Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies.

The BCL2 inhibitor venetoclax has been approved to treat different hematological malignancies. Because there is no common genetic alteration causing resistance to venetoclax in chronic lymphocytic leukemia (CLL) and B-cell lymphoma, we asked if epigenetic events might be involved in venetoclax resistance. Therefore, we employed whole-exome sequencing, methylated DNA immunoprecipitation sequencing, and genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 screening to investigate venetoclax resistance in aggressive lymphoma and high-risk CLL patients. We identified a regulatory CpG island within the PUMA promoter that is methylated upon venetoclax treatment, mediating PUMA downregulation on transcript and protein level. PUMA expression and sensitivity toward venetoclax can be restored by inhibition of methyltransferases. We can demonstrate that loss of PUMA results in metabolic reprogramming with higher oxidative phosphorylation and adenosine triphosphate production, resembling the metabolic phenotype that is seen upon venetoclax resistance. Although PUMA loss is specific for acquired venetoclax resistance but not for acquired MCL1 resistance and is not seen in CLL patients after chemotherapy-resistance, BAX is essential for sensitivity toward both venetoclax and MCL1 inhibition. As we found loss of BAX in Richter's syndrome patients after venetoclax failure, we defined BAX-mediated apoptosis to be critical for drug resistance but not for disease progression of CLL into aggressive diffuse large B-cell lymphoma in vivo. A compound screen revealed TRAIL-mediated apoptosis as a target to overcome BAX deficiency. Furthermore, antibody or CAR T cells eliminated venetoclax resistant lymphoma cells, paving a clinically applicable way to overcome venetoclax resistance.

Overall survival in the OlympiA phase III trial of adjuvant olaparib in patients with germline pathogenic variants in BRCA1/2 and high-risk, early breast cancer.

BACKGROUND: The randomized, double-blind OlympiA trial compared 1 year of the oral poly(adenosine diphosphate-ribose) polymerase inhibitor, olaparib, to matching placebo as adjuvant therapy for patients with pathogenic or likely pathogenic variants in germline BRCA1 or BRCA2 (gBRCA1/2pv) and high-risk, human epidermal growth factor receptor 2-negative, early breast cancer (EBC). The first pre-specified interim analysis (IA) previously demonstrated statistically significant improvement in invasive disease-free survival (IDFS) and distant disease-free survival (DDFS). The olaparib group had fewer deaths than the placebo group, but the difference did not reach statistical significance for overall survival (OS). We now report the pre-specified second IA of OS with updates of IDFS, DDFS, and safety. PATIENTS AND METHODS: One thousand eight hundred and thirty-six patients were randomly assigned to olaparib or placebo following (neo)adjuvant chemotherapy, surgery, and radiation therapy if indicated. Endocrine therapy was given concurrently with study medication for hormone receptor-positive cancers. Statistical significance for OS at this IA required P < 0.015. RESULTS: With a median follow-up of 3.5 years, the second IA of OS demonstrated significant improvement in the olaparib group relative to the placebo group [hazard ratio 0.68; 98.5% confidence interval (CI) 0.47-0.97; P = 0.009]. Four-year OS was 89.8% in the olaparib group and 86.4% in the placebo group (Δ 3.4%, 95% CI -0.1% to 6.8%). Four-year IDFS for the olaparib group versus placebo group was 82.7% versus 75.4% (Δ 7.3%, 95% CI 3.0% to 11.5%) and 4-year DDFS was 86.5% versus 79.1% (Δ 7.4%, 95% CI 3.6% to 11.3%), respectively. Subset analyses for OS, IDFS, and DDFS demonstrated benefit across major subgroups. No new safety signals were identified including no new cases of acute myeloid leukemia or myelodysplastic syndrome. CONCLUSION: With 3.5 years of median follow-up, OlympiA demonstrates statistically significant improvement in OS with adjuvant olaparib compared with placebo for gBRCA1/2pv-associated EBC and maintained improvements in the previously reported, statistically significant endpoints of IDFS and DDFS with no new safety signals.

Clinical features of gastroenteritis during a large waterborne Campylobacter outbreak in Askøy, Norway.

PURPOSE: Outbreaks of Campylobacter infection are common, but studies exploring the clinical features of acute illness in the outbreak setting are scarce in existing literature. The main purpose of the present study was to investigate the clinical features of self-reported acute illness in gastroenteritis cases during a large waterborne Campylobacter outbreak in Askøy municipality, Norway, in 2019. METHODS: A web-based self-administered questionnaire, and invitation to participate was sent by the municipality of Askøy as text message to mobile phones using the municipality's warning system to the inhabitants during the ongoing outbreak. RESULTS: Out of 3624 participants, 749 (20.7%) were defined as cases, of which 177 (23.6%) reported severe gastroenteritis. The most common symptoms were loose stools (90.7%), abdominal pain (89.3%) and diarrhea (88.9%), whereas 63.8% reported fever, 50.2% joint pain and 14.2% bloody stools. Tiredness, a symptom non-specific to gastroenteritis, was the overall most common symptom (91.2%). CONCLUSION: About one in four of the cases reported symptoms consistent with severe gastroenteritis. We found more joint pain and less bloody stools than reported in published studies of laboratory confirmed campylobacteriosis cases. Tiredness was common in the current study, although rarely described in previous literature of acute illness in the outbreak setting.

Exploring the multi-fractal nature of the air flow and pollutant dispersion in a turbulent urban atmosphere and its implications for long range pollutant transport.

This work investigates the multi-fractal nature of a turbulent urban atmosphere using high-resolution atmospheric data. Meteorological and concentration measurements of passive and reactive pollutants collected over a 3-year period in a sub-urban high-Reynolds number atmospheric field were analyzed. Scaling laws characterizing the self-similarity and thereby depicting the multi-fractal nature are determined by calculating the singularity spectra, where a range of Hölder exponents, h, are estimated. In doing so, the complexity of the urban atmosphere entailing different stability regimes was addressed. Using the Monin-Obukhov length (LMO) as a marker of atmospheric stability and thereby an indication of the magnitude of anisotropy, we find where and how self-similarity is manifested relative to the different regimes and we estimate corresponding appropriate scaling laws. We find that the wind speed obeys the -5/3 law suggested by Kolmogorov only when the atmosphere lies within the stable regime as defined by Monin-Obukhov theory. Specifically, when the ratio of the atmospheric boundary layer height (Hb.l) over LMO is greater than 15, and at the same time, the ratio of the height above ground of the wind measurements (z0) over LMO is higher than 3 (i.e., in stable regime), then the singularity spectra of wind speed time series indicate that the dominant Hölder exponent, hmax, coincides with Kolmogorov's second hypothesis. On the contrary under unstable regimes in the atmosphere where the anisotropy is approached, different scaling laws are estimated. In detail, when z0/LMO<0, the dominant Hölder exponent, hmax, of the singularity spectra of the wind speed time series is either negative or close to zero, which is an indication of an impulse-like singularity, that is associated with rapid changes. For the ambient temperature and air quality measurements such as of carbon monoxide and particulate matter concentrations, it was found that they obey different laws, which are related with the long-term correlation of their data fluctuation.

[Renal abscess in children].

INTRODUCTION: Renal abscesses are relatively rare in children, but they can lead to prolonged hospital stay and life-threatening complications. Scrutiny of the literature over the past two decades indicates the absence of a unified tactic for the treatment of purulent-destructive forms of pyelonephritis in children, while more and more articles are appearing in terms of a low -key approach to the treatment of renal abscess in children. MATERIALS AND METHODS: From 2005 to 2019, we treated 59 children with the renal abscess. Among the patients were 22 (37.3%) boys and 37 (62.3%) girls. The location of the abscess on the right was determined in 30 (50.8%) children, on the left, in 29 (49.2%). The average age of the patients was 109 months. The median size of kidney abscess among all patients was 29 [21; 42] mm (range from 12 to 69 mm). RESULTS: The results of treatment were evaluated in the period from 3 months to 5 years. In 27 (45.8%) patients, conservative treatment gave a positive effect, while in 32 (54.2%) abscess puncture was performed under ultrasound guidance. The median hospitalization in patients after an abscess puncture was 15 [14; 18] days, against 13 [9; 17] days for children receiving only antibiotic therapy. The duration of hospital stay was significantly longer in the group of patients who underwent puncture (p=0.019). The effectiveness of conservative therapy was a lot lower in patients with a kidney abscess of more than 3 cm, 60% versus 31% (p=0.026). All 59 patients recovered completely, and none of them required an open surgery to drain a suppurative focus of a kidney or nephrectomy. CONCLUSION: Conclusion. Our experience confirms the literature data, indicating the need to use a conservative approach to the treatment of patients with the renal abscess as a first-line therapy. Identification of an abscess with a diameter of more than 3 cm in patients considerably increases the likelihood of using an abscess puncture with the absence of the efficacy of a conservative approach.

Declining levels of serum chemokine (C-X-C motif) ligand 10 over time are associated with new onset of psoriatic arthritis in patients with psoriasis: a new biomarker?

BACKGROUND: We previously found that serum levels of chemokine (C-X-C motif) ligand 10 (CXCL10) decreased after the onset of psoriatic arthritis (PsA). OBJECTIVES: We measured CXCL10 levels over time in patients with psoriasis who developed PsA to determine whether the drop in CXCL10 was specific to these patients and further assess its association with PsA development. METHODS: Prospectively followed patients with psoriasis without arthritis [cutaneous psoriasis (PsC)] were assessed yearly by rheumatologists for the presence of PsA. Patients with PsC who developed PsA (converters) were matched to those that did not develop PsA (nonconverters) based on psoriasis duration and the interval between follow-up visits. The duration between baseline and the first visit postconversion in converters was used to assign a pseudoconversion date in nonconverters. Linear mixed-effects models were used to model the expression of CXCL10 over time. RESULTS: CXCL10 significantly declined over time in converters prior to PsA development with a significant difference in the trend over time between converters (n = 29) and nonconverters (n = 52; P < 0·001). CXCL10 continued to decline after PsA onset in a subset of converters. There was a significant difference in the trend of CXCL10 levels between converters (n = 24) and nonconverters (n = 16; P = 0·01) preconversion/pseudoconversion. This difference remained postconversion (P = 0·006) and was not different from the preconversion period (P = 0·75). CONCLUSIONS: A large difference in CXCL10 was identified in patients with PsC that are destined to develop PsA over time. This exploratory analysis supports the association of CXCL10 with PsA development in patients with PsC and warrants further study of the predictive ability of this chemokine. What is already known about this topic? Chemokine (C-X-C motif) ligand 10 (CXCL10) is elevated in psoriatic affected tissues and serum and/or plasma. Patients with psoriasis that develop psoriatic arthritis (PsA) have elevated CXCL10 levels at baseline and these levels drop after arthritis onset. What does this study add? By monitoring levels of CXCL10 in serum over multiple visits in patients with psoriasis that develop PsA as well as those that do not develop PsA, an association was identified between CXCL10 and PsA development. What is the translational message? CXCL10 is a strong candidate for use by physicians for the detection of patients with psoriasis that are at risk of developing PsA. Linked Comment: Kirby and Fitzgerald. Br J Dermatol 2020; 183:805-806.

Age and Tumor Location Predict Survival in Nonmetastatic Osteosarcoma in Upper Egypt.

AIM: The aims of this study were to assess survival outcome of pediatric patients with localized osteosarcoma of the extremities in Upper Egypt, identify factors of prognostic significance for survival, and to determine factors predictive of surgical methods used in these patients, as well as developing a clinical model for risk prediction. PATIENTS AND METHODS: A retrospective analysis of data assembled from medical records of 30 pediatric patients with a histologically verified nonmetastatic osteosarcoma of the extremities treated at South Egypt Cancer Institute with a unified chemotherapy protocol between January 2001 and December 2015 was carried out. Prognostic factors were determined using univariable and multivariable methods. A model for surgical outcomes in these patients based on the baseline clinical factors, and the parameters predictive of their tumor response to chemotherapy, was developed. RESULTS: With a median follow-up of 63 months for the study population, the estimates for event-free survival and overall survival (OS) at 3 and 5 years were 69.5% and 79% and 65.2% and 65.3%, respectively. Age 16 years or above was independently associated with both worse metastasis-free survival (hazard ratio [HR]=6.05, 95% confidence interval [CI]: 1.43-25.6, P=0.015) and OS (HR=7.9, 95% CI: 1.71-36.2, P=0.008). In the multivariable analysis, a proximal location within the limb gained a statistical significance to be independently associated with worse OS (HR=2.4, 95% CI: 1.13-22.1, P=0.003). Poor response to chemotherapy was marginally associated with worse metastasis-free survival (HR=4.9, 95% CI: 1.02-23.8, P=0.047) only in the univariable analysis. The patients found to be more likely to undergo an amputation surgery (odds ratio=14.1, 95% CI: 1.34-149.4, P=0.028) were those in whom a tumor was poorly responding to chemotherapy. CONCLUSION: In Upper Egypt, despite the reasonable survival outcomes in nonmetastatic osteosarcoma, a relatively high limb amputation rate has been encountered. The development of a clinical prediction model for future planning of possible outcome improvement in these patients, however, is still feasible.

Diagnostic value of ultrasound in calcium pyrophosphate deposition disease of the knee joint.

OBJECTIVE: To assess the diagnostic performance of ultrasound (US) for calcium pyrophosphate deposition (CPPD) at the level of menisci, hyaline cartilage (HC), tendons, and synovial fluid (SF) of the knee, and to examine inter- and intra-observer reliability. DESIGN: We consecutively included patients with knee effusion over a 2-year period (43 patients with CPPD and 131 controls). All patients underwent SF analysis, conventional radiography (CR), and US examination using the Outcome Measures in Rheumatology (OMERACT) definition of the US characteristics of CPPD. Two independent operators performed the US, and inter-observer agreement was calculated. Intra-observer agreement was examined with static images obtained for all enrolled patients. RESULTS: US revealed calcium pyrophosphate (CPP) deposits in menisci, HC, and tendon more frequently in patients with CPPD than in control patients. The presence of US CPP deposits in SF was not significantly different between the two groups. Combined US evaluation of the three components (menisci, HC, and tendon) showed the best diagnostic performance. The sensitivity and specificity for US evaluation of the three components were 74.4% and 77.1%, respectively, while for CR evaluation, the sensitivity and specificity were 44.2% and 96.9%, respectively. Inter- and intra-observer agreement were excellent for medial (κ = 0.930, 0.972) and lateral menisci (κ = 0.905, 0.942), HC (κ = 0.844, 0.957), and SF (κ = 0.817, 0.925). Tendon showed fair inter-observer (κ = 0.532) and good intra-observer reliability (κ = 0.788). CONCLUSIONS: Based on the OMERACT definition, US demonstrated better diagnostic capacity than CR to diagnose CPPD, with excellent reliability. Combined evaluation of menisci, HC, and tendon showed the best diagnostic accuracy.

OTTO: a new strategy to extract mental disease-relevant combinations of GWAS hits from individuals.

Despite high heritability of schizophrenia, genome-wide association studies (GWAS) have not yet revealed distinct combinations of single-nucleotide polymorphisms (SNPs), relevant for mental disease-related, quantifiable behavioral phenotypes. Here we propose an individual-based model to use genome-wide significant markers for extracting first genetic signatures of such behavioral continua. 'OTTO' (old Germanic=heritage) marks an individual characterized by a prominent phenotype and a particular load of phenotype-associated risk SNPs derived from GWAS that likely contributed to the development of his personal mental illness. This load of risk SNPs is shared by a small squad of 'similars' scattered under the genetically and phenotypically extremely heterogeneous umbrella of a schizophrenia end point diagnosis and to a variable degree also by healthy subjects. In a discovery sample of >1000 deeply phenotyped schizophrenia patients and several independent replication samples, including the general population, a gradual increase in the severity of 'OTTO's phenotype' expression is observed with an increasing share of 'OTTO's risk SNPs', as exemplified here by autistic and affective phenotypes. These data suggest a model in which the genetic contribution to dimensional behavioral traits can be extracted from combinations of GWAS SNPs derived from individuals with prominent phenotypes. Even though still in the 'model phase' owing to a world-wide lack of sufficiently powered, deeply phenotyped replication samples, the OTTO approach constitutes a conceptually novel strategy to delineate biological subcategories of mental diseases starting from GWAS findings and individual subjects.

Pediatric Osteosarcoma of Extremities: A 15-year Experience From a Tertiary Care Cancer Center in Upper Egypt.

AIM: To assess the outcome and determine predictors of survival in pediatric patients with osteosarcoma of the extremities treated with a unified chemotherapy protocol at a single institution over a 15-year period. MATERIALS AND METHODS: We performed a retrospective analysis of medical records of 48 pediatric patients with histologically verified osteosarcoma of the extremities diagnosed at South Egypt Cancer Institute and received treatment between January 2001 and December 2015. RESULTS: With a median follow-up of 61 months for the entire cohort, estimates of overall survival (OS) for 3- and 5-year were 50.9% and 42.1%, respectively. While the estimates of OS for 3- and 5-year in the nonmetastatic group were 79% and 65.2%, respectively. In the multivariable analysis, both metastatic disease at diagnosis and poor response to chemotherapy retained their statistical significance as independent predictors for event-free survival. Whereas for OS, a metastatic disease at diagnosis remained as the lone predictor of a dismal outcome, while a poor response to chemotherapy became marginally associated with an inferior outcome. CONCLUSIONS: In Upper Egypt, whereas slightly less than two thirds of children with localized osteosarcoma of extremities survives their disease, metastasis at presentation remains the key predictor of dismal survival outcomes.

Penetration depth of irrigants into root dentine after sonic, ultrasonic and photoacoustic activation.

AIM: To compare penetration depths of endodontic irrigants into the dentinal tubules of extracted teeth when using several activation methods. METHODOLOGY: The root canals of 90 extracted human teeth were prepared to size 40, .06 taper. The straight and round-shaped root canals were distributed randomly into six groups, and final irrigation was performed with EDTA and sodium hypochlorite as follows: (I) manual dynamic activation, (II) Ultrasonic, (III) Sonic, (IV) PIPS (photon-induced photoacoustic streaming, (V) SWEEPS (shock-wave enhanced emission photoacoustic streaming) and (0) control without final irrigation or activation. Subsequently, methylene blue was inserted into the canals and activated according to the groups (I-V). Teeth were sectioned horizontally, imaged under a light microscope, and dye penetration depths were measured in six sections per tooth and 24 points on a virtual clock-face per section. Data were analysed statistically by nonparametric tests for whole teeth and separately for coronal, middle and apical thirds. RESULTS: Penetration of dye into the dentinal tubules was lowest for the controls. Median penetration depths amounted to 700-900 µm for groups I-V with differences in the apical thirds between group I and the other test groups. Minimum penetration depths were significantly greater for PIPS in the apical thirds (P ≤ 0.046). CONCLUSIONS: Greater penetration depths occurred in the apical thirds for ultrasonic, sonic and laser-induced activation compared to manual dynamic activation. PIPS was associated with deeper penetration of irrigants. The novel SWEEPS mode did not increase irrigant penetration.

Detection of pulsed blood flow through a molar pulp chamber and surrounding tissue in vitro.

OBJECTIVES: Due to severe limitations of dental pulp sensitivity tests, the direct recording of pulsed blood flow, using photoplethysmography (PPG), has been proposed. In vivo evaluation is methodologically difficult and in vitro models have hitherto been adversely influenced by shortcomings in emulating the in vivo situation. Consequently, the aim of this study was to test an improved data acquisition system and to use this configuration for recording pulsed blood in a new model. MATERIALS AND METHODS: We introduced a PPG signal detection system by recording signals under different blood flow conditions at two wavelengths (625 and 940 nm). Pulsed blood flow signals were measured using an in vitro model, containing a molar with a glass pulp and a resin socket, which closely resembled in vivo conditions with regard to volumetric blood flow, pulp anatomy, and surrounding tissue. RESULTS: The detection system showed improved signal strength without stronger blanketing of noise. On the tooth surface, it was possible to detect signals emanating from pulsed blood flow from the glass pulp and from surrounding tissue at 625 nm. At 940 nm, pulp derived signals were recorded, without interference signals from surrounding tissue. CONCLUSION: The PPG-based method has the potential to detect pulsed blood flow in small volumes in the pulp and (at 625 nm) also in adjacent tissues. CLINICAL RELEVANCE: The results show the need for clear differentiation of the spatial origins of blood flow signals of any vitality test method to be applied to teeth.

Biotechnological Method of Preparation and Characterization of Recombinant Antimicrobial Peptide Avicin A from Enterococcus avium.

Avicin A is a bacteriocin from the gram-positive bacterium Enterococcus avium. It exhibits a high microbicidal activity against bacteria of the genus Listeria, a causative agent of the severe human infection listeriosis. We developed a biotechnological method for obtaining avicin A and characterized its structure and biological activity. We also proposed a possible mechanism of the antimicrobial action of avicin A.

Developing and testing a consensus-based core set of outcome measures for rehabilitation in musculoskeletal diseases.

OBJECTIVES: Rehabilitation is important for people with musculoskeletal diseases (MSDs), and evaluating the effect of rehabilitation on both an individual and group level is advocated. A consensus concerning use of outcome measures will improve collaboration between healthcare providers, and increase the possibility of conducting meta-analyses in future research. The aim of this study was to develop a consensus-based core set of outcome measures for rehabilitation in MSDs, and to test the feasibility and responsiveness of the set. METHOD: The core set was developed through a stepwise process comprising a Delphi consensus procedure, systematic literature searches, and a pilot study, including 386 patients, to test the feasibility and responsiveness of the set. RESULTS: The following aspects and outcome measures were selected: pain [numeric rating scale (NRS)], fatigue (NRS), physical fitness (the 30-second Sit to Stand test), mental health (Hopkins Symptom Checklist 5), daily activities (Hannover Functional Questionnaire), goal attainment (Patient-Specific Functional Scale including motivation score for baseline assessment), quality of life (5-level EuroQol 5 Dimensions), social participation (the social participation item from COOP/WONCA) and coping (Effective Musculoskeletal Consumer Scale-17). All tested outcome measures were found to be feasible, with high completion rates and acceptable score distribution. Standard response means varied from 0.3 to 0.9. CONCLUSIONS: A consensus-based core set of patient reported outcome measures is presented for evaluating rehabilitation in MSDs. The core set is feasible and responsive for use in Norway, but needs further testing in other countries.

Sampling and inactivation of wet disseminated spores from flooring materials, using commercially available robotic vacuum cleaners.

AIMS: Four commercially available robotic vacuum cleaners were assessed for sampling efficiency of wet disseminated Bacillus atrophaeus spores on carpet, polyvinyl chloride (PVC) and laminate flooring. Furthermore, their operability was evaluated and decontamination efficiency of one robot was assessed, using a sodium hypochlorite solution. METHODS AND RESULTS: In an environmental chamber, robots self-navigated around 4 m2 of flooring containing a single contaminated 0·25 m2 tile (c. 104 spores per cm2 ). Contamination levels at predetermined locations were assessed by macrofoam swabs (PVC and laminate) or water soluble tape (carpet), before and after sampling. Robots were dismantled postsampling and spore recoveries assessed. Aerosol contamination was also measured during sampling. Robot sampling efficiencies were variable, however, robots recovered most spores from laminate (up to 17·1%), then PVC and lastly the carpet. All robots spread contamination from the 'hotspot' (all robots spread <0·6% of the contamination to other areas) and became surface contaminated. Spores were detected at low levels during air sampling (<5·6 spores per litre). Liquid decontamination inactivated 99·1% of spores from PVC. CONCLUSIONS: Robotic vacuum cleaners show promise for both sampling and initial decontamination of indoor flooring. SIGNIFICANCE AND IMPACT OF THE STUDY: In the event of a bioterror incident, e.g. deliberate release of Bacillus anthracis spores, areas require sampling to determine the magnitude and extent of contamination, and to establish decontamination efficacy. In this study, we investigate robotic sampling methods against high concentrations of bacterial spores applied by wet deposition to different floorings, contamination spread to other areas, potential transfer of spores to the operators and assessment of a wet vacuum robot for spore inactivation. The robots' usability was evaluated and how they can be employed in real life scenarios. This will help to reduce the economic cost of sampling and the risk to sampling/decontamination teams.

Isolation of phage-display library-derived scFv antibody specific to Listeria monocytogenes by a novel immobilized method.

AIMS: To select Listeria monocytogenes-specific single-chain fragment variable (scFv) antibodies from a phage-display library by a novel simple and cost-effective immobilization method. METHODS AND RESULTS: Light expanded clay aggregate (LECA) was used as biomass support matrix for biopanning of a phage-display library to select L. monocytogenes-specific scFv antibody. Four rounds of positive selection against LECA-immobilized L. monocytogenes and an additional subtractive panning against Listeria innocua were performed. The phage clones selected using this panning scheme and LECA-based immobilization method exhibited the ability to bind L. monocytogenes without cross-reactivity toward 10 other non-L. monocytogenes bacteria. One of the selected phage clones was able to specifically recognize three major pathogenic serotypes (1/2a, 1/2b and 4b) of L. monocytogenes and 11 tested L. monocytogenes strains isolated from foods. CONCLUSIONS: The LECA-based immobilization method is applicable for isolating species-specific anti-L. monocytogenes scFv antibodies by phage display. SIGNIFICANCE AND IMPACT OF THE STUDY: The isolated scFv antibody has potential use in development of immunoassay-based methods for rapid detection of L. monocytogenes in food and environmental samples. In addition, the LECA immobilization method described here could feasibly be employed to isolate specific monoclonal antibodies against any given species of pathogenic bacteria from phage-display libraries.

Dentine matrix proteins: isolation and effects on human pulp cells.

AIM: To establish a simplified and efficient protocol for the isolation and concentration of matrix proteins from human dentine, and to assess the effects of extracted dentine matrix proteins (eDMP) on the behaviour of human pulp cells. METHODOLOGY: Matrix proteins were isolated from human dentine, purified, concentrated and characterized with protein and enzyme-linked immunosorbent assays (ELISA). Culture media were supplemented with eDMP in different concentrations, referred to as eDMP 1-10 000, to assess viability and proliferation of human pulp cells by DNA and MTT assays; apoptotic events were quantified by flow cytometry. Chemotactic effects of eDMP were assessed in a modified Boyden chamber assay. Expression levels of odontoblastic marker genes in pulp cells cultured with eDMPs were determined by real-time quantitative PCR, and the ability to induce mineralization was demonstrated by alizarin red staining. Nonparametric statistical analysis was performed to pairwise compare different groups at all time-points (Mann-Whitney U-test, α = 0.05). RESULTS: High concentrations of eDMP exhibited significant antiproliferative effects (P ≤ 0.023) after 5 (eDMP 1000) and 7 days (eDMP 500) without affecting cell viability. Apoptosis was barely influenced (P ≥ 0.089). eDMP exerted a concentration-dependent chemotactic stimulus on dental pulp cells with statistical significance already at low dosage (P = 0.006 at eDMP 10). Changes in gene expression indicated a differentiation into odontoblast-like cells, which was corroborated by findings of mineral nodule formation. CONCLUSIONS: A novel, effective and time-saving protocol for isolation and concentration of dentine matrix proteins is presented. As eDMP stimulates chemotaxis, differentiation and mineralization without affecting viability, endogenous dentine matrix proteins might be valuable for approaches to regenerate or engineer dental pulp.

Interactive effects of LPS and dentine matrix proteins on human dental pulp stem cells.

AIM: To investigate the combinatorial effects of lipopolysaccharide (LPS) and extracted dentine matrix proteins (eDMP) on regenerative and inflammatory responses in human dental pulp stem cells (DPSCs). METHODOLOGY: Culture media were supplemented with several concentrations of LPS, eDMP and combinations of both. Cell viability was assessed over 1 week by MTT assay; cell survival was evaluated after 24 h and 7 days by flow cytometry. The expression of mineralization-associated marker genes was determined by real-time quantitative polymerase chain reaction (RT-qPCR). To analyse the inflammatory response, secretion of interleukin 6 (IL-6) was quantified in the initial and the late phase of cell culture by enzyme-linked immunosorbent assay (ELISA). Data were treated nonparametrically and Mann-Whitney U-tests were performed to compare all experimental groups (α = 0.05). RESULTS: Whereas LPS had no impact on viability, eDMP led to a concentration-dependent decrease, which was significant after 7 days (P ≤ 0.024). A moderate decline of cell survival induced by LPS was detected after 48 h (P ≤ 0.026), whereas eDMP was able to reverse this effect. eDMP alone caused increased expression of tested marker genes, LPS had no regulatory effect. Combined eDMP and LPS induced an upregulation of collagen type I and osteocalcin, whereas expression levels of dentine matrix acidic phosphoprotein and dentine sialophosphoprotein were similar to the control. IL-6-secretion was increased by LPS over time. eDMP markedly elevated initial production of IL-6 (P ≤ 0.002), but suppressed LPS-induced cytokine production in the later phase. CONCLUSIONS: Lipopolysaccharide did not affect cell viability but interfered with odontoblast-like cell differentiation of DPSCs. Proteins from the dentine matrix may have a protective effect, attenuate the detrimental impact of LPS and thus play an important role during pulp repair.

[The role of PSMA PET-CT in patients with metastatic prostate cancer]. / Die Rolle des PSMA-PET/CT bei Patienten mit metastasiertem Prostatakarzinom.

Prostate-specific membrane antigen (PSMA) positron emission tomography-computed tomography (PET-CT) imaging for the localization of prostate cancer is increasingly available in Germany. The advances and limitations in different disease stages are reviewed. As the clinical relevance of oligometastatic disease in primary cancer detected by PSMA PET-CT imaging is not yet completely understood, it should only be used in clinical trials. In recurrent prostate cancer after therapy with curative intent, PSMA PET-CT shows encouraging potential for the planning of salvage therapy. In metastatic castration-resistant prostate cancer evidence for its use is not available.