Sustained Liver HBsAg Loss and Clonal T- and B-Cell Expansion upon Therapeutic DNA Vaccination Require Low HBsAg Levels.

BACKGROUND: Suppression of HBV DNA, inhibition of HBV surface (HBsAg) production and therapeutic vaccination to reverse HBV-specific T-cell exhaustion in chronic HBV patients are likely required to achieve a functional cure. In the AAV-HBV mouse model, therapeutic vaccination can be effective in clearing HBV when HBsAg levels are low. Using a single-cell approach, we investigated the liver immune environment with different levels of HBsAg and sustained HBsAg loss through treatment with a GalNAc-HBV-siRNA followed by therapeutic vaccination. METHODS: AAV-HBV-transduced C57BL/6 mice were treated with GalNAc-HBV-siRNA to lower HBsAg levels and then vaccinated using a DNA vaccine. We used single-cell RNA and V(D)J sequencing to understand liver immune microenvironment changes. RESULTS: GalNAc-HBV-siRNA, followed by therapeutic vaccination, achieved sustained HBsAg loss in all mice. This was accompanied by CD4 follicular helper T-cell induction, polyclonal activation of CD8 T cells and clonal expansion of plasma cells that were responsible for antibody production. CONCLUSIONS: This study provides novel insights into liver immune changes at the single-cell level, highlighting the correlation between induced reduction of HBsAg levels and clonal expansion of CD4, CD8 T cells and plasma cells in the liver upon HBV siRNA and subsequent therapeutic vaccination.

GSDMD drives canonical inflammasome-induced neutrophil pyroptosis and is dispensable for NETosis.

Neutrophils are the most prevalent immune cells in circulation, but the repertoire of canonical inflammasomes in neutrophils and their respective involvement in neutrophil IL-1ß secretion and neutrophil cell death remain unclear. Here, we show that neutrophil-targeted expression of the disease-associated gain-of-function Nlrp3A350V mutant suffices for systemic autoinflammatory disease and tissue pathology in vivo. We confirm the activity of the canonical NLRP3 and NLRC4 inflammasomes in neutrophils, and further show that the NLRP1b, Pyrin and AIM2 inflammasomes also promote maturation and secretion of interleukin (IL)-1ß in cultured bone marrow neutrophils. Notably, all tested canonical inflammasomes promote GSDMD cleavage in neutrophils, and canonical inflammasome-induced pyroptosis and secretion of mature IL-1ß are blunted in GSDMD-knockout neutrophils. In contrast, GSDMD is dispensable for PMA-induced NETosis. We also show that Salmonella Typhimurium-induced pyroptosis is markedly increased in Nox2/Gp91Phox -deficient neutrophils that lack NADPH oxidase activity and are defective in PMA-induced NETosis. In conclusion, we establish the canonical inflammasome repertoire in neutrophils and identify differential roles for GSDMD and the NADPH complex in canonical inflammasome-induced neutrophil pyroptosis and mitogen-induced NETosis, respectively.

Infective vegetative endocarditis of the mitral, aortic, and pulmonary valves due to Enterococcus hirae in a cat with a ventricular septal defect.

A 2.5-year-old female intact British Shorthair was presented for progressive complaints of abdominal distention, increased respiratory effort, lethargy and hyporexia. Based on the clinical presentation and a loud heart murmur, a cardiac cause was suspected. An echocardiogram was performed and the presumptive diagnosis of infective endocarditis of the aortic, mitral and pulmonic valves was made. Antemortem blood culture and postmortem valve biopsy confirmed bacterial endocarditis with Enterococcus hirae as etiological agent. To the authors' best knowledge, this case report is the first to describe an infective endocarditis with vegetative lesions on three cardiac valves associated with a ventricular septal defect in a cat, and Enterococcus hirae as causative agent for endocarditis in small animals.

Truncating SLC12A6 variants cause different clinical phenotypes in humans and dogs.

Clinical, pathological, and genetic findings of a primary hereditary ataxia found in a Malinois dog family are described and compared with its human counterpart. Based on the family history and the phenotype/genotype relationships already described in humans and dogs, a causal variant was expected to be found in KCNJ10. Rather surprisingly, whole-exome sequencing identified the SLC12A6 NC_006612.3(XM_014109414.2): c.178_181delinsCATCTCACTCAT (p.(Met60Hisfs*14)) truncating variant. This loss-of-function variant perfectly segregated within the affected Malinois family in an autosomal recessive way and was not found in 562 additional reference dogs from 18 different breeds, including Malinois. In humans, SLC12A6 variants cause "agenesis of the corpus callosum with peripheral neuropathy" (ACCPN, alias Andermann syndrome), owing to a dysfunction of this K+-Cl- cotransporter. However, depending on the variant (including truncating variants), different clinical features are observed within ACCPN. The variant in dogs encodes the shortest isoform described so far and its resultant phenotype is quite different from humans, as no signs of peripheral neuropathy, agenesis of the corpus callosum nor obvious mental retardation have been observed in dogs. On the other hand, progressive spinocerebellar ataxia, which is the most important feature of the canine phenotype, hindlimb paresis, and myokymia-like muscle contractions have not been described in humans with ACCPN so far. As this is the first report of a naturally occurring disease-causing SLC12A6 variant in a non-human species, the canine model will be highly valuable to better understand the complex molecular pathophysiology of SLC12A6-related neurological disorders and to evaluate novel treatment strategies.

25-OH-vitamin D, parathyroid hormone, and calcium serum levels in captive common marmosets (Callithrix jacchus): Reference values and effect of age, sex, season, and closure of long bone epiphyses.

BACKGROUND: To date, reference values for 25-OH-vitamin D, parathyroid hormone (PTH), and calcium in serum of common marmosets (Callithrix jacchus) based on a large sample size are not available. METHODS: Serum reference values for these parameters were determined and correlated with sex, age, season of sampling, and time of long bone epiphyseal closure in captive-housed marmosets. RESULTS AND CONCLUSIONS: The 90% reference range for serum 25-OH-vitamin D is 47.40-370.4 nmol/L, for PTH 2.10-30.51 pmol/L, and for calcium 2.08-2.63 mmol/L. Lower levels of vitamin D were measured in fall compared with the other seasons. Levels of PTH were higher in males than in females, and calcium levels were lower in younger animals compared with older marmosets. No other effects of age, sex, season, or timing of growth plate closure were found.

Encephalomyocarditis virus in a captive Malayan tapir (Tapirus indicus).

A 5-month-old female captive Malayan tapir (Tapirus indicus) died suddenly without preceding symptoms. Gross necropsy revealed numerous white circular and linear foci in the myocard. Differential diagnosis all turned out negative, except for encephalomyocarditis virus. Histopathology revealed mineralisation of myocardial cells and interstitial infiltration of lymphocytes, plasma cells and less neutrophils. Encephalomyocarditis virus was detected by PCR. Although encephalomyocarditis virus occurs in many mammals, this is the first published description of this virus in a Malayan tapir.

Salmonella Enteritidis flagellar mutants have a colonization benefit in the chicken oviduct.

Egg borne Salmonella Enteritidis is still a major cause of human food poisoning. Eggs can become internally contaminated following colonization of the hen's oviduct. In this paper we aimed to analyze the role of flagella of Salmonella Enteritidis in colonization of the hen's oviduct. Using a transposon library screen we showed that mutants lacking functional flagella are significantly more efficient in colonizing the hen's oviduct in vivo. A micro-array analysis proved that transcription of a number of flagellar genes is down-regulated inside chicken oviduct cells. Flagella contain flagellin, a pathogen associated molecular pattern known to bind to Toll-like receptor 5, activating a pro-inflammatory cascade. In vitro tests using primary oviduct cells showed that flagellin is not involved in invasion. Using a ligated loop model, a diminished inflammatory reaction was seen in the oviduct resulting from injection of an aflagellated mutant compared to the wild-type. It is hypothesized that Salmonella Enteritidis downregulates flagellar gene expression in the oviduct and consequently prevents a flagellin-induced inflammatory response, thereby increasing its oviduct colonization efficiency.

The novel homozygous KCNJ10 c.986T>C (p.(Leu329Pro)) variant is pathogenic for the SeSAME/EAST homologue in Malinois dogs.

SeSAME/EAST syndrome is a multisystemic disorder in humans, characterised by seizures, sensorineural deafness, ataxia, developmental delay and electrolyte imbalance. It is exclusively caused by homozygous or compound heterozygous variations in the KCNJ10 gene. Here we describe a similar syndrome in two families belonging to the Malinois dog breed, based on clinical, neurological, electrodiagnostic and histopathological examination. Genetic analysis detected a novel pathogenic KCNJ10 c.986T>C (p.(Leu329Pro)) variant that is inherited in an autosomal recessive way. This variant has an allele frequency of 2.9% in the Belgian Malinois population, but is not found in closely related dog breeds or in dog breeds where similar symptoms have been already described. The canine phenotype is remarkably similar to humans, including ataxia and seizures. In addition, in half of the dogs clinical and electrophysiological signs of neuromyotonia were observed. Because there is currently no cure and treatment is nonspecific and unsatisfactory, this canine translational model could be used for further elucidating the genotype/phenotype correlation of this monogenic multisystem disorder and as an excellent intermediate step for drug safety testing and efficacy evaluations before initiating human studies.

Basal cell carcinoma in two Hermann's tortoises (Testudo hermanni).

Neoplastic disorders are frequently encountered in the practice of reptile medicine. Herein we report the clinical behavior, antemortem diagnosis, and histopathologic characteristics of a recurrent intraoral keratinizing basal cell carcinoma (BCC) and a metastatic BCC of the carapace in 2 Hermann's tortoises (Testudo hermanni). Although squamous cell carcinomas (SCCs) in tortoises show similar predilection sites and gross pathologic features, the BCCs described in our report were characterized by a remarkably fast and highly infiltrative growth in comparison to SCCs. Accordingly, early diagnosis including reliable discrimination from SCC is essential toward the management of this neoplastic entity in tortoises.

Micromelic dysplasia-like syndrome in a captive colony of common marmosets (Callithrix jacchus).

Over several years, 0% to 5% of adolescent animals in a captive colony of common marmosets (Callithrix jacchus) showed severely bended arms and legs over several years. The animals showed no pain, discomfort, or altered behavior but were unable to stretch their distal limbs to their full extent. To characterize the lesion morphologically, the bones of 4 affected marmosets were compared macroscopically and radiographically with those of 6 unaffected animals. The deformities were characterized by mid- to distal diaphyseal bending and pronounced shortening of long bones. The morphology and density of other bones including the skull and vertebrae were unaffected. Although vitamin D values were low in a fifth affected marmoset during 10 to 16 mo of age, lesions associated with rickets were not observed. To our knowledge, this report is the first to describe a micromelic dysplasia-like syndrome comprising severe, idiopathic bending and shortening of long bones in a colony of marmosets.