Utilization of a highly adaptable murine air pouch model for minimally invasive testing of the inflammatory potential of biomaterials.

Introduction: The biocompatibility of an implanted material strongly determines the subsequent host immune response. After insertion into the body, each medical device causes tissue reactions. How intense and long-lasting these are is defined by the material properties. The so-called foreign body reaction is a reaction leading to the inflammation and wound healing process after implantation. The constantly expanding field of implant technology and the growing areas of application make optimization and adaptation of the materials used inevitable. Methods: In this study, modified liquid silicone rubber (LSR) and two of the most commonly used thermoplastic polyurethanes (TPU) were compared in terms of induced inflammatory response in the body. We evaluated the production of inflammatory cytokines, infiltration of inflammatory cells and encapsulation of foreign bodies in a subcutaneous air-pouch model in mice. In this model, the material is applied in a minimally invasive procedure via a cannula and in one piece, which allows material testing without destroying or crushing the material and thus studying an intact implant surface. The study design includes short-term (6 h) and long-term (10 days) analysis of the host response to the implanted materials. Air-pouch-infiltrating cells were determined by flow cytometry after 6 h and 10 days. Inflammation, fibrosis and angiogenesis markers were analyzed in the capsular tissue by qPCR after 10 days. Results: The foreign body reaction was investigated by macroscopic evaluation and scanning electron microscopy (SEM). Increased leukocyte infiltration was observed in the air-pouch after 6 h, but it markedly diminished after 10 days. After 10 days, capsule formations were observed around the materials without visible inflammatory cells. Discussion: For biocompatibility testing materials are often implanted in muscle tissue. These test methods are not sufficiently conclusive, especially for materials that are intended to come into contact with blood. Our study primarily shows that the presented model is a highly adaptable and minimally invasive test system to test the inflammatory potential of and foreign body reaction to candidate materials and offers more precise analysis options by means of flow cytometry.

Suppression of the growth and metastasis of mouse melanoma by Taenia crassiceps and Mesocestoides corti tapeworms.

Cancer is still one of the leading causes of death, with an estimated 19.3 million new cases every year. Our paper presents the tumor-suppressing effect of Taenia crassiceps and Mesocestoides corti on B16F10 melanoma, the intraperitoneal application of which followed the experimental infection with these tapeworms, resulting in varying degrees of effectiveness in two strains of mice. In the case of M. corti-infected ICR mice, a strong tumor growth suppression occurred, which was accompanied by a significant reduction in the formation of distant metastases in the liver and lung. Tapeworm-infected C57BL/6J mice also showed a suppression of tumor growth and, in addition, the overall survival of infected C57BL/6J mice was significantly improved. Experiments with potential cross-reaction of melanoma and tapeworm antigens with respective specific antibodies, restimulation of spleen T cells, or the direct effect of tapeworm excretory-secretory products on melanoma cells in vitro could not explain the phenomenon. However, infections with T. crassiceps and M. corti increased the number of leukocytes possibly involved in anti-tumor immunity in the peritoneal cavity of both ICR and C57BL/6J mice. This study unveils the complex interplay between tapeworm infections, immune responses, and melanoma progression, emphasizing the need for further exploration of the mechanisms driving observed tumor-suppressive effects.

The neurotropic schistosome vs experimental autoimmune encephalomyelitis: are there any winners?

The incidences of multiple sclerosis have risen worldwide, yet neither the trigger nor efficient treatment is known. Some research is dedicated to looking for treatment by parasites, mainly by helminths. However, little is known about the effect of helminths that infect the nervous system. Therefore, we chose the neurotropic avian schistosome Trichobilharzia regenti, which strongly promotes M2 polarization and tissue repair in the central nervous system, and we tested its effect on the course of experimental autoimmune encephalomyelitis (EAE) in mice. Surprisingly, the symptoms of EAE tended to worsen after the infection with T. regenti. The infection did not stimulate tissue repair, as indicated by the similar level of demyelination. Eosinophils heavily infiltrated the infected tissue, and the microglia number increased as well. Furthermore, splenocytes from T. regenti-infected EAE mice produced more interferon (IFN)-γ than splenocytes from EAE mice after stimulation with myelin oligodendrocyte glycoprotein. Our research indicates that the combination of increased eosinophil numbers and production of IFN-γ tends to worsen the EAE symptoms. Moreover, the data highlight the importance of considering the direct effect of the parasite on the tissue, as the migrating parasite may further tissue damage and make tissue repair even more difficult.

Bsep/Abcb11 knockout ameliorates Schistosoma mansoni liver pathology by reducing parasite fecundity.

BACKGROUND AND AIMS: Schistosoma mansoni infection is one of the worldwide leading causes of liver fibrosis and portal hypertension. The objective of this study was to evaluate whether polyhydroxylated bile acids (BAs), known to protect mice from the development of acquired cholestatic liver injury, counteract S. mansoni-induced inflammation and fibrosis. METHODS: Adult FVB/N wild type (WT) and Abcb11/Bsep-/- mice were infected with either 25 or 50 S. mansoni cercariae. Eight weeks post infection, effects on liver histology, serum biochemistry, gene expression profile of proinflammatory cytokines and fibrotic markers, hepatic hydroxyproline content and FACS analysis were performed. RESULTS: Bsep-/- mice infected with S. mansoni showed significantly less hepatic inflammation and tendentially less fibrosis compared to infected WT mice. Despite elevated alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase levels in infected Bsep-/- mice, inflammatory cells such as M2 macrophages and Mac-2/galectin-3+ cells were reduced in these animals. Accordingly, mRNA-expression levels of anti-inflammatory cytokines (IL-4 and IL-13) were increased in Bsep-/- mice upon infection. Furthermore, infected Bsep-/- mice exhibited decreased hepatic egg load and parasite fecundity, consequently affecting the worm reproduction rate. This outcome could arise from elevated serum BA levels and lower blood pH in Bsep-/- mice. CONCLUSIONS: The loss of Bsep and the resulting changes in bile acid composition and blood pH are associated with the reduction of parasite fecundity, thus attenuating the development of S. mansoni-induced hepatic inflammation and fibrosis.

Infectious origin of Alzheimer's disease: Amyloid beta as a component of brain antimicrobial immunity.

The amyloid cascade hypothesis, focusing on pathological proteins aggregation, has so far failed to uncover the root cause of Alzheimer's disease (AD), or to provide an effective therapy. This traditional paradigm essentially explains a mechanism involved in the development of sporadic AD rather than its cause. The failure of an overwhelming majority of clinical studies (99.6%) demonstrates that a breakthrough in therapy would be difficult if not impossible without understanding the etiology of AD. It becomes more and more apparent that the AD pathology might originate from brain infection. In this review, we discuss a potential role of bacteria, viruses, fungi, and eukaryotic parasites as triggers of AD pathology. We show evidence from the current literature that amyloid beta, traditionally viewed as pathological, actually acts as an antimicrobial peptide, protecting the brain against pathogens. However, in case of a prolonged or excessive activation of a senescent immune system, amyloid beta accumulation and aggregation becomes damaging and supports runaway neurodegenerative processes in AD. This is paralleled by the recent study by Alam and colleagues (2022) who showed that alpha-synuclein, the protein accumulating in synucleinopathies, also plays a critical physiological role in immune reactions and inflammation, showing an unforeseen link between the 2 unrelated classes of neurodegenerative disorders. The multiplication of the amyloid precursor protein gene, recently described by Lee and collegues (2018), and possible reactivation of human endogenous retroviruses by pathogens fits well into the same picture. We discuss these new findings from the viewpoint of the infection hypothesis of AD and offer suggestions for future research.

Toxocara canis infection worsens the course of experimental autoimmune encephalomyelitis in mice.

Toxocara canis, a gastrointestinal parasite of canids, is also highly prevalent in many paratenic hosts, such as mice and humans. As with many other helminths, the infection is associated with immunomodulatory effects, which could affect other inflammatory conditions including autoimmune and allergic diseases. Here, we investigated the effect of T. canis infection on the course of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Mice infected with 2 doses of 100 T. canis L3 larvae 5 weeks prior to EAE induction (the Tc+EAE group) showed higher EAE clinical scores and greater weight loss compared to the non-infected group with induced EAE (the EAE group). Elevated concentrations of all measured serum cytokines (IL-1α, IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ and TNF-α) were observed in the Tc+EAE group compared to the EAE group. In the CNS, the similar number of regulatory T cells (Tregs; CD4+FoxP3+Helios+) but their decreased proportion from total CD4+ cells was found in the Tc+EAE group compared to the EAE group. This could indicate that the group Tc+EAE harboured significantly more CD4+ T cells of non-Treg phenotype within the affected CNS. Altogether, our results demonstrate that infection of mice with T. canis worsens the course of subsequently induced EAE. Further studies are, therefore, urgently needed to reveal the underlying pathological mechanisms and to investigate possible risks for the human population, in which exposure to T. canis is frequent.

Comparative proteome analysis of the tegument of male and female adult Schistosoma mansoni.

The tegument, as the surface layer of adult male and female Schistosoma spp. represents the protective barrier of the worms to the hostile environment of the host bloodstream. Here we present the first comparative analysis of sex-specific tegument proteins of paired or virgin Schistosoma mansoni. We applied a new and highly sensitive workflow, allowing detection of even low abundance proteins. Therefore, a streptavidin-biotin affinity purification technique in combination with single pot solid-phase enhanced sample preparation was established for subsequent LC-MS/MS analysis. We were able to identify 1519 tegument proteins for male and female virgin and paired worms and categorized them by sex. Bioinformatic analysis revealed an involvement of female-specific tegument proteins in signaling pathways of cellular processes and antioxidant mechanisms. Male-specific proteins were found to be enriched in processes linked to phosphorylation and signal transduction. This suggests a task sharing between the sexes that might be necessary for survival in the host. Our datasets provide a basis for further studies to understand and ultimately decipher the strategies of the two worm sexes to evade the immune system.

Mechanisms of the host immune response and helminth-induced pathology during Trichobilharzia regenti (Schistosomatidae) neuroinvasion in mice.

Helminth neuroinfections represent serious medical conditions, but the diversity of the host-parasite interplay within the nervous tissue often remains poorly understood, partially due to the lack of laboratory models. Here, we investigated the neuroinvasion of the mouse spinal cord by Trichobilharzia regenti (Schistosomatidae). Active migration of T. regenti schistosomula through the mouse spinal cord induced motor deficits in hindlimbs but did not affect the general locomotion or working memory. Histological examination of the infected spinal cord revealed eosinophilic meningomyelitis with eosinophil-rich infiltrates entrapping the schistosomula. Flow cytometry and transcriptomic analysis of the spinal cord confirmed massive activation of the host immune response. Of note, we recorded striking upregulation of the major histocompatibility complex II pathway and M2-associated markers, such as arginase or chitinase-like 3. Arginase also dominated the proteins found in the microdissected tissue from the close vicinity of the migrating schistosomula, which unselectively fed on the host nervous tissue. Next, we evaluated the pathological sequelae of T. regenti neuroinvasion. While no demyelination or blood-brain barrier alterations were noticed, our transcriptomic data revealed a remarkable disruption of neurophysiological functions not yet recorded in helminth neuroinfections. We also detected DNA fragmentation at the host-schistosomulum interface, but schistosomula antigens did not affect the viability of neurons and glial cells in vitro. Collectively, altered locomotion, significant disruption of neurophysiological functions, and strong M2 polarization were the most prominent features of T. regenti neuroinvasion, making it a promising candidate for further neuroinfection research. Indeed, understanding the diversity of pathogen-related neuroinflammatory processes is a prerequisite for developing better protective measures, treatment strategies, and diagnostic tools.

Nitric oxide debilitates the neuropathogenic schistosome Trichobilharzia regenti in mice, partly by inhibiting its vital peptidases.

BACKGROUND: Avian schistosomes, the causative agents of human cercarial dermatitis (or swimmer's itch), die in mammals but the mechanisms responsible for parasite elimination are unknown. Here we examined the role of reactive nitrogen species, nitric oxide (NO) and peroxynitrite, in the immune response of mice experimentally infected with Trichobilharzia regenti, a model species of avian schistosomes remarkable for its neuropathogenicity. METHODS: Inducible NO synthase (iNOS) was localized by immunohistochemistry in the skin and the spinal cord of mice infected by T. regenti. The impact of iNOS inhibition by aminoguanidine on parasite burden and growth was then evaluated in vivo. The vulnerability of T. regenti schistosomula to NO and peroxynitrite was assessed in vitro by viability assays and electron microscopy. Additionally, the effect of NO on the activity of T. regenti peptidases was tested using a fluorogenic substrate. RESULTS: iNOS was detected around the parasites in the epidermis 8 h post-infection and also in the spinal cord 3 days post-infection (dpi). Inhibition of iNOS resulted in slower parasite growth 3 dpi, but the opposite effect was observed 7 dpi. At the latter time point, moderately increased parasite burden was also noticed in the spinal cord. In vitro, NO did not impair the parasites, but inhibited the activity of T. regenti cathepsins B1.1 and B2, the peptidases essential for parasite migration and digestion. Peroxynitrite severely damaged the surface tegument of the parasites and decreased their viability in vitro, but rather did not participate in parasite clearance in vivo. CONCLUSIONS: Reactive nitrogen species, specifically NO, do not directly kill T. regenti in mice. NO promotes the parasite growth soon after penetration (3 dpi), but prevents it later (7 dpi) when also suspends the parasite migration in the CNS. NO-related disruption of the parasite proteolytic machinery is partly responsible for this effect.

Isoforms of Cathepsin B1 in Neurotropic Schistosomula of Trichobilharzia regenti Differ in Substrate Preferences and a Highly Expressed Catalytically Inactive Paralog Binds Cystatin.

Schistosomula (the post-infective stages) of the neurotropic schistosome Trichobilharzia regenti possess multiple isoforms of cathepsin B1 peptidase (TrCB1.1-TrCB1.6) with involvement in nutrient digestion. The comparison of substrate preferences of TrCB1.1 and TrCB1.4 showed that TrCB1.4 had a very narrow substrate specificity and after processing it was less effective toward protein substrates when compared to TrCB1.1. Self-processing of both isoforms could be facilitated by sulfated polysaccharides due to a specific binding motif in the pro-sequence. Trans-activation by heterologous enzymes was also successfully employed. Expression profiling revealed a high level of transcription of genes encoding the enzymatically inactive paralogs TrCB1.5 and TrCB1.6. The transcription level of TrCB1.6 was comparable with that of TrCB1.1 and TrCB1.2, the most abundant active isoforms. Recombinant TrCB1.6wt, a wild type paralog with a Cys29-to-Gly substitution in the active site that renders the enzyme inactive, was processed by the active TrCB1 forms and by an asparaginyl endopeptidase. Although TrCB1.6wt lacked hydrolytic activity, endopeptidase, but not dipeptidase, activity could be restored by mutating Gly29 to Cys29. The lack of exopeptidase activity may be due to other mutations, such as His110-to-Asn in the occluding loop and Asp224-to-Gly in the main body of the mature TrCB1.6, which do not occur in the active isoforms TrCB1.1 and TrCB1.4 with exopeptidase activity. The catalytically active enzymes and the inactive TrCB1.6 paralog formed complexes with chicken cystatin, thus supporting experimentally the hypothesis that inactive paralogs could potentially regulate the activity of the active forms or protect them from being inhibited by host inhibitors. The effect on cell viability and nitric oxide production by selected immune cells observed for TrCB1.1 was not confirmed for TrCB1.6. We show here that the active isoforms of TrCB1 have different affinities for peptide substrates thereby facilitating diversity in protein-derived nutrition for the parasite. The inactive paralogs are unexpectedly highly expressed and one of them retains the ability to bind cystatins, likely due to specific mutations in the occluding loop and the enzyme body. This suggests a role in sequestration of inhibitors and protection of active cysteine peptidases.

The peripheral immune response of mice infected with a neuropathogenic schistosome.

Trichobilharzia regenti (Schistosomatidae) percutaneously infects birds and mammals and invades their central nervous system (CNS). Here, we characterized the peripheral immune response of infected mice and showed how it was influenced by the parasite-induced inflammation in the skin and the CNS. As revealed by flow cytometry, T cells expanded in the spleen and the CNS-draining lymph nodes 7-14 days post-infection. Both T-bet+ and GATA-3+ T cells were markedly elevated suggesting a mixed type 1/2 immune response. However, it dropped after 7 dpi most likely being unaffected by the neuroinflammation. Splenocytes from infected mice produced a high amount of IFN-γ and, to a lesser extent, IL-10, IL-4 and IL-17 after in vitro stimulation by cercarial homogenate. Nevertheless, it had only a limited capacity to alter the maturation status of bone marrow-derived dendritic cells (BMDCs), contrary to the recombinant T. regenti cathepsin B2, which also strongly augmented expression of Ccl5, Cxcl10, Il12a, Il33 and Il10 by BMDCs. Taken together, mice infected with T. regenti developed the mixed type 1/2 immune response, which was driven by the early skin inflammation rather than the late neuroinflammation. Parasite peptidases might play an active role in triggering the host immune response.

Cercarial dermatitis: a systematic follow-up study of human cases with implications for diagnostics.

Cercarial dermatitis (CD) is an allergic skin disease that rises in consequence of infection by invasive stages (cercariae) of trematodes of the family Schistosomatidae. CD has been considered a re-emerging disease, human cases have been reported from all continents, and tourism-threatening outbreaks occur even in frequented recreational areas. Although the symptoms of CD are generally known, the data on immune response in human patients are sporadic and incomprehensive. In the present study, we attempted to correlate the symptoms, personal history, and time course of CD in human patients with differential cell counts, dynamics of selected cytokines, and dynamics and quality of antibody response. By a systematic follow-up, we obtained a uniquely complex dataset from ten persons accidentally and concurrently infected by the same parasite species in the same locality. The onset of CD was significantly faster, and the symptoms were heavier in participants with a history of CD if compared to naive ones, who, however, also developed some of the symptoms. The repeatedly infected persons had elevated proportion of eosinophils 1 week post exposure (p.e.) and a stronger specific IgG but not IgM response, whereas specific IgE response was not observed. Increased serum levels of IL-4 occurred 1 and 3 week(s) p.e. in all participants. There was high variability in individual immunoblot patterns of IgG response, and no antigen with a universal diagnostic potential was confirmed. The presented analyses suggested that a complex approach can improve the accuracy of the diagnosis of CD, but component data should be interpreted carefully.

Humoral response of mice infected with Toxocara canis following different infection schemes.

The study was focused on the dynamics of humoral response to Toxocara canis excretory-secretory antigens (TES antigens) in mice experimentally infected by T. canis L3 larvae in different ways. In particular, we compared the effect of infection with two doses of 1000 larvae vs. repeated infections with a low number of larvae (daily infection with 10 larvae and weekly infection with 100 larvae in the course of 22 weeks). In ELISA, all infections, including both schemes with lower larval doses, elicited significant antibody response. Elevated levels of total IgE and TES-antigen-specific IgM were detected on day 12 after the first infection, followed by IgG and IgG1, and later by IgG3, IgG2a and IgG2b; specific IgE response was not detected. It seems that the high levels of IgM and IgG1 represent the best markers of infection. In addition, gradual increase of IgG2a and IgG2b could help in determination of the infection course. As a byproduct of our work, a new method of infection by repeated drinking of larvae was introduced; it minimizes the pain and discomfort for the experimental mice.

Nitric oxide and cytokine production by glial cells exposed in vitro to neuropathogenic schistosome Trichobilharzia regenti.

BACKGROUND: Helminth neuroinfections represent a serious health problem, but host immune mechanisms in the nervous tissue often remain undiscovered. This study aims at in vitro characterization of the response of murine astrocytes and microglia exposed to Trichobilharzia regenti which is a neuropathogenic schistosome migrating through the central nervous system of vertebrate hosts. Trichobilharzia regenti infects birds and mammals in which it may cause severe neuromotor impairment. This study was focused on astrocytes and microglia as these are immunocompetent cells of the nervous tissue and their activation was recently observed in T. regenti-infected mice. RESULTS: Primary astrocytes and microglia were exposed to several stimulants of T. regenti origin. Living schistosomulum-like stages caused increased secretion of IL-6 in astrocyte cultures, but no changes in nitric oxide (NO) production were noticed. Nevertheless, elevated parasite mortality was observed in these cultures. Soluble fraction of the homogenate from schistosomulum-like stages stimulated NO production by both astrocytes and microglia, and IL-6 and TNF-α secretion in astrocyte cultures. Similarly, recombinant cathepsins B1.1 and B2 triggered IL-6 and TNF-α release in astrocyte and microglia cultures, and NO production in astrocyte cultures. Stimulants had no effect on production of anti-inflammatory cytokines IL-10 or TGF-ß1. CONCLUSIONS: Both astrocytes and microglia are capable of production of NO and proinflammatory cytokines IL-6 and TNF-α following in vitro exposure to various stimulants of T. regenti origin. Astrocytes might be involved in triggering the tissue inflammation in the early phase of T. regenti infection and are proposed to participate in destruction of migrating schistosomula. However, NO is not the major factor responsible for parasite damage. Both astrocytes and microglia can be responsible for the nervous tissue pathology and maintaining the ongoing inflammation since they are a source of NO and proinflammatory cytokines which are released after exposure to parasite antigens.

Trichobilharzia regenti (Schistosomatidae): 3D imaging techniques in characterization of larval migration through the CNS of vertebrates.

Migration of parasitic worms through the host tissues, which may occasionally result in fatal damage to the internal organs, represents one of the major risks associated with helminthoses. In order to track the parasites, traditionally used 2D imaging techniques such as histology or squash preparation do not always provide sufficient data to describe worm location/behavior in the host. On the other hand, 3D imaging methods are widely used in cell biology, medical radiology, osteology or cancer research, but their use in parasitological research is currently occasional. Thus, we aimed at the evaluation of suitability of selected 3D methods to monitor migration of the neuropathogenic avian schistosome Trichobilharzia regenti in extracted spinal cord of experimental vertebrate hosts. All investigated methods, two of them based on tracking of fluorescently stained larvae with or without previous chemical clearing of tissue and one based on X-ray micro-CT, exhibit certain limits for in vivo observation. Nevertheless, our study shows that the tested methods as ultramicroscopy (used for the first time in parasitology) and micro-CT represent promising tool for precise analyzing of parasite larvae in the CNS. Synthesis of these 3D imaging techniques can provide more comprehensive look at the course of infection, host immune response and pathology caused by migrating parasites within entire tissue samples, which would not be possible with traditional approaches.