Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method.

PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

Influence of selected stool concentration techniques on the effectiveness of PCR examination in Giardia intestinalis diagnostics.

Giardia intestinalis is a widespread parasitic protozoa which has great significance as a public health threat. Molecular diagnostics of stool sample can be unreliable because of the presence of inhibitors of enzymatic reactions. The aim of this study was to determine the effectiveness of selected pre-treatment methods of fecal samples for further PCR-based diagnostics of G. intestinalis, and the effect of each component of pre-treatment solutions on PCR reactions. Seven stool concentration techniques were compared. The results showed that the most efficient concentration method for stool sample preparation for detection of G. intestinalis by PCR is centrifugal flotation with Percoll (with saturated NaNO3 as the flotation solution). This method is relatively inexpensive, less labor-intensive, and suitable for epidemiological monitoring and clinical investigations.

Molecular diagnostics of Sarcocystis spp. infections.

Protozoa of the genus Sarcocystis (phylum Apicomplexa, family Sarcocystidae) is one of the most common parasites affecting animals. Interspecies diagnostic of Sarcocystis genus was based on electron microscopy for many years. Because of absence of visible differences between species with reachable magnifications, light microscopy is useless. In many cases serological diagnostic method have lack of sensitivity. A variety of molecular methods have been developed and used to detect and identify Sarcocystis spp. and to assess the genetic diversity among this protozoan from different population/hosts. Nowadays, molecular diagnostic is the common, time/cost effective method used all over the world to interspecies differentiation.

Intracellular adhesion molecule-1 in patients developing pulmonary insufficiency after cardiopulmonary bypass.

OBJECTIVES: Cardiopulmonary bypass activates adhesion molecules, which are associated with systemic inflammation and organ dysfunction. The intracellular adhesion molecule-1 (ICAM-1) has been evaluated in patients presenting pulmonary dysfunction after cardiac surgery. MATERIALS AND METHODS: Postoperative serum levels of the ICAM-1 were measured in 40 patients who underwent isolated coronary artery bypass grafting, in 28 with uneventful postoperative recovery (70 %) (Group 1), and in 12 (30 %) with postoperative respiratory insufficiency (Group 2), defined by the need for prolonged (> 24 hours) mechanical ventilation using a fractional oxygen concentration of > 40 %. RESULTS: Patients in group 1 were ventilated for 12.21 +/- 4.86 hours and those in group 2 for 92.91 +/- 48.14 hours (p < 0.001). ICAM-1 decreased from 145.98 +/- 73.40 ng/ml to 81.15 +/- 114.82 ng/ml in group 1, while in group 2 ICAM-1 showed a significant higher level and increased to 435.01 +/- 130.02 ng/ml (p < 0.001). The leukocyte count increased in both groups as well as the C-reactive protein (CRP) during the postoperative course. The CRP behaves similar in both groups (p = 0.636) in contrast to the leukocyte count which was significantly higher in group 2 (p < 0.01). While none of the patients in group 1 died the mortality in group 2 was 50 % (p < 0.001). CONCLUSION: Respiratory insufficiency after cardiopulmonary bypass is associated with a distinct increase in the ICAM-1. The reason for the increase of the ICAM-1 in this small subset of patients has not been clarified.

Phenotype modulation in non-adherent and adherent sublines of Walker carcinosarcoma cells: the role of cell-substratum contacts and microtubules in controlling cell shape, locomotion and cytoskeletal structure.

We characterised two sublines of Walker carcinosarcoma cells generated by epigenetic changes. Subline 1 cells were mostly polarised and made no or only non-adhesive cell-substratum contacts. Subline 2 cells were spread, adhesive and mainly non-polar. Subline 1 cells migrate in a non-adhesive mode which is very efficient but operates only in a 3D environment, whereas subline 2 cells migrate in an adhesive mode, which is less efficient but works on 2D and 3D substrata. Nocodazole had little or no effect on shape, polarity and locomotion of subline 1 cells. In glass-adherent subline 2 cells, 10(-6)M nocodazole increased the proportion of polarised cells migrating in an adhesive mode and decreased adhesion to the substratum, whereas 10(-5)M nocodazole further reduced the contacts and the cells reverted to a non-adhesive mode of locomotion. When non-polar subline 2 cells were detached mechanically or by nocodazole, they became polarised and morphologically indistinguishable from non-adherent subline 1 cells. On more adhesive plastic substrata, subline 2 cells produced heterogeneous responses to nocodazole including loss of polarity. The phenotypes of Walker carcinosarcoma sublines have similarities with a broad range of cell types ranging from leucocytes to fibroblast-like cells, suggesting that these phenotypic differences can be controlled by the adhesive and contractile state rather than the cell type. Adhesion modulates contractility (isometric or isotonic contraction) and vice versa and this determines morphology (shape, F-actin, myosin and alpha-actinin), locomotion and responses to microtubule-disassembly. The model may be applied to analyse the mechanisms controlling the phenotype of cells in general.

Contact-activated migration of melanoma B16 and sarcoma XC cells.

During migration, tumour cells interact with neighbouring neoplastic and normal host cells, and such interaction may influence their motile activity. We investigated the effect of homotypic collisions on the motile activity of two tumour cell lines, mouse melanoma B16 and rat sarcoma XC, and nontransformed human skin fibroblasts. It was found that the tumour cells show only limited motile activity when moving as single cells without contact with neighbours. At a higher density of the culture (and also at a greater number of cell to cell contacts) the activation of motility of investigated tumour cells was observed. On the other hand, the normal human skin fibroblasts showed a typical reaction of density-dependent inhibition of motility. The motile activity of tumour cells was not affected by conditioned media and was visibly dependent on a direct physical contact among colliding cells. The activation of cell movement was observed about 40-50 min after the initial contact between tumour cells. Contact-activated migration of neoplastic cells was inhibited by 50 microM verapamil (a selective voltage-gated calcium channel inhibitor) and 10 microM gadolinium chloride (a nonspecific blocker of mechanosensitive ion channels) but not by pertussis toxin. The observation that homotypic collisions among tumour cells strongly increase their motile activity suggests that contact-activated migration may play a significant role in tumour invasion and metastasis.

Seroepidemiology of toxoplasmosis in the Lublin region.

Reported are results of serologic examinations for the presence of anti-Toxoplasma antibodies by direct agglutination in 1,497 people: 1,327 forestry workers and 86 farmers occupationally exposed to T. gondii from the Lublin region (eastern Poland) and 84 inhabitants of the city of Lublin examined as the control group, including 50 blood donors and 34 workers from forestry headquarters. 58.5% positive results in forestry workers, 56.9% in farmers and 46.4% in the control group were obtained. The highest percentages of positive results were obtained in Sosnowica, Wlodawa and Sobibor, all localities in the Chelm district. This finding and the prevalence of clinical cases may suggest that the Chelm district (easternmost area of the Lublin region, bordering Ukraine) is an endemic area of toxoplasmosis. A case of toxoplasmosis in a 39 year old farmer is described in whom reinfection was identified 20 years after primary diagnosis. Rapid increase in specific serologic titres and symptoms typical for toxoplasmosis were noted. The rest of the family and household animals were also found to be positive which supports the suggestion of a family-environmental case of toxoplasmosis. Survey for anti-Toxoplasma antibodies in various domestic and wild animals comprised sera from 262 cows, 120 pigs, 34 geese, 65 chickens, 3 roe deer and 10 sheep from the Lublin region. High percentages of positive results were found in cattle (53.8%) and in pigs (15%). Fowl were positive only in 0-5.9%. The cattle and pigs from the Chelm district are most probably the main sources of toxoplasmosis threatening humans in this area.

Contact-mediated acceleration of migration of melanoma B16 cells depends on extracellular calcium ions.

The escape of malignant cells from primary tumour and their active migration to the surrounding tissues are among the most important steps in the metastatic process. During migration, tumour cells interact with neighbouring neoplastic and normal cells and such interactions may affect their motile activity. We investigated the effect of extracellular calcium ions on migration of mouse melanoma B16 cells stimulated by homotypic cell-to-cell contacts. It was found that the decreasing of extracellular Ca2+ influx into B16 cells by lowering Ca2+ concentration in culture medium, or by the application of 0.5 mM La3+ (non-selective inorganic Ca2+ channels blocker), reduced the contact-mediated acceleration of migration of melanoma cells but only slightly affected the basal motile activity of non-stimulated single, separated cells moving without contacts with neighbouring ones in sparse culture. Since it was suggested that contact-mediated acceleration of migration of melanoma B16 cells may be controlled by mechanosensitive and/or voltage-gated ion channels, the presented data support the concept that these channels may affect cell migration by regulation of extracellular Ca2+ influx into stimulated cell.

Homotypic cell-cell contacts stimulate the motile activity of rat prostate cancer cells.

OBJECTIVE: To characterize the effect of homotypic cell-to-cell collisions upon the motile activities of two rat prostatic cancer cell lines of markedly different metastatic potential. MATERIALS AND METHODS: The movements of strongly and weakly metastatic MAT-LyLu and AT-2 cells, respectively, were recorded under an inverted microscope at 37 degrees C. The motile activities of the cells at various cell densities were characterized quantitatively by computer-aided tracking methods and image analysis. The following variables were assessed: speed of movement, final displacement, coefficient of movement efficiency, diffusion constant and positive flow. RESULTS: MAT-LyLu and AT-2 cells showed only limited motile activity in sparse cultures where there was little contact amongst the cells. However, under these and all other subsequent conditions tested, the motile activity of the MAT-LyLu cells was higher than the AT-2 cells. As the density of the cultured cells was increased (leading to more cell-to-cell contacts) there was a significant increase in motility. This effect was more pronounced for the AT-2 than for the MAT-LyLu cells, resulting in visible acceleration of movement by direct physical contact among the colliding cells. The motile activities of the tumour cells was only slightly affected by conditioned media. CONCLUSION: Homotypic collisions between migrating prostatic cancer cells can strongly stimulate their motility. The effect of increased contact is greater on the weakly metastatic cells, such that at high cell density, the difference in the motilities of weak and strong metastatic cells is greatly reduced.

Protein kinase C isoforms involved in regulation of cell shape and locomotion of human fibrosarcoma HT1080 cells.

The role of protein kinase C (PKC) isoforms in the regulation of cell shape [switch between fibroblast-like and crescent shape (CS)] and of locomotion of human fibrosarcoma HT1080 cells has been investigated. The PKC activator phorbol myristate acetate (PMA) induced the transition of elongated fibroblast-like cells into CS cells and stimulated locomotion. Both responses to PMA were inhibited by the PKC inhibitor Ro 31-8220. Analysis of the time course showed that stimulation of shape changes (formation of CS cells) and locomotor activity (increase in the proportion and speed of locomoting cells) was maximal in the early phase of the response (up to 2.5 hr) and significantly decreased later (15 to 21 hr). CS formation and stimulated locomotion correlated closely with a marked redistribution from the cytosol to the membrane of PKC isoforms alpha, beta1 and gamma in the early phase (0.5 to 2 hr) following activation with PMA. The subsequent reduction of the proportion of CS cells and of cell locomotion correlated with down-regulation of these isoforms in the second phase (16 to 21 hr). In contrast, the total amount and distribution of PKC beta2 remained almost unchanged with 10(-8) M PMA up to 21 hr. Furthermore, changes in shape and locomotion did not correlate with the responses of PKC delta to PMA. Inhibition of PMA-stimulated locomotion by the more specific inhibitor Gö 6976 is consistent with a role of PKC alpha and beta1 in this response. Ro 31-8220 alone induced a moderate down-regulation of PKC isoforms alpha and delta, but it also inhibited the more pronounced down-regulation of these isoforms by PMA. Our results indicate that activation of PKC isoforms alpha, gamma and beta1, but not beta2 or delta, stimulates locomotion and formation of CS cells in human fibrosarcoma HT1080 cells.

[Risk of tick-borne encephalitis (TBE) virus infection among people occupationally exposed to tick bites]. / Ocena narazenia na zakazenie wirusem kleszczowego zapalenia mózgu osób zawodowo eksponowanych na kontakt z kleszczami.

The paper presents the results of seroepidemiologic studies concerning tick-borne encephalitis (TBE) in 1583 persons (1261 forestry workers and 233 farmers) from the Lublin region (eastern Poland) occupationally exposed to ticks and in 130 healthy blond donors (a control group). The mean percentage of seropositive reactions in forestry workers amounted 19.8% and in farmers 32.0%. Based on 5-year research (1994-1998) conducted in 5 districts of the Lublin region, an existence of endemic foci of TBE was detected in the district of Biala Podlaska, on the areas of Radzyñ Podlaski and Parczew, where the percentage of seropositive reactions in forestry workers exceeded 50%. Statistical analysis showed that the frequency of seropositive reactions in forestry workers and farmers was significantly greater compared to control group (p<0.001 and p<0.05, respectively). It indicates that these groups are occupationally exposed to TBE wirus. Totally, in the years 1994-98 nine clinical cases of TBE (acute neuroinfection) in forestry workers and fourteen clinical cases in farmers were confirmed serologically. The effectiveness of specific immunization against TBE was proved on the brie of 100% seroconversion in 56 earlier seronegative forestry workers. The obtained results proved that forestry workers and farmers in Poland are under increased risk of infection with TBE virus.

Seroepidemiologic study on tick-borne encephalitis among forestry workers and farmers from the Lublin region (eastern Poland).

The paper presents the results of seroepidemiologic studies concerning tick-borne encephalitis (TBE) in 1,583 persons (1,261 forestry workers and 233 farmers) from the Lublin region (eastern Poland) occupationally exposed to ticks and in 130 healthy blood donors (a control group). The mean percentage of seropositive reactions in forestry workers amounted to 19.8% and in farmers 32.0%. Based on 5-year research (1994-1998) conducted in 5 districts of the Lublin region, the existence of endemic foci of TBE was detected in the district of Bia a Podlaska, on the areas of Radzyn Podlaski and Parczew, where the percentage of seropositive reactions in forestry workers exceeded 50%. Statistical analysis showed that the frequency of seropositive reactions in forestry workers and farmers was significantly greater compared to control group (p < 0.001 and p < 0.05, respectively). It indicates that these groups are occupationally exposed to TBE virus. In the years 1994-98, a total of nine clinical cases of TBE (acute neuroinfection) in forestry workers and fourteen clinical cases in farmers were confirmed serologically. The effectiveness of specific immunization against TBE was proved on the basis of 100% seroconversion in 56 earlier seronegative forestry workers. The obtained results proved that forestry workers and farmers in Poland are under increased risk of infection with TBE virus.

Chemotaxis of Amoeba proteus in the developing pH gradient within a pocket-like chamber studied with the computer assisted method.

A new "U" shaped, pocket-like chamber was used to observe the chemotactic responses of individual cells. This method permits monitoring of both the development of the concentration gradient of a tested substance and cell locomotion. We investigated the chemotactic responses of Amoeba proteus and observed that the amoebae moved in positively and negatively developing [H+] gradients towards the solution of lower pH in a pH range 5.75-7.75. The chemotactic response of amoebae to [H+] gradients required the presence of extracellular calcium ions. It was blocked and random locomotion was restored by the replacement of calcium with magnesium in the cell medium. Time-lapse video recording and data processing were accomplished with computer-assisted methods. This made it possible to compare selected methods of data presentation and analysis for cells locomoting in isotropic and anisotropic conditions. The cell trajectories were determined and displayed in circular diagrams, lengths of cell tracks and final cell displacements were estimated and a few parameters characterizing cell locomotion were computed.

Isolation, spreading, locomotion on various substrata, and the effect of hypotonicity on locomotion of fish keratinocytes.

The paper describes improved methods for the isolation of fish skin keratinocytes, which spread and locomote 15 min after trypsinization, in the absence of extracellular matrix proteins. The random locomotion of these keratinocytes under isotropic conditions on glass, plastic (polystyrene), and glass covered with poly-L-lysine or collagen IV was studied with computer-aided methods. Several methods for quantitative description of random cell locomotion were compared. The values of some parameters commonly computed showed non-Gaussian distribution. A comparison of keratinocyte locomotion under isotonic and hypotonic conditions revealed that the hypotonic conditions increased cell displacement (net migration) owing to the klinokinetic and not the orthokinetic effect.

Comparison of Bay K 8644, nitrendipine and atropine on spontaneous and pelvic-nerve-induced bladder contractions on rat bladder in vivo.

The effects of the dihydropyridine-type calcium antagonist (nitrendipine) and agonist (Bay K 8644) in comparison to atropine have been studied after intravenous administration on spontaneous and pelvic-nerve-induced contraction of rat urinary bladder. Bay K 8644 increased the basal internal bladder pressure as well as the amplitude of the spontaneous bladder contractions in a dose-dependent manner. In addition, an increase in systemic arterial blood pressure was noted for a period of about 20 min. In the presence of atropine the effects of Bay K 8644 on the urinary bladder were almost completely antagonized. Both nitrendipine and atropine reduced in a dose-dependent manner the amplitude of spontaneous and nerve-induced bladder contraction. The spontaneous and nerve-induced bladder contractions were significantly reduced by atropine or nitrendipine. Only nitrendipine caused a reduction of the spontaneous bladder contraction frequency. The systemic blood pressure was decreased significantly by nitrendipine but not after atropine administration. We suggest that both calcium antagonist and agonist can change the tension of the urinary bladder in vivo. As a side-effect the systemic blood pressure is altered. Atropine can antagonize the effect of BayK 8644 on the urinary bladder and reduces spontaneous and nerve-induced bladder contractions more specifically than nitrendipine.

Latex test for quantitative determination of Escherichia coli antibody.

A latex agglutination test was developed for assay of anti-Escherichia coli antisera. The test is simple, specific, sensitive, and reproducible.