Quantification of clesrovimab, an investigational, half-life extended, anti-respiratory syncytial virus protein F human monoclonal antibody in the nasal epithelial lining fluid of healthy adults.

BACKGROUND: Clesrovimab (MK-1654) is an investigational, half-life extended human monoclonal antibody (mAb) against RSV F glycoprotein in clinical trials as a prophylactic agent against RSV infection for infants. METHODS: This adult study measured clesrovimab concentrations in the serum and nasal epithelial lining fluid (ELF) to establish the partitioning of the antibody after dosing. Clesrovimab concentrations in the nasal ELF were normalized for sampling dilution using urea concentrations from ELF and serum. Furthermore, in vitro RSV neutralization of human nasal ELF following dosing was also measured to examine the activity of clesrovimab in the nasal compartment. FINDINGS: mAbs with YTE mutations are reported in literature to partition ∼1-2 % of serum antibodies into nasal mucosa. Nasal: serum ratios of 1:69-1:30 were observed for clesrovimab in two separate adult human trials after urea normalization, translating to 1.4-3.3 % of serum concentrations. The nasal PK and estimates of peripheral volume of distribution correlated with higher extravascular distribution of clesrovimab. These higher concentration of the antibody in the nasal ELF corroborated with the nasal sample's ability to neutralize RSV ex vivo. An overall trend of decreased viral plaque AUC was also noted with increasing availability of clesrovimab in the nasal ELF from a human RSV challenge study. INTERPRETATION: Along with its extended half-life, the higher penetration of clesrovimab into the nasal epithelial lining fluid and the associated local increase in RSV neutralization activity could offer infants better protection against RSV infection.

Vericiguat: A Randomized, Phase Ib, Placebo-Controlled, Double-Blind, QTc Interval Study in Patients with Chronic Coronary Syndromes.

BACKGROUND: Vericiguat is indicated for the treatment of symptomatic chronic heart failure in adult patients with reduced ejection fraction who are stabilized after a recent decompensation event. OBJECTIVE: To investigate the effects of vericiguat on QT interval in patients with chronic coronary syndromes (CCS). METHODS: This was a randomized, phase Ib, placebo-controlled, double-blind, double-dummy, multicenter study. Vericiguat once daily was up-titrated from 2.5 mg to 5 mg and then to 10 mg (treatments A, B, and C) at 14-day intervals. Positive control was moxifloxacin 400 mg (single dose on day 8 or day 50; placebo on other days [treatment D]). We evaluated the placebo-adjusted change from baseline of the Frederica-corrected QTc interval (QTcF), pharmacokinetics, safety, and tolerability of vericiguat. RESULTS: In total, 74 patients with CCS, with mean (standard deviation) age 63.4 (8.0) years, were included and 72 patients completed the study. At each timepoint up to 7 h after administration, mean placebo-corrected change in QTcF from baseline was < 6 ms and the upper limit of the two-sided 90% confidence interval of the mean was below the 10-ms threshold for clinical relevance. Moxifloxacin confirmed the assay sensitivity. Median time of maximum concentration of vericiguat was 4.5 h post-dose. The adverse event profile of vericiguat was consistent with its mechanism of action, and the findings did not indicate any safety concerns. CONCLUSIONS: As part of an integrative risk assessment, this study demonstrated no clinically relevant corrected QT prolongation with vericiguat 10 mg once daily at steady state. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov number, NCT03504982.

Vericiguat is approved for treating worsening heart failure with reduced ejection fraction. As part of the safety evaluation of vericiguat, this study assessed its effect on the QT interval of the electrocardiogram. An electrocardiogram measures electrical activity of the heart. The QT interval is the time from the start of the Q wave to the end of the T wave. A longer than normal QT interval indicates an increased chance for abnormal heart rhythms. Usually, a QT study is conducted at high doses in healthy volunteers. Previous studies indicated that high doses of vericiguat may cause increased changes in blood pressure in healthy volunteers. Therefore, this study was performed in patients at a normal therapeutic dose. Patients with chronic coronary syndromes were enrolled rather than patients with heart failure with reduced ejection fraction, because they have fewer electrocardiogram abnormalities. The starting dose of vericiguat was 2.5 mg once daily, and the dose was increased to 5 mg and then to 10 mg at 14-day intervals. Placebo was tested for comparison and moxifloxacin (400 mg), a drug known to increase the QT interval, was tested to confirm that the study could detect a change in the QT interval. An increase in the QT interval of more than 10 ms was considered clinically relevant. Of 74 patients included, 72 completed the study. At each timepoint (up to 7 h after dosing), the difference between the QT change for vericiguat and placebo was less than 10 ms; therefore, vericiguat does not prolong the QT interval to a clinically relevant extent.

A phase I study to evaluate safety, pharmacokinetics, and pharmacodynamics of respiratory syncytial virus neutralizing monoclonal antibody MK-1654 in healthy Japanese adults.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection among all infants worldwide and remains a significant cause of morbidity and mortality. To address this unmet medical need, MK-1654, a half-life extended RSV neutralizing monoclonal antibody, is in clinical development for the prevention of RSV disease in infants. This was a phase I, randomized, placebo-controlled, single-site, double-blind trial of MK-1654 in 44 healthy Japanese adults. The safety, tolerability, pharmacokinetics, antidrug antibodies (ADAs), and serum neutralizing antibody (SNA) titers against RSV were evaluated for 1 year after a single intramuscular (i.m.) or intravenous (i.v.) dose of MK-1654 or placebo in five groups (100 mg i.m., 300 mg i.m., 300 mg i.v., 1000 mg i.v., or placebo). MK-1654 was generally well-tolerated in Japanese adults. There were no serious drug-related adverse events (AEs) reported in any MK-1654 recipient and no discontinuations due to any AEs in the study. The half-life of MK-1654 ranged from 76 to 91 days across dosing groups. Estimated bioavailability was 86% for 100 mg i.m. and 77% for 300 mg i.m. One participant out of 33 (3.0%) developed detectable ADA with no apparent associated AEs. The RSV SNA titers increased in a dose-dependent manner among participants who received MK-1654. These data support the development of MK-1654 for use in Japanese infants.

Safety and immunogenicity of the measles vector-based SARS-CoV-2 vaccine candidate, V591, in adults: results from a phase 1/2 randomised, double-blind, placebo-controlled, dose-ranging trial.

BACKGROUND: We report on the safety and immunogenicity of V591, a measles vector-based SARS-CoV-2 vaccine candidate. METHODS: In this multicentre, randomised, placebo-controlled, double-blind, phase 1/2 trial, healthy adults with no history of COVID-19 disease were assigned to intramuscular injection of V591 or placebo (4:1 ratio). In part 1, younger adults (18-55 years) received V591 median tissue culture infectious dose (TCID50)-levels of 1×105 or 1×106 or placebo, 56 days apart. In part 2, younger and older (>55 years) adults received a single dose of one of four (104/105/106/107) or one of two (105/106) V591 TCID50 levels, respectively, or placebo. PRIMARY OUTCOME: safety/tolerability. Secondary outcome: humoral immunogenicity. ClinicalTrials.gov: NCT04498247. FINDINGS: From August-December 2020, 444 participants were screened and 263 randomised (210 V591; 53 placebo); 262 received at least one and 10 received two doses of V591 or placebo. Adverse events were experienced by 140/209 (67.0%) V591 dose-group participants and 37/53 (69.8%) placebo-group participants following injection 1; most frequent were fatigue (57 [27.3%] vs 20 [37.7%]), headache (57 [27.3%] vs 19 [35.8%]), myalgia (35 [16.7%] vs 10 [18.9%]), and injection-site pain (35 [16.7%] vs 4 [7.5%]). No deaths nor vaccine-related serious adverse events occurred. At Day 29, no anti-SARS-CoV-2 spike serum neutralising antibody and IgG-responses were identified in placebo or the three lower V591 dose-groups; responses were detected with V591 1×107 TCID50, although titres were lower than convalescent serum. INTERPRETATION: V591 was generally well tolerated, but immunogenicity was insufficient to warrant continued development. FUNDING: Merck Sharp & Dohme, Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA.

Forward and reverse translational approaches to predict efficacy of neutralizing respiratory syncytial virus (RSV) antibody prophylaxis.

BACKGROUND: Neutralizing mAbs can prevent communicable viral diseases. MK-1654 is a respiratory syncytial virus (RSV) F glycoprotein neutralizing monoclonal antibody (mAb) under development to prevent RSV infection in infants. Development and validation of methods to predict efficacious doses of neutralizing antibodies across patient populations exposed to a time-varying force of infection (i.e., seasonal variation) are necessary. METHODS: Five decades of clinical trial literature were leveraged to build a model-based meta-analysis (MBMA) describing the relationship between RSV serum neutralizing activity (SNA) and clinical endpoints. The MBMA was validated by backward translation to animal challenge experiments and forward translation to predict results of a recent RSV mAb trial. MBMA predictions were evaluated against a human trial of 70 participants who received either placebo or one of four dose-levels of MK-1654 and were challenged with RSV [NCT04086472]. The MBMA was used to perform clinical trial simulations and predict efficacy of MK-1654 in the infant target population. FINDINGS: The MBMA established a quantitative relationship between RSV SNA and clinical endpoints. This relationship was quantitatively consistent with animal model challenge experiments and results of a recently published clinical trial. Additionally, SNA elicited by increasing doses of MK-1654 in humans reduced RSV symptomatic infection rates with a quantitative relationship that approximated the MBMA. The MBMA indicated a high probability that a single dose of ≥ 75 mg of MK-1654 will result in prophylactic efficacy (> 75% for 5 months) in infants. INTERPRETATION: An MBMA approach can predict efficacy of neutralizing antibodies against RSV and potentially other respiratory pathogens.

A phase 1, randomized, placebo-controlled study to evaluate the safety and immunogenicity of an mRNA-based RSV prefusion F protein vaccine in healthy younger and older adults.

Respiratory Syncytial Virus (RSV) causes lower respiratory tract infections that can be severe and sometimes fatal. The risk for severe RSV infection is highest in infants and older adults. A safe and effective RSV vaccine for older adults represents a serious unmet medical need due to higher morbidity and mortality in this age group. In this randomized, partially double-blind, placebo-controlled, phase 1 dose-escalation study, we evaluated the safety, tolerability and immunogenicity of an investigational messenger ribonucleic acid (mRNA) vaccine encoding the RSV fusion protein (F) stabilized in the prefusion conformation. The study was conducted in healthy younger adults (ages ≥18 and ≤49 years) and healthy older adults (ages ≥60 and ≤79 years). Participants received mRNA-1777 (V171) or placebo as a single intramuscular dose. For each dose level, three sentinel participants were administered open-label mRNA-1777 (V171). Seventy-two younger adults were randomized and administered 25, 100, or 200 µg mRNA-1777 (V171) or placebo, and 107 older adults were randomized and administered 25, 100, 200 or 300 µg mRNA-1777 (V171) or placebo. Primary objectives were safety and tolerability and secondary objectives included humoral and cell-mediated immunogenicity. All dose levels of mRNA-1777 (V171) were generally well tolerated and no serious adverse events related to the vaccine were reported. Immunization with mRNA-1777 (V171) elicited a humoral immune response as measured by increases in RSV neutralizing antibody titers, serum antibody titers to RSV prefusion F protein, D25 competing antibody titers to RSV prefusion F protein, and cell-mediated immune responses to RSV-F peptides.

A Phase 1 Randomized, Double-Blind, Placebo-Controlled Trial to Assess the Safety, Tolerability, and Pharmacokinetics of a Respiratory Syncytial Virus Neutralizing Monoclonal Antibody MK-1654 in Healthy Adults.

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infection and related morbidity and mortality in infants. Passive immunization with an RSV-neutralizing antibody can provide rapid protection to this vulnerable population. Proof-of-concept for this approach has been demonstrated by palivizumab; however, the use of this antibody is generally restricted to the highest-risk infants due to monthly dosing requirements and its cost. To address the large unmet medical need for most infants, we are evaluating MK-1654, a fully human RSV-neutralizing antibody with half-life extending mutations targeting site IV of the fusion protein. In this 2-part, placebo-controlled, double-blind, first-in-human study, 152 healthy adults were randomized 3:1 to receive a single dose of MK-1654 or placebo in 5 cohorts (100 or 300 mg as an intramuscular dose or 300, 1000, or 3000 mg as an intravenous dose). Safety, pharmacokinetics, antidrug antibodies, and RSV serum-neutralizing antibody titers were evaluated through 1 year. MK-1654 serum concentrations increased proportionally with dose and resulted in corresponding elevations in RSV serum-neutralizing antibody titers. The antibody displayed a half-life of 73 to 88 days and an estimated bioavailability of 69% at the 300-mg dose. The overall safety profile of MK-1654 was similar to placebo, and treatment-emergent antidrug antibodies were low (2.6%) with no associated adverse events. These data support the continued development of MK-1654 for the prevention of RSV disease in infants.

Rhinovirus-16 induced temporal interferon responses in nasal epithelium links with viral clearance and symptoms.

BACKGROUND: The temporal in vivo response of epithelial cells to a viral challenge and its association with viral clearance and clinical outcomes has been largely unexplored in asthma. OBJECTIVE: To determine gene expression profiles over time in nasal epithelial cells (NECs) challenged in vivo with rhinovirus-16 (RV16) and compare to nasal symptoms and viral clearance. METHODS: Patients with stable mild to moderate asthma (n = 20) were challenged intranasally with RV16. Nasal brush samples for RNA sequencing were taken 7 days prior to infection and 3, 6 and 14 days post-infection, and blood samples 4 days prior to infection and day 6 post-infection. Viral load was measured in nasal lavage fluid at day 3, 6 and 14. RESULTS: Top differentially (>2.5-fold increase) expressed gene sets in NECs post-RV16 at days 3 and 6, compared with baseline, were interferon alpha and gamma response genes. Patients clearing the virus within 6 days (early resolvers) had a significantly increased interferon response at day 6, whereas those having cleared the virus by day 14 (late resolvers) had significantly increased responses at day 3, 6 and 14. Interestingly, patients not having cleared the virus by day 14 (non-resolvers) had no enhanced interferon responses at any of these days. The daily Cold Symptom Scores (CSS) peaked at days 3 to 5 and correlated positively with interferon response genes at day 3 (R = 0.48), but not at other time-points. Interferon response genes were also enhanced in blood at day 6 after RV16 challenge. CONCLUSION AND CLINICAL RELEVANCE: This study shows that viral load and clearance varies markedly over time in mild to moderate asthma patients exposed to a fixed RV16 dose. The host's nasal interferon response to RV16 at day 3 is associated with upper respiratory tract symptoms. The temporal interferon response in nasal epithelium associates with viral clearance in the nasal compartment.

Author Correction: Towards an arthritis flare-responsive drug delivery system.

In the original version of this Article, financial support was not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to include support from the National Football League Players Association.

Towards an arthritis flare-responsive drug delivery system.

Local delivery of therapeutics for the treatment of inflammatory arthritis (IA) is limited by short intra-articular half-lives. Since IA severity often fluctuates over time, a local drug delivery method that titrates drug release to arthritis activity would represent an attractive paradigm in IA therapy. Here we report the development of a hydrogel platform that exhibits disassembly and drug release controlled by the concentration of enzymes expressed during arthritis flares. In vitro, hydrogel loaded with triamcinolone acetonide (TA) releases drug on-demand upon exposure to enzymes or synovial fluid from patients with rheumatoid arthritis. In arthritic mice, hydrogel loaded with a fluorescent dye demonstrates flare-dependent disassembly measured as loss of fluorescence. Moreover, a single dose of TA-loaded hydrogel but not the equivalent dose of locally injected free TA reduces arthritis activity in the injected paw. Together, our data suggest flare-responsive hydrogel as a promising next-generation drug delivery approach for the treatment of IA.

Stretching Reduces Skin Thickness and Improves Subcutaneous Tissue Mobility in a Murine Model of Systemic Sclerosis.

OBJECTIVE: Although physical therapy can help preserve mobility in patients with systemic sclerosis (SSc), stretching has not been used systematically as a treatment to prevent or reverse the disease process. We previously showed in rodent models that stretching promotes the resolution of connective tissue inflammation and reduces new collagen formation after injury. Here, we tested the hypothesis that stretching would impact scleroderma development using a mouse sclerodermatous graft-versus-host disease (sclGvHD) model. METHODS: The model consists in the adoptive transfer (allogeneic) of splenocytes from B10.D2 mice (graft) into Rag2-/- BALB/c hosts (sclGvHD), resulting in skin inflammation followed by fibrosis over 4 weeks. SclGvHD mice and controls were randomized to stretching in vivo for 10 min daily versus no stretching. RESULTS: Weekly ultrasound measurements of skin thickness and subcutaneous tissue mobility in the back (relative tissue displacement during passive trunk motion) successfully captured the different phases of the sclGvHD model. Stretching reduced skin thickness and increased subcutaneous tissue mobility compared to no stretching at week 3. Stretching also reduced the expression of CCL2 and ADAM8 in the skin at week 4, which are two genes known to be upregulated in both murine sclGvHD and the inflammatory subset of human SSc. However, there was no evidence that stretching attenuated inflammation at week 2. CONCLUSION: Daily stretching for 10 min can improve skin thickness and mobility in the absence of any other treatment in the sclGvHD murine model. These pre-clinical results suggest that a systematic investigation of stretching as a therapeutic modality is warranted in patients with SSc.

Gut microbiota induce IGF-1 and promote bone formation and growth.

Appreciation of the role of the gut microbiome in regulating vertebrate metabolism has exploded recently. However, the effects of gut microbiota on skeletal growth and homeostasis have only recently begun to be explored. Here, we report that colonization of sexually mature germ-free (GF) mice with conventional specific pathogen-free (SPF) gut microbiota increases both bone formation and resorption, with the net effect of colonization varying with the duration of colonization. Although colonization of adult mice acutely reduces bone mass, in long-term colonized mice, an increase in bone formation and growth plate activity predominates, resulting in equalization of bone mass and increased longitudinal and radial bone growth. Serum levels of insulin-like growth factor 1 (IGF-1), a hormone with known actions on skeletal growth, are substantially increased in response to microbial colonization, with significant increases in liver and adipose tissue IGF-1 production. Antibiotic treatment of conventional mice, in contrast, decreases serum IGF-1 and inhibits bone formation. Supplementation of antibiotic-treated mice with short-chain fatty acids (SCFAs), products of microbial metabolism, restores IGF-1 and bone mass to levels seen in nonantibiotic-treated mice. Thus, SCFA production may be one mechanism by which microbiota increase serum IGF-1. Our study demonstrates that gut microbiota provide a net anabolic stimulus to the skeleton, which is likely mediated by IGF-1. Manipulation of the microbiome or its metabolites may afford opportunities to optimize bone health and growth.

RANK-Independent Osteoclast Formation and Bone Erosion in Inflammatory Arthritis.

OBJECTIVE: Proinflammatory molecules promote osteoclast-mediated bone erosion by up-regulating local RANKL production. However, recent evidence suggests that combinations of cytokines, such as tumor necrosis factor (TNF) plus interleukin-6 (IL-6), induce RANKL-independent osteoclastogenesis. The purpose of this study was to better understand TNF/IL-6-induced osteoclast formation and to determine whether RANK is absolutely required for osteoclastogenesis and bone erosion in murine inflammatory arthritis. METHODS: Myeloid precursors from wild-type (WT) mice or mice with either germline or conditional deletion of Rank, Nfatc1, Dap12, or Fcrg were treated with either RANKL or TNF plus IL-6. Osteoprotegerin, anti-IL-6 receptor (anti-IL-6R), and hydroxyurea were used to block RANKL, the IL-6R, and cell proliferation, respectively. Clinical scoring, histologic assessment, micro-computed tomography, and quantitative polymerase chain reaction (qPCR) were used to evaluate K/BxN serum-transfer arthritis in WT and RANK-deleted mice. Loss of Rank was verified by qPCR and by osteoclast cultures. RESULTS: TNF/IL-6 generated osteoclasts in vitro that resorbed mineralized tissue through a pathway dependent on IL-6R, NFATc1, DNAX-activation protein 12, and cell proliferation, but independent of RANKL or RANK. Bone erosion and osteoclast formation were reduced, but not absent, in arthritic mice with inducible deficiency of RANK. TNF/IL-6, but not RANKL, induced osteoclast formation in bone marrow and synovial cultures from animals deficient in Rank. Multiple IL-6 family members (IL-6, leukemia inhibitory factor, oncostatin M) were up-regulated in the synovium of arthritic mice. CONCLUSION: The persistence of bone erosion and synovial osteoclasts in Rank-deficient mice, and the ability of TNF/IL-6 to induce osteoclastogenesis, suggest that more than one cytokine pathway exists to generate these bone-resorbing cells in inflamed joints.

IL4RA on lymphatic endothelial cells promotes T cell egress during sclerodermatous graft versus host disease.

Systemic sclerosis (SSc) is a potentially fatal autoimmune disorder with limited therapeutic options. Sclerodermatous graft versus host disease (sclGvHD), induced by transfer of B10.D2 splenocytes into BALB/c Rag2-/- mice, models an inflammatory subset of SSc characterized by a prominent IL13-induced gene expression signature in the skin. Host mice deficient in IL4RA, a subunit of the type II IL4/IL13 receptor, are protected from sclGvHD. While IL4RA has a well-established role in Th2 differentiation and alternative macrophage activation, we report here a previously unappreciated function for IL4RA in lymphatic endothelial cells (LECs): regulation of activated T cell egress. Seven days after splenocyte transfer, Il4ra-/- hosts had increased numbers of activated graft CD4+ T cells in skin draining lymph nodes (dLNs) but fewer T cells in efferent lymph, blood, and skin. Sphingosine-1 phosphate (S1P), master regulator of lymphocyte egress from LNs, was lower in dLNs of Il4ra-/- hosts with a corresponding decrease of S1P kinase 1 (Sphk1) expression in LECs. Bypassing the efferent lymphatics via i.v. injection of CD4+ T cells from dLNs of Il4ra-/- sclGvHD mice restored clinical GvHD in secondary Il4ra-/- recipients. These results identify a role for IL4RA and suggest that modulation of lymphocyte egress from LNs may be effective in SSc and GvHD.

Anion Exchanger 2 Regulates Dectin-1-Dependent Phagocytosis and Killing of Candida albicans.

Anion exchanger 2 (Ae2; gene symbol, Slc4a2) is a plasma membrane Cl-/HCO3- exchanger expressed in the gastrointestinal tract, kidney and bone. We have previously shown that Ae2 is required for the function of osteoclasts, bone resorbing cells of the macrophage lineage, to maintain homeostatic cytoplasmic pH and electroneutrality during acid secretion. Macrophages require endosomal acidification for pathogen killing during the process known as phagocytosis. Chloride is thought to be the principal ion responsible for maintaining electroneutrality during organelle acidification, but whether Cl-/HCO3- exchangers such as Ae2 contribute to macrophage function is not known. In this study we investigated the role of Ae2 in primary macrophages during phagocytosis. We find that Ae2 is expressed in macrophages where it regulates intracellular pH and the binding of Zymosan, a fungal cell wall derivative. Surprisingly, the transcription and surface expression of Dectin-1, the major phagocytic receptor for Candida albicans (C. albicans) and Zymosan, is reduced in the absence of Ae2. As a consequence, Zymosan-induced Tnfα expression is also impaired in Ae2-deficient macrophages. Similar to Ae2 deficiency, pharmacological alkalinization of lysosomal pH with bafilomycin A decreases both Dectin-1 mRNA and cell surface expression. Finally, Ae2-deficient macrophages demonstrate defective phagocytosis and killing of the human pathogenic fungus C. albicans. Our results strongly suggest that Ae2 is a critical factor in the innate response to C. albicans. This study represents an important contribution to a better understanding of how Dectin-1 expression and fungal clearance is regulated.

Sex-Specific Protection of Osteoarthritis by Deleting Cartilage Acid Protein 1.

Cartilage acidic protein 1 (CRTAC1) was recently identified as an elevated protein in the synovial fluid of patients with osteoarthritis (OA) by a proteomic analysis. This gene is also upregulated in both human and mouse OA by transcriptomic analysis. The objective of this study was to characterize the expression and function of CRTAC1 in OA. Here, we first confirm the increase of CRTAC1 in cartilage biopsies from OA patients undergoing joint replacement by real-time PCR and immunohistochemistry. Furthermore, we report that proinflammatory cytokines interleukin-1beta and tumor necrosis factor alpha upregulate CRTAC1 expression in primary human articular chondrocytes and synovial fibroblasts. Genetic deletion of Crtac1 in mice significantly inhibited cartilage degradation, osteophyte formation and gait abnormalities of post-traumatic OA in female, but not male, animals undergoing the destabilization of medial meniscus (DMM) surgery. Taken together, CRTAC1 is upregulated in the osteoarthritic joint and directly induced in chondrocytes and synovial fibroblasts by pro-inflammatory cytokines. This molecule is necessary for the progression of OA in female mice after DMM surgery and thus represents a potential therapy for this prevalent disease, especially for women who demonstrate higher rates and more severe OA.

NFAT restricts osteochondroma formation from entheseal progenitors.

Osteochondromas are common benign osteocartilaginous tumors in children and adolescents characterized by cartilage-capped bony projections on the surface of bones. These tumors often cause pain, deformity, fracture, and musculoskeletal dysfunction, and they occasionally undergo malignant transformation. The pathogenesis of osteochondromas remains poorly understood. Here, we demonstrate that nuclear factor of activated T cells c1 and c2 (NFATc1 and NFATc2) suppress osteochondromagenesis through individual and combinatorial mechanisms. In mice, conditional deletion of NFATc1 in mesenchymal limb progenitors, Scleraxis-expressing (Scx-expressing) tendoligamentous cells, or postnatally in Aggrecan-expressing cells resulted in osteochondroma formation at entheses, the insertion sites of ligaments and tendons onto bone. Combinatorial deletion of NFATc1 and NFATc2 gave rise to larger and more numerous osteochondromas in inverse proportion to gene dosage. A population of entheseal NFATc1- and Aggrecan-expressing cells was identified as the osteochondroma precursor, previously believed to be growth plate derived or perichondrium derived. Mechanistically, we show that NFATc1 restricts the proliferation and chondrogenesis of osteochondroma precursors. In contrast, NFATc2 preferentially inhibits chondrocyte hypertrophy and osteogenesis. Together, our findings identify and characterize a mechanism of osteochondroma formation and suggest that regulating NFAT activity is a new therapeutic approach for skeletal diseases characterized by defective or exaggerated osteochondral growth.

Identification of Optimal Mouse Models of Systemic Sclerosis by Interspecies Comparative Genomics.

OBJECTIVE: Understanding the pathogenesis of systemic sclerosis (SSc) is confounded by considerable disease heterogeneity. Animal models of SSc that recapitulate distinct subsets of disease at the molecular level have not been delineated. We applied interspecies comparative analysis of genomic data from multiple mouse models of SSc and patients with SSc to determine which animal models best reflect the SSc intrinsic molecular subsets. METHODS: Gene expression measured in skin from mice with sclerodermatous graft-versus-host disease (GVHD), bleomycin-induced fibrosis, Tsk1/+ or Tsk2/+ mice was mapped to human orthologs and compared to SSc skin biopsy-derived gene expression. Transforming growth factor ß (TGFß) activation was assessed using a responsive signature in mice, and tumor necrosis factor receptor superfamily member 12A (TNFRSF12A) expression was measured in SSc patient and mouse skin. RESULTS: Gene expression in skin from mice with sclerodermatous GVHD and bleomycin-induced fibrosis corresponded to that in SSc patients in the inflammatory molecular subset. In contrast, Tsk2/+ mice showed gene expression corresponding to the fibroproliferative SSc subset. Enrichment of a TGFß-responsive signature was observed in both Tsk2/+ mice and mice with bleomycin-induced skin fibrosis. Expression of TNFRSF12A (the TWEAK receptor/fibroblast growth factor-inducible 14) was elevated in skin from patients with fibroproliferative SSc and the skin of Tsk2/+ mice. CONCLUSION: This study reveals similarities in cutaneous gene expression between distinct mouse models of SSc and specific molecular subsets of the disease. Different pathways underlie the intrinsic subsets including TGFß, interleukin-13 (IL-13), and IL-4. We identify a novel target, Tnfrsf12a, with elevated expression in skin from patients with fibroproliferative SSc and Tsk2/+ mice. These findings will inform mechanistic and translational preclinical studies in SSc.

Cryopreserved Amniotic Suspension for the Treatment of Knee Osteoarthritis.

There are few treatment options for symptomatic knee osteoarthritis (OA). Human amniotic suspension allografts (ASA) have anti-inflammatory and chondroregenerative potential and thus represent a promising treatment strategy. In anticipation of a large, placebo-controlled trial of intra-articular ASA for symptomatic knee OA, an open-label prospective feasibility study was performed. Six patients with Kellgren-Lawrence grades 3 and 4 tibiofemoral knee OA were administered a single intra-articular ASA injection containing cryopreserved particulated human amnion and amniotic fluid cells. Patients were followed for 12 months after treatment. No significant injection reactions were noted. Compared with baseline there were (1) no significant effect of the ASA injection on blood cell counts, lymphocyte subsets, or inflammatory markers and (2) a small, but statistically significant increase in serum IgG and IgE levels. Patient-reported outcomes including International Knee Documentation Committee, Knee Injury and Osteoarthritis Outcome, and Single Assessment Numeric Evaluation scores were collected throughout the study and evaluated for up to 12 months. Overall, this study demonstrates the feasibility of a single intra-articular injection of ASA for the treatment of knee OA and provides the foundation for a large placebo-controlled trial of intra-articular ASA for symptomatic knee OA.

The intestinal microbiome and skeletal fitness: Connecting bugs and bones.

Recent advances have dramatically increased our understanding of how organ systems interact. This has been especially true for immunology and bone biology, where the term "osteoimmunology" was coined to capture this relationship. The importance of the microbiome to the immune system has also emerged as a driver of health and disease. It makes sense therefore to ask the question: how does the intestinal microbiome influence bone biology and does dysbiosis promote bone disease? Surprisingly, few studies have analyzed this connection. A broader interpretation of this question reveals many mechanisms whereby the microbiome may affect bone cells. These include effects of the microbiome on immune cells, including myeloid progenitors and Th17 cells, as well as steroid hormones, fatty acids, serotonin and vitamin D. As mechanistic interactions of the microbiome and skeletal system are revealed within and without the immune system, novel strategies to optimize skeletal fitness may emerge.