Pathogenic potential to humans of Shiga toxin-producing Escherichia coli isolated from wild boars in Poland.

The aim of the study was to investigate the presence of Shiga toxin-producing Escherichia coli (STEC) in the wild boar population of north-eastern Poland, and to evaluate the potential health risk associated with wild boars carrying STEC/AE-STEC strains. In Poland, the African Swine Fever (ASF) virus has been a growing problem in domestic pigs and wild boars, one of the main reservoirs of the virus, because of this hunters, veterinary practitioners and foresters thus face a greater risk of coming into contact with animals. Rectal swabs samples were obtained from 152 wild boars hunter-harvested in the 2017/2018 season (autumn-winter) in north-eastern Poland. The samples were enrichment in modified buffered peptone water. Polymerase chain reaction (PCR) assays were conducted to determine the virulence profile of stx1, stx2 and eae and aggR genes, identify subtypes of stx1 and stx2 genes, and perform O and H serotyping. STEC/AE-STEC virulence genes were detected in 43 isolates (28.29%): STEC in 17 isolates (11.18%) and AE-STEC in 26 isolates (17.11%), respectively. None of the tested isolates carried the aggR gene. The most dangerous AE-STEC virulence profile associated with HUS was found in 2 isolates (1.32%): stx1NS/stx2a/d/eae serotype ONT:H7 and stx2a/eae serotype O146:H7. Six of the 152 tested samples belonged to serogroup O157 (3.95%), including one AE-STEC isolate with virulence profile stx2g/eae and five EPEC isolates. The results of this study suggest that wild boars in north-eastern Poland can carry STEC/AE-STEC strains that are potentially pathogenic for humans. This is the first report documenting the virulence of STEC and AE-STEC isolates from wild boars in Poland.

Influence of Cryopreservation on Structural, Chemical, and Immunoenzymatic Properties of Aortic Valve Allografts.

OBJECTIVES: The problems in preparing (including cryopreservation) and implanting aortic valve allografts (AVAs) is widely elaborated, but some issues need explanation. MATERIAL AND METHODS: Twenty AVAs cryopreserved in dimethylsulphoxide/RPMI solution under -160°C for 1-15 years and 3 controls stored at +4°C up to 2 weeks, from 19 male and 4 female donors, aged 20-51, ±30.8 years, were examined using light (LM), digital (DM), and scanning electron microscopy (SEM), energy dispersion X-ray spectroscopy (EDS), and enzyme-linked immunosorbent assay immunoenzymatic tests (PECAM1, CD34). RESULTS: All AVAs were macroscopically correct. LM revealed normal structure of leaflets but massive endothelial decellularization (±59 cells remained on the surface of 5 mm scraps). DM and SEM demonstrated generally normal collagen structures, but local alterations, probably influenced by freezing-thawing (gaps, separated plates) or being initial phase of native degeneration (grains). EDS detected a little elevated calcium amount in 1 specimen only. The mean PECAM1 and CD34 concentrations were at similar low level in all probes. CONCLUSIONS: Fresh and cryopreservation technologies did not significantly influence the basic properties of AVA leaflets; however, massive endothelial decellularization was present in both groups. Therefore, no endocardial cell activity nor signs of inflammation were observed. These results were independent of donors' age and sex, processing technology, and time of storage of cryopreserved AVAs.

Transcription factor GATA3 expression is induced by GLS2 overexpression in a glioblastoma cell line but is GLS2-independent in patient-derived glioblastoma.

Phosphate-activated glutaminase (GA), a ubiquitous glutamine-metabolizing enzyme, is encoded by two genes, GLS and GLS2. In mammalian cancers, GLS isoforms are perceived as molecules promoting cell proliferation and invasion, whereas the role of GLS2 isoforms seems to be more complex and cell type-specific. Previous studies have shown abundance of GLS and lack of GLS2 transcripts in T98G human glioblastoma (GBM) cell line and patient-derived GBM. Transfection with GAB sequence, the whole GLS2 cDNA transcript, suppressed malignant phenotype of T98G cells. Microarray analysis revealed upregulation of GATA3, the product of which has been implicated in suppressing growth of some peripheral cancers. In this study we confirmed a significant upregulation of GATA3 expression in the transfected cells both at mRNA and protein level. Considerable expression of GATA3 was also observed in GBM tissues (previously shown as not expressing GLS2), while only traces or no GATA3 was detected in (GLS2-expressing) non-tumorigenic brain samples. In conclusion, while mechanistic relation between GAB and GATA3 expression is evident following in vitro manipulation of GBM cell line, it does not appear to be an intrinsic property of GBM nor non-tumorigenic brain tissue.

Effect of feed supplementation with kynurenic acid on the morphology of the liver, kidney and gills in rainbow trout (Oncorhynchus mykiss Walbaum, 1792), healthy and experimentally infected with Yersinia ruckeri.

Kynurenic acid (KYNA) is an endogenous substance produced on the kynurenine pathway which is primarily known for its neuroactive properties. Recently, it has been proven that KYNA is a selective ligand for G protein-coupled receptor (GPR 35), presented on immunocompetent cells such as T lymphocytes. This opens up new possibilities of its application as an immunostimulating substance in aquaculture. Thus far, no histopathological investigations in fish have been completed to evaluate influence of KYNA supplementation in feed. This study has been undertaken to determine the effect of feed supplementation with KYNA (2.5, 25, 250 mg kg-1 of feed) for 28 days on the liver, gills and kidney in healthy fish and experimentally infected with Yersinia ruckeri. In a control group were observed a fatty liver, which is natural for this fish species in the autumn and winter season. As the dose of the supplement was increased, the fat liver changed, it decreased or completely disappeared. Additionally, inflammatory changes occurred in all the analysed organs, and their intensification was dose dependent. In the fish experimentally infected, KYNA caused aggravation of the signs in the liver, kidneys and gills, and the effect was dose dependent. The results implicate that KYNA may be a stressor for fish.

Characterization of Yersinia enterocolitica strains potentially virulent for humans and animals in river water.

AIMS: The aim of this study was to isolate and identify potentially pathogenic strains of Yersinia enterocolitica in water samples collected from the upstream section of the Drweca River in Poland. METHODS AND RESULTS: Thirty-nine water samples were collected. Strains were isolated, identified with the use of the API(®) 20E test kit (Biomerieux, Marcy l'Etoile, France) at 37°C, serotyped and subjected to a molecular analysis. Multiplex PCR was carried out to amplify three virulence genes: ail, ystA and ystB. Fragments of ail and ystA genes were not identified in the genetic material of the analysed strains. The ystB gene was identified in four strains. Yersinia enterocolitica strains of biotype 1A, which contain the ystB gene, may cause gastrointestinal problems. CONCLUSIONS: In our study, Y. enterocolitica strains of biotype 1A/ystB with serotypes 0 : 3, 0 : 5 and 0 : 8 were identified in samples collected from the Drweca River which flows through the areas protected by Natura 2000, one of the largest networks of nature conservation areas in the European Union. The presence of Y. enterocolitica in the Drweca River indicates that the analysed bacteria colonize natural water bodies. SIGNIFICANCE AND IMPACT OF THE STUDY: Most research focuses on food or sewage as a source of Y. enterocolitica infections. Little is known about the occurrence of this pathogen in natural waters. Our results show that natural waters are also a potential threat to human and animal health.

Regioselective hydrolysis of acetates in the presence of different yeast strains.

The model compound, hexane-1,2-diol diacetate, was hydrolyzed in the presence of supernatant obtained after cultivation of 4 yeast strains: Pichia jadinii, Rhodotorula glutinis and Yarrowia lipolytica KKP 379 and Saccharomyces cerevisiae 102 to evaluate the type of catalysis. The regioselectivity of extracellular enzymes as a function of hydrolysis towards primary and secondary acetic acid ester groups was monitored. The enzymes secreted by P. jadinii, R. glutinis and Y. lipolytica KKP 379 exhibited high regioselectivity towards primary position, while those from S. cerevisiae showed practically no discrimination between the ester groups.

Application of DNA vaccines in fish.

Vaccination is a most cost-effective way of controlling infectious diseases in fish. However, some vaccination techniques when applied to hatchery conditions are not as effective as we expect them to be. Modern molecular biology techniques offer a number of opportunities for improving existing bacterial or viral vaccines or creating new ones. One of the most promising trends in vaccinology is development of DNA vaccination. DNA vaccines are based on the gene encoding specific antigen, which is expressed in vaccinated organism and induces the host immune system. DNA vaccines, compared to conventional vaccines, have many advantages including ability to trigger wider immune response, bigger stability and possibility of large-scale production. To date, there are several reports indicating effectiveness of DNA vaccines used against fish pathogens.

Modulative influence of lysozyme dimer on defence mechanisms in the carp (Cyprinus carpio) and European sheatfish (Silurus glanis) after suppression induced by herbicide Roundup.

Immunomodulation is a commonly used method of prophylaxis in humans and animals. Lysozyme dimer (KLP-602) was used at a dose of 50 ug/kg b.w. in order to correct the immunosuppression caused by the action of herbicide glyphosate (Roundup- Monsanto), which was used in a single bath for 10 minutes in a concentration of 100 mg/l of water. The investigations were carried out on 2 species of fish: the carp (Cyprinus carpio L.) and european catfish (Silurus glanis L.). Herbicide glyphosate caused a decrease in metabolic and phagocytic activity (RBA and PKA) and in proliferative response stimulated by Con A and LPS in carp and european catfish. The immunosuppression sustained for about 2 weeks. The results obtained indicate the possibility of correction of immunosuppression applying lysozyme dimmer (KLP-602) after use of which, the level of the studied indexes increased.

Activation of blood platelets after percutaneous transluminal coronary angioplasty and coronary artery bypass graft surgery.

We have evaluated the activation of platelets in blood samples taken from patients with stable angina undergoing balloon angioplasty (percutaneous transluminal coronary angioplasty [PTCA]) (n=11) or coronary artery bypass grafting (CABG) under hypothermic (n=11) or normothermic conditions (n=11). We have found that surface expression of P-selectin on platelets in whole blood from PTCA patients upon thrombin treatment was significantly reduced, as compared with control platelets from healthy subjects. This effect was partially reversed when platelets washed from the same blood sample were used, but even then P-selectin expression was significantly lower in PTCA patients than it was in control subjects. There was a significant increase in basal expression of P-selectin in blood platelets taken from patients who underwent CABG under normothermic conditions (warm blood cardioplegia) as opposed to hypothermic patients (cold crystalloid cardioplegia). These platelets retain the ability to respond to agonists, although to a much lower extent than do those from healthy control donors. The surface exposure of P-selectin on resting and thrombin-treated platelets isolated from CABG surgery patients was not different from that of the control platelets. The adhesion to fibrinogen of resting and thrombin-treated platelets from patients who underwent balloon angioplasty as well as CABG surgery under normothermic and hypothermic conditions was significantly reduced when compared with the fibrinogen of the control platelets. These results suggest that the function of platelet fibrinogen receptor is impaired in patients with stable angina pectoris and that PTCA and CABG surgery activates platelets.

Does potassium dichromate induce apoptosis in lymphocytes?

The aim of this work was to investigate the possibility of apoptosis in the human peripheral blood lymphocytes after treatment with potassium dichromate (K2Cr2O7), a potential occupational carcinogenic and mutagenic agent. Lymphocytes were stimulated by phytohemagglutinin, cultured for 72 h and incubated with either 0.2 mM or 0.4 mM K2Cr2O7 for the last 24 h or 48 h of culture. The condensation and margination of chromatin with emerging 'half-moon' structure, characteristic of apoptosis were observed. Phosphatidylserine displaced from the inner to outer side of the cellular membrane in 54% of cells after a 48-h incubation with 0.4 mM K2Cr2O7 (annexin-V+/PI-); 39% of these cells were of late apoptotic--secondary necrotic form (annexin-V+/PI+). Following the agarose gel electrophoresis of DNA, a 'ladder pattern' typical of apoptosis, was found. The results of the present study demonstrate that K2Cr2O7 can induce in the human peripheral blood lymphocytes changes similar to apoptotic ones.

Spontaneous apoptosis of neutrophils in whole blood and its relation to apoptosis gene proteins.

Apoptosis of neutrophils limits their pro-inflammatory potential. We tested the ability of fresh and cultured whole blood neutrophils to undergo spontaneous apoptosis and expression of p53, Fas/Apo-1, bcl-2 protein in the cells using flow cytometry. Neutrophil apoptosis was estimated using Annexin V and propidium iodide binding and verified under light microscopy. The percentage of early and late apoptotic neutrophils in the blood samples increased significantly after 20 h culture from 12.3 +/- 14.2% and 4.3 +/- 4.2% to 39.5 +/- 14% and 15.3 +/- 9.6%, respectively. The majority of late apoptotic neutrophils had altered morphology in FSC/SSC dot plot compared to alive or early apoptotic neutrophils. Cultured neutrophils presented markedly lower expression of bcl-2 protein compared to fresh blood cells: 211 +/- 321 median of fluorescence intensity (MFI) and 787 +/- 1152 MFI, respectively. The increased percentage of late apoptotic cells after culture paralleled the increase in the Fas/Apo-1 expression and negatively correlated with bcl-2 expression. We noted intracellular expression of p53 protein in neutrophils, although the expression did not correlate neither to the percentage of the apoptotic neutrophils, nor to the Fas/Apo-1 or bcl-2 expression. Our results suggested that neutrophil apoptosis is gene regulated, moreover, we present a possibility to assess the neutrophil apoptosis and cellular expression of the proteins of apoptosis related genes in whole blood samples.

The effect of air and nitrox divings on platelet activation tested by flow cytometry.

BACKGROUND: Nitrogen (N2) microbubbles activate the blood platelets and coagulaltion system. HYPOTHESIS: Breathing nitrox rather than air may reduce the level of platelet activation associated with decompression. METHODS: We tested platelet counts and the expression of functional membrane molecules on platelets in 10 divers subjected to saturated compression in nitrox at 4 ATA and in 9 divers subjected to compression in air at 2.8 ATA. Blood samples were taken before and immediately after the test. We measured the percentages of microplatelets, platelet aggregates and platelets bearing the activation marker C-D62P, and bearing molecules forming receptors for fibrinogen (CD61) and for von Willebrand factor (CD42b) using flow cytometry and specific monoclonal antibodies. Symptoms for DCS were also evaluated. RESULTS: DCS symptoms were not noted in either the nitrox or air group. In both groups we observed a marked increase in the percentage of activated platelets bearing CD62P molecules and an enhanced number of microplatelets and a marked drop in the platelets count in the blood of (divers in the air group. CONCLUSION: In all divers we observed certain changes in the platelet system, nevertheless decompression in nitrox resulted in a lesser degree of platelet activation. Though this study cannot exclude platelet activation as an etiological factor in DCS, the findings suggest platelet activation can occur in the absence of observable sign of DCS. Thus, platelet activation may be too sensitive a marker to serve as a predictor of DCS.

Comparison of platelet aggregability and P-selectin surface expression on platelets isolated by different methods.

Three methods commonly used for isolation of blood platelets from plasma were compared. Platelets were isolated by: 1) a washing method; 2) a method of metrizamide-gradient centrifugation; 3) a modified method of gel-filtration. The last method employed BSA-Sepharose gel instead of routinely used Sepharose gel saturated with BSA. BSA-Sepharose gel was prepared by covalent binding of thermally deactivated BSA to CNBr-activated Sepharose 2B. In contrast to platelets isolated by the other methods, an aggregability of the gel-filtered platelets and control platelets in plasma, both activated with ADP, were comparable. When expression of P-selectin on the surface of freshly isolated platelets was examined, the gel-filtered platelets exhibited the same extent of fluorescence signal as platelets in the citrated blood, whereas platelets isolated by the other methods exhibited twice the extent of the signal. The methods involving the centrifugation process cause a low but a significant platelet activation.

Effects of the herbicide Roundup on the ultrastructural pattern of hepatocytes in carp (Cyprinus carpio).

Experimental studies were performed on healthy, 80-100 g carp (Cyprinus carpio). Fish were exposed by emersion in Roundup (205 mg of glyphosate/l or 410 mg of glyphosate/l) in concentrations of 40- to 20-fold lower than those used in practice. Electron microscopy revealed that the herbicide caused appearance of myelin-like structures in carp hepatocytes, swelling of mitochondria and disappearance of internal membrane of mitochondria in carp at both exposure concentrations. It means that Roundup was harmful to carp when used in applied concentrations. Results of these studies enhance our knowledge of ultrastructural pathomorphology of fish organs following exposure to Roundup.

Effects of iridovirus-like agent on the cell-mediated immunity in sheatfish (Silurus glanis)--an in vitro study.

An iridovirus-like agent was tested for the first time in vitro on cell-mediated immunity in sheatfish (Silurus glanis). The influence of the iridovirus-like agent on pronephric macrophage metabolism was examined at two temperatures, 20 and 30 degrees C, by studying the respiratory burst activity stimulated by phorbol myristate acetate as well as the proliferative ability of lymphocytes stimulated by concanavalin A (ConA) and lipopolysaccharide (LPS) measured by MTT assay. The results showed that the iridovirus-like agent decreased the macrophage activity at incubation temperatures of 20 and 30 degrees C. The highest inhibitory effect was observed at 30 degrees C. The proliferative ability of pronephric lymphocytes had a similar pattern. The results showed that applying a virus at the same time or after the mitogen at 20 and 30 degrees C decreased the lymphocyte proliferation that was stimulated by either ConA or LPS. The highest suppressive effect was observed when virus was applied 14 h after the mitogen. This preliminary in vitro study demonstrated a strong suppressive influence of the iridovirus-like agent on pronephric macrophage and lymphocyte activity in sheatfish.

[Immunologic characteristics women with recurrent spontaneous abortion of unknown etiology. I. Cellular immune response]. / Immunologiczne odrebnosci kobiet z poronieniami nawykowymi o niewyjasnionej etiologii. I. Charakterystyka stanu odpornosci komórkowej i nieswoistej.

The purpose of this study was to analyze the cellular immune response of women with recurrent spontaneous abortion (RSA) of unknown etiology. The study group was consisted of 117 nonpregnant women with RSA and 44 healthy, nonpregnant multigravidas (as a control). The following immunological parameters were analyzed in peripheral blood of two groups of women: percentage of CD3+, CD4+, CD8+, CD3+/HLA-DR+ and CD16+/CD56+ cells, lymphocyte proliferative response to mitogen and allogenic, and chemiluminescence of neutrophils. The results show that nonpregnant women with RSA of unknown etiology differ in some parameters of immune response form controls. The changes of peripherial blood T lymphocytes subpopulation were observed, consisted on lower CD8+ percentage and higher CD4+ to CD8+ T cells ratio, but lymphocytes and neutrophils activity seems to be unchanged. It seems, that among couples experiencing RSA of unknown etiology higher evidence of the same HLA antigents is not observed, when compared with couples of normal fertility.

Role of tyrosine kinase enzymes in TNF-alpha and IL-1 induced expression of ICAM-1 and VCAM-1 on human umbilical vein endothelial cells.

The aim of this study was to analyse the potential roles of protein kinase enzymes in tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) induced expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on human umbilical vein endothelial cells (HUVEC). The authors observed a marked increase in ICAM-1 and VCAM-1 expression on HUVEC stimulated for 24 h by TNF-alpha (10 ng/ml) or IL-1 (20 ng/ml). Pre-treatment of HUVEC for 30 min with protein tyrosine kinase (PTK) inhibitors genistein and herbimycin A (10 micrograms/ml and 0.5 microgram/ml, respectively) before stimulation with IL-1 did not affect the expression of these molecules. Similar results were observed with respect to VCAM-1 expression on HUVEC stimulated by TNF-alpha. In contrast, pre-incubation of HUVEC with PTK inhibitors prior to the addition of TNF-alpha significantly enhanced subsequent expression of ICAM-1, although spontaneous expression of ICAM-1 on unstimulated HUVEC was unaffected. Western blot analysis demonstrated a significant increase in phosphorylated tyrosine protein levels in HUVEC stimulated by TNF-alpha, and significantly lower levels of these proteins in TNF-alpha stimulated HUVEC pre-treated with PTK inhibitors. These results demonstrate that IL-1 induced ICAM-1 and VCAM-1 expression does not result from activation of PTK-dependent pathways. In the case of TNF-alpha induced responses, the selective co-stimulatory effect of this cytokine in combination with PTK inhibitors on ICAM-1 expression suggests a complicated intracellular pathway of TNF-alpha induced ICAM-1 expression, possibly involving down-modulation of increases in ICAM-1 by PTK enzymes.

Immunological status of competitive cyclists before and after the training season.

The total number of leucocytes, T lymphocyte subsets, mitogen induced proliferation of lymphocytes, Il-2 generation, adherence capacity and chemiluminescence of granulocytes were measured and a leukergy test performed in fifteen young cyclists. The investigations were carried out at rest at the beginning of a training season and after six months of intensive training and a racing season, cycling approximately 500 km a week. Baseline values of the tested immune parameters were within the range observed in 16 non-trained healthy controls except significantly increased non stimulated neutrophil chemiluminescence. The second cyclo-ergometer test in August showed a marked improvement in the performance capacity of the cyclists. Significant decrease in absolute numbers of CD3+ and CD4+ cells, diminished IL-2 generation and fMLP and PMA stimulated chemiluminescence of neutrophils were noted. Surprisingly, a marked increase in lymphocyte proliferation induced by PHA and anti-CD3 MoAb and normalisation in non stimulated neutrophil chemiluminescence were also observed at rest after the training season. We conclude that long-lasting intensive training may result in significant alterations in lymphocyte number and composition and in neutrophil oxidative burst capacity, but their actual significance for immunity is seen controversially.

The effect of chronic exposure to formaldehyde, phenol and organic chlorohydrocarbons on peripheral blood cells and the immune system in humans.

The aim of this study was to evaluate immunological, hematological and biochemical parameters in subjects chronically exposed to inhaled formaldehyde (F), phenol (Ph) and isomers of organic chlorohydrocarbons (Chc) released from Ksylamit. Twenty-two office workers had been exposed for 6 months to vapors of Ksylamit, used for protection of felt plates inside the office building. The concentration of Ksylamit vapor was measured at the end of the 6-month period and the level of Ph and F in the air inside the building was 1.3 mg/m3 and 0.8 mg/m3, respectively. Most of the workers had ailments due to the irritant effect of inhaled Ksylamit, but no remarkable increase in morbidity was found during the 6 months of exposure and in a 3-year follow-up. Morphological parameters of blood and the number of natural killer (NK) cells in the group of exposed subjects were within the range observed in healthy subjects matched for age and sex. The number of T-lymphocytes and NK cell cytotoxicity were significantly decreased. Phytohemagglutinin (PHA)- and alloantigen-induced lymphocyte proliferation was diminished. Some biochemical parameters suggested liver damage, although these parameters did not correlate with the levels of Ph and methanol excreted in the urine. Eight subjects with the highest levels of Ph in the urine had decreased erythrocyte and T-helper lymphocyte numbers, and increased numbers of eosinophils and monocytes. The results obtained prove that the functions of both the immune and hematopoietic systems could be affected by chronic exposure to these toxic substances.

The immunoregulatory granulocyte factor (GF). III. Characteristics of target cells for GF.

Granulocyte factor (GF) is a substance secreted selectively from the specific granules of granulocytes in the first minutes of adherence or phagocytosis. GF possesses many immunoregulatory functions. We applied flow cytometric analysis to evaluate the target cells responsible for GF action. Resting lymphocytes did not display binding sites for GF, but 93 +/- 4.6% of the neutrophils isolated from peripheral blood bound GF (mean fluorescence = 122 +/- 11). GF was bound by 26 +/- 7.6% of the lymphocytes from nonstimulated and 42.6 +/- 12% of the lymphocytes from PHA-stimulated cultures, and the mean fluorescence intensity was 14.5 +/- 1 and 54 +/- 10.6, respectively. PHA-stimulated CD8+ cells presented higher expression of binding sites for GF than CD4+ cells. Binding sites for GF were found on 66 +/- 5.1% of activated cells expressing the receptor molecule to IL-2 (CD25) and 50.3 +/- 7.4% of cultured lymphocytes expressing the early activation marker CD69. These values were significantly higher in comparison to the percentage of CD25- and CD69- cells that bound FITC-labeled GF (10.3 +/- 3.3% and 11.3 +/- 3.5%, respectively). The binding was specific, and preincubation of the cells with GF almost completely abolished FITC-labeled GF binding. It has also been shown that GF binds to lymphocytes via molecules other than CD16. High-performance liquid chromatography (HPLC) with fractionated GF showed three separate fractions and two of them had GF-like activity, as demonstrated in the mixed lymphocyte reaction (MLR).