Natalia Lanocha

Hymenolepis diminuta: Analysis of the expression of Toll-like receptor genes and protein (TLR3 and TLR9) in the small and large intestines of rats

Toll-like receptors (TLRs) play a fundamental role in the rapid activation of innate immune responses to a variety of pathogen-associated molecular patterns (PAMPs). In a previous study we observed an increase in the level of expression of TLR2 and TLR4 mRNA in the jejunum and colon during experimental hymenolepidosis in rats. In this study, we performed a quantitative real-time polymerase chain reaction (qRT-PCR), Western blot analysis and immunohistochemical staining of TLR3 and TLR9 receptors during experimental hymenolepidosis in rats. The levels of mRNA and protein expression of TLR3 and TLR9 in the jejunum had increased at 16 days post Hymenolepis diminuta infection (dpi) in the case of TLR3 and at 16 and 25 dpi in the case of TLR9. In the colon the expression of TLR3 and TLR9 had increased at 16, 25 and 40 dpi. The results of the immunohistochemical reactions showed that H. diminuta infected rats (16, 25, 40 and 60 dpi) exhibited changes in TLR3 and TLR9 localization and intensity in the epithelial cells of the jejunum and colon. The changes in the level of TLR3 and TLR9 expression may confirm involvement of the innate immune system in the pathomechanism of hymenolepidosis.

Mercury and selenium in the muscle of piscivorous common mergansers (Mergus merganser) from a selenium-deficient European country

Although the relationship between mercury (Hg) and selenium (Se) has been studied in wild birds in areas with sufficient or excessive Se levels, little is known about this relationship in areas where the supply of Se is limited. As Hg detoxification is based on the production of biologically inactive Hg-Se complexes, the aim of this study was to investigate the relationships between the concentrations of total mercury (THg), methylmercury (MeHg), inorganic mercury (InHg=THg-MeHg), percent MeHg of THg, Se and molar ratios (THg:Se, MeHg:Se, InHg:Se) in the breast muscle (n=16) of the piscivorous common mergansers (Mergus merganser) from a Se-deficient and moderately Hg-polluted area in Poland. Mergansers were divided into two groups differing in condition (A-very good condition; C-moderate condition). Concentrations of THg, MeHg and Se were determined by atomic absorption spectrometry, modified gas chromatography atomic fluorescence spectroscopy, and spectrofluorometric methods, respectively. In all studied mergansers, mean concentrations of THg, MeHg, InHg, and Se in muscle were 2.63, 1.92, 0.46, and 0.54µgg(-1)dw, respectively. THg and MeHg concentrations in the muscle of group A mergansers were greater than in group C. The ratio of THg:Se was higher in group A than in group C (2.32 vs 1.36; p<0.01), as well as the molar ratio of MeHg:Se (A vs C: 1.98 vs 1.03; p<0.05). Comparisons between mergansers from Poland and Canada showed similar THg and percent MeHg in the muscle, but mergansers from Poland had several times lower Se levels and higher THg:Se ratios (>2.0) than the Canadian mergansers and other European and North American waterbirds. We found statistically significant positive correlations (MeHg-THg, percent MeHg/THg-MeHg, percent InHg/THg-InHg, THg:Se-THg, MeHg:Se-THg, THg:Se-MeHg, MeHg:Se-MeHg, InHg:Se-InHg, MeHg:Se-THg:Se) and some negative correlations (percent InHg/THg-MeHg, percent MeHg/THg-InHg, THg:Se-Se, MeHg:Se-Se). As THg and percent MeHg in the studied mergansers were similar to populations living in non-Se-deficient areas, it is likely that different mechanisms of muscle Hg detoxification have evolved in mergansers populations living in Se-deficient areas.

Concentrations of trace elements in bones of the hip joint from patients after hip replacement surgery

The aim of this research was to determine the concentrations of two essential elements (copper (Cu) and zinc (Zn)) and three toxic elements (lead (Pb), cadmium (Cd) and mercury (Hg)) in the hip joint bones of patients from the Chair and Clinic of Orthopaedics and Traumatology at the Pomeranian Medical University in Szczecin. We examined 111 samples of hip joint bones obtained from patients from north-western Poland who had undergone arthroplasty of the femoral head. In cartilage with the adjacent compact bone, and in spongy bone from the examined patients, the concentrations (medians) determined were placed in the following descending sequence Zn>Pb>Cu>Cd>Hg. The concentrations ranged from 86 mg Zn/kg to 0.0020 mg Hg/kg dw. It was found that the concentration of lead in the cartilage with adjacent compact bone was higher in men than in women. In conclusion, it seems that in addition to routine monitoring of the abiotic environment, it is essential to monitor concentrations of heavy metals having a long-term impact in humans.

Thermophilic potentially pathogenic amoebae isolated from natural water bodies in Poland and their molecular characterization

The free-living amoebae (FLA) may live in the environment and also within other organisms as parasites and then they are called amphizoic. They are potentially pathogenic for humans and animals and are found in water that is a source of infection. The aim of this study was molecular detection and identification of these FLA in natural water bodies in North-Western Poland to evaluate the risk of the pathogenic amoebae infections. We examined surface water samples collected from 50 sites and first, the tolerance thermic test was performed in order to select thermophilic, potentially pathogenic strains. For molecular identification of FLA, regions of 18S rDNA, 16S rDNA and intergenic spacers were amplified. Acanthamoeba T4 and T16 genotypes of 18S rDNA gene and 18S rDNA of H. vermiformis were detected. We identified two variants of Acanthamoeba T4 genotype, two variants of Acanthamoeba T16 genotype and one variant of H. vermiformis. Identification of the T16 genotype and H. vermiformis in water was for the first time in Poland. Additionally, we made attempts to adapt the RLB method for detection and differentiation of FLA species and strains. PCR seems to be more sensitive than RLB hybridization, though.

Comparison of concentrations of lead and cadmium in various parts of the femur head in patients after arthroplasty of the hip joint in Northwest Poland.

OBJECTIVE To determine the concentrations of lead (Pb) and cadmium (Cd) in three kinds of materials (cartilage, cortical bone, and cancellous bone) of the femur head obtained from patients in the process of operation. METHODS Concentrations of Pb and Cd were determined in selected parts of the femur head of 30 patients after total hip arthroplasty, using ICP-AES (atomic absorption spectrophotometry). RESULTS Pb contained the highest concentration in cortical bone, while Cd did so in cancellous bone. There were statistically significant differences in the concentrations of both elements between the cartilage and cortical bone, and also differences in the concentration of Pb between the cartilage and cancellous bone. There were no significant differences in the concentrations of Pb or Cd between cortical and cancellous bone. CONCLUSION Comparative studies on toxic metals should take into account both analogous bones and their fragments, as even if they come from the same kind of bones (e.g. femur head), clear differences exist in concentrations of heavy metals related to the sampling site and type of tissue (cartilage, cortical bone, and cancellous bone).

Role of Demodex folliculorum in the pathogenesis of blepharitis.

Editor, T he ectoparasite Demodex folliculorum (Simon 1843) is most common parasite of humans. In the eye, D. folliculorum is found in the eyelash follicle. The aim of this study was to examine the prevalence of D. folliculorum in the eyelashes in patients with haematologic disorders. Eyelashes were collected from 95 patients of the Clinic of Haematology Pomeranian Medical University, Szczecin, Poland, and from 1091 healthy controls. There were 47 (49%) women and 48 (51%) men in the patients group (age range: 58.6 ± 13.1) and 759 (69.6%) women and 332 (30.4%) men in the control group (age range: 58.6 ± 13.0). From each patient, we aseptically collected two lashes from the upper right eyelid and two lashes from the upper left eyelid. The collected material was used in preparations examined using light microscopy (magnified 100 times). Infection of the examined subjects was confirmed by the detection of adult and larval forms of D. folliculorum. Demodex folliculorum is found in 21.8% of healthy controls and 18.9% of haematologic patients (Fig. 1; Table 1). In the control group, D. folliculorum occurred in 22.3% of women and in 20.8% of men. In patients with haematologic disorders, D. folliculorum occurred in 17.5% of women and in 25% of men. A much lower rate of infestation of D. folliculorum (12%) was found in immunosuppressed patients with rheumatoid arthritis (Ciftci et al. 2006). On the other hand, in patients with final-stage renal failure (ESRF) on dialysis treatment, D. folliculorum found in 44.4% of patients (Karincaoglu et al. 2005). In our study, the percentage of D. folliculorum infestation in haematologic patients ranged from 16% of patients with acute leukaemia, MPS and other haematologic diseases, and 21.7% with multiple myeloma, to about 24.1% in patients with nonHodgkin’s lymphoma and CLL. Seyhan et al. (2004) found D. folliculorum in the skin of patients with acute lymphocytic leukaemia (12%), acute myelocytic leukaemia (32%), chronic lymphocytic leukaemia (4%), chronic myelocytic leukaemia (10%), Hodgkin’s lymphoma (4%) and nonHodgkin’s lymphoma (38%). Demodex folliculorum seem to be a mediator of chronic blepharitis; some recommend that mites be sought in cilia of chronic blepharitis patients (Czepita et al. 2007). Elevated subjective symptoms of the ocular surface were caused by increased levels of D. folliculorum. Itching, burning, foreign body sensation, crusting and redness of the lid margin, and blurry vision are the main symptoms. Signs include disorders of eyelashes, lid margin inflammation, meibomian gland dysfunction, blepharoconjunctivitis and blepharokeratitis (Czepita et al. 2007). In the control group, the most common symptoms related to D. folliculorum infestation included sensitivity to light, dacryorrhea and itching of the eyelids; loss of eyelashes was the least common. In the group of haematology patients, dacryorrhea was most frequent, without any reports of sensitivity to smoke, light, foreign body sensation under the eyelids or loss of eyelashes. A 61.7% of D. folliculorum-infected patients in the ophthalmology clinic reported itching, and 59.1% reported red eyes (Inceboz et al. 2009). In our study, we found that D. folliculorum infestation was correlated with cylindrical dandruff in the lash roots, both in the control group and in haematologic patients. In patients with D. folliculorum infestation in eyelashes, we found trichiasis, meibomian gland dysfunction with lipid tear deficiency and conjunctival inflammation (Czepita et al. 2007). The prevalence of D. folliculorum in patients with haematologic diseases was similar to that of the control group. Demodicosis may cause itchiness in the eyelids and cylindrical dandruff in the lash roots.

The concentration of manganese, iron, and strontium in hip joint bone obtained from patients undergoing hip replacement surgery

The aim of this study was to determine the concentrations of manganese (Mn), iron (Fe) and strontium (Sr) in the cartilage with adjacent compact bone and spongy bone collected from patients after total hip replacement surgery. In addition, we examined relations between the concentrations of the metals in the bone and selected environmental factors. The concentration of Fe was the highest while Mn concentration was the lowest. The concentrations of Fe in the spongy bone in patients from larger cities were higher than in those living in smaller towns and villages. Significant correlations were found between Fe and Mn concentrations in the cartilage with adjacent compact bone and in the spongy bone, and between Mn and Sr in the spongy bone. In general, Mn, Fe and Sr concentrations in the bones of patients from NW Poland were lower than in other Polish regions and Europe, especially in industrialized countries. In conclusion, it seems that in addition to routine monitoring of the abiotic environment, it is essential to monitor concentrations of heavy metals having a long-term impact in humans.

Hymenolepis diminuta: analysis of the expression of Toll-like receptor genes (TLR2 and TLR4) in the small and large intestines of rats. Part II.

Toll-like receptors in the gastrointestinal tract can influence intestinal homeostasis and play a role in the repair and restitution of intestinal epithelium following tissue damage. In our previous study a statistically significant increase in the level of TLR4 and TLR2 gene expression was observed in rats in early stages of hymenolepidosis. Moreover, the immunopositive cell number and the intensity of immunohistochemical staining (indicating the presence of TLRs within intestinal epithelial cells) increased over the infection period. In this paper, we determined changes in the expression of TLR2 and TLR4 and the number of anaerobic intestinal commensal bacteria in Hymenolepis diminuta infected rats. In the isolated jejunum of infected rats at 16 days post infection (dpi), the expression of TLR4 and TLR2 was significantly higher than uninfected rats. In the colon, a statistically significantly increased expression of TLR2 was observed from 16 to 40 dpi, and TLR4 from 16 to 60 dpi. The jejunum and colon of infected rats contained Gram-negative bacteria (Escherichia coli), Gram-positive bacteria (Enterococcus, Streptococcus, Staphylococcus, Bacillus, Lactobacillus) and Candida. The total number of intestinal bacteria was higher in H. diminuta infected rats, but the observed microbiota had only minor effects on the expression of TLR2 and TLR4. Toll-like receptors play a role in maintaining epithelial barrier function in response to enteric pathogens and parasites. In our study, the alteration of TLR2 and TLR4 expression in the infected rats indicates the potential role of the innate immune system in the pathomechanism of this infection.

The effect of L-ascorbic acid and/or tocopherol supplementation on electrophysiological parameters of the colon of rats chronically exposed to lead

Summary Background The aim of this study was to assess the effect of diet supplementation with L-ascorbic acid (500 mg/L), tocopherol (3 mg/kg b.w.), and/or a water soluble analog of tocopherol (Trolox) (48 mg/L) on ion transport in the colon of rats subjected to a chronic exposure (9 months) to 0.1% lead acetate in drinking water. Material/Methods The electrophysiological parameters of the colon wall were measured with Ussing methods. Lead content in the whole blood was analyzed by graphite furnace atomic absorption spectrometry (GFAAS) using Zeeman correction. L-ascorbic acid and tocopherol in plasma was measured by high performance liquid chromatography. Immunohistochemical reaction was carried out for visualization of occludin, the intracellular tight junction protein. Results We showed a strong inhibitory effect of lead on the electrophysiological parameters, changes in intestinal permeability, disappearance of junctional occludin, decreased amount of mucus covering the colon surface, and the accumulation of PAS-positive substance in the apical region of the cytoplasm in the absorptive cells. Conclusions Supplementation with tocopherol or Trolox did not exert a beneficial influence on the studied parameters. L-ascorbic acid positively influenced the examined electrophysiological parameters, as it cancelled the inhibitory influence of lead on ion transport in the rat colon. L-ascorbic acid also protected against tight junction disruption of epithelial cells in the colon of the lead-treated rats. A similar effect was observed in the group of rats receiving lead and supplemented with L-ascorbic acid plus Trolox.

Hepatic and nephric mercury and selenium concentrations in common mergansers, mergus merganser, from baltic region, Europe

The subject of the present study was the piscivorous common mergansers (Mergus merganser). The total mercury (THg), methylmercury (MeHg), selenium (Se) inorganic mercury (InHg; THg - MeHg), percentage of THg that is MeHg (%MeHg), molar ratios (THg:Se, MeHg:Se, InHg:Se), and their mutual relations in livers and kidneys were determined in ducks from an Se-deficient area in Poland. The authors verified a hypothesis that, as a result of living in an Se-deficient region, mergansers from Poland should have higher THg:Se ratios than other waterbirds with similar THg tissue levels. Although a comparison of healthy mergansers from Poland and Canada showed similar THg tissue contents, the group in the present study had a few times lower Se levels and higher THg:Se ratios (overall means >1.7 in both livers and kidneys in all studied individuals) than the Canadian group and other European and North American waterbirds. The authors found significant correlations between various relations, including MeHg-THg, InHg-THg, Se-THg, %MeHg-THg, InHg/%THg-THg, %MeHg/THg-InHg, %InHg/THg-InHg, MeHg:Se-THg:Se, InHg:Se-THg:Se, InHg:Se-MeHg:Se in liver and InHg-THg, Se-THg, Se-InHg, %MeHg-THg, %MeHg-InHg, %InHg/THg-InHg, THg:Se-THg, InHg:Se-THg, MeHg:Se-MeHg, THg:Se-InHg, InHg:Se-InHg, and InHg:Se-THg:Se in kidney. It is likely that the main factor responsible for the high value of THG:Se ratio (>1) in mergansers from Poland is Se deficiency in central and northern Europe. Therefore, this element is unlikely to participate in the detoxification of Hg in these birds.