Monika Derda

The sustainable production of plant bioactive compounds is increasingly important as natural habitats decline. This study investigates whether in vitro cell cultures of Eryngium planum, Lychnis flos-cuculi, and Kickxia elatine can serve as alternative sources of biologically active biomass with antimicrobial and anti-Acanthamoeba properties. Callus cultures were established under optimized and controlled conditions, and metabolomic profiling was completed using UPLC-HRMS/MS. In silico analysis, using a molecular docking approach, was applied to understand the interaction between target compounds and Acanthamoeba profilin and identify possible targets for antimicrobial properties. Untargeted metabolomic analysis confirmed the presence of valuable compounds in the callus cultures of the studied species. Biological activity was assessed through anti-Acanthamoeba and antimicrobial assays. Lychnis flos-cuculi and Kickxia elatine callus extracts showed significant inhibitory effects on Acanthamoeba trophozoites, with 87.5% and 80.1% inhibition at 10 mg/mL, respectively. In contrast, E. planum extract stimulated amoebic growth. The anti-Acanthamoeba activity correlated with the presence of ferulic acid and p-coumaric acid in L. flos-cuculi extract, and acteoside in K. elatine extract. Antibacterial testing revealed moderate activity of E. planum and K. elatine extracts against Staphylococcus spp., while Gram-negative bacteria and fungi were largely resistant. These findings highlight the potential of in vitro cultures—particularly those from L. flos-cuculi and K. elatine—as promising, sustainable sources of anti-Acanthamoeba and antimicrobial agents, warranting further investigation into their pharmacologically active constituents.

Objectives: The aim was to present the complicated diagnostic and therapeutic process of atypical, painless keratitis caused by a cosmopolitan protozoan of the genus Acanthamoeba. Methods: This Case Report describes a medical case involving a 48-year-old woman who occasionally wears soft contact lenses and was referred to our hospital for treatment due to deteriorating visual acuity in her left eye. The diagnostic process included the isolation of amoebae from corneal scrapings and the morphological and molecular identification of the etiological agent of the infection. Results: After examination, painless atypical keratitis was diagnosed, initially considered recurrent herpetic keratitis. However, antiviral treatment did not bring about any improvement. Further observation revealed a dense, central, annular infiltrate on the periphery of the cornea. Despite treatment, the corneal infiltrate did not improve and the patient required therapeutic penetrating keratoplasty. Ultimately, the patient underwent combined surgery: corneal transplantation with cataract phacoemulsification and intraocular lens implantation. The postoperative course was uneventful. Conclusions: Acanthamoeba keratitis should be included in the differential diagnosis of keratitis, even in the absence of its characteristic feature of severe ocular pain, especially in contact lens wearers and patients who have had herpetic keratitis. Infection of the cornea with the Herpes simplex type 1 virus causes nerve degeneration, which probably translates into a painless course of Acanthamoeba castellanii infection.

(1) Background: The aim of the study was to compare the potency of Plantago media L. (Plantaginaceae) extracts on Acanthamoeba sp. trophozoites, which are opportunistic protozoan parasites leading to several dangerous diseases; (2) Methods: The chromatographically (TLC, HPLC-DAD) characterized water fractions of the extracts from biomass from in vitro cultures (shoots and roots), leaves, and inflorescences from field cultivation were used for the study of the acanthamoebic activity in a Thoma haemocytometer chamber; (3) Results: The anti-amoebic effect at the lowest concentration (1.0 mg/mL) was demonstrated only by the extract of the leaves from the cultivation (50.50% inhibition). The remaining samples inhibited the growth of parasites from a concentration of 5.0 mg/mL in the range of 41.36% inflorescences to 63.89% shoots in vitro. Quantitative determinations of phenolic compounds in the tested extracts indicate a tendency to increase the potency of the anti-amoebic effect with the content of a phenylethanoid glycoside—acteoside. The maximum content of this compound was determined in leaves from field cultivation (6.64%) and the minimum in inflorescences (0.65%). This is confirmed by the range of the lowest IC₅₀ values (the strongest biological activity) for the tested samples, 0.95–1.80 mg/mL for leaves from cultivation, and the high values, 9.70–5.30 mg/mL for inflorescences and in-vitro-derived roots. The strength of the biological activity of the extracts correlated with the content of acteoside, which constituted 84–93% of the sum of phenolic compounds determined; (4) Conclusions: The performed investigations proved the anti-acanthamoebic efficacy of Plantago media organs, including those obtainable by biotechnological methods, and indicated phenylethanoid glycosides, their main phenolic constituents, to be responsible for the activity. To our knowledge, this is the first report on the amoebicidal activity of Plantago media extracts from biomass produced by biotechnological methods and organs of an intact plant.

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.

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 IC₅₀ of 0.06 mg/mL at the 3rd day of treatment. Both ecdysteroids show comparable activity at IC₅₀ 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.

Introduction: 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. Material and Methods: The experimental material consisted of isolates prepared from the intestines (jejunum and colon) of BALB/c mice infected with T. spiralis taken at 4, 8, and 16 days post infection. Results: Our results based on quantitative real-time PCR showed that the mRNA level for TLR2 was statistically significantly higher in the jejuna of mice infected with T. spiralis than in this tissue of uninfected mice. In addition, the presence of TLR2 protein in the intestinal phase of trichinellosis was confirmed by a strong positive immunohistochemical reaction. Conclusion: Our results indicate that infection with T. spiralis changes the expression of TLR2 in the small intestine of the mouse host and suggest a contribution of these receptors to the host defence mechanisms during experimental trichinellosis.

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.