Probes for INS

Purpose: The integration of external cues, such as mechanics, with intrinsic cell signalling programmes, such as hedgehog (Hh) signalling, is crucial for the development, maturation and homeostasis of articular cartilage. Activation of Hh signalling in adulthood and pathophysiological mechanics, have both been associated with the development of murine and human OA. But, how chondrocytes might transduce and integrate these cues remains unknown. A microtubule-based organelle, the primary cilium, most noted for its crucial role in Hh signalling, is assembled by chondrocytes and possesses a devoted trafficking machinery, IntraFlagellar Transport or IFT. In vitro studies indicate chondrocyte helps tune the anabolic matrix response to compression and the response to Hh ligand. In vivo, the primary cilium has been proposed to be a platform for the integration of mechanics and Hh signalling in musculoskeletal tissues. While constitutive and peri-natal disruption of ciliary proteins, Hh signalling and altered mechanics, all drastically alter joint development in vivo, the influence of IFT in adult cartilage homeostasis remains unknown. Methods: IFT88 was targeted using a cartilage-specific, inducible mouse line (ACANCreERT2;Ift88fl/fl : cKO hereafter). Cre activity was validated by qPCR, RNA scope and a ROSA26tdtomato reporter line. Ift88fl/fl mice, also receiving I.P injections of tamoxifen, were used as controls. Tibial articular cartilage was assessed 2, 14 or 26 weeks-post tamoxifen, at 8, 10, 22 and 34 weeks of age respectively, using histomorphometric analyses, including measurements of articular cartilage thickness, relative calcification, subchondral bone thickness, and OARSI score by means of immunohistochemistry (IHC). The surgical DMM model, which destabilises the joint, was performed at 10 weeks of age. To explore the role of physiological mechanics, mice were allowed two weeks of voluntary wheel exercise immediately following tamoxifen administration at 8 weeks of age. qPCR was performed on micro dissected articular cartilage at 10 weeks of age in control and cKO. RNAscope was performed on cryosections of articular cartilage from 10 week old mice control and cKO. Means ± S.D are quoted throughout, Mann-Whitney U-test or Fisher’s test were used for statistical comparisons. Results: In our previous OARSI abstract of 2020 we described the phenotype arising in IFT88 cKO mice. Here we outline this in further detail and with an exploration of underlying mechanisms. Tamoxifen treatment of cKO mice resulted in a 50% reduction of Ift88 mRNA in articular cartilage (p=0.02, n=6 control, 14 cKO). Ift88 (cKO) mice had thinner medial articular cartilage (MAC), compared with controls, at all 5 time-points (Fig 1.A) In control mice, MAC thickness increased from 102.57μm (95% CI [94.30, 119.80]) at 8 weeks of age to 108.68 +/- (95% CI [101.32, 116.42]) at 10 weeks of age. Tamoxifen treatment, at 8 weeks of age, inhibited this increase in cKO mice (MC thickness at 10 weeks was 96.20 μm (95% CI [90.04, 102.36]), p=0.02, compared with 10 week ctrl, n=7). By 22 weeks of age mean MAC thickness in cKO was 90.16μm (95% CI [87.11, 93.22]) compared with 111.60μm (95% CI [104.34, 118.79]) in control animals (p=0.0002, n= 7 and 10 respectively). By 34 weeks MAC had continued to thin to 84.55μm (95% CI [75.43, 93.67]) in cKO, but this was now associated with surface damage and osteophyte formation. In the most extreme case, MAC was completely lost (Fig.1B). In contrast, lateral plateau thickness and OARSI score were unaffected. Calcified cartilage (below the tidemark) progressively increases on both plateaus, between 6 and 22 weeks of age and at all time-points thinning was attributable to the relative loss of calcified cartilage implying a failure of calcification. IHC analyses revealed no striking differences in collagen X expression, NITEGE neoepitope. There were no measurable increases in subchondral bone thickness or changes in osteoclastic activity in cKO mice. 12 weeks post DMM, OARSI scores were statistically significantly higher in cKO (29.83 +/- 7.69) than control (22.08 +/- 9.30, p< 0.05, n= 15 both groups). Two weeks of voluntary wheel exercise rescued cartilage atrophy in cKO mice (p< 0.0001), whilst no change was observed in controls. RNA isolated from microdissected articular cartilage of 10 week old control and cKO mice, two weeks post tamoxifen, revealed a statistically significant correlation between Ift88 and Tcf7l2 expression after Bonferroni correction (p=0.026). Ctgf, Gli2 and Enpp1 were also positively correlated with Ift88 expression before correction (p=0.002, p=0.0037, and p=0.009 respectively). RNA scope analysis of AC found a statistically significant (p< 0.0001, n=4 in both groups) decrease in Ift88 positive cells in cKO (27.78%) compared with controls (45.18%), whilst also showing an increase in Gli1 positive cells in cKO (50.42%) compared with controls (23.63%) (p< 0.0001, n=4 in both groups). Conclusions: Progressive thickening and calcification in the mouse medial compartment illustrates the continued mechanoadaptation of adolescent and adult articular cartilage. Depletion of the ciliary gene Ift88 inhibits medial articular cartilage thickening, leading to atrophy, which then predisposes the joint to spontaneous OA. The lateral compartment is relatively unaffected. We propose this may be due, in part, to disruption of mechanotransduction and downstream anabolic remodelling in medial cartilage. Deletion of Ift88 impairs the progressive calcification of articular cartilage, in both compartments, which may be due to disruption of Hh signalling, which is also mechanosensitive. Ift88 expression was correlated with Tcf7l2, previously shown to interact and influence Hh signalling pathways in cartilage. On-going experiments are aiming to dissect the relative roles of IFT, mechanics and Hh in the context of adult cartilage. We conclude that Ift88 is crucial to post-natal articular cartilage homeostasis and chondroprotective against OA.

Fetal responses to adverse pregnancy environments are sex-specific. In fetal guinea pigs (GPs), we assessed morphology and messenger RNA (mRNA) expression in fetal growth-restricted (FGR) tissues at midpregnancy. Female GPs were assigned either an ad libitum diet (C) or 30% restricted diet (R) prior to pregnancy to midpregnancy. At midpregnancy, a subset of R females underwent ultrasound-guided nanoparticle (NP) injection to enhance placental function. Five days later, fetuses were sampled. Fetal brain, heart, and liver were assessed for morphology (hematoxylin and eosin), proliferation (Ki67), and vascularization (CD31), as well as expression of inflammatory markers. R fetuses were 19% lighter with reduced organ weights and evidence of brain sparing compared to controls. No increased necrosis, proliferation, or vascularization was found between C and R nor male or female fetal organs. Sexual dimorphism in mRNA expression of Tgfβ and Ctgf was observed in R but not C fetal brains: increased expression in females. NP treatment increased fetal brain mRNA expression of Tgfβ and Ctgf in R males, abolishing the significant difference observed in untreated R fetuses. Sex-specific differences in mRNA expression in the fetal brain with FGR could impart a potential survival bias and may be useful for the development of treatments for obstetric diseases. Male and female fetuses respond differently to adverse pregnancy environments. Under fetal growth restriction conditions, inflammatory marker mRNA expression in the fetal brain was higher in females compared to males. Differences in gene expression between males and females may confer a selective advantage/disadvantage under adverse conditions. Better characterization of sexual dimorphism in fetal development will aid better development of treatments for obstetric diseases.

Erethizon dorsatum papillomavirus 1 (EdPV1) and Erethizon dorsatum papillomavirus 2 (EdPV2) are associated with cutaneous papillomas in North American porcupines (Erethizon dorsatum). This study defined gross, histopathologic, and molecular characteristics of viral papillomas in 10 North American porcupines submitted to the New York State Animal Health Diagnostic Center. Investigation for the presence of EdPV1 and EdPV2 DNA via polymerase chain reaction (PCR) was performed in 9 of the 10 (90.0%) porcupines, and all porcupines were investigated for the detection and localization of EdPV1 and EdPV2 E6 and E7 nucleic acid via chromogenic in situ hybridization (CISH). Next-generation sequencing (NGS) was performed in 2 porcupines. Papillomas were diagnosed on the muzzle (n = 4), caudal dorsum (n = 1), upper lip (n = 1), chin (n = 1), gingiva (n = 2), and nasal planum (n = 1). Histologically, the lesions consisted of hyperplastic epidermis or epithelium with orthokeratotic keratin, prominent keratohyalin granules, and intranuclear inclusion bodies. PCR identified EdPV1 in 6 of 9 samples and EdPV2 in the remaining 3 samples. NGS resulted in 100% genome coverage of EdPV1 and 76.20% genome coverage of EdPV2 compared with GenBank reference sequences, with 99.8% sequence identity to the complete EdPV2 L1 gene of a novel subtype recently identified in France. Hybridization patterns in 9 of the 10 (90.0%) porcupines were characterized by strong nuclear signals in the superficial epidermis, with strong nuclear and punctate cytoplasmic signals in the stratum spinosum and basale. In one animal, CISH suggested dual EdPV1 and EdPV2 infection.

The identification of additional lipid mediators, enzymes, and receptors revealed an expanded endocannabinoid system (ECS) called the endocannabinoidome (eCBome). Furthermore, eCBome research using wild type and genetically modified mice indicate the involvement of this system in modulating alcohol induced neuroinflammatory alterations associated with behavioral impairments and the release of proinflammatory cytokines. We investigated the role of cannabinoid type 2 receptors (CB2Rs) in modulating behavioral and neuro-immune changes induced by alcohol using conditional knockout (cKO) mice with selective deletion of CB2Rs in dopamine neurons (DAT-Cnr2) and in microglia (Cx3Cr1-Cnr2) cKO mice. We used a battery of behavioral tests including locomotor and wheel running activity, rotarod performance test, and alcohol preference tests to evaluate behavioral changes induced by alcohol. ELISA assay was used, to detect alterations in IL-6, IL-1α, and IL-1β in the prefrontal cortex, striatum, and hippocampal regions of mice to investigate the role of CB2Rs in neuroinflammation induced by alcohol in the brain. The involvement of cannabinoid receptors in alcohol-induced behavior was also evaluated using the non-selective cannabinoid receptor mixed agonist WIN 55,212-2. The results showed that cell-type specific deletion of CB2Rs in dopamine neurons and microglia significantly and differentially altered locomotor activity and rotarod performance activities. The result also revealed that cell-type specific deletion of CB2Rs enhanced alcohol-induced inflammation, and WIN significantly reduced alcohol preference in all genotypes compared to the vehicle controls. These findings suggest that the involvement of CB2Rs in modulating behavioral and neuroinflammatory alterations induced by alcohol may be potential therapeutic targets in the treatment of alcohol use disorder.

Antisense oligonucleotides (AONs) are short, synthetic nucleic acid sequences that work by modulating exon incorporation at the level of pre-mRNA. In Duchenne muscular dystrophy (DMD), a fatal muscle degenerative disorder caused by mutations in the DMD gene, AONs skip specific exons to correct the reading frame, producing an internally shortened but partly functional dystrophin protein. Golodirsen is an approved AON phosphorodiamidate morpholino oligomer (PMO) that specifically targets DMD exon 53. In the clinical study 4053-101, we demonstrated that intravenous golodirsen administration induces an unequivocal exon skipping and protein restoration in all the treated patients, but with inter-patient variability. We used fibroblasts isolated from the patients in this clinical trial, that were induced to undergo myogenic differentiation in vitro by expression of MyoD, to better understand the reasons behind the observed variability. We evaluated the amount and the molecular weight of dystrophin protein in treated and non-treated patient cells, by an automated capillary-based immunoassay (WES) system. In these in-vitro studies we demonstrated that the amount of protein was comparable to the previous in-vivo study and that the size of the restored protein was compatible with the different genomic deletions carried by patients. Next, we used an in-situ RNA hybridization technique, BaseScope, to investigate the sub-cellular localization of the DMD transcript in treated and non-treated differentiated patient-derived myogenic cells in vitro, which allowed us to assess the ratio of skipped and unskipped products. Our study provides additional information on the dynamics of DMD mRNA in patients and may help to better understand the biological reasons underpinning variability in dystrophin restoration that can be seen in AON clinical trials.

Rationale DNA damage accumulation is a hallmark of vascular smooth muscle cell (VSMC) ageing. Importantly, VSMC DNA damage accumulation and ageing has been implicated in the progression of cardiovascular disease (CVD), including atherosclerosis and vascular calcification. Chemotherapy drugs used in the treatment of many cancers are known to induce DNA damage in cardiovascular cells and accelerate CVD. Histone deacetylase (HDAC) inhibitors are drugs being investigated for novel treatments of many cancers. HDACs perform many vital functions in cells; HDAC6 is known to deacetylate alpha-tubulin to regulate microtubule stability and flexibility. We have recently shown that microtubule stability regulates both VSMC morphology and contractility. Therefore, in this study we investigate the impact of HDAC6 inhibition upon VSMC function. Methodology We use polyacrylamide hydrogels (PAHs) of physiological aortic stiffness to investigate the impact of HDAC6 inhibition on the contractile response of angiotensin II stimulated quiescent VSMC function. In this study, we utilise HDAC6 inhibitor BRD 9757 and Tubastatin a HDAC6 inhibitor inducing tubulin hyperacetylation. Results Our data shows that HDAC6 inhibition resulted in increased alpha-tubulin acetylation and decreased VSMC area. Further analysis revealed that although VSMC volume was unaltered, nuclear volume was decreased. Immunofluorescence microscopy revealed that HDAC6 inhibitor treatment resulted in DNA damage accumulation in VSMCs. We hypothesised that altered microtubule stability participated in this phenotype. To test this possibility, we performed a cold-stable microtubule stability assay, which revealed that HDAC6 inhibitor treated VSMCs possessed decreased microtubule stability. To test whether changes in microtubule stability induced DNA damage accumulation, we used the microtubule destabilising agents colchicine and demecholcine, and the microtubule stabilising agent paclitaxel. Importantly, either colchicine or demecholcine treatment increased DNA damage accumulation in VSMCs. In contrast, paclitaxel treatment had no effect on DNA damage levels.

Many patients with sickle cell disease (SCD) suffer from chronic pain, the underlying causes of which are unclear. Recent 16s ribosomal RNA sequencing studies revealed differences in the number and types of bacteria in the gastrointestinal tract of patients and mouse models of SCD relative to controls, but it is unclear if or how these changes contribute to symptomology. In these experiments, we used transgenic SCD mice to determine the extent to which disease related gut dysbiosis contributes to persistent pain. Reflexive pain behaviors were first measured in SCD mice following longitudinal probiotic or antibiotic treatment. Vehicle-treated SCD mice displayed significant mechanical allodynia relative to vehicle-treated wildtype mice, and antibiotic treatment further exacerbated mechanical allodynia in both genotypes. In contrast, probiotic treatment completely reversed persistent touch hypersensitivity in SCD mice. Persistent touch pain was also transiently reversed in SCD mice following fecal material transplant from healthy mice. In complementary experiments, wildtype recipient mice developed cold and touch hypersensitivity that persisted for several weeks after fecal material transplant from SCD donors. Using whole-cell patch clamp recordings, we further determined that these behavioral observations were accompanied by altered intrinsic plasticity in a select class of nodose ganglia sensory neurons, the peripheral terminals of which are well positioned to detect sensory information in the gut. Nodose ganglia neurons isolated from animals that received sickle cell fecal material transplants were hyperexcitable relative to those isolated from animals that received control fecal material transplants. These data are the first to suggest that disease-related gut dysbiosis induces pain through changes in vagal nerve activity. Ongoing studies are examining specific bacterial populations and/or metabolites responsible for these functional changes in order to develop novel therapeutics for chronic SCD pain management. Grant support from National Institutes of Health grants K99HL155791 and R01NS070711.

Aims: the knowledge of the pathogenic mechanisms of severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) is certainly a priority for the scientific community. Two main elements are involved in the biology of the most severe forms of Coronavirus disease 2019 (COVID-19): the direct cytopathic effect of the virus against the host tissues, and a dysfunction of the immune system, characterized by the exhaustion of T lymphocytes. The exhaustion of T cells in COVID-19 is poorly understand, but some data could suggest a possible role of PD-1/PD-L1 axis. The aim of this study was to evaluate the possible role of PD-L1 expression in the pulmonary tissue in subjects affected by COVID-19. Methods and results: the presence of SARS-CoV-2 in the pulmonary tissue, and its exact location, was indagated by in-situ hybridization; the expression of PD-L1 and CD8 in the same tissue was indagated by immunohistochemistry. Overall, PD-L1 resulted diffusely expressed in 70% of the cases, and an intense expression was observed in 43.5% of cases. Diffuse and intense presence of SARS-CoV-2 by in situ hybridization (ISH) significantly correlated with an intense PD-L1 expression, and with expression of PD-L1 by pneumocytes. Conclusions: PD-L1 is overexpressed in the pulmonary tissue of subjects died for COVID-19, and mainly in subjects with a high viral load. These data suggest a possible role of PD-L1 in the immune system exhaustion at the basis of the severe forms of the disease.

Despite evidence of multi-organ tropism of SARS-CoV-2 in patients with COVID-19, direct viral kidney invasion has been difficult to demonstrate. The question of whether SARS-CoV-2 can directly infect the kidney is relevant to the understanding of pathogenesis of acute kidney injury and collapsing glomerulopathy in COVID-19. Methodologies to document SARS-CoV-2 infection that have been used include immunohistochemistry, immunofluorescence, reverse transcriptase polymerase chain reaction (RT-PCR), in situ hybridization and electron microscopy. In our review of studies to date we found that SARS-CoV-2 in the kidney of patients with COVID-19 was detected in 18 of 94 (19%) by immuno-histochemistry, 71 of 144 (49%) by RT-PCR and 11 of 84 (13%) by in situ hybridization. In a smaller number of patients with COVID-19 examined by immunofluorescence, SARS-CoV-2 was detected in 10 of 13 (77%). In total, in kidneys from 102 of 235 patients (43.4%), the presence of SARS-CoV-2 was suggested by at least one of the methods used. Despite these positive findings, caution is needed as many other studies have been negative for SARS-CoV-2 presence and it should be noted that when detected it was only in kidneys obtained at autopsy. There is a clear need for studies from kidney biopsies, including those performed at early stages of the COVID-19 associated kidney disease. Development of tests to detect kidney viral infection in urine samples would be more practical as a non-invasive way to evaluate SARS CoV-2 infection during the evolution of COVID-19-associated kidney disease.

Regulator of G protein signaling 4 (RGS4) is a potent negative regulator of G-protein coupled receptor signaling duration. Recent studies have identified RGS4 as a key gene in the maintenance of mechanical and cold allodynia associated with chronic pain states in mice. RGS4 is abundantly expressed across the pain matrix, and several studies have demonstrated upregulation of RGS4 mRNA in response to both peripheral inflammation and nerve injury. Here, we tested the hypothesis that peripheral nerve injury alters RGS4 neuronal expression broadly, and with specific expression-patterning among excitatory and inhibitory interneuron populations within the superficial dorsal horn of the mouse spinal cord. Chronic neuropathic pain was induced in C57BL/6 male mice via spared-nerve injury (SNI). At 2.5 months post-surgery (SNI vs sham), animals were sacrificed and lumbar spinal cords were harvested for tissue processing. RNAScope, an ultrasensitive RNA ISH method, was used to visualize and quantify single-molecule expression of RGS4, prodynorphin/PDYN (exclusively expressed by inhibitory dorsal horn interneurons), and somatostatin/SST (expressed by excitatory interneurons) within the cellular topography of the lumbar superficial dorsal horn. RGS4 expression increased significantly in the dorsal horn following spared-nerve injury (P =0.0384). RGS4 mRNA remained unchanged (SNI vs sham) among expression-positive dorsal horn neurons (p=0.6327). In sham-treated mice, there were no differences in cell-type specific (PDYN vs. SST) RGS4 expression patterns in the dorsal horn (p=0.4170). Cell-type specific RGS4 expression in the dorsal horn was significantly different (F (2,27) = 8.353; P =0.0015; one-way ANOVA) following spared-nerve injury. SNI-induced RGS4 expression was significantly enriched in PDYN vs SST-expressing interneurons (p=0.0300), and overall RGS4 expression was significantly downregulated in SST-expressing cells compared to all cells (p=0.0012). These data demonstrate that nerve injury induces upregulation of RGS4 in the superficial dorsal horn, and suggest that RGS4-positive inhibitory interneurons may be implicated in influencing persistent pain signaling within the dorsal horn of the spinal cord.

Purpose : Soluble epoxide hydrolase (sEH) metabolizes pro-resolving epoxy fatty acids into diols. sEH is a potential therapeutic target for choroidal neovascularization (CNV) in wet age-related macular degeneration (AMD) and other eye diseases. Localization of sEH in the retina is contentious and cross-interpretation among different studies is complicated due to antibody limitations. This study aimed to define the localization of sEH through co-staining with retinal cell type markers and RNAscope in situ hybridization in human AMD and control eyes, and in mouse eyes with and without laser-induced CNV. Methods : Paraffin sections of eyes from anonymized human wet AMD and control subjects were obtained from the National Disease Research Interchange. 7-week old C57BL/6J mice underwent laser-induced CNV and on day 3 post laser, enucleated eyes were fixed and cryosectioned. Coimmunostaining was done for sEH, retinal pigment epithelium (RPE), and photoreceptor markers. RNAscope was performed using target specific probes for EPHX2 (encoding sEH) and images were acquired by confocal microscopy. Results : Costaining of sEH with cell type markers revealed that sEH is overexpressed in photoreceptors and RPE cells in areas with degenerative changes. By RNAscope, EPHX2 mRNA was also highly expressed in the pathological conditions compared to controls. EPHX2 mRNA was seen in the inner nuclear layer, outer nuclear layer and RPE of the normal and diseased human and mouse retina. Conclusions : Previous data showed sEH expression in vasculature, Müller glia, and inner and outer segments of photoreceptors. Here, we also revealed sEH protein and mRNA expression in the RPE. Overexpression of sEH at the protein and mRNA level in CNV and disease-relevant cell types indicates a functional role of sEH in AMD pathophysiology and provides a context to target these cell types for developing pharmacotherapies.

The loss of GABAergic inhibition is a mechanism that underlies neuropathic pain. Therefore, rescuing the GABAergic inhibitory tone through the activation of GABA A receptors is a strategy to reduce neuropathic pain. This study was designed to elucidate the function of the spinal α 6 -containing GABA A receptor in physiological conditions and neuropathic pain in female and male rats. Results show that α 6 -containing GABA A receptor blockade or transient α 6 -containing GABA A receptor knockdown induces evoked hypersensitivity and spontaneous pain in naive female rats. The α 6 subunit is expressed in IB4 + and CGRP + primary afferent neurons in the rat spinal dorsal horn and dorsal root ganglia but not astrocytes. Nerve injury reduces α 6 subunit protein expression in the central terminals of the primary afferent neurons and dorsal root ganglia, whereas intrathecal administration of positive allosteric modulators of the α 6 -containing GABA A receptor reduces tactile allodynia and spontaneous nociceptive behaviors in female, but not male, neuropathic rats and mice. Overexpression of the spinal α 6 subunit reduces tactile allodynia and restores α 6 subunit expression in neuropathic rats. Positive allosteric modulators of the α 6 -containing GABA A receptor induces a greater antiallodynic effect in female rats and mice compared with male rats and mice. Finally, α 6 subunit is expressed in humans. This receptor is found in CGRP + and P2X3 + primary afferent fibers but not astrocytes in the human spinal dorsal horn. Our results suggest that the spinal α 6 -containing GABA A receptor has a sex-specific antinociceptive role in neuropathic pain, suggesting that this receptor may represent an interesting target to develop a novel treatment for neuropathic pain.