Analysis of the role of Dirofilaria repens macrophage migration inhibitory factors in host-parasite interactions.

Introduction: Dirofilaria repens is a zoonotic parasitic filarial nematode that infects carnivores and occasionally humans. Knowledge of the host-parasite molecular interactions enabling the parasite's avoidance of the host immune response in subcutaneous dirofilariasis remains limited. Parasitic orthologues of host macrophage migration inhibitory factor (MIF) are molecules potentially involved in this process. Material and Methods: Complementary DNA encoding two D. repens MIF orthologues (rDre-MIF-1 and rDre-MIF-2) was cloned into a pET-28a expression vector. The recombinant proteins were produced in Escherichia coli and purified using affinity nickel chromatography. The reactivity of both recombinant proteins was analysed with infected dog and immunised mouse sera. Results: Stronger antibody production was induced by rDre-MIF-1 in mice, as evidenced by significantly higher levels of anti-rDre-MIF-1 total IgG, IgG2 and IgE antibodies than of anti-rDre-MIF-2 immunoglobulins. Additionally, a significantly different level of antibodies specific to both proteins was noted between the sera of infected dogs and those of uninfected dogs. Conclusion: This study is the first attempt to characterise MIF orthologues from the filarial parasite D. repens, which may affect the immune response during infection.

Human Adenovirus Entry and Early Events during Infection of Primary Murine Neurons: Immunofluorescence Studies In Vitro.

Human adenovirus (HAdV) is a common pathogen, which can lead to various clinical symptoms and-in some cases-central nervous system (CNS) dysfunctions, such as encephalitis and meningitis. Although the initial events of virus entry have already been identified in various cell types, the mechanism of neuronal uptake of adenoviruses is relatively little understood. The aim of this study was to investigate early events during adenoviral infection, in particular to determine the connection between cellular coxsackievirus and adenovirus receptor (CAR), clathrin, caveolin, and early endosomal proteins (EEA1 and Rab5) with the entry of HAdVs into primary murine neurons in vitro. An immunofluorescence assay and confocal microscopy analysis were carried out to determine HAdV4, 5, and 7 correlation with CAR, clathrin, caveolin, and early endosomal proteins in neurons. The quantification of Pearson's coefficient between CAR and HAdVs indicated that the HAdV4 and HAdV5 types correlated with CAR and that the correlation was more substantial for HAdV5. Inhibition of clathrin-mediated endocytosis using chlorpromazine limited the infection with HAdV, whereas inhibition of caveolin-mediated endocytosis did not affect virus entry. Thus, the entry of tested HAdV types into neurons was most likely associated with clathrin but not caveolin. It was also demonstrated that HAdVs correlate with the Rab proteins (EEA1, Rab5) present in early vesicles, and the observed differences in the manner of correlation depended on the serotype of the virus. With our research, we strove to expand knowledge regarding the mechanism of HAdV entry into neurons, which may be beneficial for developing potential therapeutics in the future.

Neurons cytoskeletal architecture remodeling during the replication cycle of mouse coronavirus MHV-JHM: a morphological in vitro study.

Nowadays, the population is still struggling with a post-COVID19 syndrome known as long COVID, including a broad spectrum of neurological problems. There is an urgent need for a better understanding and exploration of the mechanisms of coronavirus neurotropism. For this purpose, the neurotropic strain of mouse hepatitis virus (MHV-JHM) originating from the beta-coronavirus genus, the same as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been used. The role of the cytoskeleton during virus replication in neurons in vitro was determined to understand the mechanisms of MHV-JHM neuroinfection. We have described for the first time the changes of actin filaments during MHV-JHM infection. We also observed productive replication of MHV-JHM in neurons during 168 h p.i. and syncytial cytopathic effect. We discovered that the MHV-JHM strain modulated neuronal cytoskeleton during infection, which were manifested by: (i) condensation of actin filaments in the cortical layer of the cytoplasm, (ii) formation of microtubule cisternae structures containing viral antigen targeting viral replication site (iii) formation of tunneling nanotubes used by MHV-JHM for intercellular transport. Additionally, we demonstrated that the use of cytoskeletal inhibitors have reduced virus replication in neurons, especially noscapine and nocodazole, the microtubule shortening factors.

Identification of intestinal parasites in wild American mink (Neovison vison) from Biebrza and Narew national parks (Poland).

American mink (Neovison vison) is an invasive species in the sylvatic environment of Poland. Mink are exposed to different parasite infections as their preys serve as intermediate and/or paratenic hosts. The study aimed to discriminate the pattern of intestinal parasite infections in mink inhabiting Biebrza (BNP) and Narew (NNP) national parks. Gastrointestinal tract examinations revealed Coccidia, Echinostomatidae, Taenidae, and Capillariidae parasites. There was no significant difference in the parasite burden of mink, but patterns of infections varied between both localizations. Coccidia were found in 3.8% of BNP vs. 6.7% of NNP mink. Fluke prevalence was significantly higher in NNP 27.5% compared to 7.7% in BNP mink. Tapeworms were only found in 3.4% of NNP mink. Significantly more Aonchotheca eggs were found in BNP 34.6% vs. 11.4% in NNP mink. The intensity of coccidiosis and aonchothecosis was low in both parks. Fluke intensity varied between low to moderate (ranging from 1 to 16) in BNP and low to massive (ranging from 1 to 117) in NNP mink. Coinfections of various parasite species were noted in both areas. Morphological and DNA analysis revealed that flukes belonged to Isthiomorpha melis and tapeworms to Versteria mustelae. It was the first isolation of V. mustelae in mink of those localizations. In conclusion, our study showed that mink indwelling Biebrza and Narew national parks are moderately infested with parasites. Results suggest that mink play an important role as a reservoir for parasites endangering endemic mustelids, becoming also a potential risk factor in case of accidental transmissions to farm mink. That is why, more strict biosecurity measures are required to protect farm mink.

The Roles of Various Immune Cell Populations in Immune Response against Helminths.

Helminths are multicellular parasites that are a substantial problem for both human and veterinary medicine. According to estimates, 1.5 billion people suffer from their infection, resulting in decreased life quality and burdens for healthcare systems. On the other hand, these infections may alleviate autoimmune diseases and allergy symptoms. The immune system is programmed to combat infections; nevertheless, its effector mechanisms may result in immunopathologies and exacerbate clinical symptoms. This review summarizes the role of the immune response against worms, with an emphasis on the Th2 response, which is a hallmark of helminth infections. We characterize non-immune cells (enteric tuft cells-ETCs) responsible for detecting parasites, as well as the role of hematopoietic-derived cells (macrophages, basophils, eosinophils, neutrophils, innate lymphoid cells group 2-ILC2s, mast cells, T cells, and B cells) in initiating and sustaining the immune response, as well as the functions they play in granulomas. The aim of this paper is to review the existing knowledge regarding the immune response against helminths, to attempt to decipher the interactions between cells engaged in the response, and to indicate the gaps in the current knowledge.

The Role of Nuclear Factor Kappa B (NF-κB) in the Immune Response against Parasites.

The immune system consists of various cells, organs, and processes that interact in a sophisticated manner to defend against pathogens. Upon initial exposure to an invader, nonspecific mechanisms are raised through the activation of macrophages, monocytes, basophils, mast cells, eosinophils, innate lymphoid cells, or natural killer cells. During the course of an infection, more specific responses develop (adaptive immune responses) whose hallmarks include the expansion of B and T cells that specifically recognize foreign antigens. Cell to cell communication takes place through physical interactions as well as through the release of mediators (cytokines, chemokines) that modify cell activity and control and regulate the immune response. One regulator of cell states is the transcription factor Nuclear Factor kappa B (NF-κB) which mediates responses to various stimuli and is involved in a variety of processes (cell cycle, development, apoptosis, carcinogenesis, innate and adaptive immune responses). It consists of two protein classes with NF-κB1 (p105/50) and NF-κB2 (p100/52) belonging to class I, and RelA (p65), RelB and c-Rel belonging to class II. The active transcription factor consists of a dimer, usually comprised of both class I and class II proteins conjugated to Inhibitor of κB (IκB). Through various stimuli, IκB is phosphorylated and detached, allowing dimer migration to the nucleus and binding of DNA. NF-κB is crucial in regulating the immune response and maintaining a balance between suppression, effective response, and immunopathologies. Parasites are a diverse group of organisms comprised of three major groups: protozoa, helminths, and ectoparasites. Each group induces distinct effector immune mechanisms and is susceptible to different types of immune responses (Th1, Th2, Th17). This review describes the role of NF-κB and its activity during parasite infections and its contribution to inducing protective responses or immunopathologies.

Selection of new diagnostic markers for Dirofilaria repens infections with the use of phage display technology.

Dirofilaria repens is a parasitic nematode causing vector-borne disease (dirofilariasis), considered an emerging problem in veterinary and human medicine. Although main hosts are carnivores, particularly dogs, D. repens shows high zoonotic potential. The disease spreads uncontrollably, affecting new areas. Since there is no vaccine against dirofilariasis, the only way to limit disease transmission is an early diagnosis. Currently, diagnosis depends on the detection of microfilariae in the host bloodstream using modified Knott's test or multiplex PCR. However, the efficacy of tests relying on microfilariae detection is limited by microfilariae periodic occurrence. Therefore, a new reliable diagnostic test is required. Our study aimed to select new diagnostic markers for dirofilariasis with potential application in diagnostics. We focused on single epitopes to ensure high specificity of diagnosis and avoid cross-reactivity with the other parasite infections common in dogs. Using phage display technology and 12-mer peptides library, we selected epitopes highly reactive with IgG from sera of infected dogs. Additionally, our study presents the possibility of detecting D. repens specific cell-free DNA in dogs with no microfilaria but high IgG and IgM antibody levels against parasite somatic antigen.

Nematode Orthologs of Macrophage Migration Inhibitory Factor (MIF) as Modulators of the Host Immune Response and Potential Therapeutic Targets.

One of the adaptations of nematodes, which allows long-term survival in the host, is the production of proteins with immunomodulatory properties. The parasites secrete numerous homologs of human immune mediators, such as macrophage migration inhibitory factor (MIF), which is a substantial regulator of the inflammatory immune response. Homologs of mammalian MIF have been recognized in many species of nematode parasites, but their role has not been fully understood. The application of molecular biology and genetic engineering methods, including the production of recombinant proteins, has enabled better characterization of their structure and properties. This review provides insight into the current state of knowledge on MIF homologs produced by nematodes, as well as their structure, enzymatic activity, tissue expression pattern, impact on the host immune system, and potential use in the treatment of parasitic, inflammatory, and autoimmune diseases.

The Role of the Intestinal Epithelium in the "Weep and Sweep" Response during Gastro-Intestinal Helminth Infections.

Helminths are metazoan parasites infecting around 1.5 billion people all over the world. During coevolution with hosts, worms have developed numerous ways to trick and evade the host immune response, and because of their size, they cannot be internalized and killed by immune cells in the same way as bacteria or viruses. During infection, a substantial Th2 component to the immune response is evoked which helps restrain Th1-mediated tissue damage. Although an enhanced Th2 response is often not enough to kill the parasite and terminate an infection in itself, when tightly coordinated with the nervous, endocrine, and motor systems it can dislodge parasites from tissues and expel them from the gut. A significant role in this "weep and seep" response is attributed to intestinal epithelial cells (IEC). This review highlights the role of various IEC lineages (enterocytes, tuft cells, Paneth cells, microfold cells, goblet cells, and intestine stem cells) during the course of helminth infections and summarizes their roles in regulating gut architecture and permeability, and muscle contractions and interactions with the immune and nervous system.

Identification of Toxocara canis Antigen-Interacting Partners by Yeast Two-Hybrid Assay and a Putative Mechanism of These Host-Parasite Interactions.

Toxocara canis is a zoonotic roundworm that infects humans and dogs all over the world. Upon infection, larvae migrate to various tissues leading to different clinical syndromes. The host-parasite interactions underlying the process of infection remain poorly understood. Here, we describe the application of a yeast two-hybrid assay to screen a human cDNA library and analyse the interactome of T. canis larval molecules. Our data identifies 16 human proteins that putatively interact with the parasite. These molecules were associated with major biological processes, such as protein processing, transport, cellular component organisation, immune response and cell signalling. Some of these identified interactions are associated with the development of a Th2 response, neutrophil activity and signalling in immune cells. Other interactions may be linked to neurodegenerative processes observed during neurotoxocariasis, and some are associated with lung pathology found in infected hosts. Our results should open new areas of research and provide further data to enable a better understanding of this complex and underestimated disease.

Human herpesvirus type 2 infection of primary murine astrocytes causes disruption of the mitochondrial network and remodeling of the actin cytoskeleton: an in vitro morphological study.

Herpesviruses are capable of infecting not only neurons, where they establish latent infection, but also astrocytes. Since astrocytes are important for the functioning of the central nervous system (CNS), their infection may lead to serious neurological disorders. Thus, in the present study we investigated the ability of human herpesvirus type 2 (HHV-2) to infect primary murine astrocytes in vitro and the effect of infection on their mitochondrial network and actin cytoskeleton. In immunofluorescence assays, antibodies against HHV-2 antigens and glial fibrillary acidic protein (GFAP) were used to confirm that the infected cells are indeed astrocytes. Real-time PCR analysis showed a high level of HHV-2 replication in astrocytes, particularly at 168 h postinfection, confirming that a productive infection had occurred. Analysis of mitochondrial morphology showed that, starting from the first stage of infection, HHV-2 caused fragmentation of the mitochondrial network and formation of punctate and tubular structures that colocalized with virus particles. Furthermore, during the late stages of infection, the infection affected the actin cytoskeleton and induced formation of actin-based cellular projections, which were probably associated with enhanced intracellular spread of the virus. These results suggest that the observed changes in the mitochondrial network and actin cytoskeleton in productively infected astrocytes are required for effective replication and viral spread in a primary culture of astrocytes. Moreover, we speculate that, in response to injury such as HHV-2 infection, murine astrocytes cultured in vitro undergo transformation, defined in vivo as reactive astrocytosis.

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.

Anti-Inflammatory State in Arabian Horses Introduced to the Endurance Training.

Development of an anti-inflammatory state during physical training has been postulated in both human and equine athletes, but it is not completely understood. The aim of this study was to investigate whether endurance training changes pro- and anti-inflammatory cytokine profiles within a 20-week training season in young inexperienced endurance horses. Nine Arabian horses were examined in this prospective 20-week follow-up study. Blood samples were analysed 5 times monthly, at rest and after training sessions. Routine haematological examinations were performed. Cytokine patterns including IL-1ß, IL-6, TNF-α, IL-10 mRNA expression using Real Time-PCR, and serum concentrations of IL-1ß, IL-2, IL-4, IL-6, IL-17, INFγ, TNF-α, and IL-10 by ELISA test were determined. During endurance training, the most significant decrease in post-exercise cytokine type 1 levels (TNFα and IL-ß) occurred within 20 weeks, beginning from the 3rd month of training. IL-6 serum level decreased after the 4th month. The results suggest that endurance training can induce advanced overall anti-inflammatory response as an adaptation to increasing workload.

Changes in Serum Amyloid A (SAA) Concentration in Arabian Endurance Horses During First Training Season.

Sport training leads to adaptation to physical effort that is reflected by the changes in blood parameters. In equine endurance athletes, blood testing is accepted as a support in training, however, only the changes before versus after exercise in creatine phosphokinase activity (CPK) and basic blood parameters are usually measured. This study is the first longitudinal investigation of the changes in routinely measured blood parameters and, additionally, serum amyloid A (SAA), during seven months, in Arabian horses introduced to endurance training and competing in events for young horses. It has been determined that CPK, aspartate aminotransferase (AST), packed cell volume (PCV), hemoglobin concentration, red blood cell count (RBC), and concentration of total serum protein (TSP) slightly increased after training sessions and competitions in similar manner. The increase in white blood cell (WBC) count was higher after competitions and SAA increased only after competitions. Total protein concentration was the only parameter that increased with training during a 7-month program. SAA indicated only in the case of heavy effort, and, it thus may be helpful in the monitoring of training in young horses. In an optimal program, its concentration should not increase after a training session but only after heavy effort, which should not be repeated too often.

Does porcine circovirus type 3 (PCV3) interfere with porcine circovirus type 2 (PCV2) vaccine efficacy?

PCV2 is globally spread pathogen involved in a number of diseases (PCVD). Commonly used vaccines against PCV2 are proved to be highly efficacious. The role of recently discovered PCV3 for pig health and interference with PCV2 remains unknown. The study performed on serum samples from seven farms vaccinated against PCV2 and four non-vaccinated showed very low prevalence of PCV2 viremia in the former (3 out of 106 positive serum pools) and high prevalence of PCV2 viremia in the latter (35 out of 60 positive pools). Mean log10 PCV2 genome equivalents were lower in vaccinated farms (4.8 ± 0.6 log10  copies/ml) than in non-vaccinated farms (6.3 ± 1.3 log10  copies/ml). PCV3 was detected in 31 out of 106 and 12 out of 60 serum pools from vaccinated and non-vaccinated farms, respectively. Mean log10 PCV3 genome equivalents were significantly (p < 0.05) lower in vaccinated farms (3.9 ± 0.8 log10  copies/ml) than in non-vaccinated farms (4.4 ± 0.6 log10  copies/ml). Concurrent PCV2 and PCV3 infection was rare and found only in 1 out of 529 and 4 out of 292 individual serum samples from vaccinated and non-vaccinated farms, respectively. Our results showed lack of impact of PCV3 circulation on PCV2 vaccine efficacy. On the other hand, intensive PCV2 circulation and high viremia detected in non-vaccinated farms did not seem to increase the level of PCV3 infection.

Fasciola Hepatica Isolates Induce Different Immune Responses in Unmaturated Bovine Macrophages.

INTRODUCTION: Fasciola hepatica (liver fluke) is a parasite of great socioeconomic importance. A number of fluke isolates have been identified; however, to date the differences between the immunomodulatory properties of different parasite isolates have not been sufficiently investigated. The aim of this study was to explore differences between the immunomodulatory properties of two F. hepatica isolates using unmaturated bovine macrophages. MATERIAL AND METHODS: A cell line of bovine macrophages was stimulated with excretory/secretory products released by adult flukes from either a laboratory (Fh-WeyES) or wild (Fh-WildES) strain and subsequently subjected to microarray and ELISA analyses. Results: Both Fh-WeyES and Fh-WildES dampened the release of interleukin-10 by bovine macrophages, but only Fh-WildES dampened the release of proinflammatory tumour necrosis factor-α. Microarray analysis revealed that Fh-WildES down- and upregulated 90 and 18 genes, respectively, when compared to Fh-WeyES. CONCLUSION: The results indicated different impacts of the isolates on macrophages. A number of researchers use flukes obtained from local slaughterhouses for experiments. Our findings may explain some discrepancies between published results arising from parasite strain choice. The findings indicate that consideration should be given to the use of different strains, and open new and currently unexplored avenues in parasitology for controlling the parasite.

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.

Human herpesvirus type 1 and type 2 disrupt mitochondrial dynamics in human keratinocytes.

Mitochondrial movement and distribution throughout the cytoplasm is crucial for maintaining cell homeostasis. Mitochondria are dynamic organelles but can be functionally disrupted during infection. Here, we show that the ubiquitous human pathogens HHV-1 and HHV-2 induce changes in the mitochondrial morphology and distribution in the early and late phases of productive infection in human keratinocytes (HaCaT cells). We observed a decrease in the mitochondrial potential at 2 h postinfection and a decrease in cell vitality at 24 h postinfection. Moreover, we found that mitochondria migrated to the perinuclear area, where HHV-1 and HHV-2 antigens were also observed, mainly in the early stages of infection. Positive results of real-time PCR showed a high level of HHV-1 and HHV-2 DNA in HaCaT cells and culture medium. Our data demonstrate that HHV-1 and HHV-2 cause mitochondrial dysfunction in human keratinocytes.

Multifunctional Tannic Acid/Silver Nanoparticle-Based Mucoadhesive Hydrogel for Improved Local Treatment of HSV Infection: In Vitro and In Vivo Studies.

Mucoadhesive gelling systems with tannic acid modified silver nanoparticles were developed for effective treatment of herpes virus infections. To increase nanoparticle residence time after local application, semi solid formulations designed from generally regarded as safe (GRAS) excipients were investigated for their rheological and mechanical properties followed with ex vivo mucoadhesive behavior to the porcine vaginal mucosa. Particular effort was made to evaluate the activity of nanoparticle-based hydrogels toward herpes simplex virus (HSV) type 1 and 2 infection in vitro in immortal human keratinocyte cell line and in vivo using murine model of HSV-2 genital infection. The effect of infectivity was determined by real time quantitative polymerase chain reaction, plaque assay, inactivation, attachment, penetration and cell-to-cell assessments. All analyzed nanoparticle-based hydrogels exhibited pseudoplastic and thixotropic properties. Viscosity and mechanical measurements of hydrogels were found to correlate with the mucoadhesive properties. The results confirmed the ability of nanoparticle-based hydrogels to affect viral attachment, impede penetration and cell-to-cell transmission, although profound differences in the activity evoked by tested preparations toward HSV-1 and HSV-2 were noted. In addition, these findings demonstrated the in vivo potential of tannic acid modified silver nanoparticle-based hydrogels for vaginal treatment of HSV-2 genital infection.