The Development of Toxoplasma gondii Recombinant Trivalent Chimeric Proteins as an Alternative to Toxoplasma Lysate Antigen (TLA) in Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of Immunoglobulin G (IgG) in Small Ruminants.

This study presents an evaluation of seventeen newly produced recombinant trivalent chimeric proteins (containing the same immunodominant fragment of SAG1 and SAG2 of Toxoplasma gondii antigens, and an additional immunodominant fragment of one of the parasite antigens, such as AMA1, GRA1, GRA2, GRA5, GRA6, GRA7, GRA9, LDH2, MAG1, MIC1, MIC3, P35, and ROP1) as a potential alternative to the whole-cell tachyzoite lysate (TLA) used in the detection of infection in small ruminants. These recombinant proteins, obtained by genetic engineering and molecular biology methods, were tested for their reactivity with specific anti-Toxoplasma IgG antibodies contained in serum samples of small ruminants (192 samples of sheep serum and 95 samples of goat serum) using an enzyme-linked immunosorbent assay (ELISA). The reactivity of six recombinant trivalent chimeric proteins (SAG1-SAG2-GRA5, SAG1-SAG2-GRA9, SAG1-SAG2-MIC1, SAG1-SAG2-MIC3, SAG1-SAG2-P35, and SAG1-SAG2-ROP1) with IgG antibodies generated during T. gondii invasion was comparable to the sensitivity of TLA-based IgG ELISA (100%). The obtained results show a strong correlation with the results obtained for TLA. This suggests that these protein preparations may be a potential alternative to TLA used in commercial tests and could be used to develop a cheaper test for the detection of parasite infection in small ruminants.

4-Arylthiosemicarbazide Derivatives as Toxoplasmic Aromatic Amino Acid Hydroxylase Inhibitors and Anti-inflammatory Agents.

Approximately one-third of the human population is infected with the intracellular cosmopolitan protozoan Toxoplasma gondii (Tg), and a specific treatment for this parasite is still needed. Additionally, the increasing resistance of Tg to drugs has become a challenge for numerous research centers. The high selectivity of a compound toward the protozoan, along with low cytotoxicity toward the host cells, form the basis for further research, which aims at determining the molecular targets of the active compounds. Thiosemicarbazide derivatives are biologically active organic compounds. Previous studies on the initial preselection of 58 new 4-arylthiosemicarbazide derivatives in terms of their anti-Tg activity and selectivity made it possible to select two promising derivatives for further research. One of the important amino acids involved in the proliferation of Tg and the formation of parasitophorous vacuoles is tyrosine, which is converted by two unique aromatic amino acid hydroxylases to levodopa. Enzymatic studies with two derivatives (R: para-nitro and meta-iodo) and recombinant aromatic amino acid hydroxylase (AAHs) obtained in the E. coli expression system were performed, and the results indicated that toxoplasmic AAHs are a molecular target for 4-arylthiosemicarbazide derivatives. Moreover, the drug affinity responsive target stability assay also confirmed that the selected compounds bind to AAHs. Additionally, the anti-inflammatory activity of these derivatives was tested using THP1-Blue™ NF-κB reporter cells due to the similarity of the thiosemicarbazide scaffold to thiosemicarbazone, both of which are known NF-κB pathway inhibitors.

Controlling the Spatiotemporal Release of Nerve Growth Factor by Chitosan/Polycaprolactone Conduits for Use in Peripheral Nerve Regeneration.

Tubular polymeric structures have been recognized in the treatment of peripheral nerves as comparable to autologous grafting. The best therapeutic outcomes are obtained with conduits releasing therapeutic molecules. In this study, a new approach for the incorporation of biologically active agent-loaded microspheres into the structure of chitosan/polycaprolactone conduits was developed. The support of a polycaprolactone helix formed by 3D melt extrusion was coated with dopamine in order to adsorb nerve growth factor-loaded microspheres. The complex analysis of the influence of process factors on the coverage efficiency of polycaprolactone helix by nerve grow factor-loaded microspheres was analyzed. Thus, the PCL helix characterized with the highest adsorption of microspheres was subjected to nerve growth factor release studies, and finally incorporated into chitosan hydrogel deposit through the process of electrophoretic deposition. It was demonstrated by chemical and physical tests that the chitosan/polycaprolactone conduit meets the requirements imposed on peripheral nerve implants, particularly mimicking mechanical properties of surrounding soft tissue. Moreover, the conduit may support regrowing nerves for a prolonged period, as its structure and integrity persist upon incubation in lysozyme-contained PBS solution up to 28 days at body temperature. In vitro cytocompatibility toward mHippoE-18 embryonic hippocampal cells of the chitosan/polycaprolactone conduit was proven. Most importantly, the developed conduits stimulate axonal growth and support monocyte activation, the latter is advantageous especially at early stages of nerve regeneration. It was demonstrated that, through the described approach for controlling spatiotemporal release of nerve growth factors, these biocompatible structures adjusted to the specific peripheral nerve injury case can be manufactured.

The role of extracellular vesicles in parasite-host interaction.

Zewnatrzkomórkowe pecherzyki blonowe (EVs, extracellular vesicles), poczatkowo uwazane za elementy zniszczonych komórek, okazaly sie niezwykle istotnym sposobem przekazywania informacji miedzy komórkami, bez ich bezposredniego kontaktu. Ze wzgledu na powszechne wystepowanie EVs w komórkach organizmów zarówno jedno-, jak i wielokomórkowych nalezacych do róznych grup systematycznych oraz ze wzgledu na pelniona role w komunikacji miedzykomórkowej staly sie przedmiotem licznych badan i dyskusji. EVs sa uwalniane przez komórki prokariotyczne, jak i eukariotyczne, zarówno w warunkach in vivo, jak i in vitro. Chociaz uzyskiwane frakcje EVs sa zwykle mieszanina róznorodnych struktur pochodzenia blonowego wprowadzono klasyfikacje pecherzyków przede wszystkim na podstawie ich wielkosci i prawdopodobnego mechanizmu powstawania. EVs jako nosniki informacji zawieraja róznorodny material komórkowy, a jednak dzieki intensywnym pracom badawczym coraz wiecej wiadomo o ich funkcji w róznego rodzaju procesach np. nowotworowych. W pracy przedstawiono obecny stan wiedzy na temat pecherzyków blonowych bioracych udzial w szeroko pojetych interakcjach zywiciel-pasozyt, obejmujacych inwazje i kolonizacje zywiciela, ustalanie równowagi miedzy partnerami czy modulacje odpowiedzi immunologicznej zywiciela w czasie zarazenia. Poruszono kwestie potencjalnego wykorzystania pecherzyków w immunoprofilaktyce oraz diagnostyce chorób inwazyjnych. Najwiecej miejsca poswiecono inwazjom spowodowanym przez pierwotniaki, ze szczególnym uwzglednieniem parazytoz o najwiekszym znaczeniu medycznym i spolecznym w skali globalnej, co znajduje takze swoje odzwierciedlenie w literaturze swiatowej. Zebrano takze dosc skape na razie doniesienia na temat udzialu EVs w przebiegu inwazji wywolywanych przez gatunki pasozytnicze zaliczane do grupy helmintów.

The pharmaceutics from the foreign empire: the molecular pharming of the prokaryotic staphylokinase in Arabidopsis thaliana plants.

Here, we present the application of microbiology and biotechnology for the production of recombinant pharmaceutical proteins in plant cells. To the best of our knowledge and belief it is one of few examples of the expression of the prokaryotic staphylokinase (SAK) in the eukaryotic system. Despite the tremendous progress made in the plant biotechnology, most of the heterologous proteins still accumulate to low concentrations in plant tissues. Therefore, the composition of expression cassettes to assure economically feasible level of protein production in plants remains crucial. The aim of our research was obtaining a high concentration of the bacterial anticoagulant factor-staphylokinase, in Arabidopsis thaliana seeds. The coding sequence of staphylokinase was placed under control of the ß-phaseolin promoter and cloned between the signal sequence of the seed storage protein 2S2 and the carboxy-terminal KDEL signal sequence. The engineered binary vector pATAG-sak was introduced into Arabidopsis thaliana plants via Agrobacterium tumefaciens-mediated transformation. Analysis of the subsequent generations of Arabidopsis seeds revealed both presence of the sak and nptII transgenes, and the SAK protein. Moreover, a plasminogen activator activity of staphylokinase was observed in the protein extracts from seeds, while such a reaction was not observed in the leaf extracts showing seed-specific activity of the ß-phaseolin promoter.

Toxoplasma gondii: cloning, expression and immunoreactivity of recombinant ROP5 and ROP18 antigens.

Early diagnosis and determining the infective stage are critical for effective therapy of toxoplasmosis. Owing to the progress in biotechnology, commonly used native, non-standardized diagnostic antigens should be replaced by genetically engineered antigens. The recombinant proteins are also promising components of subunit vaccines against Toxoplasma gondii infections. A strategic biological role of rhoptry proteins (ROP) in parasitophorous vacuole biogenesis and virulence of the parasite creates a necessity for an intensive study on the serological activity and immunogenicity of newly developed recombinant ROP antigens. Our findings indicate that all generated preparations of recombinant ROP5 and ROP18 antigens, expressed in Escherichia coli bacteria, are recognized by specific antibodies produced during acute and chronic infections in inbred laboratory mice. We noticed, for the first time, that ROP5 IgM antibodies are an early and sensitive marker of T. gondii infection. The proven immunoreactivity of the obtained preparations has become a premise for a further study on their utility in routine diagnosis of human and animal toxoplasmosis as well as in the immunoprevention of T. gondii infection (as the main or supplementary component of the vaccine).

Towards vaccine against toxoplasmosis: evaluation of the immunogenic and protective activity of recombinant ROP5 and ROP18 Toxoplasma gondii proteins.

Toxoplasmosis is one of the most common parasitic infections worldwide. An effective vaccine against human and animal toxoplasmosis is still needed to control this parasitosis. The polymorphic rhoptry proteins, ROP5 and ROP18, secreted by Toxoplasma gondii during the invasion of the host cell have been recently considered as promising vaccine antigens, as they appear to be the major determinants of T. gondii virulence in mice. The goal of this study was to evaluate their immunogenic and immunoprotective activity after their administration (separately or both recombinant proteins together) with the poly I:C as an adjuvant. Immunization of BALB/c and C3H/HeOuJ mice generated both cellular and humoral specific immune responses with some predominance of IgG1 antibodies. The spleen cells derived from vaccinated animals reacted to the parasite's native antigens. Furthermore, the immunization led to a partial protection against acute and chronic toxoplasmosis. These findings confirm the previous assumptions about ROP5 and ROP18 antigens as valuable components of a subunit vaccine against toxoplasmosis.

Recombinant MAG1 Protein of Toxoplasma gondii as a Diagnostic Antigen.

The aim of this study was to evaluate the potential diagnostic usefulness of the full-length recombinant Toxoplasma gondii MAG1 protein by determining the levels of specific IgM and IgG antibodies in mouse and human sera obtained from individuals with acute and chronic toxoplasmosis. The obtained results revealed that IgG antibodies against MAG1 are a sensitive and specific marker of T. gondii infection since the protein was recognized by both mouse and human sera, 100% and 94.3%, respectively, rendering the full-length rMAG1 a prospective alternative for the polyvalent native antigen (TLA).

Veterinary vaccines against toxoplasmosis.

Toxoplasma gondii is a cosmopolitan protozoan parasite that infects a wide range of mammal and bird species. Common infection leads to high economic (e.g., abortions in sheep) and human (e.g., congenital toxoplasmosis or neurotoxoplasmosis in humans) losses. With one exception (Toxovax for sheep), there are no vaccines to prevent human or animal toxoplasmosis. The paper presents the current state and challenges in the development of a vaccine against toxoplasmosis, designed for farm animals either bred for consumption or commonly kept on farms and involved in parasite transmission. So far, the trials have mostly revolved around conventional vaccines and, compared with the research using laboratory animals (mainly mice), they have not been very numerous. However, the results obtained are promising and could be a good starting point for developing an effective vaccine to prevent toxoplasmosis.

Sex-dependent neurotransmitter level changes in brains of Toxoplasma gondii infected mice.

The protozoan parasite Toxoplasma gondii has the ability to alter intermediate host behavior, most impressively the natural aversion to cat scent, to favor the predation by the definitive host. However, the underlying mechanism of the observed phenomenon still remains unknown. Since changes in the neurotransmitter level are postulated as a possible contributing factor, the aim of this work was to assess the monoamine systems activity in specified brain regions involved in the natural defense behaviors, emotion evaluation, and motor and sensory stimuli integration in experimentally T. gondii infected mice compared to uninfected controls. Taking into account the natural differences between genders, the experiments were carried out on both male and female mice. Our results revealed statistically significant changes in all tested monoamine systems with regard to both gender and time after T. gondii invasion. Acute toxoplasmosis was accompanied by a decrease in noradrenergic system activity in females and its slight increase in some brain areas of males. Acute invasion also induced a rise in serotonin system activity, mostly in males. The most striking observation was an increase in the dopamine release noted in acutely infected males. We discuss our results in terms of their possible contribution to T. gondii-induced intermediate host behavior alterations and parasite transmission and with regard to postulated relationship between T. gondii seroprevalence and occurrence of certain disorders such as schizophrenia in humans.

Evaluation of long-term immunity and protection against T. gondii after immunization with multivalent recombinant chimeric T. gondii proteins.

Toxoplasmosis caused by the opportunistic, cosmopolitan protozoan Toxoplasma gondii is one of the most common parasitoses in the world. Although it may prove dangerous or even fatal for immunocompromised individuals, immunoprophylaxis for humans is still nonexistent. Thus, the aim of the current work was to assess the ability of two immunogenic recombinant chimeric T. gondii proteins, SAG2-GRA1-ROP1 (SGR) and SAG1-MIC1-MAG1-GRA2 (SMMG), selected in previous experiments to induce long-lasting immunity when administered with a safe adjuvant. Thus, the determination of immunological parameters and parasite challenge were performed both two weeks after the last boost injection and 6 months postvaccination. Both experimental vaccines triggered specific humoral and cellular responses in immunized C3H/HeOuJ male mice, characterized by the production of specific IgG (IgG1/IgG2a) antibodies in vivo and the synthesis of key Th1/Th2 cytokines by Toxoplasma lysate antigen-stimulated splenocytes in vitro. Although the levels of specific antibodies and cytokine release were in most cases lower six months postimmunization, the protection rates conferred by the vaccination were comparable regardless of the time after the administration of the last vaccine dose. The results indicate that both preparations induce long-lasting immunity, which makes them attractive candidates for further research aimed at boosting their immunogenicity and immunoprotective capacity.

The functional response of human monocyte-derived macrophages to serum amyloid A and Mycobacterium tuberculosis infection.

Introduction: In the course of tuberculosis (TB), the level of major acute phase protein, namely serum amyloid A (hSAA-1), increases up to a hundredfold in the pleural fluids of infected individuals. Tubercle bacilli infecting the human host can be opsonized by hSAA-1, which affects bacterial entry into human macrophages and their intracellular multiplication. Methods: We applied global RNA sequencing to evaluate the functional response of human monocyte-derived macrophages (MDMs), isolated from healthy blood donors, under elevated hSAA-1 conditions and during infection with nonopsonized and hSAA-1-opsonized Mycobacterium tuberculosis (Mtb). In the same infection model, we also examined the functional response of mycobacteria to the intracellular environment of macrophages in the presence and absence of hSAA-1. The RNASeq analysis was validated using qPCR. The functional response of MDMs to hSAA-1 and/or tubercle bacilli was also evaluated for selected cytokines at the protein level by applying the Milliplex system. Findings: Transcriptomes of MDMs cultured in the presence of hSAA-1 or infected with Mtb showed a high degree of similarity for both upregulated and downregulated genes involved mainly in processes related to cell division and immune response, respectively. Among the most induced genes, across both hSAA-1 and Mtb infection conditions, CXCL8, CCL15, CCL5, IL-1ß, and receptors for IL-7 and IL-2 were identified. We also observed the same pattern of upregulated pro-inflammatory cytokines (TNFα, IL-6, IL-12, IL-18, IL-23, and IL-1) and downregulated anti-inflammatory cytokines (IL-10, TGFß, and antimicrobial peptide cathelicidin) in the hSAA-1 treated-MDMs or the phagocytes infected with tubercle bacilli. At this early stage of infection, Mtb genes affected by the inside microenvironment of MDMs are strictly involved in iron scavenging, adaptation to hypoxia, low pH, and increasing levels of CO2. The genes for the synthesis and transport of virulence lipids, but not cholesterol/fatty acid degradation, were also upregulated. Conclusion: Elevated serum hSAA-1 levels in tuberculosis enhance the response of host phagocytes to infection, including macrophages that have not yet been in contact with mycobacteria. SAA induces antigen processing and presentation processes by professional phagocytes reversing the inhibition caused by Mtb infection.

Toxoplasma gondii: the vaccine potential of three trivalent antigen-cocktails composed of recombinant ROP2, ROP4, GRA4 and SAG1 proteins against chronic toxoplasmosis in BALB/c mice.

Toxoplasmosis is one of the world's most widespread zoonoses caused by protozoan parasite Toxoplasma gondii. The development of an effective vaccine for controlling toxoplasmosis is an extremely important issue due to the serious clinical and veterinary outcomes of this parasitosis. The objective of this study was evaluation of vaccine potential of three trivalent subunit recombinant vaccines composed of rROP2+rGRA4+rSAG1, rROP2+rROP4+rGRA4 and rROP2+rROP4+rSAG1 against chronic toxoplasmosis in BALB/c (H-2(d)) mice. All tested vaccines provided a partial protection against challenge with tissue cysts of the low virulence DX T. gondii strain, but the strongest level of protection was induced by the mixtures of both rhoptry proteins (rROP2 and rROP4) administered with the dense granule rGRA4 antigen or the main surface rSAG1 protein. The average parasite burden in these groups of vaccinated BALB/c mice was reduced by 84% and 77%, respectively, compared to the control PBS-injected animals. The vaccine-induced protection was correlated with the development of cellular and humoral immune responses demonstrated by the antigen-specific in vitro proliferation of spleen cells, the specific antigen-induced in vitro synthesis of Th1-type cytokines, IFN-γ and IL-2, and the generation of the high titers of systemic antigen-specific IgG1 and IgG2a antibodies. This study completed and confirmed our earlier investigations in C3H/HeJ (H-2(k)) and C57BL/6 (H-2(b)) mouse strains on the utility of the tested trivalent recombinant antigen-cocktails as potential vaccines against chronic toxoplasmosis and showed that particularly rROP2+rROP4+rGRA4 and rROP2+rROP4+rSAG1 protein-combinations are very effective in the development of a high level of protection irrespective of the genetic backgrounds and innate resistance to toxoplasmosis of the laboratory mice. It makes these two mixtures of recombinant antigens very promising for further experiments.

Behavioral changes in mice caused by Toxoplasma gondii invasion of brain.

Toxoplasma gondii, a protozoan parasite, is capable of infecting a broad range of intermediate warm-blooded hosts including humans. The parasite undergoes sexual reproduction resulting in genetic variability only in the intestine of the definitive host (a member of the cat family). The parasite seems to be capable of altering the natural behavior of the host to favor its transmission in the environment. The aim of this study was to evaluate the number of parasite cysts formed in the hippocampus and amygdala of experimentally infected mice as these regions are involved in defense behaviors control and emotion processing, and to assess the influence of the infection on mice behavior. The obtained results revealed the presence of parasite cysts both in the hippocampus and the amygdala of infected mice; however, no clear region-dependent distribution was observed. Furthermore, infected mice showed significantly diminished exploratory activity described by climbing and rearing, smaller preference for the central, more exposed part of the OF arena and engaged in less grooming behavior compared to uninfected controls.

4-Arylthiosemicarbazide derivatives - Pharmacokinetics, toxicity and anti-Toxoplasma gondii activity in vivo.

The increasing resistance of Toxoplasma gondii to drugs and side effects of therapy indicate that specific treatment for these parasites is still needed. The 4-arylthiosemicarbazide derivatives seem to be a solution to this challenge because they have low cytotoxicity against host cells and high anti-T. gondii activity. The molecular mechanism for these compounds is related to the inhibition of tyrosine amino acids involved in the proliferation and parasitophorous vacuole formation. The pharmacokinetic analysis shows that 1-(4-Methylimidazol-5-oyl)-4-(4-nitrophenyl)thiosemicarbazide and 4-(3-Iodophenyl)-1-(4-methylimidazol-5-oyl)thiosemicarbazide administered intragastrically pass into the bloodstream and cross the blood-brain barrier, and the absorption of both compounds is first-order absorption. Toxicity analysis shows that our derivatives possess lower toxicity than the routinely used drugs trimethoprim, sulfadiazine and pyrimethamine, as was observed in the level of liver enzymes and creatinine. Both derivatives are highly potent antiparasitic agents against T. gondii, prolonged survival and cure parasite-infected mice. Additionally, significant reductions in cyst formation in the brain and heart were observed, but the highest decreases were noted in muscle and the level of bradyzoites was similar to these observed in mice treated with commercially used drugs. Collectively, the obtained results support the conclusion that both compounds are highly efficacious in a mouse model of acute and chronic toxoplasmosis.

Ten-eleven translocation methylcytosine dioxygenase 3-loaded microspheres penetrate neurons in vitro causing active demethylation and neurite outgrowth.

Epigenetic processes, such as DNA methylation and other chromatin modifications, are believed to be largely responsible for establishing a reduced capacity for growth in the mature nervous system. Ten-eleven translocation methylcytosine dioxygenase 3 (Tet3)-, a member of the Tet gene family, plays a crucial role in promoting injury-induced DNA demethylation and expression of regeneration-associated genes in the peripheral nervous system. Here, we encapsulate Tet3 protein within a clinically tolerated poly(lactide-co-glycolide) microsphere system. Next, we show that Tet3-loaded microspheres are internalized into mHippoE-18 embryonic hippocampal cells. We compare the outgrowth potential of Tet3 microspheres with that of commonly used nerve growth factor (NGF)-loaded microspheres in an in vitro injury model. Tet3-containing microspheres increased levels of nuclear 5-hydroxymethylcytosine indicating active demethylation and outperformed NGF-containing microspheres in measures of neurite outgrowth. Our results suggest that encapsulated demethylases may represent a novel avenue to treat nerve injuries.

Mycobacterium tuberculosis Binds Human Serum Amyloid A, and the Interaction Modulates the Colonization of Human Macrophages and the Transcriptional Response of the Pathogen.

As a very successful pathogen with outstanding adaptive properties, Mycobacterium tuberculosis (Mtb) has developed a plethora of sophisticated mechanisms to subvert host defenses and effectively enter and replicate in the harmful environment inside professional phagocytes, namely, macrophages. Here, we demonstrated the binding interaction of Mtb with a major human acute phase protein, namely, serum amyloid A (SAA1), and identified AtpA (Rv1308), ABC (Rv2477c), EspB (Rv3881c), TB 18.6 (Rv2140c), and ThiC (Rv0423c) membrane proteins as mycobacterial effectors responsible for the pathogen-host protein interplay. SAA1-opsonization of Mtb prior to the infection of human macrophages favored bacterial entry into target phagocytes accompanied by a substantial increase in the load of intracellularly multiplying and surviving bacteria. Furthermore, binding of human SAA1 by Mtb resulted in the up- or downregulation of the transcriptional response of tubercle bacilli. The most substantial changes were related to the increased expression level of the genes of two operons encoding mycobacterial transporter systems, namely, mmpL5/mmpS5 (rv0676c), and rv1217c, rv1218c. Therefore, we postulate that during infection, Mtb-SAA1 binding promotes the infection of host macrophages by tubercle bacilli and modulates the functional response of the pathogen.

Imidazole-Thiosemicarbazide Derivatives as Potent Anti-Mycobacterium tuberculosis Compounds with Antibiofilm Activity.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogenic bacterium and the causative agent of tuberculosis. This disease is one of the most ancient and deadliest bacterial infections, as it poses major health, social and economic challenges at a global level, primarily in low- and middle-income countries. The lack of an effective vaccine, the long and expensive drug therapy, and the rapid spread of drug-resistant strains of Mtb have led to the re-emergence of tuberculosis as a global pandemic. Here, we assessed the in vitro activity of new imidazole-thiosemicarbazide derivatives (ITDs) against Mtb infection and their effects on mycobacterial biofilm formation. Cytotoxicity studies of the new compounds in cell lines and human monocyte-derived macrophages (MDMs) were performed. The anti-Mtb activity of ITDs was evaluated by determining minimal inhibitory concentrations of resazurin, time-kill curves, bacterial intracellular growth and the effect on biofilm formation. Mutation frequency and whole-genome sequencing of mutants that were resistant to ITDs were performed. The antimycobacterial potential of ITDs with the ability to penetrate Mtb-infected human macrophages and significantly inhibit the intracellular growth of tubercle bacilli and suppress Mtb biofilm formation was observed.

The effect of prolactin (PRL) on the growth of Toxoplasma gondii tachyzoites in vitro.

During the development and effector phases of the anti-Toxoplasma response, the immunological system of a host is involved in several complex interactions with the endocrine system, and prolactin (PRL) is one of the most important hormones involved in immunoregulation. In this work, the influence of the recombinant human prolactin (rhPRL) on the viability, penetration, and intensity of intracellular proliferation of Toxoplasma gondii BK strain in vitro was evaluated. Using one murine (L929) and two human cell lines (Hs27 and HeLa), no toxic effect of the rhPRL on host cells was found (by determining cellular viability using MTT assay). A similar lack of rhPRL cytotoxic activity was found in the case of the extracellular tachyzoites of T. gondii BK. Replication of parasites in the presence of rhPRL was analyzed first by simultaneous addition of the hormone and the parasites into a microculture of the host cells (treatment during infection). No statistically significant changes in the intensity of parasite proliferation in all used host cells were found for a wide range of the hormone concentrations. However, pre-incubation of the tachyzoites with rhPRL resulted in a significant reduction (up to 36.15%) in the replication abilities of the parasite. Further experiments revealed that in fact, the inhibition of replication was caused by a limited capacity of the parasites to penetrate host's cells as demonstrated by the reduced number of infected cells.

Determination of diagnostic value of Toxoplasma gondii recombinant ROP2 and ROP4 antigens in mouse experimental model.

The aim of this study was to test the potential diagnostic usefulness of recombinant Toxoplasma gondii rhoptry antigens, ROP2 and ROP4, with respect to toxoplasmosis detection and infection phase distinction in laboratory mouce by determining specific serum IgM and IgG antibodies with the use of indirect ELISA technique. The mice antibody response to ROP antigens was significantly higher in the IgM than in the IgG class with the peak on the turn of acute and latent infection, whereas the response to recombinant SAG1 antigen, used as control, revealed preferential synthesis of IgG antibodies with the highest absorbance values measured during latent toxoplasmosis.