Seroprevalence of Toxoplasma gondii among HIV Positive Patients under Surveillance in Greek Infectious Disease Units: A Screening Study with Comparative Evaluation of Serological Methods.

This study aims to screen for IgG antibodies against Toxoplasma gondii (T. gondii) in the sera of 155 newly diagnosed Human Immunodeficiency Virus (HIV) positive patients under surveillance in Greek Infectious Disease Units. Additionally, risk factors based on patient demographics were examined, and a comparative evaluation of commercially available serological methods was conducted. Three methods were employed to detect IgG antibodies against T. gondii: Enzyme-Linked Immunosorbent Assay (ELISA), Indirect Immunofluorescence Antibody Test (IFAT), and Western Blot (WB), which was used as a reference here. Forty-nine sera samples were true-positive for IgG antibodies against T. gondii, resulting in a 31.61% positivity rate, and the immunoassay test statistical reliability analysis resulted in higher IFAT accuracy (90.97%) compared to ELISA (76.26%). Furthermore, statistical analysis of demographic and immunological data included in the study placed female and foreign/non-Greek individuals at 2.24 (p = 0.0009) and 2.34 (p = 0.0006) times higher risk of positive T. gondii IgG testing compared to their male and Greek counterparts, respectively. Our findings on positivity rates and comparative serology underscore the importance of early and suitable screening measures for newly diagnosed HIV+ patients to mitigate the life-threatening outcomes that may arise from a potential subsequent T. gondii activation.

Nervous Necrosis Virus Modulation of European Sea Bass (Dicentrarchus labrax, L.) Immune Genes and Transcriptome towards Establishment of Virus Carrier State.

Viral infections of teleost fish have great environmental and economic implications in aquaculture. Nervous necrosis virus (NNV) is a pathogen affecting more than 120 different species, causing high mortality and morbidity. Herein, we studied the course of NNV experimental infection of D. labrax, focusing on survivors which indicated viral carrier state. To determine the carrier state of D. labrax head kidney, we performed a gene expression analysis of selected immune-related genes and we profiled its transcriptome 14 days post infection (dpi). All tested genes showed clear differentiations in expression levels while most of them were up-regulated 14 dpi suggesting that their role is not limited in early antiviral responses, but they are also implicated in disease persistence. To gain a better understanding of the fish that survived the acute infection but still maintained a high viral load, we studied the differential expression of 124 up-regulated and 48 down-regulated genes in D. labrax head kidney, at 14 dpi. Concluding, the NNV virus persistent profile was assessed in D. labrax, where immune-related gene modification was intense (14 dpi) and the head kidney transcriptome profile at this time point offered a glimpse into host attempts to control the infection in asymptomatic carriers.

Malaria Vaccines: From the Past towards the mRNA Vaccine Era.

Plasmodium spp. is the etiological agent of malaria, a life-threatening parasitic disease transmitted by infected mosquitoes. Malaria remains a major global health challenge, particularly in endemic regions. Over the years, various vaccine candidates targeting different stages of Plasmodium parasite life-cycle have been explored, including subunit vaccines, vectored vaccines, and whole organism vaccines with Mosquirix, a vaccine based on a recombinant protein, as the only currently approved vaccine for Plasmodium falciparum malaria. Despite the aforementioned notable progress, challenges such as antigenic diversity, limited efficacy, resistant parasites escaping protective immunity and the need for multiple doses have hindered the development of a highly efficacious malaria vaccine. The recent success of mRNA-based vaccines against SARS-CoV-2 has sparked renewed interest in mRNA vaccine platforms. The unique mRNA vaccine features, including their potential for rapid development, scalability, and flexibility in antigen design, make them a promising avenue for malaria vaccine development. This review provides an overview of the malaria vaccines' evolution from the past towards the mRNA vaccine era and highlights their advantages in overcoming the limitations of previous malaria vaccine candidates.

Transcriptomic Analysis of Fish Hosts Responses to Nervous Necrosis Virus.

Nervous necrosis virus (NNV) has been responsible for mass mortalities in the aquaculture industry worldwide, with great economic and environmental impact. The present review aims to summarize the current knowledge of gene expression responses to nervous necrosis virus infection in different fish species based on transcriptomic analysis data. Four electronic databases, including PubMed, Web of Science, and SCOPUS were searched, and more than 500 publications on the subject were identified. Following the application of the appropriate testing, a total of 24 articles proved eligible for this review. NNV infection of different host species, in different developmental stages and tissues, presented in the eligible publications, are described in detail, revealing and highlighting genes and pathways that are most affected by the viral infection. Those transcriptome studies of NNV infected fish are oriented in elucidating the roles of genes/biomarkers for functions of special interest, depending on each study's specific emphasis. This review presents a first attempt to provide an overview of universal host reaction mechanisms to viral infections, which will provide us with new perspectives to overcome NNV infection to build healthier and sustainable aquaculture systems.

Leishmania Protein Kinases: Important Regulators of the Parasite Life Cycle and Molecular Targets for Treating Leishmaniasis.

Leishmania is a protozoan parasite of the trypanosomatid family, causing a wide range of diseases with different clinical manifestations including cutaneous, mucocutaneous and visceral leishmaniasis. According to WHO, one billion people are at risk of Leishmania infection as they live in endemic areas while there are 12 million infected people worldwide. Annually, 0.9-1.6 million new infections are reported and 20-50 thousand deaths occur due to Leishmania infection. As current chemotherapy for treating leishmaniasis exhibits numerous drawbacks and due to the lack of effective human vaccine, there is an urgent need to develop new antileishmanial therapy treatment. To this end, eukaryotic protein kinases can be ideal target candidates for rational drug design against leishmaniasis. Eukaryotic protein kinases mediate signal transduction through protein phosphorylation and their inhibition is anticipated to be disease modifying as they regulate all essential processes for Leishmania viability and completion of the parasitic life cycle including cell-cycle progression, differentiation and virulence. This review highlights existing knowledge concerning the exploitation of Leishmania protein kinases as molecular targets to treat leishmaniasis and the current knowledge of their role in the biology of Leishmania spp. and in the regulation of signalling events that promote parasite survival in the insect vector or the mammalian host.

Leishmania dual-specificity tyrosine-regulated kinase 1 (DYRK1) is required for sustaining Leishmania stationary phase phenotype.

Although the multiplicative and growth-arrested states play key roles in Leishmania development, the regulators of these transitions are largely unknown. In an attempt to gain a better understanding of these processes, we characterised one member of a family of protein kinases with dual specificity, LinDYRK1, which acts as a stasis regulator in other organisms. LinDYRK1 overexpressing parasites displayed a decrease in proliferation and in cell cycle re-entry of arrested cells. Parasites lacking LinDYRK1 displayed distinct fitness phenotypes in logarithmic and stationary growth phases. In logarithmic growth phase, LinDYRK1-/- parasites proliferated better than control lines, supporting a role of this kinase in stasis, while in stationary growth phase, LinDYRK1-/- parasites had important defects as they rounded up, accumulated vacuoles and lipid bodies and displayed subtle but consistent differences in lipid composition. Moreover, they expressed less metacyclic-enriched transcripts, displayed increased sensitivity to complement lysis and a significant reduction in survival within peritoneal macrophages. The distinct LinDYRK1-/- growth phase phenotypes were mirrored by the distinct LinDYRK1 localisations in logarithmic (mainly in flagellar pocket area and endosomes) and late stationary phase (mitochondrion). Overall, this work provides first evidence for the role of a DYRK family member in sustaining promastigote stationary phase phenotype and infectivity.

A Radioactive-free Kinase Inhibitor Discovery Assay Against the Trypanosoma brucei Glycogen Synthase Kinase-3 short (TbGSK-3s).

The identification of small molecules possessing inhibitory activity in vitro, against a given target kinase, is the first step in the drug discovery process. Herein, we describe a non radioactive protocol using luciferase-based ATP assay for the identification of inhibitors for the short isoform of the Trypanosoma brucei's Glycogen Synthase Kinase-3 (TbGSK-3s). TbGSK-3s represents a potential drug target as it is essential for parasite survival. Small molecules used in our study are indirubin analogues possessing substitutions in different positions in the bis-indole backbone. Presently, the standard laboratory practice for the kinase assays is the incorporation of radiolabeled phosphate from [gamma-32P]ATP as the efforts for developing non-radioactive assays (ELISA-based assays, fluorescence quenching assays, etc.) exhibit limitations such as lack in sensitivity or limitations for broad applications. This protocol can be a useful starting point for lead discovery, as it surpasses the drawbacks of radioactive kinase assays and it allows for relatively sensitive measurements of kinase inhibition for TbGSK-3s.

Indirubin Analogues Inhibit Trypanosoma brucei Glycogen Synthase Kinase 3 Short and T. brucei Growth.

The protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis (HAT). The disease is fatal if it remains untreated, whereas most drug treatments are inadequate due to high toxicity, difficulties in administration, and low central nervous system penetration. T. brucei glycogen synthase kinase 3 short (TbGSK3s) is essential for parasite survival and thus represents a potential drug target that could be exploited for HAT treatment. Indirubins, effective leishmanicidals, provide a versatile scaffold for the development of potent GSK3 inhibitors. Herein, we report on the screening of 69 indirubin analogues against T. brucei bloodstream forms. Of these, 32 compounds had potent antitrypanosomal activity (half-maximal effective concentration = 0.050 to 3.2 µM) and good selectivity for the analogues over human HepG2 cells (range, 7.4- to over 641-fold). The majority of analogues were potent inhibitors of TbGSK3s, and correlation studies for an indirubin subset, namely, the 6-bromosubstituted 3'-oxime bearing an extra bulky substituent on the 3' oxime [(6-BIO-3'-bulky)-substituted indirubins], revealed a positive correlation between kinase inhibition and antitrypanosomal activity. Insights into this indirubin-TbGSK3s interaction were provided by structure-activity relationship studies. Comparison between 6-BIO-3'-bulky-substituted indirubin-treated parasites and parasites silenced for TbGSK3s by RNA interference suggested that the above-described compounds may target TbGSK3s in vivo To further understand the molecular basis of the growth arrest brought about by the inhibition or ablation of TbGSK3s, we investigated the intracellular localization of TbGSK3s. TbGSK3s was present in cytoskeletal structures, including the flagellum and basal body area. Overall, these results give insights into the mode of action of 6-BIO-3'-bulky-substituted indirubins that are promising hits for antitrypanosomal drug discovery.

Indirubin derivatives are potent and selective anti-Trypanosoma cruzi agents.

Current treatment for combatting Chagas disease, a life-threatening illness caused by the kinetoplastid protozoan parasite Trypanosoma cruzi is inadequate, and thus the discovery of new antiparasitic compounds is of prime importance. Previous studies identified the indirubins, a class of ATP kinase inhibitors, as potent growth inhibitors of the related kinetoplastid Leishmania. Herein, we evaluated the inhibitory activity of a series of 69 indirubin analogues screened against T. cruzi trypomastigotes and intracellular amastigotes. Seven indirubins were identified as potent T. cruzi inhibitors (low µΜ, nM range). Cell death analysis of specific compounds [3'oxime-6-bromoindirubin(6-BIO) analogues 10, 11 and 17, bearing a bulky extension on the oxime moiety and one 7 substituted analogue 32], as evaluated by electron microscopy and flow cytometry, showed a different mode of action between compound 32 compared to the three 6-BIO oxime- substituted indirubins, suggesting that indirubins may kill the parasite by different mechanisms dependent on their substitution. Moreover, the efficacy of four compounds that show the most potent anti-parasitic effect in both trypomastigotes and intracellular amastigotes (10, 11, 17, 32), was evaluated in a mouse model of T. cruzi infection. Compound 11 (3'piperazine-6-BIO) displayed the best in vivo efficacy (1/6 mortality, 94.5% blood parasitaemia reduction, 12 dpi) at a dose five times reduced over the reference drug benznidazole (20 mg/kg vs100 mg/kg). We propose 3'piperazine-6-BIO as a potential lead for the development of new treatments of Chagas disease.

Lipophilic conformationally constrained spiro carbocyclic 2,6-diketopiperazine-1-acetohydroxamic acid analogues as trypanocidal and leishmanicidal agents: An extended SAR study.

We have previously described a number of lipophilic conformationally constrained spiro carbocyclic 2,6-diketopiperazine (2,6-DKP)-1-acetohydroxamic acids as potent antitrypanosomal agents. In this report, we extend the SAR analysis in this class of compounds with respect to in vitro growth inhibition of Trypanosoma and Leishmania parasites. Introduction of bulky hydrophobic substituents at the vicinal position of the basic nitrogen atom in the spiro carbocyclic 2,6-DKP ring system can provide analogues which are potently active against bloodstream form Trypanosoma brucei and exhibit significant activities toward Trypanosoma cruzi epimastogotes and Leishmania infantum promastigotes and intracellular amastigotes. In particular, compounds possessing a benzyl or 4-chlorobenzyl substituent were found to be the most active growth inhibitors, with activities in the low nanomolar and low micromolar ranges for T. brucei and L. infantum, respectively. The benzyl-substituted (S)-enantiomer was the most potent derivative against T. brucei (IC50  = 6.8 nm), T. cruzi (IC50  = 0.21 µm), and L. infantum promastigotes (IC50  = 2.67 µm) and intracellular amastigotes (IC50  = 2.60 µm). Moreover, the (R)-chiral benzyl-substituted derivative and its racemic counterpart displayed significant activities against L. donovani. Importantly, the active compounds show high selectivity in comparison with two mammalian cell lines.

A Nitrile Glucoside and Biflavones from the Leaves of Campylospermum excavatum (Ochnaceae).

The study of the MeOH extract of the leaves of Campylospermum excavatum led to the isolation of a nitrile glucoside, named campyloside C (1) and an original derivative of ochnaflavone, 7-O-methylochnaflavone (2), along with three known biflavonoids, amentoflavone, sequoiaflavone, and sotetsuflavone (3 - 5). The linkage site of the sub-units of 2 was confirmed by chemical correlation, after semi-synthesis of a trimethoxylated derivative of ochnaflavone (2a). The structures of these compounds as well as their relative and absolute configurations were assigned by 1D- and 2D-NMR experiments, HR-ESI-MS and Electronic Circular Dichroism (ECD) calculations. A low-pass J filter HMBC experiment was performed in order to define the configuration of the double bond of 1. All of the biflavonoids were evaluated against protozoan parasites. Amentoflavone moderately inhibited the promastigote form of Leishmania infantum.

Three new trixane glycosides obtained from the leaves of Jungia sellowii Less. using centrifugal partition chromatography.

Jungia sellowii (Asteraceae) is a shrub that grows in Southern Brazil and polar extract of its leaves presents anti-inflammatory properties. Cyperane, guaiane, nortrixane, and trixane sesquiterpene types were reported as the main metabolites in Jungia species. This work aims to describe the isolation and identification of sesquiterpenes in the leaves of J. sellowii using liquid-liquid partition and centrifugal partition chromatography. Thus, the crude extract of fresh leaves of J. sellowii was partitioned with hexane, dichloromethane, ethyl acetate and butanol, respectively. The butanol fraction was then subjected to a selected ternary system optimized for the CPC (centrifugal partition chromatography): ethyl acetate-ethanol-water (9:2:10, v/v/v). The separation was carried out isocratically at a flow rate of 25 mL/min at 1200 rpm, affording seven fractions A to G. TLC of fractions B, C and F displayed a single spot corresponding to three new glycosylated sesquiterpenoids. Their structures were established by using spectroscopic data in comparison to those reported in the literature. Furthermore, the isolates were evaluated for their leishmanicidal and cytotoxic effects. No cytotoxic effect was observed against the three cancer cell lines (HL60, JURKAT and REH), but compound 1 showed a weak antiprotozoal activity. Liquid-liquid partition and CPC turned to be a versatile technique of glycoside purification which is environmentally friendly and requires a limited amount of organic solvents.

Potential Involvement of Jagged1 in Metastatic Progression of Human Breast Carcinomas.

BACKGROUND: Jagged1, the ligand of Notch, has been shown to be involved in formation of bone metastases in an experimental study. Here, clinical relevance of Jagged1 expression in tumor progression was assessed in human breast carcinomas. METHODS: Jagged1 expression was evaluated by immunohistochemistry in 228 tumor tissue samples and compared to clinicopathologic parameters and patients' outcomes. Furthermore, circulating tumor cells (CTCs) from peripheral blood of 100 unmatched metastatic cancer patients with progressive disease were enriched using Ficoll density gradient centrifugation and detected by pan-keratin/Jagged1/CD45 immunofluorescent staining. RESULTS: Jagged1 expression was detected in 50% of 228 tumors. Jagged1 expression was correlated with higher tumor grade (P = 0.047), vascular invasion (P = 0.026), luminal B subtype (P = 0.016), overexpression of Her-2 (P = 0.001), high Ki-67 expression (P = 0.035), and aldehyde dehydrogenase 1 (ALDH1) positivity (P = 0.013). Jagged 1 expression indicated shorter disease-free survival (DFS) (P = 0.040) and metastasis-free survival (P = 0.048) in lymph node-negative breast cancer for which it was the only independent predictor of DFS (multivariate analysis, P = 0.046). Tumors characterized by the strongest Jagged1 staining intensity (7.5% of cases) correlated with lymph node positivity (P = 0.037), metastatic relapse (P = 0.049), and higher number of disseminated tumor cells in bone marrow aspirates (P = 0.041). Twenty-one unmatched metastatic breast cancer patients with progressive disease were positive for CTCs, and 85.7% of the CTCs also expressed Jagged1. The presence of Jagged1(+) CTCs was significantly associated with shorter progression-free survival in patients treated with bisphosphonates (P = 0.013). CONCLUSIONS: Jagged1 expression characterizes more aggressive breast carcinoma and might be involved in tumor cell dissemination, metastatic progression, and resistance to bone-targeting therapy in breast cancer patients.

An inhibitor-driven study for enhancing the selectivity of indirubin derivatives towards leishmanial Glycogen Synthase Kinase-3 over leishmanial cdc2-related protein kinase 3.

BACKGROUND: In search of new antiparasitic agents for overcoming the limitations of current leishmaniasis chemotherapy, we have previously shown that 6-bromoindirubin-3'-oxime (6BIO) and several other 6-substituted analogues of indirubin, a naturally occurring bis-indole present in mollusks and plants, displayed reverse selectivity from the respective mammalian kinases, targeting more potently the leishmanial Cyclin-Dependent Kinase-1 (CDK1) homologue [cdc2-related protein kinase 3 (LCRK3)] over leishmanial Glycogen Synthase Kinase-3 (LGSK-3). This reversal of selectivity in Leishmania parasites compared to mammalian cells makes the design of specific indirubin-based LGSK-3 inhibitors difficult. In this context, the identification of compounds bearing specific substitutions that shift indirubin inhibition towards LGSK-3, previously found to be a potential drug target, over LCRK3 is imperative for antileishmanial targeted drug discovery. METHODS: A new in-house indirubin library, composed of 35 compounds, initially designed to target mammalian kinases (CDKs, GSK-3), was tested against Leishmania donovani promastigotes and intracellular amastigotes using the Alamar blue assay. Indirubins with antileishmanial activity were tested against LGSK-3 and LCRK3 kinases, purified from homologous expression systems. Flow cytometry (FACS) was used to measure the DNA content for cell-cycle analysis and the mode of cell death. Comparative structural analysis of the involved kinases was then performed using the Szmap algorithm. RESULTS: We have identified 7 new indirubin analogues that are selective inhibitors of LGSK-3 over LCRK3. These new inhibitors were also found to display potent antileishmanial activity with GI50 values of <1.5 µΜ. Surprisingly, all the compounds that displayed enhanced selectivity towards LGSK-3, were 6BIO analogues bearing an additional 3'-bulky amino substitution, namely a piperazine or pyrrolidine ring. A comparative structural analysis of the two aforementioned leishmanial kinases was subsequently undertaken to explain and rationalize the selectivity trend determined by the in vitro binding assays. Interestingly, the latter analysis showed that selectivity could be correlated with differences in kinase solvation thermo dynamics induced by minor sequence variations of the otherwise highly similar ATP binding pockets. CONCLUSIONS: In conclusion, 3'-bulky amino substituted 6-BIO derivatives, which demonstrate enhanced specificity towards LGSK-3, represent a new scaffold for targeted drug development to treat leishmaniasis.

The loss of virulence of histone H1 overexpressing Leishmania donovani parasites is directly associated with a reduction of HSP83 rate of translation.

Overexpression of Leishmania histone H1 (LeishH1) was previously found to cause a promastigote-to-amastigote differentiation handicap, deregulation of cell-cycle progression, and loss of parasite infectivity. The aim of this study was to identify changes in the proteome of LeishH1 overexpressing parasites associated with the avirulent phenotype observed. 2D-gel electrophoresis analysis revealed only a small protein subset of differentially expressed proteins in the LeishH1 overexpressing promastigotes. Among these was the chaperone HSP83, known for its protective role in Leishmania drug-induced apoptosis, which displayed lower translational rates. To investigate if the lower expression levels of HSP83 are associated with the differentiation handicap, we assayed the thermostability of parasites by subjecting them to heat-shock (25°C→37°C), a natural stress-factor occurring during stage differentiation. Heat-shock promoted apoptosis to a greater extent in the LeishH1 overexpressing parasites. Interestingly, these parasites were not only more sensitive to heat-shock but also to drug-induced [Sb(III)] cell-death. In addition, the restoration of HSP83 levels re-established drug resistance, and restored infectivity to LeishH1 overexpressing parasites in the murine J774 macrophage model. Overall, this study suggests that LeishH1 levels are critical for the parasite's stress-induced adaptation within the mammalian host, and highlights the cross-talk between pathways involved in drug resistance, apoptosis and virulence.

Biochemical and genetic evidence for the presence of multiple phosphatidylinositol- and phosphatidylinositol 4,5-bisphosphate-specific phospholipases C in Tetrahymena.

Eukaryotic phosphoinositide-specific phospholipases C (PI-PLC) specifically hydrolyze phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)], produce the Ca(2+)-mobilizing agent inositol 1,4,5-trisphosphate, and regulate signaling in multicellular organisms. Bacterial PtdIns-specific PLCs, also present in trypanosomes, hydrolyze PtdIns and glycosyl-PtdIns, and they are considered important virulence factors. All unicellular eukaryotes studied so far contain a single PI-PLC-like gene. In this report, we show that ciliates are an exception, since we provide evidence that Tetrahymena species contain two sets of functional genes coding for both bacterial and eukaryotic PLCs. Biochemical characterization revealed two PLC activities that differ in their phosphoinositide substrate utilization, subcellular localization, secretion to extracellular space, and sensitivity to Ca(2+). One of these activities was identified as a typical membrane-associated PI-PLC activated by low-micromolar Ca(2+), modestly activated by GTPγS in vitro, and inhibited by the compound U73122 [1-(6-{[17ß-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. Importantly, inhibition of PI-PLC in vivo resulted in rapid upregulation of PtdIns(4,5)P(2) levels, suggesting its functional importance in regulating phosphoinositide turnover in Tetrahymena. By in silico and molecular analysis, we identified two PLC genes that exhibit significant similarity to bacterial but not trypanosomal PLC genes and three eukaryotic PI-PLC genes, one of which is a novel inactive PLC similar to proteins identified only in metazoa. Comparative studies of expression patterns and PI-PLC activities in three T. thermophila strains showed a correlation between expression levels and activity, suggesting that the three eukaryotic PI-PLC genes are functionally nonredundant. Our findings imply the presence of a conserved and elaborate PI-PLC-Ins(1,4,5)P(3)-Ca(2+) regulatory axis in ciliates.