Preanalytical stability of molecular forms of prostate-specific antigen in serum samples (PSA, free PSA, [-2]proPSA) and their impact on fPSA/tPSA ratio and PHI.

BACKGROUND: Prostate cancer is a common cancer in men. Detection methods include the measurement of biomarkers: prostate-specific antigen (PSA), free PSA, [-2]proPSA, and the calculated indices: fPSA/tPSA ratio and Prostate Health Index (PHI). Proper preanalytical conditions are crucial for precise measurement and failure to adhere to protocols or regulations can influence the diagnostic algorithm. We assessed the stability of the above-mentioned biomarkers, fPSA/tPSA ratio and PHI, under various pre-analytical conditions. METHODS: Serum samples from 45 males were collected and stored under specific conditions before tPSA, fPSA, and [-2]proPSA were measured. Subsequently, the fPSA/tPSA and PHI were calculated. RESULTS: tPSA, fPSA, and [-2]proPSA remained stable during the two freeze-thaw cycles. Storage at 4°C and 22°C resulted in stable tPSA concentrations. However, fPSA levels decreased and [-2]proPSA levels increased over time. The fPSA/tPSA ratio remained stable for 72 h, at which point a decrease was observed in the samples kept at 4°C and 22°C. A gradual increase in PHI was observed in the samples kept at 4°C and 22°C. CONCLUSIONS: All biomarkers remained stable during two freeze-thaw cycles. tPSA was the most stable analyte when stored at 4°C, as well as at RT. A gradual increase of [-2]proPSA and a slight decrease in fPSA were observed during the storage test. This led to a decrease in the fPSA/tPSA ratio and an elevation in the PHI. We therefore recommend measuring prostate biomarkers promptly following blood collection. IMPACT: Understanding the pre-analytical stability of prostate biomarkers helps prevent false positive results and improve the accuracy of diagnostics for prostate cancer.

Ibrutinib in mantle cell lymphoma: a real-world retrospective multi-center analysis of 77 patients treated in the Czech Republic.

Ibrutinib revolutionized therapy for relapsed/refractory (R/R) mantle cell lymphoma (MCL). Real-world data on the outcome of unselected patients are still limited. We analyzed 77 R/R MCL patients receiving ibrutinib with at least one prior systemic anti-lymphoma therapy. After a median follow-up of 14.0 months, 56 patients relapsed/progressed, and 45 died. The overall response rate was 66%, with 31% of complete metabolic remissions on PET/CT. The median progression-free and overall survival (OS) rates were 10.3 and 23.1 months, respectively. The median OS from ibrutinib failure was 3.7 months. High proliferation rate by Ki67 (≥ 30%) and two or more previous therapy lines both negatively correlated with outcome (HR = 2.2, p = 0.04, and HR = 2.06, p = 0.08, respectively). Female gender borderline correlated with better outcome (HR = 0.53, p = 0.08). In multivariate analysis, Ki67 and response to ibrutinib both correlated with OS (p < 0.05). Importantly, ibrutinib appeared to better control nodal and extranodal lymphoma than bone marrow (BM) involvement. From 20 patients with detectable BM infiltration (before ibrutinib initiation) achieving complete (n = 13) or partial (n = 7) metabolic remission, none achieved remission in BM. We confirmed good efficacy of ibrutinib in unselected heavily pre-treated MCL patients. Our findings support the use of a combination of ibrutinib and rituximab in patients with BM involvement.

COVID-19 infection elicits textbook immune response.

Imunita po prodelané infekci SARS-CoV-2 byla dlouhodobe zpochybnována. Z jiných typu respiracních onemocnení vsak víme, ze bunky, které se vytvorí pri první infekci, dlouhodobe prezívají a pri opakované infekci je díky nim imunitní reakce rychlejsí a úcinnejsí. Popisuje se zvýsení hladin protilátek, jejich lepsí avidita a vznik nových variant. Jiz existující pametové B a T lymfocyty jsou pouzity jako "prototyp", který se dále vylepsuje. U reinfikovaných osob tak logicky klesá riziko vázných prubehu onemocnení. V predkládaném clánku jsou uvedeny výsledky dlouhodobého merení hladiny IgG protilátek proti proteinum S a N a IgA protilátek proti proteinu S u 4 osob, které prodelaly opakované infekce SARS-CoV-2 spojené s nárustem hladin protilátek a s méne závazným prubehem, nez mela primoinfekce. Tyto zkusenosti potvrzují i závery z nasí predchozí dlouhodobé studie imunity u senioru z roku 2020, v níz byla také pozorována reaktivace imunity u rekonvalescentu po pravdepodobné nové expozici SARS-CoV-2, ale bez prodelaného onemocnení. Prezentované výsledky potvrzují jiz dríve publikovaná fakta, tedy ze prodelání nemoci neposkytuje dlouhodobou ochranu pred reinfekcí, zvláste novou variantou viru, ale pokud jiz k reinfekci dojde, bývá její prubeh méne závazný nez pri primoinfekci.

Antibodies from previous infection bring sufficient and long-term protection against COVID-19.

One of the basic manifestations of an adaptive anti-infective immunity is the production of specific antibodies. The presence of antibodies can be detected in serum and serves as one of the diagnostic tools used to confirm past infection. Very often it also serves as a confirmation that the body has acquired immunity to the disease. The appearance of COVID-19 has cast a shadow of doubt on these textbook examples of antibody behavior. Information that repeatedly questions antibody measurement and its significance has been circulating among professionals and the general public. The aim of our article is to summarize the current knowledge on the immunity acquired following SARS-CoV-2 infection and to present the results of antibody testing from four Czech laboratories which have been measuring these antibodies for over one year. Our data suggest that commonly available diagnostic methods reliably predict the results of a virus neutralization test, which is the gold standard of immunity detection. By acknowledging those with naturally acquired anti-infective immunity, in addition to vaccinated individuals, we will significantly increase the perceived level of collective immunity.

NFKB1 gene single-nucleotide polymorphisms: implications for graft-versus-host disease in allogeneic hematopoietic stem cell transplantation.

Graft-versus-host disease (GVHD) represents a significant cause of mortality after allogeneic hematopoietic stem cell transplantation (HSCT). NF-kB system is a master regulator of innate immunity responses. It controls the expression of various cytokines and chemokines many of which are involved in GVHD pathogenesis. Chemo(radio) therapy administered during conditioning induces DNA damage and activates DNA damage response (DDR) signaling resulting in irreversible cell cycle arrest - cellular senescence which has been described to be associated with robust pro-inflammatory secretion mostly controlled by NF-kB. The NFKB1 gene encodes the DNA-binding subunit of the NF-kB complex. Using the candidate gene approach, we analyzed possible association of two single-nucleotide polymorphisms (SNPs) rs3774937 C/T and rs3774959 A/G of the NFKB1 gene with GVHD and transplant-related mortality (TRM) occurrence in 109 recipients allografted from HLA-identical donor. Both SNPs in recipients were found to be strongly associated with acute GVHD. Nevertheless, no significant association with chronic GVHD and TRM was found. Presented pilot results contribute to pre-clinical observations and suggest that NF-kB may be an important regulator of HSCT-related inflammatory reactions such as acute GVHD. Novel pathogenic mechanisms of GVHD may arise from perspectives of DDR and cellular senescence where NF-kB plays an essential role.

Age is not a disease: Evolution of protective antibodies against SARS-CoV-2 in seniors from the Břevnice nursing home.

In the nursing home in Břevnice, 21 out of 23 seniors were infected with the SARS-CoV-2 virus in the spring of 2020. Three of them died from the infection. This study monitors the antibodies formation in group of seniors who have overcome the infection. Between April and November (1-8 months after the infection), the antibodies were measured repeatedly within the period of several weeks. The virus neutralizing tests was used as well as the ECLIA assay by Roche (total immunoglobulins), CMIA assay by Abbott (IgG) and ELISA assay by Euroimmun (IgG and IgA). Six months from the infection, neutralizing antibodies were detected in 18 out of 20 seniors. High levels of neutralizing antibodies correlated with higher levels of immunoglobulins and were a good predictor of an increase of IgG in the autumn during the second wave of the epidemic in the Czech Republic. During the autumn wave, neither any of the clients, nor any staff contracted the virus, although the prevalence of PCR positivity in the Vysočina region reached around 5 %. The antigen tests also came out all negative. This study shows that in the senior population, the production of protective antibodies follows a normal pattern, and the seniors are probably immune to a repeated infection for at least 8 months from the first infection. Based on these results, we would like to open the discussion on the testing for seroprevalence in nursing homes, possible changes to the epidemiologic regime in relation to the risk of infection, and about vaccination schemes in these centers.

Testing for COVID-19: a few points to remember.

Diagnostic approaches to COVID-19 include clinical history, PCR tests for the presence of SARS-CoV-2 virus and detection of antibodies. By combining these three approaches, the seroprevalence of anti-SARS-CoV-2 antibodies can be examined in healthcare teams. The aim of the study was to examine the seroprevalence of anti-SARS-CoV-2 antibodies in a population of healthcare professionals 6 - 8 weeks after the first COVID-19 case was detected in the Czech Republic. A total of 269 subjects were enrolled in the study (187 women, 82 men) with a median age of 45.9 years (21 - 71 years). We used a questionnaire to ascertain travel history and clinical signs of any respiratory tract infection. Blood samples were collected, and IgG levels were analysed in all samples. The level of IgA antibodies was analysed in those positive for IgG. PCR testing was performed in cases testing positive for presence of antibodies. The enzyme-linked immunosorbent assay (ELISA) test system for SARS-CoV-2 from Euroimmun (Germany) was used to analyse immunoglobulin levels. 17 % of the tested cohort reported symptoms compatible with COVID-19 and 35.8 % reported history of international travel. There were 5 subjects positive IgG cases (of 269; 1.85 %), and one IgA positive and IgG borderline positive subject (0.37 %). There was only one PCR positive subject. Anti SARS-CoV-2 antibodies were thus detected in 2.22% of participating health professionals. This article shows the pitfalls of the testing methods and highlights the necessity of using a correct testing algorithm, considering the character of the tested population and the expected low prevalence.

Exploratory drilling: how to set up, carry out, and evaluate a seroprevalence study.

Seroprevalence studies represent a very important tool to find out what fraction of population has already met with the new type of coronavirus (e.g. SARS-CoV-2). Without these data, it is almost impossible for the state authorities to manage the epidemic and adopt rational measures. This article brings the results of a medium-sized seroprevalence study which was carried out in the spring of 2020 in South Bohemia. In the Strakonice and Písek regions, the ELISA method was used to test the prevalence of IgA and IgG antibodies in 2011 subjects, volunteers from general public and selected professions working in areas with a higher exposure to the infection. The study showed that already in May 2020, 2.9% of inhabitants of the Strakonice region and 1.9% of inhabitants of the Písek region had antibodies against the coronavirus. These numbers imply that for each PCR positive person, there were at least fifty others who had probably already undergone the infection. The article points out three types of problems that might occur in such a study. First, the study must be planned correctly, and possible outcomes must be pre-assessed. Second, an appropriate test must be selected with known parameters. This enables us to correctly estimate the share of false positive and false negative results. Third, the data must be evaluated in a reasonable way and correct inference must be performed. We offer a set of recommendations how to manage these issues and how to solve problems that inevitably arise in such a large-scale testing.

Experimental hybrid quantum-classical reinforcement learning by boson sampling: how to train a quantum cloner.

We report on experimental implementation of a machine-learned quantum gate driven by a classical control. The gate learns optimal phase-covariant cloning in a reinforcement learning scenario having fidelity of the clones as reward. In our experiment, the gate learns to achieve nearly optimal cloning fidelity allowed for this particular class of states. This makes it a proof of present-day feasibility and practical applicability of the hybrid machine learning approach combining quantum information processing with classical control. The quantum information processing performed by the setup is equivalent to boson sampling, which, in complex systems, is predicted to manifest quantum supremacy over classical simulation of linear-optical setups.

Influence of female hormones on fascia elasticity: An elastography study.

The aim of this study was to examine the influence of hormonal changes during the menstrual cycle on deep fasciae. A total of 29 women, 17 users, and 12 nonusers of hormonal contraceptives were examined clinically and by ultrasound, including shear wave elastography, at two phases of the menstrual cycle. The thickness and elasticity of the fascia lata, thoracolumbar fascia, and plantar fascia were measured, compared between hormonal contraceptive users and nonusers, and correlated with clinical data. There were statistically significant differences between users and nonusers of hormonal contraceptives: the thoracolumbar fascia was thicker in nonusers (P = 0.011), and nonusers had higher maximal and mean stiffnesses of the fascia lata (P = 0.01 and 0.0095, respectively). Generally, nonusers had a higher body mass index (BMI). The elasticity of the thoracolumbar and the plantar fasciae did not differ significantly between the groups. We found no correlation between thickness and elasticity in the fasciae. There were no statistically significant differences in hypermobility, cephalgia, or dysmenorrhea between users and nonusers of hormonal contraceptives. The results of this pilot study suggest that deep fasciae can be evaluated by shear wave elastography. Nonusers of contraceptives had greater stiffness of the fascia lata and higher BMI. Clin. Anat. 32:941-947, 2019. © 2019 Wiley Periodicals, Inc.

Estimating heat tolerance of plants by ion leakage: a new method based on gradual heating.

Heat tolerance of plants related to cell membrane thermostability is commonly estimated via the measurement of ion leakage from plant segments after defined heat treatment. To compare heat tolerance of various plants, it is crucial to select suitable heating conditions. This selection is time-consuming and optimizing the conditions for all investigated plants may even be impossible. Another problem of the method is its tendency to overestimate basal heat tolerance. Here we present an improved ion leakage method, which does not suffer from these drawbacks. It is based on gradual heating of plant segments in a water bath or algal suspensions from room temperature up to 70-75°C. The electrical conductivity of the bath/suspension, which is measured continuously during heating, abruptly increases at a certain temperature TCOND (within 55-70°C). The TCOND value can be taken as a measure of cell membrane thermostability, representing the heat tolerance of plants/organisms. Higher TCOND corresponds to higher heat tolerance (basal or acquired) connected to higher thermostability of the cell membrane, as evidenced by the common ion leakage method. The new method also enables determination of the thermostability of photochemical reactions in photosynthetic samples via the simultaneous measurement of Chl fluorescence.

Evaluation of the Effect of Tissue Compression on the Results of Shear Wave Elastography Measurements.

Shear wave imaging is considered to be more precise and less operator dependent when compared with strain imaging. It enables quantitative and reproducible data (Young's modulus of the imaged tissue). However, results of shear wave imaging can be affected by a variety of different factors. The aim of this study is to evaluate the effect of the pressure applied by the ultrasound probe during examination on the measured values of Young's modulus. The effect of the tissue compression on the results of the real-time shear wave elastography was evaluated via the gelatine phantom measurements, via the ex vivo experiments with pig liver, and via the in vivo measurements of the thyroid gland stiffness on healthy volunteers. The results of our measurements confirmed that the measured value of Young's modulus increases with the increasing pressure applied on the imaged object. The highest increase was observed during the ex vivo experiments (400%), and the lowest increase was detected in the case of the phantom measurements (8%). A two- to threefold increase in Young's modulus was observed between the minimum and maximum pressure in the case of the in vivo elastography measurements of thyroid gland. The Veronda-Westman theoretical model was used for the description of the tissue nonlinearity. We conclude that tissue compression by the force exerted on the probe can significantly affect the results of the real-time shear wave elastography measurements. Minimum pressure should be used when measuring the absolute value of Young's modulus of superficial organs.

Role of DNA Repair Factor Xeroderma Pigmentosum Protein Group C in Response to Replication Stress As Revealed by DNA Fragile Site Affinity Chromatography and Quantitative Proteomics.

Replication stress (RS) fuels genomic instability and cancer development and may contribute to aging, raising the need to identify factors involved in cellular responses to such stress. Here, we present a strategy for identification of factors affecting the maintenance of common fragile sites (CFSs), which are genomic loci that are particularly sensitive to RS and suffer from increased breakage and rearrangements in tumors. A DNA probe designed to match the high flexibility island sequence typical for the commonly expressed CFS (FRA16D) was used as specific DNA affinity bait. Proteins significantly enriched at the FRA16D fragment under normal and replication stress conditions were identified using stable isotope labeling of amino acids in cell culture-based quantitative mass spectrometry. The identified proteins interacting with the FRA16D fragment included some known CFS stabilizers, thereby validating this screening approach. Among the hits from our screen so far not implicated in CFS maintenance, we chose Xeroderma pigmentosum protein group C (XPC) for further characterization. XPC is a key factor in the DNA repair pathway known as global genomic nucleotide excision repair (GG-NER), a mechanism whose several components were enriched at the FRA16D fragment in our screen. Functional experiments revealed defective checkpoint signaling and escape of DNA replication intermediates into mitosis and the next generation of XPC-depleted cells exposed to RS. Overall, our results provide insights into an unexpected biological role of XPC in response to replication stress and document the power of proteomics-based screening strategies to elucidate mechanisms of pathophysiological significance.

ATM gene single nucleotide polymorphisms predict regimen-related gastrointestinal toxicity in patients allografted after reduced conditioning.

Polymorphisms of genes involved in innate and adaptive immunity have become an object of major interest in regard to hematopoietic stem cell transplantation (HSCT) complications. Regimen-related gastrointestinal toxicity (RR-GIT) is the dominant complication during the pre-engraftment period and has been linked to increased risk of graft-versus-host disease (GVHD) development. According to our hypothesis, functional variants of genes participating in DNA damage response (DDR) may have an impact on the extent of tissue damage caused by the conditioning regimen. In our single-center study, we analyzed 62 patients who underwent HSCT from HLA-identical donors after reduced conditioning. The patients were genotyped for 5 single nucleotide polymorphisms (SNPs, rs4585 T/G, rs189037 A/G, rs227092 T/G, rs228590 C/T, and rs664677 T/C) of the ATM gene-the essential member of the DDR pathways, using allele-specific matrix-assisted laser desorption/ionization, time-of-flight (MALDI-TOF) mass spectrometry assay. Because of almost absolute linkage disequilibrium observed among all 5 SNPs, association of 2 major ATM haplotypes (ATM1/ATM2) with RR-GIT and acute GVHD (aGVHD) was analyzed. Importantly, the univariate and multivariate analysis showed that patients homozygous for ATM2 haplotype (rs4585*T, rs189037*A, rs227092*T, rs228590*C, and rs664677*T) are more likely to suffer from high-grade RR-GIT than ATM1 homozygous patients. The association with aGVHD was not significant. To our knowledge, this is the first report showing the ATM gene variability in relation to RR-GIT in the allogeneic HSCT setting.

Does the healthy vaccinee bias rule them all? Association of COVID-19 vaccination status and all-cause mortality from an analysis of data from 2.2 million individual health records.

OBJECTIVES: We investigated the validity of claims of the healthy vaccinee effect (HVE) in COVID-vaccine studies by analyzing associations between all-cause mortality (ACM) and COVID-19 vaccination status. METHODS: Approximately 2.2 million individual records from two Czech health insurance companies were retrospectively analyzed. Each age group was stratified according to the vaccination status (unvaccinated vs. individuals less than 4 weeks vs. more than 4 weeks from Doses 1, 2, 3, and 4 or more doses of vaccine). ACMs in these groups were computed and compared. RESULTS: Consistently over datasets and age categories, ACM was substantially lower in the vaccinated than unvaccinated groups regardless of the presence or absence of a wave of COVID-19 deaths. Moreover, the ACMs in groups more than 4 weeks from Doses 1, 2, or 3 were consistently several times higher than in those less than 4 weeks from the respective dose. HVE appears to be the only plausible explanation for this, which is further corroborated by a created mathematical model. CONCLUSIONS: In view of the presence of HVE, the baseline difference in the frailty of vaccinated and unvaccinated populations in periods without COVID-19 must be taken into account when estimating COVID-19 vaccine effectiveness from observational data.

Prognostic value of cerebrospinal fluid biomarkers in multiple sclerosis: The key role of kappa free light chains and a multivariate predictor for disease progression.

OBJECTIVE: Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the central nervous system with varying progression rates among individuals. The ability to predict disease progression is crucial for treatment decisions with disease-modifying therapies (DMTs). A few cerebrospinal fluid (CSF) biomarkers have been investigated in relation to disease progression, but few have been effectively translated into clinical practice. The aim of this study was to evaluate the diagnostic and prognostic value of known CSF markers, to compare their sensitivity and specificity, and to develop a prognostic model using a combination of markers to predict disease progression. METHODS: This retrospective cohort study included 82 patients with a first episode of inflammatory demyelinating symptoms suggestive of MS between January 2018 and January 2021. Patients underwent diagnostic lumbar puncture and other investigations according to the multiple sclerosis (MS) protocol. They were divided into three groups according to MRI findings, relapse rate and EDSS score. CSF marker concentrations were determined by laser nephelometry and electrochemiluminescence immunoassay. RESULTS: The results showed that the number of oligoclonal bands could discriminate the progression-free group from the other groups, but had a lower discriminatory power compared to CSF marker concentrations. Among CSF markers, FLC kappa showed the best discriminatory performance. By combining FLC kappa with gender and lesion localization information, a simple predictor of progression-free group membership was proposed. This predictor showed good sensitivity (91 %) and specificity (65 %). CONCLUSION: In conclusion, CSF FLC kappa concentration, together with gender and lesion localization, may be a valuable predictor of disease progression in MS patients. This study highlights the potential of using CSF biomarkers for prognostic purposes and offers a simple approach to predicting disease progression.

Crop growth dynamics: Fast automatic analysis of LiDAR images in field-plot experiments by specialized software ALFA.

Repeated measurements of crop height to observe plant growth dynamics in real field conditions represent a challenging task. Although there are ways to collect data using sensors on UAV systems, proper data processing and analysis are the key to reliable results. As there is need for specialized software solutions for agricultural research and breeding purposes, we present here a fast algorithm ALFA for the processing of UAV LiDAR derived point-clouds to extract the information on crop height at many individual cereal field-plots at multiple time points. Seven scanning flights were performed over 3 blocks of experimental barley field plots between April and June 2021. Resulting point-clouds were processed by the new algorithm ALFA. The software converts point-cloud data into a digital image and extracts the traits of interest-the median crop height at individual field plots. The entire analysis of 144 field plots of dimension 80 x 33 meters measured at 7 time points (approx. 100 million LiDAR points) takes about 3 minutes at a standard PC. The Root Mean Square Deviation of the software-computed crop height from the manual measurement is 5.7 cm. Logistic growth model is fitted to the measured data by means of nonlinear regression. Three different ways of crop-height data visualization are provided by the software to enable further analysis of the variability in growth parameters. We show that the presented software solution is a fast and reliable tool for automatic extraction of plant height from LiDAR images of individual field-plots. We offer this tool freely to the scientific community for non-commercial use.