The In Vitro

The in vitro effect of ivermectin lethal dose on the activity of trehalose-6-phosphate synthase (TPS) and phosphatase (TPP) and the expression of their mRNA (tps1, tps2, and tpp genes) in the muscle of adult female Ascaris suum was investigated. The presence of ivermectin in the medium caused a decrease in TPS and TPP activities during the experiment compared with the start and control groups. The exception was the group of worms grown for 8 hours in a IVM solution, in which there was a little higher TPS activity than in the control. Real-time qPCR analysis showed reduced expression of tps1 and tps2, and unchanged tpp expression after 20 hours of incubation relative to the expression at time zero. Relative to the appropriate control groups, the expression of tps2 gene was slight increased but the other two genes were reduced after 8-hours of IVM-treatment. Then the expression of all three genes was lower at the end of cultivation. The level of gene expression was positively correlated with the activity of specific enzymes. In the case of tpp gene there was only a weak correlation. Prolonged exposure to ivermectin was effective in lowering TPS and TPP activity and their mRNA expression. However, the drug did not block the pathway

Alloimmunizacja u chorych na niedokrwistość autoimmunohemolityczną oraz genotypowanie krwinek czerwonych w celu udoskonalenia doboru krwi do przetoczeń

Wstęp: Autoprzeciwciała typu ciepłego występujące w surowicy chorych na niedokrwistość autoimmunohemolityczną powodują niezgodność w próbie zgodności i utrudniają wykrycie klinicznie istotnych alloprzeciwciał, stwarzając ryzyko hemolitycznego powikłania poprzetoczeniowego. Dobór krwi do przetoczenia dla tych chorych wymaga szczególnych procedur dla większego bezpieczeństwa transfuzji. Materiał i metody: Analizowano częstość alloimmunizacji u 163 chorych z NAIH (155 z autoprzeciwciałami typu ciepłego, 8 typu mieszanego). U wszystkich chorych określano fenotyp w antygenach Rh, K, konieczny dla doboru krwi do przetoczenia, a u 53 spośród nich, badano ponadto fenotyp Kidd, Duffy, S, s, k jak również stosowano metodę genotypowania krwinek czerwonych. Wyniki: Alloprzeciwciała istotne klinicznie wykryto u 31 chorych (19%). Łącznie zidentyfikowano 42 przeciwciała o różnej swoistości (u 7 chorych występowały przeciwciała o dwóch lub więcej swoistościach) z układów grupowych: Rh, Kell, Kidd, Duffy, MNS i LW. U 14 chorych z alloprzeciwciałami, wykrycie ich wymagało uprzedniego wyadsorbowania autoprzeciwciał z surowicy metodą auto- lub alloadsorpcji. W grupie 53 chorych, fenotyp Rh, K, Kidd i S udało się określić u 37 (70%) w testach serologicznych, zaś fenotyp rozszerzony o antygeny Duffy, s i k, tylko u 9 chorych (17%). Wyniki fenotypowania były zgodne z oznaczeniami genetycznymi. U pozostałych chorych o rozszerzonym fenotypie wnioskowano na podstawie genotypowania, ponieważ wyniki serologiczne nie były miarodajne - dwie populacje krwinek (przetoczenia krwi w ostatnich 3 miesiącach) lub/i autoaglutynacja krwinek chorego. Wnioski: Wprowadzenie metod genotypowania obok metod fenotypowania okazało się konieczne dla ustalenia rozszerzonego fenotypu krwinek czerwonych u chorych z NAIH. Badania te usprawniają dobór krwi do przetaczania i zwiększają bezpieczeństwo transfuzji.Background: The presence of warm autoantibodies in the sera of patients with autoimmune haemolytic anaemia are the cause of incompatibility in the compatibility test, which produces difficulties in the detection of clinically significant alloantibodies thus generating the risk of haemolytic transfusion reactions. For such patients the selection of red cells for transfusion requires special procedures to ensure safety. Material and methods:Alloimmunization was analyzed in 163 AIHA patients (155 with warm type and 8 with mixed type of autoantibodies). In all patients, red cell phenotyping for Rh, K, was performed prior to transfusion and in 53 of them, red cells were phenotyped additionally in Kidd, Duffy, S, s, k with genotyping as well. Results: Clinically significant alloantibodies were found in 31 patients (19%). In all, 42 antibodies of different specificities were identified (in 7 cases we detected antibodies with two or more specificities) within the following blood group systems: Rh, Kell, Kidd, Duffy, MNS and LW. In 14 of the alloimmunized patients, auto- or alloadsorption was required for alloantibody detection. In the group of 53 patients, phenotyping of Rh, K, Kidd and S antigens was successful in 37 patients (70%) with serological tests only, whereas the extended phenotype for Duffy, s, k antigens was successful only in 9 patients (17%). In each case, the genotype results were consistent with the phenotype. In the remaining patients, the results of extended phenotyping with serological method proved unreliable, due either to mixed-field reactions (after recent transfusion < 3 months) or autoagglutination. Genotyping was useful for predicting the phenotype in such cases. Conclusions: In addition to phenotyping, genotyping of red cells is necessary for establishing the extended phenotype in AIHA patients. Such procedure is important for blood transfusion safety

Correlation of NHR-48 Transcriptional Modulator Expression with Selected CYP Genes’ Expression during Thiabendazole Treatment of Anisakis simplex (s.l.)?—An In Vitro Study

Anisakis simplex (s.l.) is a complex of three sibling (biological) species of parasitic nematodes of marine mammals, including A. berlandi, A. pegreffii and A. simplex (s.s.). It is characterized by a complex life cycle in which humans can become accidental hosts by consuming dishes made of raw or undercooked fish containing L3 larvae, which in many regions of the world is related to the national or regional culinary tradition. This has spurred scientific efforts to develop new methods for treating the disease, called anisakiasis, and to neutralize invasive L3. Thiabendazole (TBZ) is a wide-spectrum anthelminthic with a higher efficacy than albendazole, a drug whose long-term use induces resistance in many parasitic species. Cytochromes P450 participate in TBZ metabolism, and the expression of their genes is controlled by nuclear hormone receptors (NHR). This study aimed to examine the effects of TBZ on the above-described pathway in invasive larvae of A. simplex (s.l.). The efficacy of TBZ against A. simplex (s.l.) larvae was observed for the first time. Larvae were cultured in vitro for 72 h in a medium containing TBZ at five concentrations from 0.5 to 1.5 mM. However, the survival curves did not significantly differ from each other. This means that all of the concentrations of TBZ had a similar effect on the A. simplex (s.l.) L3 larvae during in vitro culture. Nevertheless, TBZ modified the expression of nhr-48, cyp13a3 and cyp1a1 genes in the L3 of A. simplex (s.l.)

THE CONCENTRATION OF TREHALOSE AND ACITVITY OF TREHALASE FROM GALLERIA MELLONELLA LARVAE INFECTED BY STEINERNEMA AFFFINIS, BOVIEN 1937 (NEMATODA: RHABDITIDA: STEINERNEMATIDAE)

The experimental studies were conducted on caterpillars of wax moth Galleria mellonella infected with Steinernema affinis larvae. The concentration of trehalose and the activity of trehal ase were measured during the invasion lasting 48h. The level of trehalose and activity of enzyme were slightly lower in infected insects in comparison to the control animals

THE COMPARISON OF PROPERTIES OF 0-AMYLASE FROM THE THIRD AND FOURTH STAGE OF ANISAKIS SIMPLEX LARVAE

α-Amylase is present in the third (L3) and in the fourth-stage (L4) of larvae from Anisakis simplex. The enzymes from both sources differ in same of their properties. Α-Amylase from L3 showed a maximum at pH 7,8, enzyme from L4 stage at pH 6,5. The α-amylase from L3 was mainly lysosomal enzyme. The enzyme from L4 was located in the microsomal fraction. The L3 α-amylase showed the inhibition by EDT A and by -SH reagent iodoacetic acid. These agents did not change the activity of L4 enzyme. Bath izoenzymes were unaffected by calcium and magnesium ions. Generally the α-amylase from L4 stage had higher activity (3,71 u/mg) than L3 one (2,29 u/mg)

The activity of hydrolases of larval stages of Anisakis simplex [Nematoda]

Activity of hydrolases during the third and fourth larval stage of Anisakis simplex was identified by applying the API ZYM test method. In A. simplex larvae the activity of phosphatases was high, particularly that of acid phosphatase (40 nmol/mg-'). Among esterases lack of activity of lipase (Cj 4) is worth noticing while the activity of esterases (C4) and (Cg) was high. The activity of those later two enzymes was higher in L larvae than in Ly larvae. The highest activity in the subclass of glucosidases was recorded for B-fucosidase and N-acetyl-B-glucosaminidase. A higher activity in L3 larvae than in Ly larvae was recorded for: B-glucuronidase and N-acetyl-B-glucosaminidase (2-fold) and B-fucosidase (3-fold). Differently the activity of B-galactosidase and B-glucosidase was higher in L4 larvae than in L3 larvae. The tests did not show activity of o-galactosidase, 8-glucosidase and amannosidase on both larval forms

Expression of Genes Encoding the Enzymes for Glycogen and Trehalose Metabolism in L3 and L4 Larvae of Anisakis simplex

Trehalose and glycogen metabolism plays an important role in supporting life processes in many nematodes, including Anisakis simplex. Nematodes, cosmopolitan helminths parasitizing sea mammals and humans, cause a disease known as anisakiasis. The aim of this study was to investigate the expression of genes encoding the enzymes involved in the metabolism of trehalose and glycogen—trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP), glycogen synthase (GS), and glycogen phosphorylase (GP)—in stage L3 and stage L4 larvae of A. simplex. The expression of mRNA all four genes, tps, tpp, gs, and gp, was examined by real-time polymerase chain reaction. The A. simplex ribosomal gene (18S) was used as a reference gene. Enzymatic activity was determined. The expression of trehalose enzyme genes was higher in L3 than in L4 larvae, but an inverse relationship was noted for the expression of gs and gp genes

THE ENZYMES OF CARBOHYDRATES METABOLISM FROM CYSTTIDICOLA FARIONIS (CYSTIDICOLIDAE)

The content of glycogen, glucose and trehalose was measured in larvae and adults of Cystidicola farionis, the parasite isolated from the swim bladder of Osmerus eperlanus from Vistula Lagoon. Activity of glycogen phosphorylase, α-amylase, glucoamylase. maltase, trehalase. And trehalose phosphorylase were measured. The highest activity was recorded for α-amylase 10,07 ± 0,97 mu/mg and 7,47 ± 0,24 mu/mg, next maltase 1,34 ± 0,63 μmol /mg and 2,06 ± 1,65 μmol/mg respectively for larvae and adults. The activity of glucoamylase was nearly the same for adults and larvae (about 0,20 μmol/mg). The trehalase activity was higher at adults (0,49 ± 0,42 μmol/mg) than at larvae (0,18 ± 0,12 μmol/mg). The activity of glycogen phosphorylase was much higher at larvae (3,58 ± 1,49 μmol/mg) than at adults parasite (0,10 ± 0,02 μmol/mg). The trehalose phosphorylase was present in both stages of parasite, but its activity was low. The content of glycogen and glucose was two-limes higher in the adults' body than in larvae