NKG2D-mediated detection of metabolically stressed hepatocytes by innate-like T cells is essential for initiation of NASH and fibrosis.

Metabolic-associated fatty liver disease (MAFLD) is a spectrum of clinical manifestations ranging from benign steatosis to cirrhosis. A key event in the pathophysiology of MAFLD is the development of nonalcoholic steatohepatitis (NASH), which can potentially lead to fibrosis and hepatocellular carcinoma, but the triggers of MAFLD-associated inflammation are not well understood. We have observed that lipid accumulation in hepatocytes induces expression of ligands specific to the activating immune receptor NKG2D. Tissue-resident innate-like T cells, most notably γδ T cells, are activated through NKG2D and secrete IL-17A. IL-17A licenses hepatocytes to produce chemokines that recruit proinflammatory cells into the liver, which causes NASH and fibrosis. NKG2D-deficient mice did not develop fibrosis in dietary models of NASH and had a decreased incidence of hepatic tumors. The frequency of IL-17A+ γδ T cells in the blood of patients with MAFLD correlated directly with liver pathology. Our findings identify a key molecular mechanism through which stressed hepatocytes trigger inflammation in the context of MAFLD.

Serum bicarbonate stability study at room temperature - influence of time to centrifugation and air exposure on bicarbonate measurement reported according to the CRESS checklist.

OBJECTIVES: The aim was to evaluate the stability of serum bicarbonate at room temperature, depending on time to centrifugation and air exposure. METHODS: Stability study was conducted in the laboratory of Clinical Hospital Centre Rijeka, Croatia in January-February 2022. Nine samples from 10 volunteers were collected in clot activator gel tubes (Greiner Bio-One). Bicarbonate was measured on Beckman Coulter AU480 (Beckman Coulter, Brea, USA). Three tubes were left at room temperature for 30 min, three tubes for 2 h, three tubes for 4 h until centrifugation. First tube from first group (baseline) was measured immediately after centrifugation. Other measurements were expressed as percentage deviation (PD%) from baseline. First tube was remeasured after 1 and 2 h (OT_0h_1h; OT_0h_2h). Second and third tubes were opened 1 and 2 h after centrifugation (C_0h_1h; C_0h_2h). Second group of tubes was processed the same way with 2-hour centrifugation delay (WB_2h; OT_2h_1h; OT_2h_2h; C_2h_1h; C_2h_2h), and third group with 4-hour delay (WB_4h; OT_4h_1h; OT_4h_2h; C_4h_1h; C_4h_2h). PD% was compared to Maximum Permissible Difference (MPD=5.69%). MedCalc statistical software was used (MedCalc, Ostend, Belgium). RESULTS: Bicarbonate baseline mean value (range) was 27.3 (23.4-29.6) mmol/L. Obtained PD% (95%CI) were: C_0h_1h 0.46 (-1.21, 2.12); C_0h_2h 0.18 (-2.22, 2.57); OT_0h_1h -6.46 (-7.57, -5.36); OT_0h_2h -10.67 (-12.13, -9.21); WB_2h -0.15 (-2.04, 1.74); C_2h_1h 0.01 (-1.52, 1.54); C_2h_2h -0.40 (-2.65, 1.85); OT_2h_1h -5.43 (-7.30, -3.55); OT_2h_2h -11.32 (-13.57, -9.07); WB_4h -0.85 (-3.28, 1.58); C_4h_1h -2.52 (-4.93, 0.11); C_4h_2h -3.02 (-5.62, 0.43); OT_4h_1h -7.34 (-9.64, -5.05); OT_4h_2h -11.85 (-14.38, -9.33). CONCLUSIONS: Serum bicarbonate is stable for 4 h in closed uncentrifuged tubes, another 2 h in closed tubes after centrifugation, and is unstable within 1 h in opened tube.

Inherited Thrombophilia and Risk of Thrombosis in Children with Cancer: a Single-center Experience.

OBJECTIVES: Thrombosis is an increasingly recognized complication of childhood malignancy and its treatment. The incidence and etiology of pediatric cancer-related thrombosis is still not well understood. The aim of this study was to evaluate the prevalence of common prothrombotic genetic conditions in children with cancer, the frequency of thrombosis, and the role of inherited thrombophilia in the development of thrombosis in a pediatric oncology population. PATIENTS AND METHODS: Forty-seven children (36 treated for hematological malignancies and 11 for solid tumors) with a median age of 8.8. years (range 0.4 - 19.3 years) were included in the study. Genetic polymorphisms of Factor V Leiden (G1691A), prothrombin G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T were determined by real-time polymerase chain reaction-based DNA analysis. RESULTS: Four (8.5%) patients were heterozygous for Factor V Leiden, 3 (6.4%) were heterozygous for prothrombin G20210A mutation, and 3 (6.4%) were homozygous for MTHFR C677T mutation. All patients had implanted central venous catheters. Four (8.5%) children had documented thrombosis, three of which were in the upper venous system. Two of the four patients with thrombosis had Factor V Leiden heterozygosity. CONCLUSIONS: Thrombosis is an important complication of childhood cancer. The risk of thrombosis may be increased in patients with Factor V Leiden. In the absence of consensus guidelines, our results support the recommendation for thrombophilia screening in children with cancer.

An analysis of the vitamin D overtesting in a tertiary healthcare centre.

Introduction: Vitamin D testing is excessively used in clinical practice, despite of the clinical guidelines statements against population screening for vitamin D deficiency. This study aimed to assess an annual number of performed 25-hydroxy vitamin D (25(OH)D) tests that were unsupported by the national guidelines for prevention, detection and therapy of vitamin D deficiency in adults and to calculate associated financial burden for the publicly funded healthcare. Materials and methods: A representative sample of requested 25(OH)D tests in 2018 (N = 474) was formed after selection and randomisation of data set (N = 5298) collected from the laboratory information system database of the Clinical Department for Laboratory Diagnostics, the Clinical Hospital Centre Rijeka. Records were classified in two groups depending on associated medical condition(s) according to the national guidelines. An annual cost of the total and group specific vitamin D testing was calculated on the base of a single test price reimbursed by the Croatian Healthcare Insurance Fund (CHIF). Results: Medical conditions with high-risk for vitamin D deficiency were detected in 43% (206/474) of vitamin D requests (group 1). Conditions not associated with vitamin D deficiency were detected in 57% (268/474) requests (group 2). A total cost of 25(OH)D testing for the CHIF was 58,729.50 EUR (25,523.79 EUR in the group 1 and 33,205.71 EUR in the group 2). Conclusions: More than half of all 25(OH)D tests performed in the clinical laboratory represent avoidable cost for the public healthcare. Prevention of population screening by vitamin D testing is needed.

Prevalence of Aminotransferase Macroenzymes in Rheumatoid Arthritis Patients and Impact on Their Management.

INTRODUCTION: Rheumatoid arthritis (RA) treatment can be hepatotoxic, but liver enzymes can be falsely elevated due to macroenzyme presence. Macroenzymes are often found in autoimmune diseases, but prevalence and effect on treatment is unclear. This study aimed to determine aminotransferase macroenzyme prevalence and effect in RA patients. MATERIALS AND METHODS: This study included consecutive RA patients without liver disease sent for laboratory tests. Samples with elevated AST or ALT were processed for macroenzymes. Presence was determined using polyethylene glycol precipitation (PEG). RESULTS: Out of 126 patients, 21 had elevated aminotransferase levels. Due to liver disease, 6 patients were excluded, another 3 were unavailable for informed consent, leaving 12 patients for inclusion. Out of 12 patients, 1 had increased AST levels, 2 increased ALT levels, and 9 both. Macro-ALT was detected in 5/11 patients, 1 also had macro-AST. Out of 5 patients with macroenzymes, treatment change was seen in 3/5 patients, imaging in 2/5, both in 2/5. CONCLUSION: Elevated liver enzymes in RA patients is not always indicative of hepatotoxicity, as shown by the fact that about half of patients in our study had macroenzymes detected. Before assuming drug hepatotoxicity and changing treatment or ordering imaging, rheumatologists could consider macroenzyme presence.

Attitudes and practices of open data, preprinting, and peer-review-A cross sectional study on Croatian scientists.

Attitudes towards open peer review, open data and use of preprints influence scientists' engagement with those practices. Yet there is a lack of validated questionnaires that measure these attitudes. The goal of our study was to construct and validate such a questionnaire and use it to assess attitudes of Croatian scientists. We first developed a 21-item questionnaire called Attitudes towards Open data sharing, preprinting, and peer-review (ATOPP), which had a reliable four-factor structure, and measured attitudes towards open data, preprint servers, open peer-review and open peer-review in small scientific communities. We then used the ATOPP to explore attitudes of Croatian scientists (n = 541) towards these topics, and to assess the association of their attitudes with their open science practices and demographic information. Overall, Croatian scientists' attitudes towards these topics were generally neutral, with a median (Md) score of 3.3 out of max 5 on the scale score. We also found no gender (P = 0.995) or field differences (P = 0.523) in their attitudes. However, attitudes of scientist who previously engaged in open peer-review or preprinting were higher than of scientists that did not (Md 3.5 vs. 3.3, P<0.001, and Md 3.6 vs 3.3, P<0.001, respectively). Further research is needed to determine optimal ways of increasing scientists' attitudes and their open science practices.

Viral infection of the ovaries compromises pregnancy and reveals innate immune mechanisms protecting fertility.

Viral infections during pregnancy are a considerable cause of adverse outcomes and birth defects, and the underlying mechanisms are poorly understood. Among those, cytomegalovirus (CMV) infection stands out as the most common intrauterine infection in humans, putatively causing early pregnancy loss. We employed murine CMV as a model to study the consequences of viral infection on pregnancy outcome and fertility maintenance. Even though pregnant mice successfully controlled CMV infection, we observed highly selective, strong infection of corpus luteum (CL) cells in their ovaries. High infection densities indicated complete failure of immune control in CL cells, resulting in progesterone insufficiency and pregnancy loss. An abundance of gap junctions, absence of vasculature, strong type I interferon (IFN) responses, and interaction of innate immune cells fully protected the ovarian follicles from viral infection. Our work provides fundamental insights into the effect of CMV infection on pregnancy loss and mechanisms protecting fertility.

Vitamin D for treatment of non-alcoholic fatty liver disease detected by transient elastography: A randomized, double-blind, placebo-controlled trial.

AIM: To evaluate the effects of vitamin D on transient elastography (TE, FibroScan) indices of liver steatosis (controlled attenuation parameter [CAP]) and fibrosis (liver stiffness measurement [LSM]) in adults with non-alcoholic fatty liver disease (NAFLD). PATIENTS AND METHODS: In this randomized (2:1), double-blind, single-centre, 12-month trial, patients with NAFLD were treated with vitamin D (1000 IU/day) (n = 201) or a matching placebo (n = 110). Two co-primary outcomes were changes in CAP and LSM after 360 days of treatment versus baseline. Two main secondary outcomes were CAP/LSM changes after 180 days of treatment. RESULTS: Both CAP and LSM gradually decreased in vitamin D-treated patients and slightly increased in the placebo arm. Vitamin D was superior to placebo for both primary outcomes (mean differences in CAP and LSM changes (-49.5 dB/m [95% CI -59.5 to -39.4] and -0.72 kPa [95% CI -1.43 to 0.00], respectively) and both secondary outcomes (-22.1 dB/m [-32.1 to -12.1] and -0.89 kPa [-1.61 to -0.17], respectively). Of a number of exploratory outcomes (change at 12 months vs. baseline), vitamin D reduced serum uric acid (-17.9 µmol/L [-30.6 to -5.2]), gamma-glutamyltransferase (-8.9 IU/L [-15.5 to -2.3)] and fasting serum insulin levels (-5.1 pmol/L [-9.3 to -0.8]) as well as the homeostatic model assessment of insulin resistance index (-1.6 [-3.1 to -0.2]) (false discovery rate [5%]-adjusted P-values between .0572 and .0952). CONCLUSION: Low-medium dose supplementation of vitamin D (1000 IU/day) over 12 months reduces TE indices of liver steatosis (CAP) and fibrosis (LSM) in NAFLD patients.

IL-33/ST2 pathway drives regulatory T cell dependent suppression of liver damage upon cytomegalovirus infection.

Regulatory T (Treg) cells dampen an exaggerated immune response to viral infections in order to avoid immunopathology. Cytomegaloviruses (CMVs) are herpesviruses usually causing asymptomatic infection in immunocompetent hosts and induce strong cellular immunity which provides protection against CMV disease. It remains unclear how these persistent viruses manage to avoid induction of immunopathology not only during the acute infection but also during life-long persistence and virus reactivation. This may be due to numerous viral immunoevasion strategies used to specifically modulate immune responses but also induction of Treg cells by CMV infection. Here we demonstrate that liver Treg cells are strongly induced in mice infected with murine CMV (MCMV). The depletion of Treg cells results in severe hepatitis and liver damage without alterations in the virus load. Moreover, liver Treg cells show a high expression of ST2, a cellular receptor for tissue alarmin IL-33, which is strongly upregulated in the liver of infected mice. We demonstrated that IL-33 signaling is crucial for Treg cell accumulation after MCMV infection and ST2-deficient mice show a more pronounced liver pathology and higher mortality compared to infected control mice. These results illustrate the importance of IL-33 in the suppressive function of liver Treg cells during CMV infection.

Influence of a prolonged fasting and mild activity on routine laboratory tests.

OBJECTIVES: Despite the standardization of the phlebotomy procedure, blood analysis is occasionally requested after recommended hours with the excuse that the patient is still fasting. We aimed to examine the influence of prolonged fasting and mild physical activity on routine laboratory tests. DESIGN AND METHODS: The study was conducted on 30 volunteers (27 female) median age 40y (20-59). Blood samples were taken in the morning (7:00-8:00a.m.) and early afternoon (1:00-2:00p.m.) after prolonged fasting and usual daily activities. Serum glucose (GLU), urea, creatinine, triglyceride, uric acid (UA), iron and electrolytes were analyzed on Roche cobas 6000 c501 and complete blood count on Siemens ADVIA 2120i. Statistical significance between the two measurements was tested using paired t-test or Wilcoxon test according to data distribution. Clinical significance was judged against calculated reference change values (RCV). RESULTS: A statistically significant decrease was found for red blood cell count, hemoglobin, hematocrit, mean corpuscular volume (MCV), GLU, urea, creatinine, triglycerides and electrolytes, whereas white blood cell count and iron were significantly increased. Judging against desirable bias derived from biological variation, a significant change was found for all the analytes except MCV, platelet count, UA and triglycerides. A clinically significant change was not found for any of the tested analytes when compared to RCV. CONCLUSIONS: Prolonged fasting and mild activity will not influence the medical decision for healthy subjects with normal results. Despite the present statistically significant change, the clinically significant change was not shown. However, the study did not include pathological results which have to be interpreted more carefully.

How do we handle self-plagiarism in submitted manuscripts?

Self-plagiarism is a controversial issue in scientific writing and presentation of research data. Unlike plagiarism, self-plagiarism is difficult to interpret as intellectual theft under the justification that one cannot steal from oneself. However, academics are concerned, as self-plagiarized papers mislead readers, do not contribute to science, and bring undeserved credit to authors. As such, it should be considered a form of scientific misconduct. In this paper, we explain different forms of self-plagiarism in scientific writing and then present good editorial policy toward questionable material. The importance of dealing with self-plagiarism is emphasized by the recently published proposal of Text Recycling Guidelines by the Committee on Publication Ethics (COPE).

Patchwork plagiarism--a jigsaw of stolen puzzle pieces.

Plagiarism remains at the top in terms of interest to the scientific community. In its many vicious forms, patchwork plagiarism is characterized by numerous unresolved issues and often passes "below the radar" of editors and reviewers. The problem of detecting the complexity of misconduct has been partially resolved by plagiarism detection software. However, interpretation of relevant reports is not always obvious or easy. This article deals with plagiarism in general and patchwork plagiarism in particular, as well as related problems that editors must deal with to maintain the integrity of scientific journals.

Biomechanical trial of modified flexor tendon sutures: an in vitro study.

Proven benefits of early active mobilisation for intra-synovial flexor tendon repairs have inducted new criteria for a 'perfect suture'. This study has examined different variations of modified Kessler's suture, which could fulfil the new criteria. A total of 93 swine extensor tendons were transected, repaired and tested using a dynamometer with constant rate of extension. The first part of study tested clinically the most used modified Kessler suture, a variation of double modified Kessler suture and intact tendons as a control group. Further variations in the second part of study were due to type of suture, location and number of the knots and type of peripheral suture. According to the results, the tested version of double modified Kessler suture with crossed peripheral suture was the strongest one among all tested variations. The ultimate force for the authors preferred modification of the double modified Kessler (DMK) is significantly higher than modified Kessler suture. The version of DMK with crossed peripheral suture is the strongest one among all tested variations. The lowest strength manifests variation with two knots between tendon ends. The variations with interlocked and outsided knot or monofilament tread are not statistically significant regarding ultimate force. The frequency of suture failure events (suture pull out or tendon and/or suture rupture) is equal respecting braided or monofilament suture. The preferred modification of the double modified Kessler (DMK) suture with crossed peripheral suture is the strongest one among all tested variations and could achieve, concerning range of force, early active mobilisation. Further variations due to the type of thread and location, type and number of the knots did not show statistical significance.

Lamellar body count as a diagnostic test in predicting neonatal respiratory distress syndrome.

AIM: To determine the lamellar body count (LBC) cutoff value for fetal lung maturity and to evaluate the clinical usefulness of LBC in predicting the severity of neonatal respiratory distress syndrome (RDS). METHODS: A prospective study was conducted from 2002 until 2010. LBC was estimated in uncentrifugated amniotic fluid samples using Cell-Dyn 1800 analyzer. Amniotic fluid samples were obtained by amniocentesis or by puncturing embryonic membranes during cesarean section. The presence of mild, moderate, and severe RDS was assessed by neonatologist. RESULTS: A total of 313 patients with singleton pregnancies (24-41 weeks) were included in the study and 294 met the inclusion criteria. RDS was diagnosed in 28 neonates - mild in 8, moderate in 10, and severe in 10. In premature neonates (<37 gestational weeks), significant differences in LBC were only found between the subgroup without RDS and the group with moderate and the group with severe RDS (P<0.001). In all neonates, significant differences were found between neonates without RDS and neonates with RDS. Using LBC cutoff value of ≥20,000/µL, sensitivity, specificity, and positive and negative predictive values of LBC in determining mature fetal lungs were 96%, 88%, 45.6%, and 99.5%, respectively. CONCLUSION: This study suggests that LBC cutoff value of ≥20,000/µL can predict pulmonary maturity and reduce the risk of neonatal respiratory distress syndrome.

Prevalence of plagiarism in recent submissions to the Croatian Medical Journal.

To assess the prevalence of plagiarism in manuscripts submitted for publication in the Croatian Medical Journal (CMJ). All manuscripts submitted in 2009-2010 were analyzed using plagiarism detection software: eTBLAST, CrossCheck, and WCopyfind. Plagiarism was suspected in manuscripts with more than 10% of the text derived from other sources. These manuscripts were checked against the Déjà vu database and manually verified by investigators. Of 754 submitted manuscripts, 105 (14%) were identified by the software as suspicious of plagiarism. Manual verification confirmed that 85 (11%) manuscripts were plagiarized: 63 (8%) were true plagiarism and 22 (3%) were self-plagiarism. Plagiarized manuscripts were mostly submitted from China (21%), Croatia (14%), and Turkey (19%). There was no significant difference in the text similarity rate between plagiarized and self-plagiarized manuscripts (25% [95% CI 22-27%] vs. 28% [95% CI 20-33%]; U = 645.50; P = 0.634). Differences in text similarity rate were found between various sections of self-plagiarized manuscripts (H = 12.65, P = 0.013). The plagiarism rate in the Materials and Methods (61% (95% CI 41-68%) was higher than in the Results (23% [95% CI 17-36%], U = 33.50; P = 0.009) or Discussion (25.5 [95% CI 15-35%]; U = 57.50; P < 0.001) sections. Three authors were identified in the Déjà vu database. Plagiarism detection software combined with manual verification may be used to detect plagiarized manuscripts and prevent their publication. The prevalence of plagiarized manuscripts submitted to the CMJ, a journal dedicated to promoting research integrity, was 11% in the 2-year period 2009-2010.

Comparison of methods: Passing and Bablok regression.

The comparison of methods experiment is important part in process of analytical methods and instruments validation. Passing and Bablok regression analysis is a statistical procedure that allows valuable estimation of analytical methods agreement and possible systematic bias between them. It is robust, non-parametric, non sensitive to distribution of errors and data outliers. Assumptions for proper application of Passing and Bablok regression are continuously distributed data and linear relationship between data measured by two analytical methods. Results are presented with scatter diagram and regression line, and regression equation where intercept represents constant and slope proportional measurement error. Confidence intervals of 95% of intercept and slope explain if their value differ from value zero (intercept) and value one (slope) only by chance, allowing conclusion of method agreement and correction action if necessary. Residual plot revealed outliers and identify possible non-linearity. Furthermore, cumulative sum linearity test is performed to investigate possible significant deviation from linearity between two sets of data. Non linear samples are not suitable for concluding on method agreement.

Validation of methods performance for routine biochemistry analytes at Cobas 6000 analyzer series module c501.

INTRODUCTION: Cobas 6000 (Roche, Germany) is biochemistry analyzer for spectrophotometric, immunoturbidimetric and ion-selective determination of biochemical analytes. Hereby we present analytical validation with emphasis on method performance judgment for routine operation. MATERIALS AND METHODS: Validation was made for 30 analytes (metabolites, enzymes, trace elements, specific proteins and electrolytes). Research included determination of within-run (N = 20) and between-run imprecision (N = 30), inaccuracy (N = 30) and method comparison with routine analyzer (Beckman Coulter AU640) (N = 50). For validation of complete analytical process we calculated total error (TE). Results were judged according to quality specification criteria given by European Working Group. RESULTS: Within-run imprecision CVs were all below 5% except for cholesterol, triglycerides, IgA and IgM. Between-run CVs for all analytes were below 10%. Analytes that did not meet the required specifications for imprecision were: total protein, albumin, calcium, sodium, chloride, immunoglobulins and HDL cholesterol. Analytes that did not fulfill requirements for inaccuracy were: total protein, calcium, sodium and chloride. Analytes that deviated from quality specifications for total error were: total protein, albumin, calcium, sodium, chloride and IgM. Passing-Bablok regression analysis provided linear equation and 95% confidence interval for intercept and slope. Complete accordance with routine analyzer Beckman Coulter AU640 showed small number of analytes. Other analytes showed small proportional and/or small constant difference and therefore need to be adjusted for routine operation. CONCLUSIONS: Regarding low CV values, tested analyzer has satisfactory accuracy and precision and is extremely stable. Except for analytes that are coherent on both analyzers, some analytes require adjustments of slope and intercept for complete accordance.

The quality of the extra-analytical phase of laboratory practice in some developing European countries and Mexico - a multicentric study.

BACKGROUND: This cross-sectional multicentric survey study aimed to assess the quality of the extra-analytical phase of laboratory activities in some developing European countries and Mexico. We assessed the quality of the extra-analytical practices in participating laboratories regarding the: a) sample acceptance criteria; b) phlebotomy procedures; c) test results reporting and d) recording non-conformities. METHODS: A survey was performed during the April-May 2009. A total of 15 clinical laboratories from the following countries were included: Bosnia, Croatia, Czech Republic, Hungary, Mexico, Poland, Portugal, Romania, Serbia and Ukraine. Questions were scored (scores from 1-4) and average scores was calculated for each category. RESULTS: The overall score for all respondents (n = 443) was 3.10 ± 0.33. The average score was 3.11 ± 0.56 for sample acceptance criteria, 2.76 ± 0.58 for phlebotomy and 3.34 ± 0.53, for test results reporting (F = 116.49; p < 0.001). Laboratory accreditation was associated with better practices and higher overall quality of the extra-analytical procedures (F = 16.62; p < 0.001). Moreover, the highest scores for sample acceptance criteria (F = 8.32; p < 0.001), phlebotomy procedures (F = 13.28; p < 0.001) and for reporting non-conformities (F = 33.62; p < 0.001) were observed for accredited laboratories or laboratories under preparation for accreditation. CONCLUSIONS: The overall quality of the extra-analytical practices in countries in this survey is not satisfactory. Phlebotomy practices are the most critical extra-analytical activity. Since laboratory accreditation was associated with better practices and higher overall quality of the extra-analytical procedures, we believe that the most significant improvement could be made by implementing the total quality management system and standardizing laboratory procedures.