Novel nucleotide-packaging vaccine delivers antigen and poly(I:C) to dendritic cells and generate a potent antibody response in vivo.

An issue with many current vaccines is the dependency on broadly inflammatory adjuvants, such as aluminum hydroxide or aluminum salts that affect many immune- and non-immune cells. These adjuvants are not necessarily activating all antigen-presenting cells (APCs) that take up the antigen and most likely they also activate APCs with no antigen uptake, as well as many non-immune cells. Conjugation of antigen and adjuvant would enable the use of smaller amounts of adjuvant and avoid unnecessary tissue damage and activation of bystander cells. It would ensure that all APCs that take up the antigen would also become activated and avoid that immature and non-activated APCs present the antigen to T cells without a co-stimulatory signal, leading to tolerogenesis. We have developed a novel vaccine that co-deliver antigen and a nucleotide adjuvant to the same APC and lead to a strong activation response in dendritic cells and macrophages. The vaccine is constructed as a fusion-protein with an antigen fused to the DNA/RNA-binding domain from the Hc2 protein from Chlamydia trachomatis. We have found that the fusion protein is able to package polyinosinic:polycytidylic acid (poly(I:C)) or dsDNA into small particles. These particles were taken up by macrophages and dendritic cells and led to strong activation and maturation of these cells. Immunization of mice with the fusion protein packaged poly(I:C) led to a stronger antibody response compared to immunization with a combination of poly(I:C) and antigen without the Hc2 DNA/RNA-binding domain.

Cholesterol-binding motifs in STING that control endoplasmic reticulum retention mediate anti-tumoral activity of cholesterol-lowering compounds.

The cGAS-STING pathway plays a crucial role in anti-tumoral responses by activating inflammation and reprogramming the tumour microenvironment. Upon activation, STING traffics from the endoplasmic reticulum (ER) to Golgi, allowing signalling complex assembly and induction of interferon and inflammatory cytokines. Here we report that cGAMP stimulation leads to a transient decline in ER cholesterol levels, mediated by Sterol O-Acyltransferase 1-dependent cholesterol esterification. This facilitates ER membrane curvature and STING trafficking to Golgi. Notably, we identify two cholesterol-binding motifs in STING and confirm their contribution to ER-retention of STING. Consequently, depletion of intracellular cholesterol levels enhances STING pathway activation upon cGAMP stimulation. In a preclinical tumour model, intratumorally administered cholesterol depletion therapy potentiated STING-dependent anti-tumoral responses, which, in combination with anti-PD-1 antibodies, promoted tumour remission. Collectively, we demonstrate that ER cholesterol sets a threshold for STING signalling through cholesterol-binding motifs in STING and we propose that this could be exploited for cancer immunotherapy.

Cerebellar abiotrophy in an Icelandic horse.

BACKGROUND: Cerebellar abiotrophy (CA) is an uncommon hereditary neurodegenerative disorder affecting the cerebellar Purkinje cells. Equine CA has been reported in several breeds, but a genetic etiology has only been confirmed in the Arabian breed, where CA is caused by an autosomal recessive mutation. CASE PRESENTATION: Clinical and histological findings consistent with CA are reported in an 8.5-month-old Icelandic filly. The filly showed a perceived sudden onset of marked head tremor, incoordination, ataxia, lack of menace response and a broad-based stance. Cerebrospinal fluid, hematological and biochemical findings were all within the normal range, ruling out several differential diagnoses. Post mortem histopathological examination revealed Purkinje cell degeneration accompanied by astrogliosis. Assessment of the filly's pedigree revealed that its parents shared a common ancestor. CONCLUSIONS: To the authors' knowledge, this is the first report of CA in the Icelandic breed. The identification of a common parental ancestor makes autosomal recessive inheritance of CA in this filly possible, but this would need to be confirmed by further studies. Veterinarians and breeders working with Icelandic horses should be aware of this condition and report suspected cases in order to support genetic investigation.

Biomarkers of Kidney Disease in Horses: A Review of the Current Literature.

Creatinine only allows detection of kidney disease when 60 to 75% of the glomerular function is lost and is therefore not an ideal marker of disease. Additional biomarkers could be beneficial to assess kidney function and disease. The objectives are to describe new equine kidney biomarkers. This systematic review assesses the available literature, including the validation process and reference values, following which the authors suggest recommendations for clinical use. SDMA may have some potential as equine kidney biomarker, but there is currently a lack of evidence that SDMA offers any advantage compared to creatinine in detecting Acute Kidney Injury (AKI). Cystatin C and podocin show potential as biomarkers for kidney disease (including detecting AKI earlier than creatinine) and should be studied further. NGAL has potential as a biomarker of kidney disease (including detecting AKI earlier than creatinine), and potential as an inflammatory marker. Literature on MMP-9 does not allow for conclusive statements about its potential as a biomarker for kidney disease. The future may show that NAG has potential. For all biomarkers, at this stage, available scientific information is limited or too scarce to support clinical use, and only SDMA can be measured for clinical purposes. In conclusion, there are multiple new biomarkers with the potential to diagnose kidney problems. However, there are only a few studies available and more data is needed before these biomarkers can be applied and recommended in our daily practice.

Chronic Renal Failure-Causes, Clinical Findings, Treatments and Prognosis.

Chronic kidney disease (CKD) is rare in horses with an overall prevalence reported to be 0.12%. There is often a continuum from Acute Kidney Injury (AKI) to CKD, and patients with CKD may be predisposed to episodes of AKI. The most common clinical signs are non-specific with weight loss, polyuria/polydipsia and ventral edema. Less common clinical signs are poor appetite and performance, dull hair coat, oral ulcerations, gastro-intestinal ulceration, gingivitis, dental tartar and diarrhea. Rarely, horses may develop forebrain signs. Creatinine increases when at least 2/3 of kidney function have been lost and a more accurate assessment of kidney function is an estimated glomerular filtration rate measuring iohexol clearance time combined with protein content in the urine. Tubulointerstitial disease and glomerulonephritis are common causes of chronic kidney disease together with pyelonephritis and nephrolithiasis. Dietary changes and avoiding nephrotoxic drugs are key in slowing down the degenerative process.

Investigation into pathophysiology of naturally occurring palatal instability and intermittent dorsal displacement of the soft palate (DDSP) in racehorses: Thyro-hyoid muscles fatigue during exercise.

Exercise induced intermittent dorsal displacement of the soft palate (DDSP) is a common cause of airway obstruction and poor performance in racehorses. The definite etiology is still unclear, but through an experimental model, a role in the development of this condition was identified in the dysfunction of the thyro-hyoid muscles. The present study aimed to elucidate the nature of this dysfunction by investigating the spontaneous response to exercise of the thyro-hyoid muscles in racehorses with naturally occurring DDSP. Intramuscular electrodes were implanted in the thyro-hyoid muscles of nine racehorses, and connected to a telemetric unit for electromyographic monitoring implanted subcutaneously. The horses were recruited based on upper airway function evaluated through wireless endoscopy during exercise. Five horses, with normal function, were used as control; four horses were diagnosed as DDSP-affected horses based on repeated episodes of intermittent dorsal displacement of the soft palate. The electromyographic activity of the thyro-hyoid muscles recorded during incremental exercise tests on a high-speed treadmill was analyzed to measure the mean electrical activity and the median frequency of the power spectrum, thereafter subjected to wavelet decomposition. The affected horses had palatal instability with displacement on repeated exams prior to surgical implantation. Although palatal instability persisted after surgery, only two of these horses displaced the palate after instrumentation. The electromyographic traces from this group of four horses showed, at highest exercise intensity, a decrease in mean electrical activity and median power frequency, with progressive decrease in the contribution of the high frequency wavelets, consistent with development of thyro-hyoid muscle fatigue. The results of this study identified fatigue as the main factor leading to exercise induced palatal instability and DDSP in a group of racehorses. Further studies are required to evaluate the fiber type composition and metabolic characteristics of the thyro-hyoid muscles that could predispose to fatigue.

Activation of dendritic cells by targeted DNA: a potential addition to the armamentarium for anti-cancer immunotherapy.

In the past decade, remarkable progress has been made in immunotherapy against cancer. Specifically, the introduction of immune checkpoint inhibitors has revolutionized the field. However, many patients are unable to benefit significantly from this treatment option. One of the major reasons for this is most likely the absence of an adequate tumor-specific T cell response in these patients. A way to circumvent this problem might be to combine immune checkpoint inhibitor treatment with new strategies to activate tumor-specific T cells. One such strategy could be to activate and mature dendritic cells in situ. Dendritic cells carry an array of external and internal pattern recognition receptors that induce cell activation and maturation when interacting with their corresponding damage-associated or pathogen-associated molecular patterns (DAMPs or PAMPs). Targeting such molecular patterns directly to dendritic cells might be a way to evoke stronger immune responses. Here, we review our recent findings using antibody-targeted DNA. We summarize the results from our experiments showing that dendritic cells can be actively targeted in vivo through the αXß2 integrin subunit CD11c, and that DNA delivered through this receptor in vitro leads to maturation of dendritic cells via the cytosolic cGAS/STING DNA-sensing pathway.

Analysis of neurofilament concentration in healthy adult horses and utility in the diagnosis of equine protozoal myeloencephalitis and equine motor neuron disease.

Neurofilaments (NFs) are structural proteins of neurons that are released in significant quantities in the cerebrospinal fluid and blood as a result of neuronal degeneration or axonal damage. Therefore, NFs have potential as biomarkers for neurologic disorders. Neural degeneration increases with age and has the potential to confound the utility of NFs as biomarkers in the diagnosis of neurologic disorders. We investigated this relationship in horses with and without neurological diagnosis. While controlling for horse type (draft, pleasure, and racing), we evaluated the relationship between serum heavy-chain phosphorylated neurofilaments (pNF-H) and age, sex, and serum vitamin E concentrations. Serum pNF-H concentrations increased by 0.002 ng/ml for each year increase in age. There were significant differences in the serum pNF-H concentration among the type of activity performed by the horse. The highest serum pNF-H concentration was found in horses performing heavy work activity (racehorse) and with lower serum pNF-H concentration found among light (pleasure riding) and moderate (draft) activity. There was no significant association between the pNF-H concentration and sex or vitamin E concentration. Serum pNF-H concentration was elevated among horses afflicted with EMND and EPM when compared with control horses without evidence of neurologic disorders. Accordingly, serum pNF-H concentration can serve as a useful biomarker to complement the existing diagnostic work-up of horses suspected of having EPM or EMND.

Asymmetric recurrent laryngeal nerve conduction velocities and dorsal cricoarytenoid muscle electromyographic characteristics in clinically normal horses.

The dorsal cricoarytenoid (DCA) muscles, are a fundamental component of the athletic horse's respiratory system: as the sole abductors of the airways, they maintain the size of the rima glottis which is essential for enabling maximal air intake during intense exercise. Dysfunction of the DCA muscle leads to arytenoid collapse during exercise, resulting in poor performance. An electrodiagnostic study including electromyography of the dorsal cricoarytenoid muscles and conduction velocity testing of the innervating recurrent laryngeal nerves (RLn) was conducted in horses with normal laryngeal function. We detected reduced nerve conduction velocity of the left RLn, compared to the right, and pathologic spontaneous activity (PSA) of myoelectrical activity within the left DCA muscle in half of this horse population and the horses with the slowest nerve conduction velocities. The findings in this group of horses are consistent with left sided demyelination and axonal loss, consistent with Recurrent Laryngeal Neuropathy (RLN), a highly prevalent degenerative disorder of the RLn in horses that predominantly affects the left side. The detection of electromyographic changes compatible with RLN in clinically unaffected horses is consistent with previous studies that identified "subclinical" subjects, presenting normal laryngeal function despite neuropathologic changes within nerve and muscle confirmed histologically.

CD11c-targeted Delivery of DNA to Dendritic Cells Leads to cGAS- and STING-dependent Maturation.

Immunotherapeutic activation of tumor-specific T cells has proven to be an interesting approach in anticancer treatment. Particularly, anti-CTLA-4 and anti-PD-1/PD-L1 treatment looks promising, and conceivably, even better clinical results might be obtained if such treatment could be combined with boosting the existing tumor-specific T-cell response. One way to achieve this could be by increasing the level of maturation of dendritic cells locally and in the draining lymph nodes. When exposed to cancer cells, dendritic cells may spontaneously mature because of danger-associated molecular patterns derived from the tumor cells. Double-stranded DNA play a particularly important role in the activation of the dendritic cells, through engagement of intracellular DNA-sensors, and signaling through the adaptor protein STING. In the present study, we have investigated the maturational response of human monocyte-derived dendritic cells (moDC) and human monocytic THP-1 cells to targeted and untargeted DNA. We used an anti-CD11c antibody conjugated with double-stranded DNA to analyze the maturation status of human moDCs, as well as maturation using a cGAS KO and STING KO THP-1 cell maturation model. We found that dendritic cells can mature after exposure to cytoplasmic double-stranded DNA delivered through CD11c-mediated endocytosis. Moreover, we show that THP-1 cells matured using IL-4, GM-CSF, and ionomycin upregulate DC-maturation markers after CD11c-targeted delivery of double-stranded DNA. This upregulation is completely abrogated in cGAS KO and STING KO cells.

Phosphorylated neurofilament H (pNF-H) as a potential diagnostic marker for neurological disorders in horses.

The current study aimed at the investigating the potential use of phosphorylated neurofilament H (pNF-H) as a diagnostic biomarker for neurologic disorders in the horse. Paired serum and cerebrospinal fluid (CSF) samples (n=88) and serum only (n=30) were obtained from horses diagnosed with neurologic disorders and clinically healthy horses as control. The neurologic horses consisted of equine protozoal myeloencephalitis (EPM) (38 cases) and cervical vertebral malformation (CVM) (23 cases). Levels of pNF-H were determined using an ELISA. The correlation between CSF and serum concentrations of pNF-H was evaluated using Spearman's Rank test and the significance of the difference among the groups was assessed using a nonparametric test. Horses had higher pNF-H levels in the CSF than serum. Horses afflicted with EPM had significantly higher serum pNF-H levels in comparison to controls or CVM cases. The correlation between CSF and serum pNF-H levels was poor in both the whole study population and among subgroups of horses included in the study. There was significant association between the likelihood of EPM and the concentrations of pNF-H in either the serum or CSF. These data suggest that pNF-H could be detected in serum and CSF samples from neurologic and control horses. This study demonstrated that pNF-H levels in serum and CSF have the potential to provide objective information to help in the early diagnosis of horses afflicted with neurologic disorders.

Cavalier King Charles Spaniels with Chiari-like malformation and Syringomyelia have increased variability of spatio-temporal gait characteristics.

BACKGROUND: Chiari-like malformation in the Cavalier King Charles Spaniel is a herniation of the cerebellum and brainstem into or through the foramen magnum. This condition predisposes to Syringomyelia; fluid filled syrinxes within the spinal cord. The resulting pathology in spinal cord and cerebellum create neuropathic pain and changes in gait. This study aims to quantify the changes in gait for Cavalier King Charles Spaniel with Chiari-like malformation and Syringomyelia. METHODS: We compared Cavalier King Charles Spaniel with Chiari-like malformation with (n = 9) and without (n = 8) Syringomyelia to Border Terriers (n = 8). Two video cameras and manual tracking was used to quantify gait parameters. RESULTS AND CONCLUSIONS: We found a significant increase in coefficient of variation for the spatio-temporal characteristics and ipsilateral distance between paws and a wider base of support in the thoracic limbs but not in the pelvic limbs for Cavalier King Charles Spaniels compared with the border terrier.

Altered fraction of regulatory B and T cells is correlated with autoimmune phenomena and splenomegaly in patients with CVID.

Common variable immunodeficiency (CVID) is a heterogeneous primary immunodeficiency disease, leading to recurrent bacterial airway infections and often also autoimmune complications. To shed light on the regulatory lymphocytes from these patients, we analyzed the levels of regulatory B (pro-B10) cell and regulatory T (Treg) cell subpopulations in PBMCs from twenty-six patients diagnosed with CVID using multi-color flowcytometry. Pro-B10 cells were induced by 48h in vitro stimulation prior to analysis. Suppressor function was measured on a subset of patients with splenomegaly and autoimmune complications. The levels of regulatory B and T cells were correlated to clinical manifestations, including autoimmunity, splenomegaly and CVID EUROclass subgroups. We demonstrate a significant association between elevated levels of pro-B10 cells, decreased levels of Tregs and autoimmune phenomena in CVID patients. The finding of marked abnormalities in regulatory lymphocyte populations contribute to our understanding of the pathogenesis of CVID and potentially be valuable in the clinical management and treatment of patients.

Accuracy and precision of oscillometric blood pressure in standing conscious horses.

BACKGROUND: Arterial blood pressure (BP) is a relevant clinical parameter that can be measured in standing conscious horses to assess tissue perfusion or pain. However, there are no validated oscillometric noninvasive blood pressure (NIBP) devices for use in horses. ANIMALS: Seven healthy horses from a teaching and research herd. HYPOTHESIS/OBJECTIVE: To evaluate the accuracy and precision of systolic arterial pressure (SAP), diastolic arterial pressure (DAP), and mean arterial pressure (MAP) in conscious horses obtained with an oscillometric NIBP device when compared to invasively measured arterial BP. METHODS: An arterial catheter was placed in the facial or transverse facial artery and connected to a pressure transducer. A cuff for NIBP was placed around the tail base. The BP was recorded during normotension, dobutamine-induced hypertension, and subnormal BP induced by acepromazine administration. Agreement analysis with replicate measures was utilized to calculate bias (accuracy) and standard deviation (SD) of bias (precision). RESULTS: A total of 252 pairs of invasive arterial BP and NIBP measurements were analyzed. Compared to the direct BP measures, the NIBP MAP had an accuracy of -4 mm Hg and precision of 10 mm Hg. SAP had an accuracy of -8 mm Hg and a precision of 17 mm Hg and DAP had an accuracy of -7 mm Hg and a precision of 14 mm Hg. CONCLUSIONS AND CLINICAL RELEVANCE: MAP from the evaluated NIBP monitor is accurate and precise in the adult horse across a range of BP, with higher variability during subnormal BP. MAP but not SAP or DAP can be used for clinical decision making in the conscious horse.

A STAT1-gain-of-function mutation causing Th17 deficiency with chronic mucocutaneous candidiasis, psoriasiform hyperkeratosis and dermatophytosis.

During recent years, inborn errors of human IL-17 immunity have been demonstrated to underlie primary immunodeficiencies with chronic mucocutaneous candidiasis (CMC). Various defects in receptors responsible for sensing of Candida albicans or downstream signalling to IL-17 may lead to susceptibility to Candida infection. While CMC is common in patients with profound T cell immunodeficiencies, CMC is also recognised as part of other immunodeficiencies in syndromic CMC, or as relatively isolated CMC disease. We describe a 40-year-old woman with a clinical picture involving cutaneous bacterial abscesses, chronic oral candidiasis and extensive dermatophytic infection of the feet. By whole exome sequencing, we identified a STAT1-gain-of-function mutation. Moreover, the patient's peripheral blood mononuclear cells displayed severely impaired Th17 responses. The patient was treated with antifungals and prophylactic antibiotics, which led to resolution of the infection. We discuss the current knowledge within the field of Th17 deficiency and the pathogenesis and treatment of CMC.

Functional IRF3 deficiency in a patient with herpes simplex encephalitis.

Herpes simplex encephalitis (HSE) in children has previously been linked to defects in type I interferon (IFN) production downstream of Toll-like receptor 3. Here, we describe a novel genetic etiology of HSE by identifying a heterozygous loss-of-function mutation in the IFN regulatory factor 3 (IRF3) gene, leading to autosomal dominant (AD) IRF3 deficiency by haploinsufficiency, in an adolescent female patient with HSE. IRF3 is activated by most pattern recognition receptors recognizing viral infections and plays an essential role in induction of type I IFN. The identified IRF3 R285Q amino acid substitution results in impaired IFN responses to HSV-1 infection and particularly impairs signaling through the TLR3-TRIF pathway. In addition, the R285Q mutant of IRF3 fails to become phosphorylated at S386 and undergo dimerization, and thus has impaired ability to activate transcription. Finally, transduction with WT IRF3 rescues the ability of patient fibroblasts to express IFN in response to HSV-1 infection. The identification of IRF3 deficiency in HSE provides the first description of a defect in an IFN-regulating transcription factor conferring increased susceptibility to a viral infection in the CNS in humans.

Is a standalone inertial measurement unit accurate and precise enough for quantification of movement symmetry in the horse?

Standalone 'low-cost' inertial measurement units (IMUs) could facilitate large-scale studies into establishing minimal important differences (MID) for orthopaedic deficits (lameness) in horses. We investigated accuracy and limits of agreement (LoA) after correction of magnitude-dependent differences of a standalone 6 degree-of-freedom IMU compared with an established IMU-based gait analysis system (MTx) in six horses for two anatomical landmarks (sacrum and sternum). Established symmetry measures were calculated from vertical displacement: symmetry index (SI), difference between minima (MinDiff) and difference between maxima (MaxDiff). For the sacrum, LoA were ± 0.095 for SI, ± 6.6 mm for MinDiff and ± 4.3 mm for MaxDiff. For the sternum, LoA values were ± 0.088 for SI, ± 5.0 mm for MinDiff and ± 4.2 mm for MaxDiff. Compared with reference data from mildly lame horses, SI values indicate sufficient precision, whereas MinDiff and MaxDiff values are less favourable. Future studies should investigate specific calibration and processing algorithms further improving standalone IMU performance.

The DR6 protein from human herpesvirus-6B induces p53-independent cell cycle arrest in G2/M.

HHV-6B infection inhibits cell proliferation in G2/M, but no protein has so far been recognized to exert this function. Here we identify the protein product of direct repeat 6, DR6, as an inhibitor of G2/M cell-cycle progression. Transfection of DR6 reduced the total number of cells compared with mock-transfected cells. Lentiviral transduction of DR6 inhibited host cell DNA synthesis in a p53-independent manner, and this inhibition was DR6 dose-dependent. A deletion of 66 amino acids from the N-terminal part of DR6 prevented efficient nuclear translocation and the ability to inhibit DNA synthesis. DR6-induced accumulation of cells in G2/M was accompanied by an enhanced expression of cyclin B1 that accumulated predominantly in the cytoplasm. Pull-down of cyclin B1 brought down pCdk1 with the inactivating phosphorylation at Tyr15. Together, DR6 delays cell cycle with an accumulation of cells in G2/M and thus might be involved in HHV-6B-induced cell-cycle arrest.

Accuracy and precision of gait events derived from motion capture in horses during walk and trot.

This study aimed to create an evidence base for detection of stance-phase timings from motion capture in horses. The objective was to compare the accuracy (bias) and precision (SD) for five published algorithms for the detection of hoof-on and hoof-off using force plates as the reference standard. Six horses were walked and trotted over eight force plates surrounded by a synchronised 12-camera infrared motion capture system. The five algorithms (A-E) were based on: (A) horizontal velocity of the hoof; (B) Fetlock angle and horizontal hoof velocity; (C) horizontal displacement of the hoof relative to the centre of mass; (D) horizontal velocity of the hoof relative to the Centre of Mass and; (E) vertical acceleration of the hoof. A total of 240 stance phases in walk and 240 stance phases in trot were included in the assessment. Method D provided the most accurate and precise results in walk for stance phase duration with a bias of 4.1% for front limbs and 4.8% for hind limbs. For trot we derived a combination of method A for hoof-on and method E for hoof-off resulting in a bias of -6.2% of stance in the front limbs and method B for the hind limbs with a bias of 3.8% of stance phase duration. We conclude that motion capture yields accurate and precise detection of gait events for horses walking and trotting over ground and the results emphasise a need for different algorithms for front limbs versus hind limbs in trot.