The first laboratory confirmed invasive meningococcal disease of serogroup C with abdominal clinical presentation in Slovakia, 2019.

Acute abdominal clinical presentations as initial manifestation of meningococcal infection are uncommon and frequently provoked by hyperinvasive isolates of meningococci. 10% of patients infected by the meningococcal strain, that is on the rise in Europe, suffer from abdominal pain. We hereby report the first laboratory confirmed fatal case of an otherwise healthy adult male presented with acute abdominal pain during first 24-48 hours, masking Neisseria meningitidis (N. meningitidis) infection. In the National Reference Center for meningococci, in the blood of a man post-mortem, we identified N. meningitidis serogroup C using real time polymerase chain reaction (PCR). Subsequently, massivelly-parallel sequencing (MPS) was performed on isolated total DNA for pathogen confirmation and further investigation.

Evaluation of 16S rRNA primer sets for characterisation of microbiota in paediatric patients with autism spectrum disorder.

intestinal microbiota is becoming a significant marker that reflects differences between health and disease status also in terms of gut-brain axis communication. Studies show that children with autism spectrum disorder (ASD) often have a mix of gut microbes that is distinct from the neurotypical children. Various assays are being used for microbiota investigation and were considered to be universal. However, newer studies showed that protocol for preparing DNA sequencing libraries is a key factor influencing results of microbiota investigation. The choice of DNA amplification primers seems to be the crucial for the outcome of analysis. In our study, we have tested 3 primer sets to investigate differences in outcome of sequencing analysis of microbiota in children with ASD. We found out that primers detected different portion of bacteria in samples especially at phylum level; significantly higher abundance of Bacteroides and lower Firmicutes were detected using 515f/806r compared to 27f/1492r and 27f*/1495f primers. So, the question is whether a gold standard of Firmicutes/Bacteroidetes ratio is a valuable and reliable universal marker, since two primer sets towards 16S rRNA can provide opposite information. Moreover, significantly higher relative abundance of Proteobacteria was detected using 27f/1492r. The beta diversity of sample groups differed remarkably and so the number of observed bacterial genera.

Specificity of gut microbiota in children with autism spectrum disorder in Slovakia and its correlation with astrocytes activity marker and specific behavioural patterns.

Recent research suggests the involvement of bidirectional gut-brain axis in autism spectrum disorder (ASD). The aim of this study was to establish the role of changed gut microbiota in behavioural and gastrointestinal manifestations, but also in astrocyte activation in children with ASD. Distinct faecal microbiota in children with ASD was found to be more heterogeneous compared to that in neurotypical children. Different bacterial abundance and correlation with behavioural and GI manifestations revealed several bacterial genera possibly important for ASD. Microbial-neuronal cross talk could be accomplished through S100B, released by glial cells, activated by low grade inflammation. More complex studies are required to understand ASD pathogenesis.

Impact of smoking cigarette on the mRNA expression of cytokines in mucosa of inflammatory bowel disease.

It is well known that smoking is the risk factor in the development and clinical course of Crohn s disease (CD), but on the other hand, smoking is a protective factor against ulcerative colitis (UC). The pathways that are influenced by smoking in CD and UC are poorly understood. The aim of our study was to analyse the influence of smoking on the mRNA expression of cytokines in mucosa in patients with CD and UC. We performed a cross-sectional study. The cohort consisted of 86 IBD patients (48 CD patients and 38 UC patients) and took place at the IBD Centre at the University Hospital Bratislava-Ruzinov. We took the demographic and clinical data of each patient, including information about their smoking habits. We performed a colonoscopy on each patient and took biopsies from both inflamed and non-inflamed sigma (CD, UC) and terminal ileum (CD). mRNA was extracted from mucosal biopsy samples for each cytokine and was normalized to a housekeeping gene (GAPDH). Finally, we compared the mRNA expression of target cytokines in the mucosa of smokers and non-smokers in IBD patients. Smokers with Crohn s disease have a significantly higher mRNA expression of pro-inflammatory cytokine TNF ? (p=0.003) in inflamed mucosa in sigma compared with non-smokers. In smokers with ulcerative colitis, we observed significantly higher mRNA expression of anti-inflammatory cytokine IL 10 (p=0.022) in non-inflamed mucosa of sigma. Similarly, smokers with UC have a significantly decreased mRNA expression of cytokine TLR 2 (p=0.024) and CCR1 (p=0.049) in non-inflamed mucosa of sigma. Based on our results, smoking has a positive influence on cessation and the clinical course of UC due to the stimulation of anti-inflammatory cytokine IL 10 in mucosa. On the other hand, smokers with CD have a higher expression of pro-inflammatory cytokine TNF ?, which could be associated with a worsening of the disease and response to therapy.

Influence of vitamin D on the expression of mRNA of cytokines in the mucosa of inflammatory bowel disease patients.

The aim of this study was to analyze the influence of 25(OH)VD serum concentration on the expression of mRNA cytokines (IL-6, IL-8, IL-12, IL-17, IL-23, TNFα, CCR1, CCR2, CCR5, CCR9, CCL5, TLR2, TLR4, TLR5, CD207 ,CD206, FoxP3) in mucosa of IBD patients. The cohort consisted of 86 IBD patients (48 CD and 38 UC) followed at the IBD center of University Hospital Bratislava-Ruzinov. We performed colonoscopy in each patient and took biopsies from mucosa of sigma and terminal ileum. Serum concentration of 25(OH)VD was assessed at the time of colonoscopy. mRNA was extracted from mucosal biopsy samples for each cytokine. Then we analyzed the correlation between VD and the expression of mRNA of cytokines from biopsies samples.  In CD we observed a significant positive correlation of serum concentration 25(OH)VD and the expression mRNA level of IL-6. There was also trend towards significant positive correlation of the expression mRNA of TNFα, IL-10, IL-23, TLR 2 in inflamed mucosa of terminal ileum as well as the expression mRNA of CCR5 and CCR1 in non-inflamed mucosa from terminal ileum. We also found a trend towards positive correlation between 25(OH)VD and the expression mRNA of IL-23, TLR4, CD 207, CCR1, CCR5 and CD 206 in non-inflamed mucosa of sigma in UC.VD significantly correlated with the levels of expression of several inflammatory cytokines including TNFα in colonic mucosa of patients with IBD (Tab. 4, Fig. 3, Ref. 31).

Relationship between copper(ii) complexes with FomA adhesin fragments of F. nucleatum and colorectal cancer. Coordination pattern and ability to promote ROS production.

The copper(ii) ion binding of the Ac-KGHGNG-NH2 and Ac-PTVHNE-NH2 fragments of FomA adhesin from Fusobacterium nucleatum was studied using potentiometry, UV-Vis, CD, EPR and DFT techniques. The coordination pattern was described in a wide range of pH values. Ligands begin interactions with metal ions using imidazole nitrogen. At pH 6.8 (a value typical of the large intestine environment), the metal ion was coordinated by the 3N donor atoms {Nim, 2 × N-amide} in both cases. However, the copper(ii) ion was bound more effectively by the Ac-PTVHNE-NH2 peptide. The formation of reactive oxygen species (ROS) was studied by UV-Vis and fluorescence spectroscopy, as well as gel electrophoresis in the presence of H2O2 and/or ascorbic acid. The complexes generated ROS in the highest amounts among all compounds. Moreover, they stimulated the CT26 cell line (mouse colon carcinoma) to produce ROS which lead to oxidative stress. It was also determined that such radicals took part in the plasmid degradation mechanism.

Detection and molecular characterization of Slovak tomato isolates belonging to two recombinant strains of potato virus Y.

Total RNAs from a symptomless tomato plant were subjected to next-generation sequencing (NGS) analysis, revealing the presence of a single viral agent - potato virus Y (PVY). The analysis of determined full-length genome sequence assigned the PVY SL16 isolate to the recombinant PVY-N-Wi strain group. A series of primers targeting the four main recombinant junction (RJ) sites were used for characterization of additional 5 tomato PVY isolates recovered in Western Slovakia. Based on the partial sequences, the isolates could be classified as belonging to PVY-N-Wi and PVY-NTNa strain groups. Interestingly, both these distinct recombinant PVY types were identified in mixed infection in one tomato sample (SL31). Our results further reinforce the data on the complexity of PVY infection and confirm the recombination as a significant evolutionary process shaping the PVY diversity.

Studies of viomycin, an anti-tuberculosis antibiotic: copper(ii) coordination, DNA degradation and the impact on delta ribozyme cleavage activity.

Viomycin is a basic peptide antibiotic, which is among the most effective agents against multidrug-resistant tuberculosis. In this paper we provide the characteristics of its acid base properties, coordination preferences towards the Cu(ii) ions, as well as the reactivity of the resulting complexes against plasmid DNA and HDV ribozyme. Careful coordination studies throughout the wide pH range allow for the characterisation of all the Cu(ii)-viomycin complex species. The assignment of proton chemical shifts was achieved by NMR experiments, while the DTF level of theory was applied to support molecular structures of the studied complexes. The experiments with the plasmid DNA reveal that at the physiological levels of hydrogen peroxide the Cu(ii)-viomycin complex is more aggressive against DNA than uncomplexed metal ions. Moreover, the degradation of DNA by viomycin can be carried out without the presence of transition metal ions. In the studies of antigenomic delta ribozyme catalytic activity, viomycin and its complex are shown to modulate the ribozyme functioning. The molecular modelling approach allows the indication of two different locations of viomycin binding sites to the ribozyme.

Invasive candidiasis in liver transplant patients: Incidence and risk factors in a pediatric cohort.

Prolonged OR, re-transplantation, and high-volume intraoperative transfusion have been associated with increased risk for IC in adult LT recipients. Antifungal prophylaxis is recommended for adult patients with these risk factors. There are limited data on the incidence of and risk factors for IC in pediatric LT recipients. A retrospective cohort study of all pediatric LT patients at the CHOP between 2000 and 2012 and the CHP between 2004 and 2012 was performed to define the incidence of IC within 30 days of LT. A 3:1 matched case-control study with incidence density sampling was performed. Conditional logistic regression analyses were used to explore risk factors associated with IC. Among 397 recipients, the incidence of IC was 2.5%. Bivariate analyses showed that ICU admission prior to transplant, OR > 10 h, intraoperative volume infusion of >300 mL/kg, and broad-spectrum antibiotics were significantly associated with IC. In a multivariate model, only ICU admission remained significantly associated with IC. Antifungal prophylaxis was not significantly protective against IC. The low incidence of IC and lack of an identified protective effect from antifungal prophylaxis suggest that prophylaxis in pediatric LT recipients should not be routinely recommended to prevent IC events in the first 30 days post-transplant.

Liver preservation with machine perfusion and a newly developed cell-free oxygen carrier solution under subnormothermic conditions.

We describe a new preservation modality combining machine perfusion (MP) at subnormothermic conditions(21 °C) with a new hemoglobin-based oxygen carrier (HBOC) solution. MP (n=6) was compared to cold static preservation (CSP; n=6) in porcine orthotopic liver transplants after 9 h of cold ischemia and 5-day follow-up. Recipients' peripheral blood, serial liver biopsies, preservation solutions and bile specimens were collected before, during and after liver preservation. Clinical laboratorial and histological analyses were performed in addition to mitochondrial functional assays, transcriptomic, metabolomic and inflammatory inflammatory mediator analyses. Compared with CSP, MP animals had: (1) significantly higher survival (100%vs. 33%; p<0.05); (2) superior graft function (p<0.05);(3) eight times higher hepatic O2 delivery than O2 consumption (0.78 mL O2/g/h vs. 0.096 mL O2/g/h) during MP; and (4) significantly greater bile production (MP=378.5 ± 179.7; CS=151.6 ± 116.85). MP downregulated interferon (IFN)-α and IFN-γ in liver tissue. MP allografts cleared lactate, produced urea, sustained gluconeogenesis and produced hydrophilic bile after reperfusion. Enhanced oxygenation under subnormothermic conditions triggers regenerative and cell protective responses resulting in improved allograft function. MP at 21 °C with the HBOC solution significantly improves liver preservation compared to CSP.

Allospecific CD154+ T cells associate with rejection risk after pediatric liver transplantation.

Antigen-specific T cells, which express CD154 rapidly, but remain untested in alloimmunity, were measured with flow cytometry in 16-h MLR of 58 identically-immunosuppressed children with liver transplantation (LTx), to identify Rejectors (who had experienced biopsy-proven rejection within 60 days posttransplantation). Thirty-one children were sampled once, cross-sectionally. Twenty-seven children were sampled longitudinally, pre-LTx, and at 1-60 and 61-200 days after LTx. Results were correlated with proliferative alloresponses measured by CFSE-dye dilution (n = 23), and CTLA4, a negative T-cell costimulator, which antagonizes CD154-mediated effects (n = 31). In cross-sectional observations, logistic regression and leave-one-out cross-validation identified donor-specific, CD154 + T-cytotoxic (Tc)-memory cells as best associated with rejection outcomes. In the longitudinal cohort, (1) the association between CD154 + Tc-memory cells and rejection outcomes was replicated with sensitivity/specificity 92.3%/84.6% for observations at 1-60 days, and (2) elevated pre-LTx CD154 + Tc-memory cell responses were associated with significantly increased incidence (p = 0.02) and hazard (HR = 7.355) of rejection in survival/proportional hazard analysis. CD154 expression correlated with proliferative alloresponses (r = 0.835, p = 7.1e-07), and inversely with CTLA4 expression of allospecific CD154 + Tc-memory cells (r =-0.706, p = 3.0e-05). Allospecific CD154 + T-helper-memory cells, not CD154 + Tc-memory, were inhibited by increasing Tacrolimus concentrations (p = 0.026). Collectively, allospecific CD154 + T cells provide an estimate of rejection risk in children with LTx.

Late graft loss or death in pediatric liver transplantation: an analysis of the SPLIT database.

Late graft loss (LGL) and late mortality (LM) following liver transplantation (LT) in children were analyzed from the studies of pediatric liver transplantation (SPLIT) database. Univariate and multivariate associations between pre- and postoperative factors and LGL and LM in 872 patients alive with their primary allografts 1 year after LT were reviewed. Thirty-four patients subsequently died (LM) and 35 patients underwent re-LT (LGL). Patients who survive the first posttransplant year had 5-year patient and graft survival rates of 94.2% and 89.2%, respectively. Graft loss after the first year was caused by rejection in 49% of the cases with sequelae of technical complications accounting for an additional 20% of LGL. LT for tumor, steroid resistant rejection, reoperation in the first 30 days and >5 admissions during the first posttransplant year were independently associated with LGL in multivariate analysis. Malignancy, infection, multiple system organ failure and posttransplant lymphoproliferative disease accounted for 61.8% of all late deaths after LT. LT performed for FHF and tumor were associated with LM. Patients who are at or below the mean for weight at the time of transplant were also at an increased risk of dying. Frequent readmission was also found to be associated with LM.

Cryptococcosis in liver and kidney transplant recipients receiving anti-thymocyte globulin or alemtuzumab.

Rabbit anti-thymocyte globulin (ATG) and alemtuzumab have been used for induction or preconditioning and for the treatment of acute rejection in organ transplant recipients in many centers. Such regimens may lead to a substantial decline in the CD4 lymphocyte count to levels seen in other population groups at high risk of cryptococcosis. In view of this, we examined the impact of such therapy on the cumulative incidence of cryptococcosis among liver and kidney recipients. A total of 834 liver and 727 kidney transplants were performed during the study period. Seven hundred and eighty-one patients did not receive ATG or alemtuzumab; 646 received 1 dose of either drug, and 134 patients received 2 doses of either drug. The cumulative incidence of cryptococcosis was 0.26% (2/781) among those who did not receive ATG or alemtuzumab; 0.3% (2/646) among those who received only 1 dose, and 2.24% (3/134) among those who received 2 doses (P=0.03). There were 5 cases of cryptococcosis in liver recipients and 2 in kidney recipients. There were 3 cases of cryptococcal meningitis, 3 of pneumonia, and 1 of disseminated disease. The 2 kidney recipients had meningitis. Diagnosis occurred at a median of 255 days (range 7-517) after transplantation. The mortality rate was 14.2%. We conclude that the use of 1 dose of ATG or alemtuzumab is not associated with an increased cumulative incidence of cryptococcosis, but that those patients receiving 2 doses are at increased risk.

Oxidative stress in fatty livers of obese Zucker rats: rapid amelioration and improved tolerance to warm ischemia with tocopherol.

Fatty livers in humans and rats are less tolerant of ischemia, endotoxin, and alcohol. We hypothesized that fatty livers of obese (Ob) Zucker rats are oxidatively stressed and oxidative stress could be relieved by antioxidant treatment, leading to improved tolerance to ischemia. Total glutathione (GSH), tocopherol (TOC), ascorbic acid (AA), catalase (CAT), superoxide dismutase (SOD), and selenium-glutathione peroxidase (Se-GPx) were measured in the livers of Ob and lean (Ln) Zucker rats before and after treatment with high-dose TOC and ascorbate. Also, survival in treated Ob rats following a lethal 90 minutes of partial in vivo warm ischemia was examined. Fatty livers of Ob rats contained significantly less GSH, TOC, and CAT, in comparison with livers of Ln rats. Immunoblotting showed significantly decreased CAT protein without changes in mRNA in fatty livers. There were no significant differences in AA, SOD, and Se-GPx between the 2 groups. Pretreatment with TOC and ascorbate over 48 hours completely corrected the decreases in GSH, TOC, and CAT. Most importantly, TOC with or without ascorbate pretreatment significantly improved survival in Ob rats following ischemia in a dose-dependent manner. In conclusion, TOC administration corrected the oxidative stress in fatty livers of Ob Zucker rats and improved survival following lethal ischemia. Additional studies are needed to determine the efficacy of TOC-a relatively inexpensive agent-in treating patients with fatty livers in a variety of clinical conditions, possibly including liver transplantation.

Successful nonfreezing, subzero preservation of rat liver with 2,3-butanediol and type I antifreeze protein.

BACKGROUND: Organ cryopreservation is hindered by ice inflicted damage. Nonfreezing preservation of livers at subzero temperatures might offer advantages over current preservation. METHODS: Sprague-Dawley rats were divided into three groups. UW livers (n = 6) were stored in University of Wisconsin (UW) solution at +4 degrees C. UWB livers (n = 6) were perfused ex vivo with UW + 10% 2,3-butanediol at < or =7 degrees C and stored at -4 degrees C. AFP livers (n = 4) were preserved identical to UWB livers, except for addition of 1 mg/ml of type I antifreeze protein. After 24 h livers were perfused with Krebs-Henseleit buffer (37 degrees C) for 60 min. Bile production, O(2) consumption (O(2)C), taurocholate extraction, and lactate dehydrogenase (LDH) release during perfusion and liver adenine nucleotide content and energy charge at the end of perfusion were measured. Cell membrane integrity was determined by trypan blue infusion. RESULTS: Ice formation was prevented in all livers stored at -4 degrees C. Bile production, O(2)C, and taurocholate extraction were similar among three groups. Livers stored at -4 degrees C contained significantly more adenine nucleotides than livers stored at +4 degrees C but the energy charge was similar. LDH release was significantly greater (P < 0.05) in the AFP group vs UWB and UW (63 vs 28 and 21 mU/min/g liver, respectively). Hepatocyte and sinusoidal cell trypan blue uptake was similar in all three groups. CONCLUSIONS: Butanediol with or without AFP was effective in preventing ice formation up to 24 h in rat livers stored at -4 degrees C. Although as effective as current +4 degrees C protocols, subzero preservation for longer periods needs to be achieved prior to clinical application.

[Pathomorphology and pathogenesis of changes in Alzheimer's disease]. / Patomorfologia i patogeneza zmian w chorobie Alzheimera.

In this paper the actual issues of pathomorphology and pathogenesis of Alzheimer's disease are discussed. The importance of beta-amyloid is recognized. The linkage between late-onset form of Alzheimer's disease and the mutations of gene encoding the amyloid precursor protein (on chromosome 21) was found. Phosphorylation of paired helical filament (which are composed of tau protein) plays the important role. There is evidence for a strong association between apolipoprotein E genotype (on chromosome 19) and late-onset dementia of Alzheimer's type. Two more genes were recently identified: PS-1 and PS-2. Their mutations occur in 70-80% cases of early-onset form of the disease. There is much information about the role of head injury, cholinergic deficiency, estrogen, nerve growth factor and the decline in brain glucose metabolism. Our current knowledge can lead to development of prevention strategies and early recognition of Alzheimer's disease.

Pilot study of multiple chemotherapy resistance modulators plus epirubicin in the treatment of resistant malignancies.

We studied the toxicity and efficacy of adding to epirubicin five resistance modulators in the treatment of resistant solid tumors. Additional drugs were added in successive cohorts of patients, such that cohort 1 patients received two drugs along with their epirubicin, while cohort 4 patients received five modulators along with their epirubicin. Metronidazole, tamoxifen (cohort 1), dipyridamole (cohort 2), ketoconazole (cohort 3) and cyclosporin (cohort 4) were administered with epirubicin. A total of 22 patients were treated. Nausea and vomiting was usually mild to moderate. There was an unexpectedly high incidence of possible cardiac toxicity associated with treatment, although in some patients it was uncertain whether or not observed cardiac events were related to treatment. Granulocytopenia was significant in all four cohorts, but it was unclear whether it was increased by the modulators. There were two febrile neutropenic events in cohorts 1 and 2 successfully treated with antibiotics, and three septic deaths (one in each of cohorts 1, 2 and 4). It was elected to discontinue enrollment on the study prematurely in light of cardiac and other toxicity seen in the first two patients accrued in cohort 4. A single response was observed. While this approach is feasible, the observed toxicity and the difficulty patients experienced in ingesting the large number of prescribed pills will make further exploration of this approach difficult.

A phase II study of 5-fluorouracil plus high-dose folinic acid in the treatment of recurrent small cell lung cancer.

Fourteen patients with small cell lung cancer (SCLC) previously treated with one or two chemotherapy regimens were entered in this study. 5-Fluorouracil 370 mg/m2 or 300 mg/m2 was given with folinic acid 200 mg/m2 by i.v. rapid infusion on days 1 through 5. Cycles were repeated every 28 days. There were no objective responses. One patient had stable disease for 47 weeks. The overall median survival duration was 23 weeks. Toxicity was comparable to that seen in other studies of 5-fluorouracil plus folinic acid.

A phase II study of 5-fluorouracil plus folinic acid in malignant gliomas in adults.

Fourteen patients with malignant gliomas were entered on a phase II study of 5-fluorouracil 300-370 mg/m2 plus folinic acid 200 mg/m2 x 5 days q4 weeks. To be eligible, patients could not have received more than 1 prior chemotherapy regimen. A single patient with a recurrent oligodendroglioma responded. Toxicity (predominantly stomatitis, diarrhea, and granulocytopenia) was tolerable and was similar to that seen in studies of 5-fluorouracil plus folinic acid in other tumor types. This regimen has minimal activity in recurrent malignant gliomas.