Chitin-mediated blockade of chitinase-like proteins reduces tumor immunosuppression, inhibits lymphatic metastasis and enhances anti-PD-1 efficacy in complementary TNBC models.

BACKGROUND: Chitinase-like proteins (CLPs) play a key role in immunosuppression under inflammatory conditions such as cancer. CLPs are enzymatically inactive and become neutralized upon binding of their natural ligand chitin, potentially reducing CLP-driven immunosuppression. We investigated the efficacy of chitin treatment in the context of triple-negative breast cancer (TNBC) using complementary mouse models. We also evaluated the immunomodulatory influence of chitin on immune checkpoint blockade (ICB) and compared its efficacy as general CLP blocker with blockade of a single CLP, i.e. chitinase 3-like 1 (CHI3L1). METHODS: Female BALB/c mice were intraductally injected with luciferase-expressing 4T1 or 66cl4 cells and systemically treated with chitin in combination with or without anti-programmed death (PD)-1 ICB. For single CLP blockade, tumor-bearing mice were treated with anti-CHI3L1 antibodies. Metastatic progression was monitored through bioluminescence imaging. Immune cell changes in primary tumors and lymphoid organs (i.e. axillary lymph nodes and spleen) were investigated through flow cytometry, immunohistochemistry, cytokine profiling and RNA-sequencing. CHI3L1-stimulated RAW264.7 macrophages were subjected to 2D lymphatic endothelial cell adhesion and 3D lymphatic integration in vitro assays for studying macrophage-mediated lymphatic remodeling. RESULTS: Chitin significantly reduced primary tumor progression in the 4T1-based model by decreasing the high production of CLPs that originate from tumor-associated neutrophils (TANs) and Stat3 signaling, prominently affecting the CHI3L1 and CHI3L3 primary tumor levels. It reduced immunosuppressive cell types and increased anti-tumorigenic T-cells in primary tumors as well as axillary lymph nodes. Chitin also significantly reduced CHI3L3 primary tumor levels and immunosuppression in the 66cl4-based model. Compared to anti-CHI3L1, chitin enhanced primary tumor growth reduction and anti-tumorigenicity. Both treatments equally inhibited lymphatic adhesion and integration of macrophages, thereby hampering lymphatic tumor cell spreading. Upon ICB combination therapy, chitin alleviated anti-PD-1 resistance in both TNBC models, providing a significant add-on reduction in primary tumor and lung metastatic growth compared to chitin monotherapy. These add-on effects occurred through additional increase in CD8α+ T-cell infiltration and activation in primary tumor and lymphoid organs. CONCLUSIONS: Chitin, as a general CLP blocker, reduces CLP production, enhances anti-tumor immunity as well as ICB responses, supporting its potential clinical relevance in immunosuppressed TNBC patients.

Integrated gut metabolome and microbiome fingerprinting reveals that dysbiosis precedes allergic inflammation in IgE-mediated pediatric cow's milk allergy.

BACKGROUND: IgE-mediated cow's milk allergy (IgE-CMA) is one of the first allergies to arise in early childhood and may result from exposure to various milk allergens, of which ß-lactoglobulin (BLG) and casein are the most important. Understanding the underlying mechanisms behind IgE-CMA is imperative for the discovery of novel biomarkers and the design of innovative treatment and prevention strategies. METHODS: We report a longitudinal in vivo murine model, in which two mice strains (BALB/c and C57Bl/6) were sensitized to BLG using either cholera toxin or an oil emulsion (n = 6 per group). After sensitization, mice were challenged orally, their clinical signs monitored, antibody (IgE and IgG1) and cytokine levels (IL-4 and IFN-γ) measured, and fecal samples subjected to metabolomics. The results of the murine models were further extrapolated to fecal microbiome-metabolome data from our population of IgE-CMA (n = 22) and healthy (n = 23) children (Trial: NCT04249973), on which polar metabolomics, lipidomics and 16S rRNA metasequencing were performed. In vitro gastrointestinal digestions and multi-omics corroborated the microbial origin of proposed metabolic changes. RESULTS: During mice sensitization, we observed multiple microbially derived metabolic alterations, most importantly bile acid, energy and tryptophan metabolites, that preceded allergic inflammation. We confirmed microbial dysbiosis, and its associated effect on metabolic alterations in our patient cohort, through in vitro digestions and multi-omics, which was accompanied by metabolic signatures of low-grade inflammation. CONCLUSION: Our results indicate that gut dysbiosis precedes allergic inflammation and nurtures a chronic low-grade inflammation in children on elimination diets, opening important new opportunities for future prevention and treatment strategies.

Spiroplasma infection as a cause of severe congenital keratouveitis, cataract and glaucoma.

BACKGROUND: Only seven cases of ocular Spiroplasma infection have been reported to date, all presenting as congenital cataracts with concomitant intraocular inflammation. We describe the first case of Spiroplasma infection initially presenting as a corneal infiltrate. CASE PRESENTATION: A 1-month-old girl was referred for a corneal infiltrate in the left eye. She presented in our hospital with unilateral keratouveitis. Examination showed a stromal corneal infiltrate and dense white keratic precipitates in the left eye. Herpetic keratouveitis was suspected and intravenous acyclovir therapy was initiated. Two weeks later, the inflammation in the left eye persisted and was also noticed in the right eye. Acute angle-closure glaucoma and a cataract with dilated iris vessels extending onto the anterior lens capsule developed in the left eye. The inflammation resolved after treatment with azithromycin. Iridectomy, synechiolysis and lensectomy were performed. Bacterial metagenomic sequencing (16 S rRNA) and transmission electron microscopy revealed Spiroplasma ixodetis species in lens aspirates and biopsy. Consequently, a diagnosis of bilateral Spiroplasma uveitis was made. CONCLUSIONS: In cases of congenital cataract with concomitant intraocular inflammation, Spiroplasma infection should be considered. The purpose of this case report is to raise awareness of congenital Spiroplasma infection as a cause of severe keratouveitis, cataract and angle-closure glaucoma in newborns. Performing molecular testing on lens aspirates is essential to confirm diagnosis. Systemic macrolides are suggested as the mainstay of treatment.

The Attenuated Pseudorabies Virus Vaccine Strain Bartha Hyperactivates Plasmacytoid Dendritic Cells by Generating Large Amounts of Cell-Free Virus in Infected Epithelial Cells.

Pseudorabies virus (PRV) is a porcine alphaherpesvirus and the causative agent of Aujeszky's disease. Successful eradication campaigns against PRV have largely relied on the use of potent PRV vaccines. The live attenuated Bartha strain, which was produced by serial passaging in cell culture, represents one of the hallmark PRV vaccines. Despite the robust protection elicited by Bartha vaccination, very little is known about the immunogenicity of the Bartha strain. Previously, we showed that Bartha-infected epithelial cells trigger plasmacytoid dendritic cells (pDC) to produce much higher levels of type I interferons than cells infected with wild-type PRV. Here, we show that this Bartha-induced pDC hyperactivation extends to other important cytokines, including interleukin-12/23 (IL-12/23) and tumor necrosis factor alpha (TNF-α) but not IL-6. Moreover, Bartha-induced pDC hyperactivation was found to be due to the strongly increased production of extracellular infectious virus (heavy particles [H-particles]) early in infection of epithelial cells, which correlated with a reduced production of noninfectious light particles (L-particles). The Bartha genome is marked by a large deletion in the US region affecting the genes encoding US7 (gI), US8 (gE), US9, and US2. The deletion of the US2 and gE/gI genes was found to be responsible for the observed increase in extracellular virus production by infected epithelial cells and the resulting increased pDC activation. The deletion of gE/gI also suppressed L-particle production. In conclusion, the deletion of US2 and gE/gI in the genome of the PRV vaccine strain Bartha results in the enhanced production of extracellular infectious virus in infected epithelial cells and concomitantly leads to the hyperactivation of pDC. IMPORTANCE The pseudorabies virus (PRV) vaccine strain Bartha has been and still is critical in the eradication of PRV in numerous countries. However, little is known about how this vaccine strain interacts with host cells and the host immune system. Here, we report the surprising observation that Bartha-infected epithelial porcine cells rapidly produce increased amounts of extracellular infectious virus compared to wild-type PRV-infected cells, which in turn potently stimulate porcine plasmacytoid dendritic cells (pDC). We found that this phenotype depends on the deletion of the genes encoding US2 and gE/gI. We also found that Bartha-infected cells secrete fewer pDC-inhibiting light particles (L-particles), which appears to be caused mainly by the deletion of the genes encoding gE/gI. These data generate novel insights into the interaction of the successful Bartha vaccine with epithelial cells and pDC and may therefore contribute to the development of vaccines against other (alphaherpes)viruses.

Changes in intestinal morphology, number of mucus-producing cells and expression of coronavirus receptors APN, DPP4, ACE2 and TMPRSS2 in pigs with aging.

Porcine enteric viral infections cause high morbidity and mortality in young piglets (<3 weeks). Later, these rates decrease with age. This age-dependent infectivity remains largely unexplored. This study investigated the changes in intestinal morphology, number of mucus-producing cells and expression level of coronavirus receptors in three age groups of pigs. Villus height and crypt depth increased with age from 3 days to 3 months in duodenum and ileum but not in mid-jejunum, where the villus height decreased from 580 µm at 3 days to 430 µm at 3 months. Enterocyte length-to-width ratio increased from 3 days to 3 months in all intestinal regions. The number of mucus-producing cells increased with age in the intestinal villi and crypts. The Brunner's glands of the duodenum contained the highest concentration of mucus-producing cells. The expression of coronavirus receptor APN was highest in the small intestinal villi at all ages. DPP4 expression slightly decreased over time in jejunum and ileum; it was highest in the ileal villi of 3-day-old piglets (70.2% of cells). ACE2 and TMPRSS2 positive cells increased with age in jejunal and ileal crypts and were particularly dominant in the ileal crypts (> 45% of cells). Except for the expression of DPP4 in the jejunum and ileum of young pigs, the expression pattern of the selected coronavirus receptors was very different and not correlated with the age-dependent susceptibility to viral infections. In contrast, the number of mucus-producing cells increased over time and may play an essential role in protecting enteric mucosae against intestinal viruses.

The shrimp nephrocomplex serves as a major portal of pathogen entry and is involved in the molting process.

Viruses, such as white spot syndrome virus, and bacteria, such as Vibrio species, wreak havoc in shrimp aquaculture [C. M. Escobedo-Bonilla et al., J. Fish. Dis. 31, 1-18 (2008)]. As the main portal of entry for shrimp-related pathogens remain unclear, infectious diseases are difficult to prevent and control. Because the cuticle is a strong pathogen barrier, regions lacking cuticular lining, such as the shrimp's excretory organ, "the antennal gland," are major candidate entry portals [M. Corteel et al., Vet. Microbiol. 137, 209-216 (2009)]. The antennal gland, up until now morphologically underexplored, is studied using several imaging techniques. Using histology-based three-dimensional technology, we demonstrate that the antennal gland resembles a kidney, connected to a urinary bladder with a nephropore (exit opening) and a complex of diverticula, spread throughout the cephalothorax. Micromagnetic resonance imaging of live shrimp not only confirms the histology-based model, but also indicates that the filling of the diverticula is linked to the molting cycle and possibly involved therein. Based on function and complexity, we propose to rename the antennal gland as the "nephrocomplex." By an intrabladder inoculation, we showed high susceptibility of this nephrocomplex to both white spot syndrome virus and Vibrio infection compared to peroral inoculation. An induced drop in salinity allowed the virus to enter the nephrocomplex in a natural way and caused a general infection followed by death; fluorescent beads were used to demonstrate that particles may indeed enter through the nephropore. These findings pave the way for oriented disease control in shrimp.

Equine Herpesvirus 1 Bridles T Lymphocytes To Reach Its Target Organs.

Equine herpesvirus 1 (EHV1) replicates in the respiratory epithelium and disseminates through the body via a cell-associated viremia in leukocytes, despite the presence of neutralizing antibodies. "Hijacked" leukocytes, previously identified as monocytic cells and T lymphocytes, transmit EHV1 to endothelial cells of the endometrium or central nervous system, causing reproductive (abortigenic variants) or neurological (neurological variants) disorders. In the present study, we questioned the potential route of EHV1 infection of T lymphocytes and how EHV1 misuses T lymphocytes as a vehicle to reach the endothelium of the target organs in the absence or presence of immune surveillance. Viral replication was evaluated in activated and quiescent primary T lymphocytes, and the results demonstrated increased infection of activated versus quiescent, CD4+ versus CD8+, and blood- versus lymph node-derived T cells. Moreover, primarily infected respiratory epithelial cells and circulating monocytic cells efficiently transferred virions to T lymphocytes in the presence of neutralizing antibodies. Albeit T-lymphocytes express all classes of viral proteins early in infection, the expression of viral glycoproteins on their cell surface was restricted. In addition, the release of viral progeny was hampered, resulting in the accumulation of viral nucleocapsids in the T cell nucleus. During contact of infected T lymphocytes with endothelial cells, a late viral protein(s) orchestrates T cell polarization and synapse formation, followed by anterograde dynein-mediated transport and transfer of viral progeny to the engaged cell. This represents a sophisticated but efficient immune evasion strategy to allow transfer of progeny virus from T lymphocytes to adjacent target cells. These results demonstrate that T lymphocytes are susceptible to EHV1 infection and that cell-cell contact transmits infectious virus to and from T lymphocytes.IMPORTANCE Equine herpesvirus 1 (EHV1) is an ancestral alphaherpesvirus that is related to herpes simplex virus 1 and causes respiratory, reproductive, and neurological disorders in Equidae. EHV1 is indisputably a master at exploiting leukocytes to reach its target organs, accordingly evading the host immunity. However, the role of T lymphocytes in cell-associated viremia remains poorly understood. Here we show that activated T lymphocytes efficiently become infected and support viral replication despite the presence of protective immunity. We demonstrate a restricted expression of viral proteins on the surfaces of infected T cells, which prevents immune recognition. In addition, we indicate a hampered release of progeny, which results in the accumulation of nucleocapsids in the T cell nucleus. Upon engagement with the target endothelium, late viral proteins orchestrate viral synapse formation and viral transfer to the contact cell. Our findings have significant implications for the understanding of EHV1 pathogenesis, which is essential for developing innovative therapies to prevent the devastating clinical symptoms of infection.

Pseudorabies Virus US3-Induced Tunneling Nanotubes Contain Stabilized Microtubules, Interact with Neighboring Cells via Cadherins, and Allow Intercellular Molecular Communication.

Tunneling nanotubes (TNTs) are long bridge-like structures that connect eukaryotic cells and mediate intercellular communication. We found earlier that the conserved alphaherpesvirus US3 protein kinase induces long cell projections that contact distant cells and promote intercellular virus spread. In this report, we show that the US3-induced cell projections constitute TNTs. In addition, we report that US3-induced TNTs mediate intercellular transport of information (e.g., green fluorescent protein [GFP]) in the absence of other viral proteins. US3-induced TNTs are remarkably stable compared to most TNTs described in the literature. In line with this, US3-induced TNTs were found to contain stabilized (acetylated and detyrosinated) microtubules. Transmission electron microscopy showed that virus particles are individually transported in membrane-bound vesicles in US3-induced TNTs and are released along the TNT and at the contact area between a TNT and the adjacent cell. Contact between US3-induced TNTs and acceptor cells is very stable, which correlated with a marked enrichment in adherens junction components beta-catenin and E-cadherin at the contact area. These data provide new structural insights into US3-induced TNTs and how they may contribute to intercellular communication and alphaherpesvirus spread.IMPORTANCE Tunneling nanotubes (TNT) represent an important and yet still poorly understood mode of long-distance intercellular communication. We and others reported earlier that the conserved alphaherpesvirus US3 protein kinase induces long cellular protrusions in infected and transfected cells. Here, we show that US3-induced cell projections constitute TNTs, based on structural properties and transport of biomolecules. In addition, we report on different particular characteristics of US3-induced TNTs that help to explain their remarkable stability compared to physiological TNTs. In addition, transmission electron microscopy assays indicate that, in infected cells, virions travel in the US3-induced TNTs in membranous transport vesicles and leave the TNT via exocytosis. These data generate new fundamental insights into the biology of (US3-induced) TNTs and into how they may contribute to intercellular virus spread and communication.

Intussusceptive Pillar Formation in Developing Porcine Glomeruli.

BACKGROUND/AIMS: Intussusceptive angiogenesis (IA) is a dynamic process which contributes to vascular expansion and remodeling. Intraluminal pillars have long been the distinctive structural indicator of IA. However, the mechanism of their formation has not been fully elucidated. METHODS: Using light and electron microscopy, we studied intussusceptive vascular growth in the developing porcine metanephric kidney. RESULTS: We observed intraluminal pillars formed by endothelial cells in the vasculature of developing glomeruli. Their diameter was < 2.5 µm, consistent with the diameter of nascent pillars. TEM revealed that the majority of these pillars consisted only of endothelium. However, a central core of extracellular matrix (ECM) covered by endothelium, reminiscent of a more mature intussusceptive pillar, was also found in the lumen of a glomerular capillary. Perivascular cells or pericytes were not involved in the pillar structure during these stages of formation. CONCLUSION: This study shows ECM presence in a mature intussusceptive pillar without any perivascular cell involvement in the structure. This leads to the hypothesis that ECM deposition precedes the participation of these cells in the formation of intraluminal pillars during IA in porcine metanephric glomerular capillaries.

A preliminary study of pulmonary vein implant applicability and safety as a potential ablation platform in a follow-up study in pigs.

BACKGROUND: Recurrence of atrial fibrillation after an ablation procedure remains a major problem which emphasizes the need for improved pulmonary vein isolation techniques. AIMS: The aim of this study was to describe an implantation procedure of a pulmonary vein-stent which may possibly serve as an ablation technique in the future and to examine stent safety in a follow-up study in pigs. METHODS AND RESULTS: Eight pigs were catheterized and nine self-expanding nitinol stents were implanted through a transfemoral or transatrial approach into the antra of the pulmonary veins. After 3 months' follow-up, the animals were euthanized for further examination. During the follow-up phase, no complications were observed. Absence of thrombus formation or pulmonary vein wall dissection was noticed during anatomical and histological evaluation of the heart-lung packages. All implants were almost completely covered by neo-intima, of which thickness varied between 0.2 and 3.9 mm. CONCLUSIONS: Stents can safely be positioned and deployed into the antra of the pulmonary veins without any acute or long-term (3 months) adverse effects. In the future, these implants could function as a permanently implanted ablation device and provide new therapeutic strategies for pulmonary vein isolation in patients with atrial fibrillation.

Isolation and Characterization of Functionally Active Extracellular Vesicles from Culture Medium Conditioned by Bovine Embryos In Vitro.

Extracellular vesicles (EVs) play a possible role in cell⁻cell communication and are found in various body fluids and cell conditioned culture media. The aim of this study was to isolate and characterize EVs in culture medium conditioned by bovine embryos in group and to verify if these EVs are functionally active. Initially, ultracentrifuged bovine serum albumin (BSA) containing medium was selected as suitable EV-free embryo culture medium. Next, EVs were isolated from embryo conditioned culture medium by OptiPrepTM density gradient ultracentrifugation. Isolated EVs were characterized by nanoparticle tracking analysis, western blotting, transmission, and immunoelectron microscopy. Bovine embryo-derived EVs were sizing between 25⁻230 nm with an average concentration of 236.5 ± 1.27 × 108 particles/mL. Moreover, PKH67 EV pre-labeling showed that embryo-secreted EVs were uptaken by zona-intact bovine embryos. Since BSA did not appear to be a contaminating EV source in culture medium, EV functionality was tested in BSA containing medium. Individual embryo culture in BSA medium enriched with EVs derived from conditioned embryo culture medium showed significantly higher blastocyst rates at day 7 and 8 together with a significantly lower apoptotic cell ratio. In conclusion, our study shows that EVs play an important role in inter embryo communication during bovine embryo culture in group.

Cellular and transcriptomic response to treatment with the probiotic candidate Vibrio lentus in gnotobiotic sea bass (Dicentrarchus labrax) larvae.

The present study aimed at evaluating the cellular and transcriptomic responses induced by the probiotic candidate Vibrio lentus with gnotobiotic European sea bass (Dicentrarchus labrax, Linnaeus 1785) larvae. For this, a histomorphological analysis was performed using the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) and the anti-proliferating cell nuclear antigen (PCNA) assay. In addition, a global transcriptomic approach was adopted to study the whole body mRNA changes upon administration of V. lentus by microarrays with the custom Agilent sea bass oligonucleotide-microarray v2.0 (4 × 44 K). Following V. lentus administration, the apoptotic and cell proliferative indexes did not show significant differences between treatments for hindgut nor for midgut. However, V. lentus treatment did significantly modify the gene expression related not only to cell proliferation and cell death, but also to cell adhesion, reactive oxygen species metabolism, iron transport, and immune response. Our data represent the first global analysis of the effects of the probiotic candidate V. lentus on the gene expression profile in gnotobiotic European sea bass, and as such, provides a first delineation of the mechanisms by which this agent interacts with its host and exerts its beneficial effects.

Divergence between the Highly Virulent Zoonotic Pathogen Helicobacter heilmannii and Its Closest Relative, the Low-Virulence "Helicobacter ailurogastricus" sp. nov.

Helicobacter heilmannii naturally colonizes the stomachs of dogs and cats and has been associated with gastric disorders in humans. Nine feline Helicobacter strains, classified as H. heilmannii based on ureAB and 16S rRNA gene sequences, were divided into a highly virulent and a low-virulence group. The genomes of these strains were sequenced to investigate their phylogenetic relationships, to define their gene content and diversity, and to determine if the differences in pathogenicity were associated with the presence or absence of potential virulence genes. The capacities of these helicobacters to bind to the gastric mucosa were investigated as well. Our analyses revealed that the low-virulence strains do not belong to the species H. heilmannii but to a novel, closely related species for which we propose the name Helicobacter ailurogastricus. Several homologs of H. pylori virulence factors, such as IceA1, HrgA, and jhp0562-like glycosyltransferase, are present in H. heilmannii but absent in H. ailurogastricus. Both species contain a VacA-like autotransporter, for which the passenger domain is remarkably larger in H. ailurogastricus than in H. heilmannii. In addition, H. ailurogastricus shows clear differences in binding to the gastric mucosa compared to H. heilmannii. These findings highlight the low-virulence character of this novel Helicobacter species.

A Preclinical Study of an Implanted Device in the Pulmonary Veins, Intended for the Treatment of Atrial Fibrillation in an Ovine Model.

BACKGROUND: Atrial fibrillation is the most frequent arrhythmia in adults of which the interventional cure is hampered by high recurrence rates. Recurrence after ablation is due to an incomplete isolation of the pulmonary veins. A new ablation technique was performed, in the antra of ovine pulmonary veins, by device implantation, which was heated through a wireless heat-generating system. METHODS AND RESULTS: Implants were placed transatrially in the pulmonary veins of sheep. Using a wireless heating system, the energy was afterward transferred through wires to the implanted device according to a defined protocol. The position of the implant and the applied lesions were macroscopically evaluated. Samples of the ablated tissue of the atrio-pulmonary vein junction were histologically and immunohistochemically examined. CONCLUSIONS: Six ablation procedures in four sheep were successfully performed without adverse cardiac reactions. Implantation of the device and the wireless heat generation was feasible. Sufficient heat was produced at the level of the antra of the pulmonary veins to create ablation lesions, which were histologically and immunohistochemically confirmed.

Comparative immune responses against Psoroptes ovis in two cattle breeds with different susceptibility to mange.

The sheep scab mite, Psoroptes ovis, is a major problem in the beef cattle industry, especially in Belgian Blue (BB) cattle. This breed is naturally more predisposed to psoroptic mange but reasons for this high susceptibility remain unknown. Different immune responses could be a potential cause; thus in this study, the cutaneous immune response and in vitro cellular immune response after antigen re-stimulation were examined in naturally infested BB. Cytokine production in the skin and in circulating re-stimulated peripheral blood mononuclear cells (PBMC) demonstrated a mixed pro-inflammatory Th2/Th17 profile, with transcription of IL-4, IL-13, IL-6 and IL-17. Strong IL-17 up-regulation in the skin of BB was associated with an influx of eosinophils and other immune cells, potentially leading towards more severe symptoms. Virtually no changes in cutaneous IFN-γ transcription were detected, while there was substantial IFN-γ up-regulation in re-stimulated PBMC from infested and uninfested animals, potentially indicating a role of this pro-inflammatory cytokine in the innate immune response. In Holstein-Friesian (HF) cattle, generally more resistant to P. ovis infection, a largely similar immunologic response was observed. Differences between HF and BB were the lack of cutaneous IL-17 response in infested HF and low transcription levels of IFN-γ and high IL-10 transcription in re-stimulated PBMC from both infested and uninfested animals. Further research is needed to identify potential cell sources and biological functions for these cytokines and to fully unravel the basis of this different breed susceptibility to P. ovis.

Interactions of highly and low virulent Flavobacterium columnare isolates with gill tissue in carp and rainbow trout.

The interactions of Flavobacterium columnare isolates of different virulence with the gills of carp (Cyprinus carpio L.) and rainbow trout (Oncorhynchus mykiss Walbaum) were investigated. Both fish species were exposed to different high (HV) or low virulence (LV) isolates and sacrificed at seven predetermined times post-challenge. Histopathological and ultrastructural examination of carp and rainbow trout inoculated with the HV-isolate disclosed bacterial invasion and concomitant destruction of the gill tissue, gradually spreading from the filament tips towards the base, with outer membrane vesicles surrounding most bacterial cells. In carp, 5-10% of the fish inoculated with the LV-isolate became moribund and their gill tissue displayed the same features as described for the HV-isolate, albeit to a lesser degree. The bacterial numbers retrieved from the gill tissue were significantly higher for HV- compared to LV-isolate challenged carp and rainbow trout. TUNEL-stained and caspase-3-immunostained gill sections demonstrated significantly higher apoptotic cell counts in carp and rainbow trout challenged with the HV-isolate compared to control animals. Periodic acid-Schiff/alcian blue staining demonstrated a significantly higher total gill goblet cell count for HV- and LV-isolate challenged compared to control carp. Moreover, bacterial clusters were embedded in a neutral matrix while being encased by acid mucins, resembling biofilm formation. Eosinophilic granular cell counts were significantly higher in the HV-isolate compared to LV-isolate inoculated and control carp. The present data indicate a high colonization capacity, and the destructive and apoptotic-promoting features of the HV-isolate, and point towards important dynamic host mucin-F. columnare interactions warranting further research.

Mimicking herpes simplex virus 1 and herpes simplex virus 2 mucosal behavior in a well-characterized human genital organ culture.

We developed and morphologically characterized a human genital mucosa explant model (endocervix and ectocervix/vagina) to mimic genital herpes infections caused by herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). Subsequent analysis of HSV entry receptor expression throughout the menstrual cycle in genital tissues was performed, and the evolution of HSV-1/-2 mucosal spread over time was assessed. Nectin-1 and -2 were expressed in all tissues during the entire menstrual cycle. Herpesvirus entry mediator expression was limited mainly to some connective tissue cells. Both HSV-1 and HSV-2 exhibited a plaque-wise mucosal spread across the basement membrane and induced prominent epithelial syncytia.

Ultrastructural evidence of telocytes in the embryonic chick heart.

The cardiac telocyte (TC) is a novel interstitial cell type with a unique ultrastructure and great potential in therapy. The present study examined its presence in the heart of chicken embryos ageing 7-15 days old (Hamburger-Hamilton [HH] stages 31-41) using transmission electron microscopy. TCs were identified across all stages in the atrial and ventricular myocardium, close to maturing cardiomyocytes, blood vessels and lymphatics. Early-stage TCs have immature features resembling mesenchymal cells. Late-stage TCs were distinct, possessing the cytoplasmic prolongations termed telopodes (Tps), which are very long and thin, usually 1-3 in number, and display a moniliform appearance and have an average thickness below 0.2 µm. TCs residing in the epicardium and endocardium were also detected. In the subepicardium near developing coronary vessels, they were localized in the cardiac stem cell niches, coexisting with cardiac stem cells and cardiomyocyte progenitors. Electron-dense structures and the release of extracellular vesicles were observed between embryonic TCs and surrounding structures, suggesting roles in intercellular communication, cardiomyocyte differentiation and maturation, angiogenesis, and stem cell nursing and guidance.

Experimental Infection Models and Their Usefulness for White Spot Syndrome Virus (WSSV) Research in Shrimp.

White spot syndrome virus (WSSV) is marked as one of the most economically devastating pathogens in shrimp aquaculture worldwide. Infection of cultured shrimp can lead to mass mortality (up to 100%). Although progress has been made, our understanding of WSSV's infection process and the virus-host-environment interaction is far from complete. This in turn hinders the development of effective mitigation strategies against WSSV. Infection models occupy a crucial first step in the research flow that tries to elucidate the infectious disease process to develop new antiviral treatments. Moreover, since the establishment of continuous shrimp cell lines is a work in progress, the development and use of standardized in vivo infection models that reflect the host-pathogen interaction in shrimp is a necessity. This review critically examines key aspects of in vivo WSSV infection model development that are often overlooked, such as standardization, (post)larval quality, inoculum type and choice of inoculation procedure, housing conditions, and shrimp welfare considerations. Furthermore, the usefulness of experimental infection models for different lines of WSSV research will be discussed with the aim to aid researchers when choosing a suitable model for their research needs.

Granule exocytosis of granulysin and granzyme B as a potential key mechanism in vaccine-induced immunity in cattle against the nematode Ostertagia ostertagi.

Ostertagia ostertagi is considered one of the most economically important bovine parasites. As an alternative to anthelmintic treatment, an experimental host-protective vaccine was previously developed on the basis of ASP proteins derived from adult worms. Intramuscular injection of this vaccine, combined with QuilA as an adjuvant, significantly reduced fecal egg counts by 59%. However, the immunological mechanisms triggered by the vaccine are still unclear. Therefore, in this study, the differences in immune responses at the site of infection, i.e., the abomasal mucosa, between ASP-QuilA-vaccinated animals and QuilA-vaccinated control animals were investigated on a transcriptomic level by using a whole-genome bovine microarray combined with histological analysis. Sixty-nine genes were significantly impacted in animals protected by the vaccine, 48 of which were upregulated. A correlation study between the parasitological parameters and gene transcription levels showed that the transcription levels of two of the upregulated genes, those for granulysin (GNLY) and granzyme B (GZMB), were negatively correlated with cumulative fecal egg counts and total worm counts, respectively. Both genes were also positively correlated with each other and with another upregulated gene, that for the IgE receptor subunit (FCER1A). Surprisingly, these three genes were also correlated significantly with CMA1, which encodes a mast cell marker, and with counts of mast cells and cells previously described as globule leukocytes. Furthermore, immunohistochemical data showed that GNLY was present in the granules of globule leukocytes and that it was secreted in mucus. Overall, the results suggest a potential role for granule exocytosis by globule leukocytes, potentially IgE mediated, in vaccine-induced protection against O. ostertagi.