related, but equally important serotypes 6A and 6B.

Inhibition of the renin-angiotensin and endothelin (ET) systems prevents the development of obliterative airway disease (OAD) in rat tracheal allografts. In this study, we assessed whether these therapeutic approaches are effective even when the same were started after signs of OAD were already manifest. Rat tracheas were heterotopically transplanted from Brown-Norway donors into Brown-Norway or Lewis recipients. Allograft recipients received bosentan, ramipril, bosentan plus ramipril or vehicle from day 10 to 24. Untreated allografts and isografts were harvested at day 10 or 24. In tracheal grafts, morphometric studies together with molecular analysis by real-time PCR were performed. Fibroproliferative process in untreated tracheal allografts but not in isografts started already at day 10. Neither bosentan nor ramipril treatment alone as monotherapy could modify the development of OAD when administered only between day 10 and day 24. By contrast, the combination treatment of bosentan and ramipril ameliorated airway obstruction by day 24, which was accompanied by reduced mRNA expression of intragraft transforming growth factor-beta1 and platelet-derived growth factor-A and -B chains. Only the combined blockade with angiotensin-converting enzyme inhibitors and ET receptor antagonists can reduce the progression of OAD in this model if the treatment is initiated late in the disease course.

Detection of HPV infections requires a robust time-effective single-step method for efficient screening. A molecular beacon-based one-step multiplex real-time PCR system was developed to detect 15 high-risk (HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) and 5 low-risk HPV types (HPV types 6, 11, 42, 43, 44). Molecular beacons detecting high-risk types are 5'-FAM-3'-DABCYL-labelled, molecular beacons for low-risk detection are 5'-TET-3'-DABCYL-labelled, while the internal control added before sample DNA extraction is detected by a 5'-FAM-TexasRed-3'-DABCYL wavelength-shifting molecular beacon. Accordingly, fluorescent data for HPV detection are collected at 530 nm for high-risk types, 560 nm in case of low-risk types and the reaction internal control is detected at 610 nm on a Roche LightCycler 2.0 instrument. The sensitivity for detected types varies between 22 and 700 copies/reaction. The clinical performance was tested on 161 clinical sample DNAs. The MB-RT PCR results were compared to the typing results obtained by the L1F/L1R PCR and hybridization-based system described previously, and the concordance rate between the two systems was 89.44%. The favorable characteristics shown by this multiplex single-step real-time HPV detection system make this promising approach worthy for further development and application for clinical screening.

Pregnant women were examined following healthy pregnancies at term. Amniotic fluids were sampled before arteficial rupture of membranes using closed vacutainer system. Blood samples were also taken from the pregnants simultaneously. Endotoxin concentrations of amniotic fluids were tested by the semiquantitative Limulus amebocyte lysate. Both amniotic fluids and blood samples were tested for the presence of DNA of lymphotropic human herpesviruses. The DNA of human papillomaviruses were tested only in the amniotic fluid samples. One-third of the amniotic fluids tested were found to contain measurable amounts of endotoxin. Lymphotropic herpesvirus DNA was deteced in every fourth amniotic fluid sample and in every 8th blood sample. The prevalence of papillomaviruses was 7 of 96 samples. No significant correlation was found between the presence of endotoxin and viruses in the amniotic fluids. Epstein-Barr virus, human cytomegalovirus and human herpesvirus type 7 were found more frequently in the amniotic fluids than in blood samples (7 to 1). The prevalence of human herpesvirus 6 and 8 was higher in the blood samples than that in the amniotic fluids. The mean weight of the neonates were not impaired significantly by the presence of either viruses or endotoxin. Possible post partum consequences, i.e. partial immunotolerance to viruses is discussed.

There are several genital HPV DNA detection systems described, however most of them present different disadvantages regarding the number of amplified and detected types, sensitivity, type specificity. A new PCR and hybridization based detection method was developed for sensitive and balanced amplification and specific typing of HPV DNA from clinical samples. The technique amplifies and detects 46 HPV types: 2a, 3, 6, 7, 10, 11, 13, 16, 18, 26, 27, 28, 29, 30, 31, 33, 34, 35, 39, 40, 42, 43, 44/55, 45, 51, 52, 53, 54, 56, 57, 58, 59, 61, 66, 67, 68, 70, 72, 73(MM9), 74, 82(MM4), 84(MM8), 89, 90, 91. Key elements of the L1F/L1R PCR and hybridization system are: the special selection of the amplified region, a novel and optimized amplification primer set, circumspectly designed general and type-specific oligonucleotide probes. Detection following a multiplex PCR is based on solid phase hybridization in microtiter plate format using general and type-specific probes at medium stringency, which makes the detection robust in case of small sequence variations. The assay is highly reproducible and suitable for automation. The method was compared to Hybrid Capture II test, and after clarifying conflicting results, the comparison showed an excellent agreement (96.2%).

The aim of this study was to test whether donor and recipient gender influence the development of obliterative airway disease in an established rat model of human bronchiolitis obliterans. Rat tracheas were transplanted from male and female Brown-Norway donors into male and female Lewis recipients. Grafts were harvested at days 7, 14 and 21 for morphometric studies and molecular analysis by real-time PCR. At each time point, both epithelial injury and the extent of luminal occlusion of the tracheal allografts were similar between the groups. Furthermore, intragraft mRNA expression for transforming growth factor (TGF)-beta1, interleukin (IL)-2 and interferon (INF)-gamma did not differ between the groups. Our data suggest that gender has no impact on the development of obliterative airway disease in this transplant model.

Background: Blockade of the renin-angiotensin and the endothelin (ET) systems ameliorates fibrous airway obliteration in rat tracheal allografts. The aim of this study was to test which of these pharmacologic interventions attenuates the process more effectively, and whether combination therapy confers additional benefit., Methods: Rat tracheas were heterotopically transplanted from Brown-Norway donors into Lewis recipients. Recipients received the dual endothelin-1 (ET-1) receptor blocker, bosentan (100 mg/kg), the angiotensin-converting enzyme (ACE) inhibitor, ramipril (5 mg/kg), bosentan plus ramipril (doses as just noted) or vehicle. Grafts were harvested at Days 7 and 21 for morphometric studies and molecular analysis by real-time polymerase chain reaction (PCR)., Results: At Day 21, bosentan and ramipril treatment reduced the percentage of luminal occlusion compared with vehicle to a similar extent. In animals that received the combined treatment, luminal obliteration was further reduced compared with the respective monotherapies. Furthermore, rats treated with bosentan plus ramipril showed a lower percent of epithelial necrosis. The beneficial effects of bosentan and ramipril, alone or in combination, were associated with reduced mRNA levels of transforming growth factor (TGF)-beta1 and platelet-derived growth factor (PDGF)-A in the tracheal allografts., Conclusions: Our data suggest that ET receptor blockade prevents fibrous airway obliteration to a similar extent as ACE inhibition in this model. Interruption of both pathways provides superior graft protection as compared with single-system blockade.

Objective: Elaboration of an empiric antibiotic regimen for women with pelvic inflammatory disease (PID) for a geographical area in eastern Hungary., Study Design: Pathogens were identified by culturing or polymerase chain reaction (PCR) from 2215 patients with suspected PID between 1 January 1999 and 31 December 2001. Empiric guidelines for PID treatment were based on susceptibility testing of the recovered bacteria, patient acceptance and cost-effectiveness of drugs and recommendations of earlier studies., Results: Chlamydia trachomatis was detected in 11%, Neisseria gonorrhoeae in 2%, Streptococcus spp. in 17%, Enterococcus spp. 9%, genital mycoplasmas in 25%, all obligate anaerobic pathogens in 30% of the patients. All antibiotics chosen for our regimen were effective in vitro against one or more recovered pathogens at least in 80%; this regimen produced 98% clinical cure rate in mild cases of PID., Conclusion: Early detection and prompt empirical antimicrobial therapy adapted to the local microflora and its resistance pattern can lead to good clinical results.

The acquisition of human papillomavirus (HPV), the most important etiological agent of cervical cancer, does not cause clinical complaints. Although HPV spreads together with agents causing pelvic inflammatory disease (PID) with complaints forcing the patient to seek medical advice, PID has not yet been evaluated as a predictor of cervical cancer. The present study aimed to determine the relationship between PID and HPV in order to evaluate the possible risk factor role of PID for cervical cancer. Two groups of patients were studied: (i) 2,215 women with PID; (ii) 4,217 women participating in a cervical cancer screening programme who were found to have cytological atypia, mucopurulent cervicitis or other colposcopically detected disorders but were free of symptoms of PID. The presence of HPV and other STD agents in cervical smears was detected with polymerase-chain reaction. HPV prevalence was 33.74% in patients with PID and 26.40% in the group of women without PID (p < 0.001). This suggests that patients suffering from PID apparently have a higher risk of cervical cancer.

The effects of bafilomycin A1 and of the reduced level of endosomal epsilon-COP (coatomer protein) on the infectivity of human adenovirus type 5 were investigated in Coxsackie adenovirus receptor- (CAR-) transfected Chinese hamster ovary (CHO) cells. The endosomal proton pump inhibitor bafilomycin A1 was able to cause only partial inhibition. Using Id1F cells (an epsilon-COP thermosensitive mutant CHO cell line) the reduction of epsilon-COP level also had partial inhibitory effect. Based on these results and comparing them to existing models of the adenovirus entry, we propose a refined model in which there are two pathways of adenoviral entry: the first one involves the epsilon-COP as the downstream effector of the acidification and can be blocked by bafilomycin A1 and the second one is a pH-independent pathway.

To study the role of extracellular-signal-regulated kinase (ERK) cascade and the small GTP-ase proteins in the activation of the c-fos promoter by angiotensin II (AII), transient transfection experiments were performed in CHO cells stably expressing the rat AT(1A) receptor. In this system AII activated ERK in 1 min and also increased the transcriptional activity of the c-fos promoter-luciferase reporter gene construct. The activation of the promoter proved to be dependent on the Ras-Raf-ERK cascade as cotransfection of expression vectors known to specifically inhibit this cascade blocked the effect of AII. Dominant-negative p21Rac1 mutant partially blocked the activation of the c-fos promoter by AII. However, activation of the c-fos promoter was independent of protein kinase C (PKC) as bisindolylmaleimide I, a specific PKC inhibitor did not block the effect of AII. These results suggest that AII activates the transcription of the c-fos through the Ras-Raf-ERK cascade. Furthermore, p21Rac1 is involved in the modulation of the c-fos promoter by AII., (Copyright 2001 S. Karger AG, Basel)

The widespread presence of adenoviruses in various species makes it probable that infection and the carrier state also exist in cats. On the basis of these considerations, investigations were carried out to find antibodies against adenovirus in sera from different cat populations kept under different conditions. For the antibody detection, purified adenovirus was used in an indirect ELISA. To produce positive serum, SPF cats were immunized with a purified hexon preparation. Altogether 632 field sera of different origin were tested. Among field samples, adenovirus seropositivity varied between 10-26%.

Fifteen nosocomial cases of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae occurred among 132 neonates in a premature intensive care unit in Hungary in June through November 1998. Fourteen strains were indistinguishable by molecular biological typing and harbored the same single conjugative extended-spectrum beta-lactamase-encoding plasmid that was spontaneously found in a Serratia marcescens strain in the same patient.

Plasmids expressing the human cytomegalovirus (HCMV) glycoprotein B (gB) (UL55) or phosphoprotein 65 (pp65) (UL83) were constructed and evaluated for their ability to induce immune responses in mice. The full-length gB as well as a truncated form expressing amino acids 1-680 of gB, and lacking the fragment encoding amino acids 681 907 including the transmembrane domain of gB (gB680) were evaluated. Immunization of mice with plasmids coding for gB or gB680 induced ELISA and neutralizing antibodies, with the highest titres in mice immunized with the gB680 plasmid. Mice immunized with the gB plasmid predominantly produced IgG2a gB-specific antibody, while the gB680 plasmid raised mostly IgG1 anti-gB antibody. Mice immunized with the pp65 plasmid developed pp65-specific cytotoxic T lymphocytes (CTL) and ELISA antibodies. Immunization with a mixture of both gB and pp65 plasmids raised antibodies to both proteins and pp65-specific CTL, indicating a lack of interference between these two plasmids. These results suggest that DNA immunization is a useful approach for vaccination against HCMV disease.

Agarose gel electrophoresis and subsequent purification of DNA bands from the agarose gel is a widely used molecular biological method. There are different methods to achieve this goal, however they have different advantages and disadvantages. One major problem is the presence of different contaminants in the final sample. We developed a method which is effective in removal of the agarose contaminants.