Microbiota from human infants consuming secretors or non-secretors mothers' milk impacts the gut and immune system in mice.

Maternal secretor status is one of the determinants of human milk oligosaccharides (HMOs) composition, which, in turn, influences the gut microbiota composition of infants. To understand if this change in gut microbiota impacts immune cell composition, intestinal morphology, and gene expression, 21-day-old germ-free C57BL/6 mice were transplanted with fecal microbiota from infants whose mothers were either secretors (SMM) or non-secretors (NSM) or from infants consuming dairy-based formula (MFM). For each group, one set of mice was supplemented with HMOs. HMO supplementation did not significantly impact the microbiota diversity; however, SMM mice had a higher abundance of genus Bacteroides, Bifidobacterium, and Blautia, whereas, in the NSM group, there was a higher abundance of Akkermansia, Enterocloster, and Klebsiella. In MFM, gut microbiota was represented mainly by Parabacteroides, Ruminococcaceae_unclassified, and Clostrodium_sensu_stricto. In mesenteric lymph node, Foxp3+ T cells and innate lymphoid cells type 2 were increased in MFM mice supplemented with HMOs, while in the spleen, they were increased in SMM + HMOs mice. Similarly, serum immunoglobulin A was also elevated in MFM + HMOs group. Distinct global gene expression of the gut was observed in each microbiota group, which was enhanced with HMOs supplementation. Overall, our data show that distinct infant gut microbiota due to maternal secretor status or consumption of dairy-based formula and HMO supplementation impacts immune cell composition, antibody response, and intestinal gene expression in a mouse model. IMPORTANCE: Early life factors like neonatal diet modulate gut microbiota, which is important for the optimal gut and immune function. One such factor, human milk oligosaccharides (HMOs), the composition of which is determined by maternal secretor status, has a profound effect on infant gut microbiota. However, how the infant gut microbiota composition determined by maternal secretor status or consumption of infant formula devoid of HMOs impacts infant intestinal ammorphology, gene expression, and immune signature is not well explored. This study provides insights into the differential establishment of infant microbiota derived from infants fed by secretor or non-secretor mothers milk or those consuming infant formula and demonstrates that the secretor status of mothers promotes Bifidobacteria and Bacteroides sps. establishment. This study also shows that supplementation of pooled HMOs in mice changed immune cell composition in the spleen and mesenteric lymph nodes and immunoglobulins in circulation. Hence, this study highlights that maternal secretor status has a role in infant gut microbiota composition, and this, in turn, can impact host gut and immune system.

Colonic epithelial hypoxia remains constant during the progression of diabetes in male UC Davis type 2 diabetes mellitus rats.

INTRODUCTION: Colonocyte oxidation of bacterial-derived butyrate has been reported to maintain synergistic obligate anaerobe populations by reducing colonocyte oxygen levels; however, it is not known whether this process is disrupted during the progression of type 2 diabetes. Our aim was to determine whether diabetes influences colonocyte oxygen levels in the University of California Davis type 2 diabetes mellitus (UCD-T2DM) rat model. RESEARCH DESIGN AND METHODS: Age-matched male UCD-T2DM rats (174±4 days) prior to the onset of diabetes (PD, n=15), within 1 month post-onset (RD, n=12), and 3 months post-onset (D3M, n=12) were included in this study. Rats were administered an intraperitoneal injection of pimonidazole (60 mg/kg body weight) 1 hour prior to euthanasia and tissue collection to estimate colonic oxygen levels. Colon tissue was fixed in 10% formalin, embedded in paraffin, and processed for immunohistochemical detection of pimonidazole. The colonic microbiome was assessed by 16S gene rRNA amplicon sequencing and content of short-chain fatty acids was measured by liquid chromatography-mass spectrometry. RESULTS: HbA1c % increased linearly across the PD (5.9±0.1), RD (7.6±0.4), and D3M (11.5±0.6) groups, confirming the progression of diabetes in this cohort. D3M rats had a 2.5% increase in known facultative anaerobes, Escherichia-Shigella, and Streptococcus (false discovery rate <0.05) genera in colon contents. The intensity of pimonidazole staining of colonic epithelia did not differ across groups (p=0.37). Colon content concentrations of acetate and propionate also did not differ across UCD-T2DM groups; however, colonic butyric acid levels were higher in D3M rats relative to PD rats (p<0.01). CONCLUSIONS: The advancement of diabetes in UCD-T2DM rats was associated with an increase in facultative anaerobes; however, this was not explained by changes in colonocyte oxygen levels. The mechanisms underlying shifts in gut microbe populations associated with the progression of diabetes in the UCD-T2DM rat model remain to be identified.

Case report: A case of oviductal and uterine leiomyosarcoma in an 11-year-old dog.

An 11-year-old, intact female Pomeranian dog was presented for evaluation due to an 18-h history of anorexia and lethargy. Abdominal ultrasound revealed a 3×3 cm mass of mixed echogenicity at the level of the left ovary. At laparotomy, a 5 mm mass was identified at the cranial region of the right uterine horn and a 3 cm round mass was visualized near the cranial aspect of the left uterine horn. Ovariohysterectomy was performed. A diagnosis of grade 1 oviductal and uterine leiomyosarcoma was made via histopathology for both masses. Oviductal leiomyosarcomas are rare and generally locally invasive similar to other soft tissue sarcomas but do not often metastasize. Uterine leiomyosarcomas are also uncommon but are one of the more common tumors affecting the female reproductive tract. This is the only known case report of oviductal leiomyosarcoma in the dog and the only report of uterine leiomyosarcoma in addition to oviductal leiomyosarcoma as well. This case illustrates the oviduct as an additional site that can be affected by leiomyosarcoma and demonstrates surgery as a treatment option for patients diagnosed with this condition.

An FcRn-targeted mucosal vaccine against SARS-CoV-2 infection and transmission.

SARS-CoV-2 is primarily transmitted through droplets and airborne aerosols, and in order to prevent infection and reduce viral spread vaccines should elicit protective immunity in the airways. The neonatal Fc receptor (FcRn) transfers IgG across epithelial barriers and can enhance mucosal delivery of antigens. Here we explore FcRn-mediated respiratory delivery of SARS-CoV-2 spike (S). A monomeric IgG Fc was fused to a stabilized spike; the resulting S-Fc bound to S-specific antibodies and FcRn. Intranasal immunization of mice with S-Fc and CpG significantly induced antibody responses compared to the vaccination with S alone or PBS. Furthermore, we intranasally immunized mice or hamsters with S-Fc. A significant reduction of virus replication in nasal turbinate, lung, and brain was observed following nasal challenges with SARS-CoV-2 and its variants. Intranasal immunization also significantly reduced viral airborne transmission in hamsters. Nasal IgA, neutralizing antibodies, lung-resident memory T cells, and bone-marrow S-specific plasma cells mediated protection. Hence, FcRn delivers an S-Fc antigen effectively into the airway and induces protection against SARS-CoV-2 infection and transmission.

Deficiency of macrophage-derived Dnase1L3 causes lupus-like phenotypes in mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease caused by environmental factors and loss of key proteins, including the endonuclease Dnase1L3. Dnase1L3 absence causes pediatric-onset lupus in humans, while reduced activity occurs in adult-onset SLE. The amount of Dnase1L3 that prevents lupus remains unknown. To genetically reduce Dnase1L3 levels, we developed a mouse model lacking Dnase1L3 in macrophages (conditional knockout [cKO]). Serum Dnase1L3 levels were reduced 67%, though Dnase1 activity remained constant. Homogeneous and peripheral antinuclear antibodies were detected in the sera by immunofluorescence, consistent with anti-double-stranded DNA (anti-dsDNA) antibodies. Total immunoglobulin M, total immunoglobulin G, and anti-dsDNA antibody levels increased in cKO mice with age. The cKO mice developed anti-Dnase1L3 antibodies. In contrast to global Dnase1L3-/- mice, anti-dsDNA antibodies were not elevated early in life. The cKO mice had minimal kidney pathology. Therefore, we conclude that an intermediate reduction in serum Dnase1L3 causes mild lupus phenotypes, and macrophage-derived DnaselL3 helps limit lupus.

Inhibitory checkpoint molecule mRNA expression in canine soft tissue sarcoma.

Canine soft tissue sarcomas (STS) are common neoplasms and considered immune deserts. Tumour infiltrating lymphocytes are sparse in STS and, when present, tend to organize around blood vessels or at the periphery of the neoplasm. This pattern is associated with an immunosuppressive tumour microenvironment linked to overexpression of molecules of the PD-axis. PD-1, PD-L1 and PD-L2 expression correlates with malignancy and poor prognosis in other neoplasms in humans and dogs, but little is known about their role in canine STS, their relationship to tumour grade, and how different therapies affect expression. The objective of this study was to evaluate the expression of checkpoint molecules across STS tumour grades and after tumour ablation treatment. Gene expression analysis was performed by reverse-transcriptase real-time quantitative PCR in soft tissue sarcomas that underwent histotripsy and from histologic specimens of STS from the Virginia Tech Animal Laboratory Services archives. The expression of PD-1, PD-L1 and PD-L2 was detected in untreated STS tissue representing grades 1, 2, and 3. Numerically decreased expression of all markers was observed in tissue sampled from the treatment interface relative to untreated areas of the tumour. The relatively lower expression of these checkpoint molecules at the periphery of the treated area may be related to liquefactive necrosis induced by the histotripsy treatment, and would potentially allow TILs to infiltrate the tumour. Relative increases of these checkpoint molecules in tumours of a higher grade and alongside immune cell infiltration are consistent with previous reports that associate their expression with malignancy.

Deficiency of macrophage-derived Dnase1L3 causes lupus-like phenotypes in mice.

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease caused by environmental factors and loss of key proteins. One such protein is a serum endonuclease secreted by macrophages and dendritic cells, Dnase1L3. Loss of Dnase1L3 causes pediatric-onset lupus in humans is Dnase1L3. Reduction in Dnase1L3 activity occurs in adult-onset human SLE. However, the amount of Dnase1L3 necessary to prevent lupus onset, if the impact is continuous or requires a threshold, and which phenotypes are most impacted by Dnase1L3 remain unknown. To reduce Dnase1L3 protein levels, we developed a genetic mouse model with reduced Dnase1L3 activity by deleting Dnase1L3 from macrophages (cKO). Serum Dnase1L3 levels were reduced 67%, though Dnase1 activity remained constant. Sera were collected weekly from cKO and littermate controls until 50 weeks of age. Homogeneous and peripheral anti-nuclear antibodies were detected by immunofluorescence, consistent with anti-dsDNA antibodies. Total IgM, total IgG, and anti-dsDNA antibody levels increased in cKO mice with increasing age. In contrast to global Dnase1L3 -/- mice, anti-dsDNA antibodies were not elevated until 30 weeks of age. The cKO mice had minimal kidney pathology, except for deposition of immune complexes and C3. Based on these findings, we conclude that an intermediate reduction in serum Dnase1L3 causes mild lupus phenotypes. This suggest that macrophage-derived DnaselL3 is critical to limiting lupus.

The E2 glycoprotein holds key residues for Mayaro virus adaptation to the urban Aedes aegypti mosquito.

Adaptation to mosquito vectors suited for transmission in urban settings is a major driver in the emergence of arboviruses. To better anticipate future emergence events, it is crucial to assess their potential to adapt to new vector hosts. In this work, we used two different experimental evolution approaches to study the adaptation process of an emerging alphavirus, Mayaro virus (MAYV), to Ae. aegypti, an urban mosquito vector of many other arboviruses. We identified E2-T179N as a key mutation increasing MAYV replication in insect cells and enhancing transmission after escaping the midgut of live Ae. aegypti. In contrast, this mutation decreased viral replication and binding in human fibroblasts, a primary cellular target of MAYV in humans. We also showed that MAYV E2-T179N generates reduced viremia and displays less severe tissue pathology in vivo in a mouse model. We found evidence in mouse fibroblasts that MAYV E2-T179N is less dependent on the Mxra8 receptor for replication than WT MAYV. Similarly, exogenous expression of human apolipoprotein receptor 2 and Mxra8 enhanced WT MAYV replication compared to MAYV E2-T179N. When this mutation was introduced in the closely related chikungunya virus, which has caused major outbreaks globally in the past two decades, we observed increased replication in both human and insect cells, suggesting E2 position 179 is an important determinant of alphavirus host-adaptation, although in a virus-specific manner. Collectively, these results indicate that adaptation at the T179 residue in MAYV E2 may result in increased vector competence-but coming at the cost of optimal replication in humans-and may represent a first step towards a future emergence event.

Fetal Loss in Pregnant Rabbits Infected with Genotype 3 Hepatitis E Virus Is Associated with Altered Inflammatory Responses, Enhanced Virus Replication, and Extrahepatic Virus Dissemination with Positive Correlations with Increased Estradiol Level.

Hepatitis E virus (HEV) causes adverse clinical outcomes in pregnant women, but the underlying mechanisms remain poorly understood. To delineate the mechanisms of pregnancy-associated adverse effects during HEV infection, we utilized a genotype 3 HEV from rabbit (HEV-3ra) and its cognate host (rabbits) to systematically investigate the clinical consequences, viral replication dynamics, and host immune and hormonal responses of HEV infection during pregnancy. We found a significant fetal loss of 23% in HEV-infected pregnant rabbits, indicating an early-stage miscarriage. HEV infection in pregnant rabbits was characterized by higher viral loads in feces, intestinal contents, liver, and spleen tissues, as well as a longer and earlier onset of viremia than in infected nonpregnant rabbits. HEV infection altered the pattern of cytokine gene expressions in the liver of pregnant rabbits and caused a transient increase of serum interferon gamma (IFN-γ) shortly after a notable increase in viral replication, which may contribute to early fetal loss. Histological lesions in the spleen were more pronounced in infected pregnant rabbits, although moderate liver lesions were seen in both infected pregnant and nonpregnant rabbits. Total bilirubin was elevated in infected pregnant rabbits. The serum levels of estradiol (E2) in HEV-infected pregnant rabbits were significantly higher than those in mock-infected pregnant rabbits at 14 days postinoculation (dpi) and correlated positively with higher viral loads in feces, liver, and spleen tissues at 28 dpi, suggesting that it may play a role in extrahepatic virus dissemination. The results have important implications for understanding the severe diseases associated with HEV infection during pregnancy. IMPORTANCE HEV causes adverse pregnancy outcomes, with a mortality rate of >30% in pregnant women, but the underlying mechanisms are poorly understood. In this study, we utilized HEV-3ra and its cognate host (pregnant rabbit) to delineate the potential underlying mechanisms of pregnancy-associated adverse outcomes during HEV infection. We found that infected pregnant rabbits had a fetal loss of 23%, which coincided with enhanced viral replication and an elevated systemic IFN-γ response, followed by longer viremia duration and extrahepatic viral dissemination. Estradiol levels were increased in infected pregnant rabbits and correlated positively with higher fecal viral shedding and higher viral loads in liver and spleen tissues. Infected pregnant rabbits had more pronounced splenic lesions, higher serum total bilirubin, and an altered cytokine gene expression profile in the liver. The results will contribute to our understanding of the mechanisms of HEV-associated adverse pregnancy outcomes.

Ribavirin Treatment Failure-Associated Mutation, Y1320H, in the RNA-Dependent RNA Polymerase of Genotype 3 Hepatitis E Virus (HEV) Enhances Virus Replication in a Rabbit HEV Infection Model.

Chronic hepatitis E virus (HEV) infection has become a significant clinical problem that requires treatment in immunocompromised individuals. In the absence of an HEV-specific antiviral, ribavirin (RBV) has been used off-label, but treatment failure may occur due to mutations in the viral RNA-dependent RNA polymerase (RdRp), including Y1320H, K1383N, and G1634R. Chronic hepatitis E is mostly caused by zoonotic genotype 3 HEV (HEV-3), and HEV variants from rabbits (HEV-3ra) are closely related to human HEV-3. Here, we explored whether HEV-3ra, along with its cognate host, can serve as a model to study RBV treatment failure-associated mutations observed in human HEV-3-infected patients. By utilizing the HEV-3ra infectious clone and indicator replicon, we generated multiple single mutants (Y1320H, K1383N, K1634G, and K1634R) and a double mutant (Y1320H/K1383N) and assessed the role of mutations on replication and antiviral activity of HEV-3ra in cell culture. Furthermore, we also compared the replication of the Y1320H mutant with the wild-type HEV-3ra in experimentally infected rabbits. Our in vitro analyses revealed that the effects of these mutations on rabbit HEV-3ra are altogether highly consistent with those on human HEV-3. Importantly, we found that the Y1320H enhances virus replication during the acute stage of HEV-3ra infection in rabbits, which corroborated our in vitro results showing an enhanced viral replication of Y1320H. Taken together, our data suggest that HEV-3ra and its cognate host is a useful and relevant naturally occurring homologous animal model to study the clinical relevance of antiviral-resistant mutations observed in human HEV-3 chronically-infected patients. IMPORTANCE HEV-3 causes chronic hepatitis E that requires antiviral therapy in immunosuppressed individuals. RBV is the main therapeutic option for chronic hepatitis E as an off-label use. Several amino acid changes, including Y1320H, K1383N, and G1634R, in the RdRp of human HEV-3 have reportedly been associated with RBV treatment failure in chronic hepatitis E patients. In this study, we utilized an HEV-3ra from rabbit and its cognate host to investigate the effect of these RBV treatment failure-associated HEV-3 RdRp mutations on viral replication efficiency and antiviral susceptibility. The in vitro data using rabbit HEV-3ra was highly comparable to those from human HEV-3. We demonstrated that the Y1320H mutation significantly enhanced HEV-3ra replication in cell culture and enhanced virus replication during the acute stage of HEV-3ra infection in rabbits. The rabbit HEV-3ra infection model should be useful in delineating the role of human HEV-3 RBV treatment failure-associated mutations in antiviral resistance.

Evaluation of a Plant-Based Infant Formula Containing Almonds and Buckwheat on Gut Microbiota Composition, Intestine Morphology, Metabolic and Immune Markers in a Neonatal Piglet Model.

A controlled-neonatal piglet trial was conducted to evaluate the impact of a plant-based infant formula containing buckwheat and almonds as the main source of protein compared to a commercially available dairy-based formula on the gut health parameters. Two day old piglets were fed either a plant-based or a dairy-based formula until day 21. Gut microbiome, cytokines, growth and metabolism related outcomes, and intestinal morphology were evaluated to determine the safety of the plant-based infant formula. This study reported that the plant-based formula-fed piglets had a similar intestinal microbiota composition relative to the dairy-based formula-fed group. However, differential abundance of specific microbiota species was detected within each diet group in the small and large intestinal regions and fecal samples. Lactobacillus delbrueckii, Lactobacillus crispatus, and Fusobacterium sp. had higher abundance in the small intestine of plant-based formula-fed piglets compared to the dairy-based group. Bacteroides nordii, Enterococcus sp., Lactobacillus crispatus, Prevotella sp., Ruminococcus lactaris, Bacteroides nordii, Eisenbergiella sp., Lactobacillus crispatus, Prevotella sp., and Akkermansia muciniphila had greater abundance in the large intestine of the plant based diet fed piglets relative to the dairy-based diet group. In the feces, Clostridiales, Bacteroides uniformis, Butyricimonasvirosa, Cloacibacillus porcorum, Clostridium clostridioforme, and Fusobacterium sp. were abundant in dairy-based group relative to the plant-based group. Lachnospiraceae, Clostridium scindens, Lactobacillus coleohominis, and Prevetolla sp. had greater abundance in the feces of the plant-based group in comparison to the dairy-based group. Gut morphology was similar between the plant and the dairy-based formula-fed piglets. Circulatory cytokines, magnesium, triiodothyronine (T3), thyroxine (T4), thyroid stimulating hormone (TSH), vitamin D, vitamin K, and IgE levels were similar among all piglets independent of dietary group. Overall, the present study demonstrated that a plant-based formula with buckwheat and almonds as the primary source of protein can support similar gut microbiota growth and health outcomes compared to a dairy-based infant formula.

A hepatitis B virus core antigen-based virus-like particle vaccine expressing SARS-CoV-2 B and T cell epitopes induces epitope-specific humoral and cell-mediated immune responses but confers limited protection against SARS-CoV-2 infection.

The hepatitis B virus core antigen (HBcAg) tolerates insertion of foreign epitopes and maintains its ability to self-assemble into virus-like particles (VLPs). We constructed a ∆HBcAg-based VLP vaccine expressing three predicted severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B and T cell epitopes and determined its immunogenicity and protective efficacy. The recombinant ∆HBcAg-SARS-CoV-2 protein was expressed in Escherichia coli, purified, and shown to form VLPs. K18-hACE2 transgenic C57BL/6 mice were immunized intramuscularly with ∆HBcAg VLP control (n = 15) or ∆HBcAg-SARS-CoV-2 VLP vaccine (n = 15). One week after the 2nd booster and before virus challenge, five ∆HBcAg-SARS-CoV-2 vaccinated mice were euthanized to evaluate epitope-specific immune responses. There is a statistically significant increase in epitope-specific Immunoglobulin G (IgG) response, and statistically higher interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) expression levels in ∆HBcAg-SARS-CoV-2 VLP-vaccinated mice compared to ∆HBcAg VLP controls. While not statistically significant, the ∆HBcAg-SARS-CoV-2 VLP mice had numerically more memory CD8+ T-cells, and 3/5 mice also had numerically higher levels of interferon gamma (IFN-γ) and tumor necrosis factor (TNF). After challenge with SARS-CoV-2, ∆HBcAg-SARS-CoV-2 immunized mice had numerically lower viral RNA loads in the lung, and slightly higher survival, but the differences are not statistically significant. These results indicate that the ∆HBcAg-SARS-CoV-2 VLP vaccine elicits epitope-specific humoral and cell-mediated immune responses but they were insufficient against SARS-CoV-2 infection.

Maternal antibody repertoire restriction modulates the development of lupus-like disease in BXSB offspring.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease that has a strong preference for women of child-bearing age. Maternal factors play an essential role in shaping the immune system of the newborn, yet it is unknown whether maternal factors could modulate the development of SLE in the offspring. Activation-induced cytidine deaminase (AID) is an enzyme required for somatic hypermutation and class switch recombination. Given that IgG and IgA isotypes account for the vast majority of passive immunity in rodents, our previously established AID-deficient BXSB mice provide a model in which maternal antibodies that can be transferred to the offspring are greatly diminished and have restricted repertoire. In this study, we compared genotypically identical mice born to either AID-sufficient dams or AID-deficient dams and evaluated the effects of maternal antibodies in disease progression. Offspring from knockout dams developed disease at a faster rate, as shown by more severe nephritis and elevated pathogenic autoantibodies compared to their counterparts born to wild-type dams. When immune competent pups were cross fostered onto AID-deficient dams, these mice exhibited more severe disease characteristics, including exacerbated lupus nephritis, increased levels of circulating antinuclear antibodies, and more activated T cells. These results suggest that a protective antibody effect contributes to the modulation of SLE progression in postnatal period. Overall, these findings highlight the importance of maternal antibodies in programming the immune system and altering SLE development in offspring.

In-Transit Metastasis in a Dog with High-Grade Soft Tissue Sarcoma: A Case Report.

A 6 yr old male castrated American Staffordshire terrier was referred for a nonhealing wound at the site of a previously incompletely excised, high-grade soft tissue sarcoma. Physical examination revealed right popliteal lymphadenopathy and a fungating mass of the right pelvic limb at the level of the hock. Thoracic and abdominal computed tomography revealed mild lymphadenopathy of multiple iliac and inguinal lymph nodes. Right pelvic limb amputation and inguinal lymphadenectomy were performed. Histopathology was consistent of a high-grade soft tissue sarcoma with diffuse spread through the lymphatic vessels of the right pelvic limb up to the right inguinal lymph node but not affecting the lymph node itself. Doxorubicin chemotherapy was elected postoperatively as adjuvant therapy. Approximately 4 mo following initiation of chemotherapy, the patient developed a firm, tubular subcutaneous mass starting near the previous amputation site with tracking toward the thorax. Fine needle aspiration of the new mass was consistent with atypical spindle cell proliferation. Palliative care was elected, and the patient was euthanized 3 mo later because of progressive disease. In-transit metastasis is a rare behavior for soft tissue sarcomas across all species, and this is the first report of such a presentation for canine soft tissue sarcoma.

An FcRn-targeted mucosal vaccine against SARS-CoV-2 infection and transmission.

SARS-CoV-2 and its variants cause COVID-19, which is primarily transmitted through droplets and airborne aerosols. To prevent viral infection and reduce viral spread, vaccine strategies must elicit protective immunity in the airways. FcRn transfers IgG across epithelial barriers; we explore FcRn-mediated respiratory delivery of SARS-CoV-2 spike (S). A monomeric IgG Fc was fused to a stabilized S protein; the resulting S-Fc bound to S-specific antibodies (Ab) and FcRn. A significant increase in Ab responses was observed following the intranasal immunization of mice with S-Fc formulated in CpG as compared to the immunization with S alone or PBS. Furthermore, we intranasally immunize adult or aged mice and hamsters with S-Fc. A significant reduction of virus replication in nasal turbinate, lung, and brain was observed following nasal challenges with SARS-CoV-2, including Delta and Omicron variants. Intranasal immunization also significantly reduced viral transmission between immunized and naive hamsters. Protection was mediated by nasal IgA, serum-neutralizing Abs, tissue-resident memory T cells, and bone marrow S-specific plasma cells. Hence FcRn delivers an S-Fc antigen effectively into the airway and induces protection against SARS-CoV-2 infection and transmission. Based on these findings, FcRn-targeted non-invasive respiratory immunizations are superior strategies for preventing highly contagious respiratory viruses from spreading.

North American House Sparrows Are Competent for Usutu Virus Transmission.

Usutu virus (USUV, Flaviviridae) is an emerging mosquito-borne virus that has been implicated in neuroinvasive disease in humans and epizootic deaths in wild birds. USUV is maintained in an enzootic cycle between ornithophilic mosquitoes, primarily Culex spp., and wild birds, predominantly passerine species. However, limited experimental data exist on the species competent for USUV transmission. Here, we demonstrate that house sparrows are susceptible to multiple USUV strains. Our study also revealed that Culex quinquefasciatus mosquitoes are susceptible to USUV, with a significantly higher infection rate for the Netherlands 2016 USUV strain compared to the Uganda 2012 USUV strain at 50% and 19%, respectively. To assess transmission between avian host and mosquito vector, we allowed mosquitoes to feed on either juvenile chickens or house sparrows inoculated with USUV. Both bird models transmitted USUV to C. quinquefasciatus mosquitoes. Linear regression analyses indicated that C. quinquefasciatus infection rates were positively correlated with avian viremia levels, with 3 to 4 log10 PFU/mL representing the minimum avian viremia threshold for transmission to mosquitoes. Based on the viremia required for transmission, house sparrows were estimated to more readily transmit the Netherlands 2016 strain compared to the Uganda 2012 strain. These studies provide insights on a competent reservoir host of USUV. IMPORTANCE Usutu virus (USUV) is a zoonotic mosquito-borne virus that can cause neuroinvasive disease, including meningitis and encephalitis, in humans and has resulted in hundreds of thousands of deaths in wild birds. The perpetuation of USUV in nature is dependent on transmission between Culex spp. mosquitoes and various avian species. To date, few experimental data exist for determining which bird species are important for the maintenance of USUV. Our studies showed that house sparrows can transmit infectious Usutu virus, indicating their role as a competent host species. By identifying reservoir species of USUV, we can predict areas of USUV emergence and mitigate its impacts on global human and wildlife health.

Human Milk Oligosaccharides Impact Cellular and Inflammatory Gene Expression and Immune Response.

Human milk harbors complex carbohydrates, including human milk oligosaccharides (HMOs), the third most abundant component after lactose and lipids. HMOs have been shown to impact intestinal microbiota, modulate the intestinal immune response, and prevent pathogenic bacterial binding by serving as decoy receptors. However, the direct effect of HMOs on intestinal function and immunity remains to be elucidated. To address this knowledge gap, 21-day-old germ-free mice (C57BI/6) were orally gavaged with 15 mg/day of pooled HMOs for 7 or 14 days and euthanized at day 28 or 35. A set of mice was maintained until day 50 to determine the persistent effects of HMOs. Control groups were maintained in the isolators for 28, 35, or 50 days of age. At the respective endpoints, intestinal tissues were subjected to histomorphometric and transcriptomic analyses, while the spleen and mesenteric lymph nodes (MLNs) were subjected to flow cytometric analysis. The small intestine (SI) crypt was reduced after HMO treatment relative to control at days 28 and 35, while the SI villus height and large intestine (LI) gland depth were decreased in the HMO-treated mice relative to the control at day 35. We report significant HMO-induced and location-specific gene expression changes in host intestinal tissues. HMO treatment significantly upregulated genes involved in extracellular matrix, protein ubiquitination, nuclear transport, and mononuclear cell differentiation. CD4+ T cells were increased in both MLNs and the spleen, while CD8+ T cells were increased in the spleen at day 50 in the HMO group in comparison to controls. In MLNs, plasma cells were increased in HMO group at days 28 and 35, while in the spleen, only at day 28 relative to controls. Macrophages/monocytes and neutrophils were lower in the spleen of the HMO group at days 28, 35, and 50, while in MLNs, only neutrophils were lower at day 50 in the 14-day HMO group. In addition, diphtheria toxoid and tetanus toxoid antibody-secreting cells were higher in HMO-supplemented group compared to controls. Our data suggest that HMOs have a direct effect on gastrointestinal tract metabolism and the immune system even in the absence of host microbiota.

Hepatitis E virus infects brain microvascular endothelial cells, crosses the blood-brain barrier, and invades the central nervous system.

Hepatitis E virus (HEV) is an important but understudied zoonotic virus causing both acute and chronic viral hepatitis. A proportion of HEV-infected individuals also developed neurological diseases such as Guillain-Barré syndrome, neuralgic amyotrophy, encephalitis, and myelitis, although the mechanism remains unknown. In this study, by using an in vitro blood-brain barrier (BBB) model, we first investigated whether HEV can cross the BBB and whether the quasi-enveloped HEV virions are more permissible to the BBB than the nonenveloped virions. We found that both quasi-enveloped and nonenveloped HEVs can similarly cross the BBB and that addition of proinflammatory cytokine tumor necrosis factor alpha (TNF-α) has no significant effect on the ability of HEV to cross the BBB in vitro. To explore the possible mechanism of HEV entry across the BBB, we tested the susceptibility of human brain microvascular endothelial cells lining the BBB to HEV infection and showed that brain microvascular endothelial cells support productive HEV infection. To further confirm the in vitro observation, we conducted an experimental HEV infection study in pigs and showed that both quasi-enveloped and nonenveloped HEVs invade the central nervous system (CNS) in pigs, as HEV RNA was detected in the brain and spinal cord of infected pigs. The HEV-infected pigs with detectable viral RNA in CNS tissues had histological lesions in brain and spinal cord and significantly higher levels of proinflammatory cytokines TNF-α and interleukin 18 than the HEV-infected pigs without detectable viral RNA in CNS tissues. The findings suggest a potential mechanism of HEV-associated neuroinvasion.

Canine melanoma: A review of diagnostics and comparative mechanisms of disease and immunotolerance in the era of the immunotherapies.

Melanomas in humans and dogs are highly malignant and resistant to therapy. Since the first development of immunotherapies, interest in how the immune system interacts within the tumor microenvironment and plays a role in tumor development, progression, or remission has increased. Of major importance are tumor-infiltrating lymphocytes (TILs) where distribution and cell frequencies correlate with survival and therapeutic outcomes. Additionally, efforts have been made to identify subsets of TILs populations that can contribute to a tumor-promoting or tumor-inhibiting environment, such as the case with T regulatory cells versus CD8 T cells. Furthermore, cancerous cells have the capacity to express certain inhibitory checkpoint molecules, including CTLA-4, PD-L1, PD-L2, that can suppress the immune system, a property associated with poor prognosis, a high rate of recurrence, and metastasis. Comparative oncology brings insights to comprehend the mechanisms of tumorigenesis and immunotolerance in humans and dogs, contributing to the development of new therapeutic agents that can modulate the immune response against the tumor. Therapies that target signaling pathways such as mTOR and MEK/ERK that are upregulated in cancer, or immunotherapies with different approaches such as CAR-T cells engineered for specific tumor-associated antigens, DNA vaccines using human tyrosinase or CGSP-4 antigen, anti-PD-1 or -PD-L1 monoclonal antibodies that intercept their binding inhibiting the suppression of the T cells, and lymphokine-activated killer cells are already in development for treating canine tumors. This review provides concise and recent information about diagnosis, comparative mechanisms of tumor development and progression, and the current status of immunotherapies directed toward canine melanoma.