Transcriptomic profiling reveals histone acetylation-regulated genes involved in somatic embryogenesis in Arabidopsis thaliana.

BACKGROUND: Somatic embryogenesis (SE) exemplifies the unique developmental plasticity of plant cells. The regulatory processes, including epigenetic modifications controlling embryogenic reprogramming of cell transcriptome, have just started to be revealed. RESULTS: To identify the genes of histone acetylation-regulated expression in SE, we analyzed global transcriptomes of Arabidopsis explants undergoing embryogenic induction in response to treatment with histone deacetylase inhibitor, trichostatin A (TSA). The TSA-induced and auxin (2,4-dichlorophenoxyacetic acid; 2,4-D)-induced transcriptomes were compared. RNA-seq results revealed the similarities of the TSA- and auxin-induced transcriptomic responses that involve extensive deregulation, mostly repression, of the majority of genes. Within the differentially expressed genes (DEGs), we identified the master regulators (transcription factors - TFs) of SE, genes involved in biosynthesis, signaling, and polar transport of auxin and NITRILASE-encoding genes of the function in indole-3-acetic acid (IAA) biosynthesis. TSA-upregulated TF genes of essential functions in auxin-induced SE, included LEC1/LEC2, FUS3, AGL15, MYB118, PHB, PHV, PLTs, and WUS/WOXs. The TSA-induced transcriptome revealed also extensive upregulation of stress-related genes, including those related to stress hormone biosynthesis. In line with transcriptomic data, TSA-induced explants accumulated salicylic acid (SA) and abscisic acid (ABA), suggesting the role of histone acetylation (Hac) in regulating stress hormone-related responses during SE induction. Since mostly the adaxial side of cotyledon explant contributes to SE induction, we also identified organ polarity-related genes responding to TSA treatment, including AIL7/PLT7, RGE1, LBD18, 40, HB32, CBF1, and ULT2. Analysis of the relevant mutants supported the role of polarity-related genes in SE induction. CONCLUSION: The study results provide a step forward in deciphering the epigenetic network controlling embryogenic transition in somatic cells of plants.

Immunomodulatory in vitro effects of Trichinella cystatin-like protein on mouse splenocytes.

Trichinella parasites have developed specific mechanisms allowing successful completion of their life cycle. These mechanisms are in a great part involved in immunomodulation and studying them may provide a valuable insight into the functioning of the immune system. Trichinella products may be also used as potential therapeutic agents to treat immune diseases. This study investigates the immunomodulatory potential of recombinant multi cystatin-like protein (CLP) derived from T. britovi to determine whether CLP has anti-inflammatory properties in vitro. CLP is a highly antigenic glycoprotein present in Trichinella excetory-secretory (ES) products. AlphaFold structure prediction confirms that it consists of three type-two cystatin-like domains. Mouse splenocytes were stimulated in vitro with lipopolysaccharide (LPS) and co-stimulated with recombinant CLP. The culture supernatants were collected and tested for secreted cytokine levels using ELISA. CLP was found to reduce LPS-induced secretion of inflammatory cytokines TNFα and IL-6. On the contrary, in some experimental groups, co-stimulation with CLP resulted in increased secretion of the regulatory cytokine IL-10. The obtained results indicate that CLP has anti-inflammatory properties and future research on its function is advisable, specifically in the context of the therapy of inflammatory disorders.

Sex-the most underappreciated variable in research: insights from helminth-infected hosts.

The sex of a host affects the intensity, prevalence, and severity of helminth infection. In many cases, one sex has been found to be more susceptible than the other, with the prevalence and intensity of helminth infections being generally higher among male than female hosts; however, many exceptions exist. This observed sex bias in parasitism results primarily from ecological, behavioural, and physiological differences between males and females. Complex interactions between these influences modulate the risk of infection. Indeed, an interplay among sex hormones, sex chromosomes, the microbiome and the immune system significantly contributes to the generation of sex bias among helminth-infected hosts. However, sex hormones not only can modulate the course of infection but also can be exploited by the parasites, and helminths appear to have developed molecules and pathways for this purpose. Furthermore, host sex may influence the efficacy of anti-helminth vaccines; however, although little data exist regarding this sex-dependent efficacy, host sex is known to influence the response to vaccines. Despite its importance, host sex is frequently overlooked in parasitological studies. This review focuses on the key contributors to sex bias in the case of helminth infection. The precise nature of the mechanisms/factors determining these sex-specific differences generally remains largely unknown, and this represents an obstacle in the development of control methods. There is an urgent need to identify any protective elements that could be targeted in future therapies to provide optimal disease management with regard to host sex. Hence, more research is needed into the impact of host sex on immunity and protection.

Generation of a single-chain variable fragment phage display antibody library from naïve mice panned against Fasciola hepatica antigens.

Monoclonal antibodies have a wide range of applications in basic and applied research as well as in the medical and pharmaceutical industries. Phage display antibody libraries offer an alternative to hybridoma technology for the generation of monoclonal antibodies and can be applied to high-throughput screening and facilitate the generation of novel antibodies. Despite their utility in several fields of research there has been limited application of antibody libraries in the study of trematode parasites. Fasciola hepatica causes considerable loss to the agriculture sector and is also a human pathogen. The parasite's excretory/secretory material contains numerous molecules that facilitate its invasion and survival within the mammalian host, including cathepsin B and L proteases. F. hepatica cathepsin B2 is expressed during the initial weeks of infection and has suspected roles in immune evasion and as a digestive enzyme in the parasite's gut; it is considered a good target for vaccination or therapeutic inhibitors. In this study, we produced a single-chain variable fragment (scFv) phage display library from naïve mice. The library was used to identify several scFv that can bind to antigens from adult F. hepatica homogenate, and a scFv that can bind to F. hepatica cathepsin B2. The results highlight the potential applicability of such a library to facilitate the study of F. hepatica and other parasites. This is the first report of the application of a naïve phage display antibody library to the study of F. hepatica.

Construction of a novel phage display antibody library against Fasciola hepatica, and generation of a single-chain variable fragment specific for F. hepatica cathepsin L1.

Phage display technology to produce recombinant monoclonal antibodies or antibody fragments permits the identification of sought after antibodies in short time frames at low cost along with direct and rapid selection for antibody characteristics. Monoclonal antibodies can facilitate the identification and characterisation of parasite molecules that function at the host-parasite interface to help understand at the molecular level the biology of the parasite and disease progression, which often leads to new drug targets, diagnostic antigens or vaccine candidates. The trematode Fasciola hepatica is an important veterinary and human parasite. In this work, we infected rats with F. hepatica and amplified the generated antibody repertoire to produce a single-chain variable fragment (scFv) phage display library. The library was used to identify a scFv that recognises cathepsin L1, a major component of the adult parasites excretory/secretory material and an important vaccine candidate. This is the first report of the construction of a phage display antibody library from a F. hepatica infected host, and also the first instance such a library has been used to identify an affinity-matured monoclonal antibody fragment that binds to a F. hepatica antigen. The scFv library and methods detailed should facilitate future research characterising F. hepatica antigens.

[Exosomal microRNAs as a part of the cell-cell communication in cancer]. / Egzosomalne mikroRNA jako element komunikacji miedzykomórkowej w nowotworach.

Exosomes are small membrane vesicles released by several types of cells into the extracellular matrix. They contain both, proteins and nucleic acids, including DNA fragments, mRNAs, microRNAs, and other non-coding RNAs, that can be transported to the recipient cells. They are one of the key elements of intercellular communication that occurs in the tumor microenvironment. Recently studies have shown that exosomal microRNAs are involved in the regulation of cell migration and invasiveness, angiogenesis, metastasis, and the modulation of immune response against cancer. Moreover, exosomal microRNAs could be also potential cancer biomarkers. This review summarizes the current knowledge about biogenesis of exosomal microRNAs and their role in the tumorigenesis.

The improvement of the in vitro plant regeneration in barley with the epigenetic modifier of histone acetylation, trichostatin A.

Genotype-limited plant regeneration is one of the main obstacles to the broader use of genetic transformation in barley breeding. Thus, developing new approaches that might improve responses of in vitro recalcitrant genotypes remains at the center of barley biotechnology. Here, we analyzed different barley genotypes, including "Golden Promise," a genotype commonly used in the genetic transformation, and four malting barley cultivars of poor regenerative potential. The expression of hormone-related transcription factor (TF) genes with documented roles in plant regeneration was analyzed in genotypes with various plant-regenerating capacities. The results indicated differential expression of auxin-related TF genes between the barley genotypes in both the explants and the derived cultures. In support of the role of auxin in barley regeneration, distinct differences in the accumulation of free and oxidized auxin were observed in explants and explant-derived callus cultures of barley genotypes. Following the assumption that modifying gene expression might improve plant regeneration in barley, we treated the barley explants with trichostatin A (TSA), which affects histone acetylation. The effects of TSA were genotype-dependent as TSA treatment improved plant regeneration in two barley cultivars. TSA-induced changes in plant regeneration were associated with the increased expression of auxin biosynthesis-involved TFs. The study demonstrated that explant treatment with chromatin modifiers such as TSA might provide a new and effective epigenetic approach to improving plant regeneration in recalcitrant barley genotypes.

Microarray analysis of rat immune responses to liver fluke infection following vaccination with Fasciola hepatica phosphoglycerate kinase.

Fasciolosis is a considerable veterinary problem, causing significant economic losses to livestock production and the food industry. Research in the area of Fasciola hepatica infection immunology is necessary to improve our knowledge about immunological mechanism evoked by the parasite and to develop new control strategies against liver fluke. In this present paper we analyzed the expression levels of cytokines in rats infected with F. hepatica following immunization with F. hepatica phosphoglycerate kinase - a novel vaccine antigen. Immune response analysis using microarray was undertaken six weeks after infection. Expression levels of INF-γ and IL-4, which are characteristic cytokines secreted during Th1-like and Th2-like immune responses, respectively, were unchanged in vaccinated animals as compared to control animals. This indicates the vaccine did not influence the major modulation of immune responses typically observed during Fasciola infections, however, other subtle but significant variations were observed that indicated altered inflammatory and possibly T helper cell responses. A significant rise in IL-12α chain expression levels was observed. Expression levels of TNF-α and some related molecules, such as ADAM17, FasL, CD40 and TRAF3 were also elevated. Expression levels of molecules involved in IL-1 signaling pathways were reduced, although a rise in IL-1α expression was noted.

Immune Response of the Host and Vaccine Development.

Vaccines are one of the greatest achievements of modern medicine, offering an effective way to fight and control infectious diseases [...].

Effect of Fasciola hepatica proteins on the functioning of rat hepatocytes.

Fasciolosis is a hepatic parasitic infection that affects many mammal species and creates a great economic and veterinary problem. Molecular mechanisms of parasite-hepatocyte interactions have not been precisely characterized yet. Therefore, the aim of the study was to investigate alterations in the metabolic activity of rat liver cells exposed to Fasciola hepatica somatic proteins. Hepatocytes were incubated with 0-1 mg/ml of fluke's somatic proteins for various periods of time. Afterward, changes in hepatocytes metabolic activity were determined with MTT and enzyme leakage tests. Hepatocytes' capacity to synthesize albumin was also investigated. It was observed that protein concentration, as well as longevity of their action, influenced metabolic activity of rat liver cells. Diminution of hepatocytes survival rate, an increase in enzyme leakage and altered synthetic capacity after treatment with parasite's proteins were reported. It is concluded that somatic proteins of F. hepatica may play an important role in liver cell damaging.

The failure of a DNA prime/protein boost regime and CTLA-4 mediated targeting to improve the potency of a DNA vaccine encoding Fasciola hepatica phosphoglycerate kinase in sheep.

DNA vaccination in large animals has often been associated with poor immunogenicity, consequently several approaches have been evaluated to enhance its efficacy. Here, we tested a cDNA encoding a phosphoglycerate kinase from Fasciola hepatica (cDNA-FhPGK/pCMV) as a vaccine against ovine fasciolosis and investigated whether a DNA prime/protein boost regime or CTLA-4 (cytotoxic lymphocyte antigen 4) mediated targeting improved DNA vaccine efficacy. No statistically significant differences in the cellular responses were seen in either vaccine trial when compared with the respective control groups. However, specific antibody responses were considerably enhanced in DNA primed/protein boosted sheep, but not among CTLA-4 targeted cDNA-FhPGK/pCMV vaccinated animals. Nevertheless, increased titers of specific IgG1 did not contribute to protection against infection, with no differences in liver fluke recoveries reported. If DNA vaccines against fasciolosis in target species are to reach the market one day, more research in this area is needed.

Vaccination against Fasciola hepatica using cathepsin L3 and B3 proteases delivered alone or in combination.

No licensed vaccine is currently available for prevention of Fasciola hepatica infections. However, considering the alarming increase in drug resistance, there is an urgent need for a safe and fully effective vaccine against fasciolosis. Here, we tested if cathepsins L (FhCL3-1, FhCL3-2) and B (FhCB3) secreted by juvenile liver flukes are viable vaccine targets when delivered alone or in combination in a rat model. Since control over the early immune response is crucial for parasite's establishment in its host, it was hypothesised that targeting fluke juvenile stages may prove beneficial. Moreover, it was assumed that selected antigens will act in a cumulative manner to interfere with liver fluke migration and thereby will reduce F. hepatica infection. Recombinant FhCL3-1 and FhCL3-2 delivered alone reduced liver fluke burdens by 47 % and 63 %, respectively. A trivalent vaccine containing rFhCL3-1/CL3-2/CB3 did not increase the protective vaccine efficacy compared to the rFhCL3-2 vaccinated group (53 %), although, reductions in liver fluke wet weight (statistically significant) and liver damage score were most pronounced. Further, the highest IgG1 and IgG2a levels were seen in rFhCL3-2 vaccinated rats, the group for which the highest reduction in worm burden was demonstrated. Moreover, IgG1 and IgG2a levels in vaccinated rats were significantly elevated compared to those reported for control groups up to 4 week post-infection. While the mechanism of protection remains unknown, it appears that it depends on vaccine-induced antibodies directed against cathepsins. The obtained results imply that F. hepatica juvenile-specific cathepsins are promising vaccine candidates that induce responses that successfully target early migratory liver fluke stages. Now, the challenge is to evaluate these juvenile-specific cathepsins for use in livestock.

Intranasal delivery of a formulation containing stage-specific recombinant proteins of Fasciola hepatica cathepsin L5 and cathepsin B2 triggers an anti-fecundity effect and an adjuvant-mediated reduction in fluke burden in sheep.

Fasciola hepatica infection continues to be a major problem in the agriculture sector, particularly in sheep and cattle. Cathepsin L and B proteases are major components of the excretory/secretory material of the parasite, and their roles in several important aspects of parasite invasion and survival has led to their use as targets in rational vaccine design. Previous studies in rats demonstrated that the use of stage-specific antigens, cathepsin B2 and cathepsin L5, as part of a multivalent vaccine, was able to confer significant protection against challenge. In the present study, recombinant versions of cathepsin L5 and cathepsin B2 produced in yeast were used in combination to vaccinate sheep. Intramuscular and intranasal forms of administration were applied, and sheep were subsequently challenged with 150 F. hepatica metacercariae. Intramuscular vaccination was able to induce a strong systemic antibody response against both antigens, but failed to confer significant protection. Conversely, no elevated antibody response was detected against the vaccine antigens following nasal vaccination; however, a reduction in parasite egg viability (>92%) and a statistically significant (p = 0.006), predominantly adjuvant-mediated reduction in worm burdens was observed.

Lymphocyte responses of rats vaccinated with cDNA encoding a phosphoglycerate kinase of Fasciola hepatica (FhPGK) and F. hepatica infection.

Lymphocyte responses in the blood, peritoneal fluid and both mesenteric and hepatic lymph nodes of cDNA-FhPGK/pCMV vaccinated and/or Fasciola hepatica infected rats of both sexes were investigated to provide an insight into the immune responses that develop in different body compartments. The immune response that developed in cDNA-FhPGK/pCMV vaccinated females contributed to partial protection against F. hepatica infection (54% reduction in fluke recovery), while more liver flukes were found in the livers and bile ducts of cDNA-FhPGK/pCMV vaccinated male rats than in unvaccinated animals (increase of 13%). Rat sex not only affected the ultimate effectiveness of vaccination but also lymphocyte responses following vaccination and/or infection. Different CD4+ and CD8+ T cell profiles were noted in peritoneal fluid and lymph nodes, but not in blood, during acute and chronic fasciolosis. Moreover, independent lymphocyte responses developed in distinct body compartments. Immune responses of rats were polarized towards Th2/Treg with lymphocytes isolated from male rats showing higher IL-4 and IL-10 production than females. Lymphocyte proliferative capacities in response to mitogen (PHA) or vaccine antigen (FhPGK) were impaired in both sexes with a considerably higher reduction observed for males and restored lymphocyte proliferative capacities reported for females vaccinated with cDNA-FhPGK/pCMV during chronic fasciolosis.

Sex and vaccination: Insights from female rats vaccinated with juvenile-specific proteases from Fasciola hepatica.

Most animal research is less evidence-based for females, with the majority of studies conducted on males. Since immune responses vary between males and females, sexual dimorphism in immunity contributes, among other things, to sex-based differences post-vaccination. However, the issue of sex effects in animal vaccine research is rarely considered in vaccine study design. Previously, we have evaluated the efficacy of cathepsin L3 (FhCL3-1 and FhCL3-2) and B3 proteases (FhCB3) from juvenile Fasciola hepatica as vaccines against fasciolosis in male rats. Their administration resulted in reductions in liver fluke recovery in the range of 47-63% when compared with an infection control group. Here, we investigated if the protective effect of vaccination with these proteins can also be observed for female rats. The data indicates females were not protected from F. hepatica infection when vaccinated with juvenile cathepsins. Only in the FhCL3-2 vaccinated group was a low, non-significant, reduction in worm burden observed (21%). Although liver fluke mean body lengths and wet weights were reduced in vaccinated animals when compared with the infection controls, these effects were adjuvant- not vaccine-induced, while for males changes in these parameters were related primarily to vaccination. Specific humoral responses throughout the study were evident; however, trends in antibody responses in females replicated trends observed previously for male humoral responses. Formerly, elevated levels of FhCL3-1 and FhCL3-2 specific IgG1 and IgG2a were suggested to be correlated with protection. Here, despite increased and clear responses of these antibodies, protection was not observed. Hence, in the present study the roles of IgG1 and IgG2 in liver fluke reduction are questionable. Results demonstrated in our study show that observations obtained in one sex are not always applicable to the other sex. Hopefully, the findings of the study will stimulate discussion of the issue of sex impacts on post-vaccination outcomes and will encourage researchers to consider sex in their future vaccine studies.

A Preliminary Study of a Lettuce-Based Edible Vaccine Expressing the Cysteine Proteinase of Fasciola hepatica for Fasciolosis Control in Livestock.

Oral vaccination with edible vaccines is one of the most promising approaches in modern vaccinology. Edible vaccines are an alternative to conventional vaccines, which are typically delivered by injection. Here, freeze-dried transgenic lettuce expressing the cysteine proteinase of the trematode Fasciola hepatica (CPFhW) was used to orally vaccinate cattle and sheep against fasciolosis, which is the most important trematode disease due to the parasite's global distribution, wide spectrum of host species and significant economic losses of farmers. In the study, goals such as reducing the intensity of infection, liver damage and F. hepatica fecundity were achieved. Moreover, we demonstrated that the host sex influenced the outcome of infection following vaccination, with female calves and male lambs showing better protection than their counterparts. Since differences occurred following vaccination and infection, different immunization strategies should be considered for different sexes and host species when developing new control methods. The results of the present study highlight the potential of oral vaccination with plant-made and plant-delivered vaccines for F. hepatica infection control.

Immune responses in rats and sheep induced by a DNA vaccine containing the phosphoglycerate kinase gene of Fasciola hepatica and liver fluke infection.

Immune responses of rats and sheep following vaccination with cDNA encoding phosphoglycerate kinase of Fasciola hepatica (cDNA-FhPGK/pCMV) and F. hepatica infection were investigated in the present study. cDNA-FhPGK/pCMV vaccinated female Sprague-Dawley rats were better protected by vaccination than their male counterparts - 48% reduction in fluke burden for females and no protection for males when compared with appropriate infection control groups. Moreover, male rats developed marked leukocytosis during the study with higher neutrophil, eosinophil and monocyte responses than females. Additionally, dynamics of eosinophil and monocyte responses varied between sexes. Increased titres of anti-FhPGK IgG1 and IgG2a correlated with the protective effect of vaccination that was observed among female rats. In the case of male sheep, no differences in worm burdens and in the course of the immune response were observed following vaccination. Titres of specific antibodies detected were low, and cellular responses were not significant. Apparently, sheep immune responses induced by cDNA-FhPGK/pCMV vaccination are not effective at controlling F. hepatica infection. Poor immunogenicity of DNA vaccines in large animals is still a major obstacle of this technology that has to be overcome.

Variations in cercarial production and the level of in vitro activation of metacercariae of two different isolates of Fasciola hepatica.

Fasciola hepatica infections cause large economic losses and are a serious veterinary medicine problem in many regions of the world. Recent studies examining fascioliasis in the bison population from Bialowieza National Park have shown that the prevalence of infection with this parasite is up to 100%. Liver flukes isolated from bison from Bialowieza National Park in Poland were compared with a fluke strain originally obtained from the Central Veterinary Laboratory, Weybridge, UK, to determine variations in cercarial production and establish the ability of their metacercariae to activate in vitro. Some small differences in cercarial production between the two isolates are shown, while significant differences in the ability of their metacercariae to activate in vitro were observed.

Urinary creatinine to serum creatinine ratio and renal failure index in dogs infected with Babesia canis.

Urinary creatinine to serum creatinine (UCr/SCr) ratio and renal failure index (RFI) are useful indices of renal damage. Both UCr/SCr ratio and RFI are used in differentiation between prerenal azotaemia and acute tubular necrosis. In this work the authors calculated the UCr/SCr ratio and RFI in dogs infected with Babesia canis and the values of these indices in azotaemic dogs infected with the parasite. The results of this study showed significantly lower UCr/SCr ratio in dogs infected with B. canis than in healthy dogs. Moreover, in azotaemic dogs infected with B. canis the UCr/SCr ratio was significantly lower and the RFI was significantly higher than in non-azotaemic dogs infected with B. canis. The calculated correlation between RFI and duration of the disease before diagnosis and treatment was high, positive and statistically significant (r = 0.89, p < 0.001). The results of this study showed that during the course of canine babesiosis caused by B. canis in Poland acute tubular necrosis may develop.

Evaluation of the immune response of male and female rats vaccinated with cDNA encoding a cysteine proteinase of Fasciola hepatica (FhPcW1).

Not only do males and females of many species vary in their responses to certain parasitic infections, but also to treatments such as vaccines. However, there are very few studies investigating differences among sexes following vaccination and infection. Here we demonstrate that female Sprague-Dawley rats vaccinated with cDNA encoding a recently discovered cysteine proteinase of Fasciola hepatica (FhPcW1) develop considerably lower liver fluke burdens after F. hepatica infection than their male counterparts. This is accompanied by differences in the course of their immune responses which involve different eosinophil and monocyte responses throughout the study as well as humoral responses. It is evident that host gender influences the outcome of parasitic infections after vaccination and research on both sexes should be considered when developing new treatments against parasites.