Long-lasting mRNA-encoded interleukin-2 restores CD8+ T cell neoantigen immunity in MHC class I-deficient cancers.

Major histocompatibility complex (MHC) class I antigen presentation deficiency is a common cancer immune escape mechanism, but the mechanistic implications and potential strategies to address this challenge remain poorly understood. Studying ß2-microglobulin (B2M) deficient mouse tumor models, we find that MHC class I loss leads to a substantial immune desertification of the tumor microenvironment (TME) and broad resistance to immune-, chemo-, and radiotherapy. We show that treatment with long-lasting mRNA-encoded interleukin-2 (IL-2) restores an immune cell infiltrated, IFNγ-promoted, highly proinflammatory TME signature, and when combined with a tumor-targeting monoclonal antibody (mAB), can overcome therapeutic resistance. Unexpectedly, the effectiveness of this treatment is driven by IFNγ-releasing CD8+ T cells that recognize neoantigens cross-presented by TME-resident activated macrophages. These macrophages acquire augmented antigen presentation proficiency and other M1-phenotype-associated features under IL-2 treatment. Our findings highlight the importance of restoring neoantigen-specific immune responses in the treatment of cancers with MHC class I deficiencies.

Morphological and phenotypical characteristics of porcine satellite glial cells of the dorsal root ganglia.

Satellite glial cells (SGCs) of the dorsal root ganglia (DRG) ensure homeostasis and proportional excitability of sensory neurons and gained interest in the field of development and maintenance of neuropathic pain. Pigs represent a suitable species for translational medicine with a more similar anatomy and physiology to humans compared to rodents, and are used in research regarding treatment of neuropathic pain. Knowledge of anatomical and physiological features of porcine SGCs is prerequisite for interpreting potential alterations. However, state of knowledge is still limited. In the present study, light microscopy, ultrastructural analysis and immunofluorescence staining was performed. SGCs tightly surround DRG neurons with little vascularized connective tissue between SGC-neuron units, containing, among others, axons and Schwann cells. DRG were mainly composed of large sized neurons (∼59%), accompanied by fewer medium sized (∼36%) and small sized sensory neurons (∼6%). An increase of neuronal body size was concomitant with an increased number of surrounding SGCs. The majority of porcine SGCs expressed glutamine synthetase and inwardly rectifying potassium channel Kir 4.1, known as SGC-specific markers in other species. Similar to canine SGCs, marked numbers of porcine SGCs were immunopositive for glial fibrillary acidic protein, 2',3'-cyclic-nucleotide 3'-phosphodiesterase and the transcription factor Sox2. Low to moderate numbers of SGCs showed aquaporin 4-immunoreactivity (AQP4) as described for murine SGCs. AQP4-immunoreactivity was primarily found in SGCs ensheathing small and medium sized neuronal somata. Low numbers of SGCs were immunopositive for ionized calcium-binding adapter molecule 1, indicating a potential immune cell character. No immunoreactivity for common leukocyte antigen CD45 nor neural/glial antigen 2 was detected. The present study provides essential insights into the characteristic features of non-activated porcine SGCs, contributing to a better understanding of this cell population and its functional aspects. This will help to interpret possible changes that might occur under activating conditions such as pain.

Intact Type I Interferon Receptor Signaling Prevents Hepatocellular Necrosis but Not Encephalitis in a Dose-Dependent Manner in Rift Valley Fever Virus Infected Mice.

Rift Valley fever (RVF) is a zoonotic and emerging disease, caused by the RVF virus (RVFV). In ruminants, it leads to "abortion storms" and enhanced mortality rates in young animals, whereas in humans it can cause symptoms like severe hemorrhagic fever or encephalitis. The role of the innate and adaptive immune response in disease initiation and progression is still poorly defined. The present study used the attenuated RVFV strain clone 13 to investigate viral spread, tissue tropism, and histopathological lesions after intranasal infection in C57BL/6 wild type (WT) and type I interferon (IFN-I) receptor I knockout (IFNAR-/-) mice. In WT mice, 104 PFU RVFV (high dose) resulted in a fatal encephalitis, but no hepatitis 7-11 days post infection (dpi), whereas 103 PFU RVFV (low dose) did not cause clinical disease or significant histopathological lesions in liver and the central nervous system (CNS). In contrast, IFNAR-/- mice infected with 103 PFU RVFV developed hepatocellular necrosis resulting in death at 2-5 dpi and lacked encephalitis. These results show that IFNAR signaling prevents systemic spread of the attenuated RVFV strain clone 13, but not the dissemination to the CNS and subsequent fatal disease. Consequently, neurotropic viruses may be able to evade antiviral IFN-I signaling pathways by using the transneuronal instead of the hematogenous route.

SARS-CoV-2 Omicron variant causes mild pathology in the upper and lower respiratory tract of hamsters.

Since its discovery in 2019, multiple variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have been identified. This study investigates virus spread and associated pathology in the upper and lower respiratory tracts of Syrian golden hamsters at 4 days post intranasal SARS-CoV-2 Omicron infection, in comparison to infection with variants of concern (VOCs) Gamma and Delta as well as ancestral strain 614 G. Pathological changes in the upper and lower respiratory tract of VOC Omicron infected hamsters are milder than those caused by other investigated strains. VOC Omicron infection causes a mild rhinitis with little involvement of the olfactory epithelium and minimal lesions in the lung, with frequent sparing of the alveolar compartment. Similarly, viral antigen, RNA and infectious virus titers are lower in respiratory tissues of VOC Omicron infected hamsters. These findings demonstrate that the variant has a decreased pathogenicity for the upper and lower respiratory tract of hamsters.

Astrocyte depletion alters extracellular matrix composition in the demyelinating phase of Theiler's murine encephalomyelitis.

Astrocytes produce extracellular matrix (ECM) glycoproteins contributing to the blood-brain barrier and regulating the immune response in the central nervous system (CNS). The aim of this study was to investigate the impact of astrocyte depletion upon the clinical outcome and the composition of ECM glycoproteins in a virus-induced animal model of demyelination. Glial fibrillary acidic protein (GFAP)-thymidine-kinase transgenic SJL (GFAP-knockout) and wildtype mice were infected with Theiler's murine encephalomyelitis virus (TMEV). Astrocyte depletion was induced during the progressive, demyelinating disease phase by ganciclovir administration once daily between 56 and 77 days post infection (dpi). At 77 dpi GFAP-knockout mice showed a significant deterioration of clinical signs associated with a reduction of azan and picrosirius red stained ECM-molecules in the thoracic spinal cord. Basement-membrane-associated ECM-molecules including laminin, entactin/nidogen-1 and Kir4.1 as well as non-basement membrane-associated ECM-molecules like collagen I, decorin, tenascin-R and CD44 were significantly reduced in the spinal cord of GFAP-knockout mice. The reduction of the investigated ECM-molecules demonstrates that astrocytes play a key role in the production of ECM-molecules. The present findings indicate that the detected loss of Kir4.1 and CD44 as well as the disruption of the integrity of perineuronal nets led to the deterioration of clinical signs in GFAP-knockout mice.

SARS-CoV-2 Infection Dysregulates Cilia and Basal Cell Homeostasis in the Respiratory Epithelium of Hamsters.

Similar to many other respiratory viruses, SARS-CoV-2 targets the ciliated cells of the respiratory epithelium and compromises mucociliary clearance, thereby facilitating spread to the lungs and paving the way for secondary infections. A detailed understanding of mechanism involved in ciliary loss and subsequent regeneration is crucial to assess the possible long-term consequences of COVID-19. The aim of this study was to characterize the sequence of histological and ultrastructural changes observed in the ciliated epithelium during and after SARS-CoV-2 infection in the golden Syrian hamster model. We show that acute infection induces a severe, transient loss of cilia, which is, at least in part, caused by cilia internalization. Internalized cilia colocalize with membrane invaginations, facilitating virus entry into the cell. Infection also results in a progressive decline in cells expressing the regulator of ciliogenesis FOXJ1, which persists beyond virus clearance and the termination of inflammatory changes. Ciliary loss triggers the mobilization of p73+ and CK14+ basal cells, which ceases after regeneration of the cilia. Although ciliation is restored after two weeks despite the lack of FOXJ1, an increased frequency of cilia with ultrastructural alterations indicative of secondary ciliary dyskinesia is observed. In summary, the work provides new insights into SARS-CoV-2 pathogenesis and expands our understanding of virally induced damage to defense mechanisms in the conducting airways.

Double-edged effects of tamoxifen-in-oil-gavage on an infectious murine model for multiple sclerosis.

Tamoxifen gavage is a commonly used method to induce genetic modifications in cre-loxP systems. As a selective estrogen receptor modulator (SERM), the compound is known to have immunomodulatory and neuroprotective properties in non-infectious central nervous system (CNS) disorders. It can even cause complete prevention of lesion development as seen in experimental autoimmune encephalitis (EAE). The effect on infectious brain disorders is scarcely investigated. In this study, susceptible SJL mice were infected intracerebrally with Theiler's murine encephalomyelitis virus (TMEV) and treated three times with a tamoxifen-in-oil-gavage (TOG), resembling an application scheme for genetically modified mice, starting at 0, 18, or 38 days post infection (dpi). All mice developed 'TMEV-induced demyelinating disease' (TMEV-IDD) resulting in inflammation, axonal loss, and demyelination of the spinal cord. TOG had a positive effect on the numbers of oligodendrocytes and oligodendrocyte progenitor cells, irrespective of the time point of application, whereas late application (starting 38 dpi) was associated with increased demyelination of the spinal cord white matter 85 dpi. Furthermore, TOG had differential effects on the CD4+ and CD8+ T cell infiltration into the CNS, especially a long lasting increase of CD8+ cells was detected in the inflamed spinal cord, depending of the time point of TOG application. Number of TMEV-positive cells, astrogliosis, astrocyte phenotype, apoptosis, clinical score, and motor function were not measurably affected. These data indicate that tamoxifen gavage has a double-edged effect on TMEV-IDD with the promotion of oligodendrocyte differentiation and proliferation, but also increased demyelination, depending on the time point of application. The data of this study suggest that tamoxifen has also partially protective functions in infectious CNS disease. These effects should be considered in experimental studies using the cre-loxP system, especially in models investigating neuropathologies.

Vascular Inflammation Is Associated with Loss of Aquaporin 1 Expression on Endothelial Cells and Increased Fluid Leakage in SARS-CoV-2 Infected Golden Syrian Hamsters.

Vascular changes represent a characteristic feature of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection leading to a breakdown of the vascular barrier and subsequent edema formation. The aim of this study was to provide a detailed characterization of the vascular alterations during SARS-CoV-2 infection and to evaluate the impaired vascular integrity. Groups of ten golden Syrian hamsters were infected intranasally with SARS-CoV-2 or phosphate-buffered saline (mock infection). Necropsies were performed at 1, 3, 6, and 14 days post-infection (dpi). Lung samples were investigated using hematoxylin and eosin, alcian blue, immunohistochemistry targeting aquaporin 1, CD3, CD204, CD31, laminin, myeloperoxidase, SARS-CoV-2 nucleoprotein, and transmission electron microscopy. SARS-CoV-2 infected animals showed endothelial hypertrophy, endothelialitis, and vasculitis. Inflammation mainly consisted of macrophages and lower numbers of T-lymphocytes and neutrophils/heterophils infiltrating the vascular walls as well as the perivascular region at 3 and 6 dpi. Affected vessels showed edema formation in association with loss of aquaporin 1 on endothelial cells. In addition, an ultrastructural investigation revealed disruption of the endothelium. Summarized, the presented findings indicate that loss of aquaporin 1 entails the loss of intercellular junctions resulting in paracellular leakage of edema as a key pathogenic mechanism in SARS-CoV-2 triggered pulmonary lesions.

Vasculitis and Neutrophil Extracellular Traps in Lungs of Golden Syrian Hamsters With SARS-CoV-2.

Neutrophil extracellular traps (NETs) have been identified as one pathogenetic trigger in severe COVID-19 cases and therefore well-described animal models to understand the influence of NETs in COVID-19 pathogenesis are needed. SARS-CoV-2 infection causes infection and interstitial pneumonia of varying severity in humans and COVID-19 models. Pulmonary as well as peripheral vascular lesions represent a severe, sometimes fatal, disease complication of unknown pathogenesis in COVID-19 patients. Furthermore, neutrophil extracellular traps (NETs), which are known to contribute to vessel inflammation or endothelial damage, have also been shown as potential driver of COVID-19 in humans. Though most studies in animal models describe the pulmonary lesions characterized by interstitial inflammation, type II pneumocyte hyperplasia, edema, fibrin formation and infiltration of macrophages and neutrophils, detailed pathological description of vascular lesions or NETs in COVID-19 animal models are lacking so far. Here we report different types of pulmonary vascular lesions in the golden Syrian hamster model of COVID-19. Vascular lesions included endothelialitis and vasculitis at 3 and 6 days post infection (dpi), and were almost nearly resolved at 14 dpi. Importantly, virus antigen was present in pulmonary lesions, but lacking in vascular alterations. In good correlation to these data, NETs were detected in the lungs of infected animals at 3 and 6 dpi. Hence, the Syrian hamster seems to represent a useful model to further investigate the role of vascular lesions and NETs in COVID-19 pathogenesis.

Amelanotic Malignant Melanoma in a Himalayan Rex Guinea Pig (Cavia porcellus).

A Himalayan Rex guinea pig was presented with a history of nodular, partially ulcerated masses in the subcutis of the left shoulder. Histological examination revealed a garland-like to nodular, infiltrative neoplastic mass of the epidermis and hair follicle epithelium, which obscured the dermoepidermal junction. Neoplastic cells were immunopositive for S100, PNL-2, vimentin and melan-A antigens. No immunolabelling of CD3, CD79, Iba-1 or pancytokeratin was observed. This is the first detailed description of spontaneous amelanotic malignant melanoma in this species.

Equine Idiopathic Systemic Granulomatous Disease With Manifestation in the Cerebellum Associated With Equid Gammaherpesvirus 2.

Idiopathic systemic granulomatous disease (ISGD), also known as equine sarcoidosis is an uncommon disease of horses, manifesting in exfoliative dermatitis and granulomatous inflammation in various organs. The current report presents a case of a 15-year-old Hanoverian mare with a 4-month history of weight loss, recurrent fever, skin lesions, and movement disorders. Pathological examination revealed granulomatous and necrotizing inflammation in the skin, regional lymph nodes, and cerebellum. Based on histological, immunohistochemical, and microbiological findings, the diagnosis of ISGD was made. Sequencing of the polymerase chain reaction product of pooled brain tissue revealed the presence of equid gammaherpesvirus 2 DNA. This case is the first description of generalized ISGD with granulomatous dermatitis simultaneously affecting the skin and cerebellum.

Persistent Bilateral Mydriasis Associated With a Pituitary Adenoma in a Horse.

Severe bilateral mydriasis and bilaterally decreased vision were observed in a 23-year-old crossbred warmblood gelding with a history of pituitary pars intermedia dysfunction. Ten years after the onset of clinical signs, it was killed humanely because of worsening of clinical signs and loss of therapeutic responsiveness. Postmortem examination of the head was performed to confirm the suspected pituitary neoplasm and to investigate secondary oculomotor lesions. Pathomorphologic examination revealed an expansile space-occupying pituitary adenoma and degenerative changes in the preganglionic oculomotor nerve, ciliary ganglion, and optic chiasm. The ocular clinical findings share features of a cavernous sinus syndrome.

Impact of Astrocyte Depletion upon Inflammation and Demyelination in a Murine Animal Model of Multiple Sclerosis.

Astrocytes play a key role in demyelinating diseases, like multiple sclerosis (MS), although many of their functions remain unknown. The aim of this study was to investigate the impact of astrocyte depletion upon de- and remyelination, inflammation, axonal damage, and virus distribution in Theiler`s murine encephalomyelitis (TME). Groups of two to six glial fibrillary acidic protein (GFAP)-thymidine-kinase transgenic SJL mice and SJL wildtype mice were infected with TME virus (TMEV) or mock (vehicle only). Astrocyte depletion was induced by the intraperitoneal administration of ganciclovir during the early and late phase of TME. The animals were clinically investigated while using a scoring system and a rotarod performance test. Necropsies were performed at 46 and 77 days post infection. Cervical and thoracic spinal cord segments were investigated using hematoxylin and eosin (H&E), luxol fast blue-cresyl violet (LFB), immunohistochemistry targeting Amigo2, aquaporin 4, CD3, CD34, GFAP, ionized calcium-binding adapter molecule 1 (Iba1), myelin basic protein (MBP), non-phosphorylated neurofilaments (np-NF), periaxin, S100A10, TMEV, and immunoelectron microscopy. The astrocyte depleted mice showed a deterioration of clinical signs, a downregulation and disorganization of aquaporin 4 in perivascular astrocytes accompanied by vascular leakage. Furthermore, astrocyte depleted mice showed reduced inflammation and lower numbers of TMEV positive cells in the spinal cord. The present study indicates that astrocyte depletion in virus triggered CNS diseases contributes to a deterioration of clinical signs that are mediated by a dysfunction of perivascular astrocytes.