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Toll-like receptors (TLRs) play an important role in fast activation of the immune response to a variety of pathogens, including parasites. In this study, we focused on TLR2, because this receptor is one of the best known and most frequently analysed members of the TLR family. The aim of this study was to assess the effect of Trichinella spiralis on expression of TLR2 during the intestinal stage of infection.

Introduction: Toll-like receptors (TLRs) play a key role in the rapid activation of the innate immune response to a variety of pathogens. The aim of this study was to evaluate the effect of Trichinella spiralis infection on the level of expression of the tlr4 gene in mouse intestines during the intestinal phase of experimental trichinellosis.

Previous research shows that posing many questions about an event may lead to asking questions about unwitnessed details and that people sometimes provide substantive and erroneous answers to them. Therefore, two experiments investigated the role of the problem-solving and judgment processes, which are unrelated to memory access, in improving responding to unanswerable questions. Experiment 1 compared the effects of a brief retrieval training with the effects of an instruction to increase the criterion of reporting. As expected, the two manipulations had different effects on participants' answers, which demonstrates that training can do more than just instigate more cautious responding. However, we found evidence against our prediction that an enhancement in metacognitive ability underlies improved responding after training. Experiment 2 investigated, for the first time, the role of constant awareness that questions can be unanswerable and that such questions should be rejected. We compared the effects of training with the effects of a small change in response format that ensured such awareness. The effects of the two manipulations were similar, which supports our prediction that the constant awareness of unanswerable questions is a key factor behind improved responding. Practical implications for the eyewitness memory domain are discussed. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Toll-like receptors (TLR) are involved in the recognition of numerous pathogens, including Acanthamoeba spp. Thanks to this, it is possible for immune cells to recognize microorganisms and trigger the body's innate immune response. The stimulation of TLRs also leads to the activation of specific immunity. The aim of the study was to determine the TLR2 and TLR4 gene expression in the skin of BALC/c mice infected with Acanthamoeba with AM22 strain isolated from a patient. Receptor expression was assessed by real-time polymerase chain reaction (qPCR) in the amoeba-infected host with normal (A) and reduced immunity (AS) as well as in the control host with normal immunity (C) and reduced immunity (CS). Statistical analysis of TLR2 gene expression in A and AS groups compared to C and CS groups, respectively, were statistically insignificant. In the A group, we found statistical upregulation of TLR4 gene expression at 8 dpi compared to the C group. While in AS group, TLR4 gene expression was at a similar level, such as in the CS group. Taking into account the host's immune status, the TLR4 gene expression was statistically higher in the skin of host from A group than in host from AS group at the beginning of the infection. Increased TLR4 gene expression in hosts with normal immunity infected with Acanthamoeba suggests the involvement of the studied receptor in the course of acanthamoebiasis. The above research results provide new data on the involvement of the studied receptor in the skin in the host's immune defense triggered during the Acanthamoeba infection.

Alveolar echinococcosis (AE) is a chronic zoonotic disease caused by the larval form of Echinococcus multilocularis . In humans, it may become a serious chronic infection of the liver which resembles a slow malignant process leading to death when untreated. The aim of the study was an assessment of the risk factors of the E. multilocularis infections and the description of AE clinical course in the group of 36 patients with confirmed AE, hospitalized at the Department and Clinic of Tropical and Parasitic Diseases, Poznan University of Medical Sciences between 2013 and 2022. Among the study participants, most patients cultivated land, bred livestock, worked in the forest, or were employed in animal shelters. The E. multilocularis infection was diagnosed based on imaging and immunoassay techniques within 6 months in the majority of patients hospitalized in the Department. All patients hospitalized in the Department initiated anti-parasitic therapy at the moment of the diagnosis. Pharmacological treatment combined with surgery was applied in most of the study participants, who were presented with more advanced stages of infection. We conclude the following: 1. For humans in the risk group, regular abdominal imaging examinations and the detection of specific antibodies against E. multilocularis are recommended. 2. Regular screening tests in the hyperendemic areas of AE would increase the early detection of the disease and to improve the clinical prognosis in this extremely life-threatening parasitic disease.

Three species from the Eryngium L. genus- E. campestre , E. maritimum , and E. planum , plants with a rich chemical composition, were selected for phytochemical and biological studies. The applied biotechnological methods allowed to obtain the biomass of these rare or protected species in the form of multiplied shoots (stationary system) and roots cultured in a liquid medium (agitated system). In the extracts from the raw material obtained under in vitro conditions, the content of selected phenolic acids and flavonoids (HPLC-DAD method) as well as the total of polyphenols (Folin-Ciocalteu assay) were quantified. The highest amount of all phenolic compounds was found in extracts from E. planum roots (950.90 ± 33.52 mg/100 g d.w.), and the lowest from E. campestre roots (285.00 ± 10.07 mg/100 g d.w.). The quantitatively dominant compound proved to be rosmarinic acid. The highest amounts were confirmed for E. planum root extract (694.58 mg/100 g d.w.), followed by E. planum (388.95 mg/100 g d.w.) and E. campestre (325.85 mg/100 g d.w.) shoot extracts. The total content of polyphenols was always increased in the biomass from in vitro cultures in comparison to the analogous organs of intact plants of each species. The obtained extracts were assessed for antiprotozoal activity against Acanthamoeba sp. The strength of biological activity of the extracts correlated with the content of phenolic compounds. To our knowledge, this is the first report on the amoebicidal activity of E. campestre , E. maritimum , and E. planum extracts from biomass produced by biotechnological methods.

Michel (The mismeasure of consciousness: a problem of coordination for the perceptual awareness scale. Philos Sci 2019;86:1239-49) claims that the Perceptual Awareness Scale (PAS) faces the problem of coordination (also known as validity). We argue that his claim holds only under certain theoretical assumptions which need to be made explicit as these are likely not in line with the PAS proponents' standpoint. We also call for terminological clarity, an example being the usage of 'levels' of consciousness. Precise terminology combined with an explicit reference to the chosen theoretical perspective is necessary conditions for making progress in consciousness research and the development of consciousness theories., (© The Author(s) 2021. Published by Oxford University Press.)

Genetically uniform plant material, derived from Lychnis flos-cuculi propagated in vitro, was used for the isolation of 20-hydroxyecdysone and polypodine B and subjected to an evaluation of the antifungal and antiamoebic activity. The activity of 80% aqueous methanolic extracts, their fractions, and isolated ecdysteroids were studied against pathogenic Acanthamoeba castellani . Additionally, a Microtox ® acute toxicity assay was performed. It was found that an 80% methanolic fraction of root extract exerts the most potent amoebicidal activity at IC 50 of 0.06 mg/mL at the 3rd day of treatment. Both ecdysteroids show comparable activity at IC 50 of 0.07 mg/mL. The acute toxicity of 80% fractions at similar concentrations is significantly higher than that of 40% fractions. Crude extracts exhibited moderate antifungal activity, with a minimum inhibitory concentration (MIC) within the range of 1.25-2.5 mg/mL. To the best of our knowledge, the present report is the first to show the biological activity of L. flos-cuculi in terms of the antifungal and antiamoebic activities and acute toxicity. It is also the first isolation of the main ecdysteroids from L. flos-cuculi micropropagated, ecdysteroid-rich plant material.

Genetically uniform shoots of Eryngium alpinum L. cultured in vitro were subjected to the qualitative analysis applying the UPLC-HESI-HRMS technique. In vitro cultures give the opportunity to perform the phytochemical studies on the protected species without harvesting the plant material from the natural environment. The phytochemical screening of the crude methanolic extracts of shoots, both from in vitro cultures and in vivo plants, revealed the presence of phenolic acids, coumarins, flavonoids, triterpenoid saponins, amino acids, or dipeptides. Active compounds detected are known to have medicinal importance, and for this reason, the present study represents a preliminary investigation of the extracts against pathogenic and opportunistic amoeba. Among the extracts tested, the extract of shoots from in vitro cultures exhibited remarkable amoebicidal action against trophozoites. On the second day of treatment, the extract at the concentrations of 5 mg/mL, 2.5 mg/mL, and 0.5 mg/mL showed the highest antiamoebicidal effect: the inhibition of trophozoites reached 81.14%, 66.38%, and 54.99%, respectively. To our best knowledge, the present report is the first to show the phytochemical screening and to discuss the antiamoebic activity of Eryngium alpinum L. shoots, both from in vitro cultures and in vivo plants.

We aimed to distinguish electrophysiological signatures of visual awareness from other task-related processes through manipulating the level of processing of visual stimuli. During an event-related EEG experiment, 36 subjects performed either color (low-level condition) or magnitude (high-level condition) evaluations of masked digits. Participants also assessed subjective visibility of each stimulus using the Perceptual Awareness Scale (PAS). Mean amplitude of the components of interest was analyzed (VAN - 140-240 ms; LP - 380-480 ms) with weighted regression mixed model. In the VAN component time window the mean amplitude correlated with PAS rating in both conditions. Mean amplitude in the LP time window correlated with PAS ratings in the high-level condition, but not in the low-level condition. Our results support the temporal unfolding of ERP makers of conscious processing, with an early component reflecting the initial perceptual experience and a late component being a correlate of the conscious experience of non-perceptual information., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The genus Acanthamoeba, which may cause different infections in humans, occurs widely in the environment. Lung inflammation caused by these parasites induces pulmonary pathological changes such as pulmonary necrosis, peribronchial plasma cell infiltration, moderate desquamation of alveolar cells and partial destruction of bronchial epithelial cells, and presence of numerous trophozoites and cysts among inflammatory cells. The aim of this study was to assess the influence of plant extracts from Artemisia annua L. on expression of the toll-like receptors TLR2 and TLR4 in lungs of mice with acanthamoebiasis. A. annua, which belongs to the family Asteraceae, is an annual plant that grows wild in Asia. In this study, statistically significant changes of expression of TLR2 and TLR4 were demonstrated. In the lungs of infected mice after application of extract from A. annua the expression of TLRs was observed mainly in bronchial epithelial cells, pneumocytes (to a lesser extent during the outbreak of infection), and in the course of high general TLR expression. TLR4 in particular was also visible in stromal cells of lung parenchyma. In conclusion, we confirmed that a plant extract of A. annua has a modulatory effect on components of the immune system such as TLR2 and TLR4., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The treatment of acanthamoebiasis is a still a problem. Our previous studies showed that the application of extracts from Artemisia annua L. significantly prolonged the survival of mice infected by Acanthamoeba. This plant has medicinal properties in the treatment of human parasitic diseases. The aim of this study was to evaluate the effects of A. annua on expression of Toll-like receptors (TLRs) 2 and 4 in brain of mice with Acanthamoeba infection. Mice were infected with Acanthamoeba sp. strain Ac309 (KY203908) by intranasal inoculation without and after application of A. annua extract. The administration of extract from A. annua significantly reduced the level of expression of TLR2 and modified the level of expression of TLR4. A. annua extract is a natural substance that is well tolerated in animals and may be considered as a combination therapy in treatment of acanthamoebiasis. Our study suggested that A. annua extract may be used as an alternative therapeutic tool., (Copyright © 2018 Elsevier Inc. All rights reserved.)

According to the levels-of-processing hypothesis, transitions from unconscious to conscious perception may depend on stimulus processing level, with more gradual changes for low-level stimuli and more dichotomous changes for high-level stimuli. In an event-related fMRI study we explored this hypothesis using a visual backward masking procedure. Task requirements manipulated level of processing. Participants reported the magnitude of the target digit in the high-level task, its color in the low-level task, and rated subjective visibility of stimuli using the Perceptual Awareness Scale. Intermediate stimulus visibility was reported more frequently in the low-level task, confirming prior behavioral results. Visible targets recruited insulo-fronto-parietal regions in both tasks. Task effects were observed in visual areas, with higher activity in the low-level task across all visibility levels. Thus, the influence of level of processing on conscious perception may be mediated by attentional modulation of activity in regions representing features of consciously experienced stimuli., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Chronic progressive diseases of the central nervous system such as granulomatous amoebic encephalitis, amoebic keratitis, amoebic pneumonitis and also skin infections caused by free-living amoebae (Acanhamoeba spp.) are a significant challenge for pharmacotherapy. This is due to the lack of effective treatment because of encystation, which makes the amoebae highly resistant to anti-amoebic drugs. A very inter- esting and promising source of future drugs in this area are plant materials obtained not only from the habitat but also from plant in vitro culture as an alternative source of biomaterials. Alcoholic extracts from leaves of Passiflora incarnata, P. caerulea, P. alata (Passifloraceae) and from callus cultures were evaluated in vito for amoebicidal activity. Phytochemical analysis showed that all extracts contained phenolic compounds including flavonoids? Biological study revealed that all extracts showed amoebostatic and amoebicidal properties in concentrations from 4 to 12 mg/mL. Extracts of P. alata leaf and callus showed the most effective activities (IC5, 4.01 mg/mL, IC,5 7.29 mg/mL, respectively) after 48 h of exposure, which was correlated with the highest concentration of total phenolics and flavonoids in comparison with other extracts.

Eye diseases caused by amoebae from the genus Acanthamoeba are usually chronic and severe, and their treatment is prolonged and not very effective. The difficulties associated with therapy have led to attempts at finding alternative treatment methods. Particularly popular is searching for cures among drugs made of plants. However, no substances with total efficacy in treating Acanthamoeba keratitis have been identified.Results of our semi in vivo studies of tea tree oil simulating eyeball infection demonstrated 100% effectiveness in the case of both trophozoites and cysts of amoebae from the genus Acanthamoeba. The action of tea tree oil indicates that this is the first substance with a potential ability to quickly and effectively remove the amoebae from the eye. Tea tree oil has the ability to penetrate tissues, which allows it to destroy amoebae in both the shallow and deep layers of the cornea. The present research into the use of tea tree oil in the therapy of Acanthamoeba infection is the first study of this type in parasitology. It offers tremendous potential for effective treatment of Acanthamoeba keratitis and other diseases caused by these protozoa.

Diseases transmitted by hygiene pests remain a very serious problem in spite of fast developments in science and medicine. The present study focuses on pests carrying germs that pose a threat to human health and life. The quick pace of life, the need to satisfy human needs and mass production of food sometimes result in flagrant sanitary, hygienic and epidemiological deficiencies. These irregularities are conducive to hygiene pests, which, when not held in check by proper control measures, may act more efficiently and quickly.

The Toll-like receptors (TLRs) of the innate immune system play an important role in the recognition of pathogens such as bacteria, viruses, fungi, and parasites. In this study, we examined the changes in the level of expression of TLR2 and TLR4 mRNA and protein in the brains of mice infected with Acanthamoeba spp. The Acanthamoeba strains were isolated from a patient with Acanthamoeba keratitis (AK) (Ac55) and Malta Lake (Ac43). In the brain isolated from mice at 2 days post-infection (dpi) with Acanthamoeba strains Ac55 and Ac43, mRNAs for TLR2 and TLR4 were significantly more strongly expressed in comparison with the uninfected mice. In Acanthamoeba-infected mice, TLR2 and TLR4 expression was detected in neurons, glial cells, and endothelial cells within the neocortex. These receptors showed more intense expression in ependymocytes of the choroid plexus of infected mice at 2 dpi. Increased levels of TLR2 and TLR4 mRNA expression in infected mice suggest the involvement of these TLRs in the recognition of Acanthamoeba spp. pathogen-associated molecular patterns (PAMPs).

Toll-like receptors (TLRs) play a key role in the innate immune responses to a variety of pathogens including parasites. TLRs are among the most highly conserved in the evolution of the receptor family, localized mainly on cells of the immune system and on other cells such as lung cells. The aim of this study was to determine for the first time the expression of TLR2 and TLR4 in the lung of Acanthamoeba spp. infected mice using quantitative real-time polymerase chain reaction (Q-PCR) and immunohistochemical (IHC) staining. The Acanthamoeba spp. were isolated from a patient with Acanthamoeba keratitis (AK) (strain Ac 55) and from environmental samples of water from Malta Lake (Poznań, Poland - strain Ac 43). We observed a significantly increased level of expression of TLR2 as well as TLR4 mRNA from 2 to 30 days post Acanthamoeba infection (dpi) in the lungs of mice infected with Ac55 (KP120880) and Ac43 (KP120879) strains. According to our observations, increased TLR2 and TLR4 expression in the pneumocytes, interstitial cells and epithelial cells of the bronchial tree may suggest an important role of these receptors in protective immunity against Acanthamoeba infection in the lung. Moreover, increased levels of TLR2 and TLR4 mRNA expression in infected Acanthamoeba mice may suggest the involvement of these TLRs in the recognition of this amoeba pathogen-associated molecular pattern (PAMP)., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The treatment of acanthamoebiasis is a great problem. Most cerebral invasions end with death, and the treatment of ocular invasions is usually long-lasting and not very effective. Numerous plant extracts and substances isolated from plants, which are effective against trophozoites or cysts, have been studied in the treatment of acanthamoebiasis. However, no agents that are simultaneously effective against both developing forms of amoebae have been discovered yet. It seems that such a plant which fulfils both tasks is Artemisia annua L. Our studies showed that water, alcohol and chloroform extracts from the herb A. annua L. can be applied in general and local treatment or in combined therapy with antibiotics in the treatment of acanthamoebiasis. Extracts from this plant show not only in vitro but also in vivo effects. Studies carried out on experimental animals infected with amoebae show that the application of these extracts significantly prolongs the survival of the animals.

Free-living amoebae belonging to the genus Acanthamoeba are the causative factor of many diseases. Among others, they cause Acanthamoeba keratitis (AK), a condition that usually occurs in contact lens wearers, though it is also observed in non-wearers. The number of diagnosed cases of AK increased more than eightfold during 8 years in the USA, and a proportional increase in frequency also occurred in Poland and Europe. Cases of AK are usually diagnosed late, and their therapy is difficult and rarely successful. AK is an uncommon diagnosis in Poland. The increased number of positive cases observed in our laboratory may reflect the growing at-risk population of contact lens wearers. Acanthamoeba as a genus of facultative human parasites is currently classified into 17 genotypes. Isolates belonging to seven genotypes were found to be associated with AK. One genotype in particular, T4, was found to be overrepresented in human disease. The main finding of our study is that in Poland, AK is almost always associated with the T4 genotype.

Amoebae from the genus Acanthamoeba are known agents leading to various diseases such as granulomatous amoebic encephalitis (GAE), a chronic progressive disease of the central nervous system, amoebic keratitis (AK), chronic eye infection, amoebic pneumitis (AP), chronic lung infection, and skin infections. It is known that various synthetic anti-Acanthamoeba substances are ineffective. Therefore, other substances, e.g., natural plant compounds, are the focus of biological investigations regarding anti-parasite activity. In this work, the ability of four abietane diterpenoids (ferruginol, salvipisone, aethiopinone, and 1-oxo-aethiopinone) to inhibit Acanthamoeba growth is reported. All investigated compounds were active against Acanthamoeba growing in vitro. Among them, ferruginol demonstrated the highest activity against Acanthamoeba. This compound inhibited Acanthamoeba growth by about 72% in a 3-day exposure period (IC50 17.45 μM), while aethiopinone and 1-oxo-aethiopinone demonstrated this activity at the level of 55-56%. Salvipisone reduced the growth of Acanthamoeba in vitro culture by 39%. For this compound, the value of IC50 was 701.94 μM after 72 h of exposure.

Despite the considerable progress of medicine, parasitic diseases still pose a great threat to human health and life. Among parasitic diseases, those transmitted by vectors, mainly arthropods, play a particular role. These diseases occur most frequently in the poorest countries and affect a vast part of the human population. They include malaria, babesiosis, trypanosomiasis, leishmaniasis and filariasis. This study presents those vector-transmitted diseases that are responsible for the greatest incidence and mortality of people on a global scale. Attention is focused primarily on diseases transmitted by mosquitoes, flies, Hemiptera and ticks.

Acanthamoeba are widespread free-living amoebae which may cause granulomatous amoebic encephalitis (GAE), keratitis, skin ulcerations and disseminated tissue infection. An important diagnostic and prognostic factor for the treatment of infection is a quick and correct diagnosis of amoebae strains. The aim of our study was to develop a rapid method for detection and identification of pathogenic Acanthamoeba spp. strains from diagnostic material collected from water. In this study we analysed five amplification-based genetic markers (Aca 16S, Ac6/210, GP, JDP, Nelson) used for identification of pathogenic Acanthamoeba spp. strains isolated in water sources in Poland, Iceland and Sweden. Our results demonstrated the presence of pathogenic Acanthamoeba strains in tap water. PCR assay appeared to be a more rapid and sensitive method to detect the presence of amoebae than the limited conventional techniques. Based on our observations, we can confirm that the use of four out of five genetic markers (Aca 16S, Ac 6/210, JDP, GP, Nelson) may be helpful in identification of Acanthamoeba spp. strains, but only one Aca 16S primer pair is a highly specific marker that distinguishes between pathogenic strains of Acanthamoeba and other free-living amoeba families.

The paper presents an overview of the use of natural therapeutic agents in combating parasitic diseases. Nowadays there is increasing demand for proven plant therapies, which often are found to be more effective than synthetic pharmaceuticals in chronic diseases. In many cases herbal preparations perfectly supplement the conventional treatment and at the same time do not cause side effects. On the pharmaceutical market there are many drugs of plant origin which have been applied in the treatment of parasitic diseases. However, researchers are still looking for new plants, or specific substances isolated from them, which can be used in therapy. In this paper, drugs of plant origin used in the treatment of amoebiasis, giardiasis, malaria, leishmaniasis, trypanosomiasis and acanthamoebiasis are described.

Selected fractions of ethanolic extracts obtained from leaves and roots of Eryngium planum (Apiaceae) were evaluated in vitro for amebicidal activity against Acanthamoeba castellanii. This free-living ameba is the cause of Acanthamoeba keratitis, which is a painful, vision-impairing disease of the eyes, and chronic granulomatous amebic encephalitis. Treatment is very difficult and not always effective because of encystation, which makes the amebae highly resistant to anti-amebic drugs. The search for novel natural amebicidal agents is still of current interest. Fractions of E. planum ethanolic extract from basal leaves: flavonoid fraction (Lf), flavonoid-saponin fraction (Lf-s), saponin fraction (Ls) and phenolic acids fraction (La) and from roots: saponin fraction (Rs) and phenolic acids fraction (Ra) were assayed for antiamebic activity. In the presence of the saponin fractions and phenolic acid fractions (ranging from 1-5 mg/mL), the number of the trofozoites of Acanthamoeba castellanii viable strain 309 decreased during the experimental period (0-72 h). On the other hand, the flavonoid fraction from leaves showed a stimulating activity on the amebae. Almost all fractions (except the flavonoid fraction) showed a time- and dose-dependent amebistatic activity on the trophozoites. Of the fractions tested, the phenolic acid fraction from roots at the concentration of 5 mg/L showed the amebicidal activity on the trophozoites.

The glutathione S-transferases (GSTs) are a family of multifunctional enzymes involved in cellular detoxification. The aim of this study was to evaluate the effect of albendazole--drug of choice for trichinellosis--on the total activity and kinetics of cytosolic GST in the mouse intestines during experimental trichinellosis. Our results showed a statistically significant decrease in the total GST activity both in the small and large intestines of the mice infected with the nematode Trichinella spiralis (Owen, 1835) and treated with albendazole, compared with the control mice that were infected but untreated with the drug. Furthermore, albendazole administration modified the kinetics of substrate saturation of GST in the intestines of the infected mice because the drug caused changes in Michaelis constant values of this enzyme. Based on our observations, we suggest that the quaternary structure of GST from the mouse intestines is impacted by this drug during trichinellosis.

The glutathione S-transferases (GSTs) are a group of multifunctional enzymes, which play a critical role in cellular detoxification. Our investigations deal with the contribution of GST in the biochemical defense against Trichinella spiralis infection. The aim of this study was to examine the effect of T. spiralis infection on the total activity and kinetic properties of cytosolic GST in the intestine during the intestinal phase of experimental trichinellosis in mice. Our results showed a statistically significant increase (relative to the uninfected control) in the total GST activity both in the small and large intestines of the infected mice. Moreover, we observed changes in the kinetics of substrate saturation of GST. Trichinellosis in the small intestine caused a 12-fold decrease in the low K (m) value and a sixfold increase in the high K (m) value. In the large intestine, infection with T. spiralis caused only a fivefold increase in the low K (m) value, whereas the high K (m) value remained unchanged. We suggest that GST from the mouse small intestine could be involved in the detoxification of parasite excretory-secretory products released to the host intestine during trichinellosis and that these products influence the quaternary structure of this enzyme.

Some protozoans are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent. Acanthamoeba is an exception since its cyst wall contains cellulose. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible due to the similarity of the constituent beta-1,4-linked hexose backbones of these molecules. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. The identification of Acanthamoeba spp., which is based primarily on morphological and biochemical features, is labor-intensive and requires cloning and axenization. We describe a novel immunocytochemical method for identification of Acanthamoeba spp. based on selective binding of Trichoderma reesei cellulase to protozoan cyst wall cellulose. A recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from T. reesei cellulases was coupled to the fluorescent dyes Alexa Fluor 350 and Alexa Fluor 568 or was labeled with biotin using EZ-Link sulfo-NHS-biotin. No staining reaction was observed with chitin-containing preparations of fungi. Thus, the recombinant CBDs can be used as a marker to distinguish between cellulose and chitin. This allows rapid identification of Acanthamoeba cyst wall cellulose in paraffin or frozen sections of infected tissues.

Strains of Acanthamoeba sp. constitute a factor contributing to the occurrence of chronic granulomatous amoebic encephalitis, keratitis, pneumonia, as well as inflammations of other organs. Treatment of these diseases is very difficult and not always effective. A majority of these infections have been fatal. The aim of our study was to examine the amoebicidal or amoebistatic activity of plant extracts from Rubus chamaemorus, Pueraria lobata, Solidago virgaurea and Solidago graminifolia. For the purpose of isolation of pharmacologically active substances, we used the aboveground parts of plants, together with flowers, roots and leaves. It was established that extracts from S. virgauera, P. lobata and R. chamaemorus displayed chemotherapeutic properties in vitro in concentrations of approximately 0.01-0.05 mg extract/mL, i.e., in concentrations of 0.350 microg/mL expressed in ellagic acid for R. chamaemorus and 0.053 microg/mL expressed in puerarin for P. lobata. Therapeutic index values is 3.5-20. As a result of in vivo experiments, it was found out that, following therapy using the extracts, animals infected with Acanthamoeba sp. survived for an extended period (2.5-3 times longer). It was determined that plant extracts may be used both externally and internally in the case of a combined therapy for acanthamoebiasis. The tested extracts are not toxic for animals.

Species identification within the genus Acanthamoeba is based predominantly on morphological and biochemical features. It is labor-intensive and requires cloning and axenization. We described a novel immunocytochemical method for the identification of Acanthamoeba spp. based on selective binding of Clostridium cellulovorans cellulase to protozoan cyst wall cellulose. Free-living amoebae isolated from different water sources by filtration and subsequent cultivation on non-nutrient agar were assigned to genera Acanthamoeba, Naegleria or Hartmannella using morphological taxonomic criteria. Tissues samples from experimentally infected mice were fixed in formalin and for sectioning embedded in paraffin or snap frozen. The Cellulose-Binding Domain of C. cellulovorans cellulase (CBD) obtained as a recombinant protein, were coupled to the fluorescent dye using Alexa Fluor350, 488, 568 - Protein Labelling Kit or labelled with the biotin using EZ-Link Sulfo-NHS-Biotin. All coupling procedures were performed according to the methods provided by manufacturers. For staining with CBD conjugate, slides containing cysts collected from the agar plates or tissue sections were immersed with PBS and incubated with CBD for 30 min at room temperature, washed 3 times with PBS. For staining with CBD-biotin slides containing cysts were incubated with biotinylated CBD for 30 min at room temperature. Subsequent washings in changes of PBS were followed by the incubation with Strept ABComplex/HRP, for 30 min at room temperature, than 3,3 diaminobenzidine tetrahydrochloride was added for 15 min. Slides were rinsed with water, dried and examined in the light microscope. We showed that cellulose could be easily detected by immunofluorescence using conjugated CBD in the inner cyst wall of Acanthamoeba spp. The reference strains of Acanthamoeba spp. and all Acanthamoeba strains isolated from water and from tissues of infected animals gave positive reaction. CBD prepared as a biotynylated protein can be also used for the demonstration of Acanthamoeba cyst in infected tissues and environmental samples.

Sulphur isotope compositions were determined in two different Polish coals (hard coal and lignite) and by-products originating from their combustion. The desulphurization process was also investigated. It was demonstrated that desulphurization changes the isotopic composition of sulphur emitted in the form of SO(2) to the atmosphere even if the process is conducted in a different way (wet lime technology and electron beam method). This fact has to be considered in the studies regarding anthropogenic sulphur genesis and its fate in the environment.

In this study, the effect of trichinellosis as well as the effect of albendazole treatment on the kinetics of substrate saturation of cytosolic glutathione transferase (GST) in the skeletal muscles from infected mice was examined because the idea of multifunctional mode of action of anthelmintic drugs was considered. Our results pointed out to the influence of trichinellosis and albendazole treatment on the quaternary structure of GST in mouse muscles. A double reciprocal plot of GST saturation in control mice was biphasic with apparent low and high K (m) values equal to 0.54 and 1.0 mM, respectively. Infection with Trichinella spiralis in the third week postinfection caused a 2.3-fold increase in the high K (m) value and at the same time a 2.8-fold decrease in the low K (m). In the sixth week postinfection, the high K (m) value was unchanged, but the low K (m) value increased 2.3 times. Calculated from the double reciprocal plot of GST substrate saturation in muscles from infected and treated mice (measured in the third week postinfection only), the high K (m) value increased 1.4 times relative to the respective controls. The normal substrate saturation plot of GST in treated mice has a clearly "double sigmoid" character. Our results suggest that despite the complicated character of the GST saturation curve, albendazole seems to act as an allosteric activator for cytosolic GST in infected mouse muscles.

The free-living amoebae are ubiquitous organisms. They are found in humid soil and all water reservoirs, i.e. fresh, sea, freezing and hot water. They mainly feed on bacteria. Pathogenic properties of amoebae and the mechanisms underlying pathological changes induced during human infection have not yet been fully elucidated. They are the causative agents of primary amoebic meningo-encephalitis (PAM), granulomatous amebic encephalitis (GAE), a chronic progressive disease of the central nervous system, amebic keratitis (AK), a chronic eye infection; amebic pneumitis (AP), a chronic lung infection, and skin infection. Only a few isolates are strongly and permanently pathogenic to humans. Some isolates lose their pathogenic properties after one passage. It has been assumed that such "temporary", unstable pathogenic properties of the amoebae may be caused by internal factors carried by them. It is generally known that the free-living amoebae may be naturally infected with pathogenic bacteria, which have the ability to survive for a long time and to proliferate in the amoebae cells. The role of the amoeba in the process of maintaining, propagating and transmitting human pathogens has not been well recognized. It has been suggested that some infections can be acquired by inhaling aerosols containing amoebae cells filled with bacteria. The presence of bacteria inside the free-living amoebae possess a great challenge to organisations responsible for testing and inspecting the quality and cleanliness of surface waters, swimming pools and drinking water intakes.

The biological reaction caused by oxygen-derived free radicals at the molecular and cellular levels involves many different biochemical components which can be directly damaged by oxidizing radicals. As such a reaction may lead to pathological processes, defence mechanisms have evolved to limit the rate of free radical production. These mechanisms employ low-molecular-weight non-enzymatic antioxidants and antioxidant enzymes which are inducible by oxidant stress. In this study, the activity of two antioxidant enzymes, superoxide dismutase (EC 1.15.1.1) and glutathione peroxidase (EC 1.11.1.9), and the level of non-enzymatic antioxidants (total antioxidant status) in the blood from mice infected with Trichinella spiralis was examined. We observed a statistically significant, up to above twofold increase (relative to the control value in uninfected mice) in the level of both enzymes as well as in the total antioxidant status. An intensification of antioxidant processes during trichinellosis could be related to the presence of T. spiralis larvae, which may induce phagocytes to generate free radicals. Our research shows that the maximum growth in antioxidant activity in the blood appears during the period of the greatest muscle damage caused by T. spiralis infection at 3-7 weeks post-infection.

The free-living amoebae from genus Acanthamoeba are the causative agents of granulomatous amebic encephalitis (GAE), a chronic progressive disease of the central nervous system; amebic keratitis (AK), a chronic eye infection; amebic pneumitis (AP), a chronic lung infection, and skin infection. Chemotherapy of Acanthamoeba infection is problematic. The majority of infections have been fatal. Only a few cases are reported to have been treated successfully with very highly toxic drugs. The therapy might be succeed, if the diagnosis and therapy is made at very early stage of infection. In our experiments we used the following plant extracts: Solidago virgaurea, Solidago graminifolia, Rubus chamaemorus, Pueraria lobata, and natural plants products as ellagic acid and puerarin. Those therapeutic agents and plants extracts have been tested in vitro for amebicidal or amebostatic activity against pathogenic Acanthamoeba spp. Our results showed that methanol extracts obtained from plants are active against axenic pathogenic Acanthamoeba sp. trophozoites in vitro at concentration below 0.1 mg/ml. Further studies are needed to investigate whether these extracts are also effective in vivo in animal model of infection with Acanthamoeba sp.