Arthrofibrosis is a prevalent condition affecting greater than 5% of the general population and leads to a painful decrease in joint range of motion (ROM) and loss of independence due to pathologic accumulation of periarticular scar tissue. Current treatment options are limited in effectiveness and do not address the underlying cause of the condition: accumulation of fibrotic collagenous tissue. Herein, the naturally occurring peptide hormone relaxin-2 is administered for the treatment of adhesive capsulitis (frozen shoulder) and to restore glenohumeral ROM in shoulder arthrofibrosis. Recombinant human relaxin-2 down-regulates type I collagen and α smooth muscle actin production and increases intracellular cAMP concentration in human fibroblast-like synoviocytes, consistent with a mechanism of extracellular matrix degradation and remodeling. Pharmacokinetic profiling of a bolus administration into the glenohumeral joint space reveals the brief systemic and intraarticular (IA) half-lives of relaxin-2: 0.96 h and 0.62 h, respectively. Furthermore, using an established, immobilization murine model of shoulder arthrofibrosis, multiple IA injections of human relaxin-2 significantly improve ROM, returning it to baseline measurements collected before limb immobilization. This is in contrast to single IA (sIA) or multiple i.v. (mIV) injections of relaxin-2 with which the ROM remains constrained. The histological hallmarks of contracture (e.g., fibrotic adhesions and reduced joint space) are absent in the animals treated with multiple IA injections of relaxin-2 compared with the untreated control and the sIA-and mIV-treated animals. As these findings show, local delivery of relaxin-2 is an innovative treatment of shoulder arthrofibrosis. relaxin | fibrosis | frozen shoulder | arthrofibrosis

The mechanisms by which bacteria sense the host environment and alter gene expression are poorly understood. LiaFSR is a gene regulatory system unique to Gram-positive bacteria including group A Streptococcus (GAS) and responds to cell envelope stress. We previously showed that LiaF acts as an inhibitor to LiaFSR activation in GAS. To better understand gene regulation associated with LiaFSR activation, we performed RNA-sequencing on isogenic deletion mutants fixed in a LiaFSR “always on” (Δ liaF ) or “always off” (Δ liaR ) state. Transcriptome analyses of Δ liaF and Δ liaR in GAS showed near perfect inverse correlation including the gene encoding the global transcriptional regulator SpxA2. In addition, mutant transcriptomes included genes encoding multiple virulence factors and showed substantial overlap with the CovRS regulon. Chromatin immunoprecipitation quantitative PCR demonstrated direct spxA2 gene regulation following activation of the response regulator, LiaR. High SpxA2 levels as a result of LiaFSR activation were directly correlated with increased CovR-regulated virulence gene transcription. Further, consistent with known virulence gene repression by phosphorylated CovR, elevated SpxA2 levels were inversely correlated with CovR phosphorylation. Despite increased transcription of several virulence factors, Δ liaF (high SpxA2) exhibited a paradoxical virulence phenotype in both in vivo mouse and ex vivo human blood models of disease. Likewise, despite decreased virulence factor transcription in Δ liaR (low SpxA2), increased virulence was observed in an in vivo mouse model of disease – a phenotype attributable, in part, to known SpxA2-associated speB transcription. Our findings provide evidence of a critical role of LiaFSR in sensing the host environment and suggest a potential mechanism for gene regulatory system crosstalk shared by many Gram-positive pathogens.

Hutchinson–Gilford progeria syndrome (HGPS) is a uniformly fatal condition that is especially prevalent in skin, cardiovascular, and musculoskeletal systems. A wide gap exists between our knowledge of the disease and a promising treatment or cure. The aim of this study was to first characterize the musculoskeletal phenotype of the homozygous G608G BAC-transgenic progeria mouse model, and to determine the phenotype changes of HGPS mice after a five-arm preclinical trial of different treatment combinations with lonafarnib, pravastatin, and zoledronic acid. Microcomputed tomography and CT-based rigidity analyses were performed to assess cortical and trabecular bone structure, density, and rigidity. Bones were loaded to failure with three-point bending to assess strength. Contrast-enhanced µCT imaging of mouse femurs was performed to measure glycosaminoglycan content, thickness, and volume of the femoral head articular cartilage. Advanced glycation end products were assessed with a fluorometric assay. The changes demonstrated in the cortical bone structure, rigidity, stiffness, and modulus of the HGPS G608G mouse model may increase the risk for bending and deformation, which could result in the skeletal dysplasia characteristic of HGPS. Cartilage abnormalities seen in this HGPS model resemble changes observed in the age-matched WT controls, including early loss of glycosaminoglycans, and decreased cartilage thickness and volume. Such changes might mimic prevalent degenerative joint diseases in the elderly. Lonafarnib monotherapy did not improve bone or cartilage parameters, but treatment combinations with pravastatin and zoledronic acid significantly improved bone structure and mechanical properties and cartilage structural parameters, which ameliorate the musculoskeletal phenotype of the disease.

LiaFSR is a gene regulatory system important for response to cell membrane stress in Gram-positive bacteria but is minimally studied in the important human pathogen group A Streptococcus (GAS). Using immunofluorescence and immunogold electron microscopy, we discovered that LiaF (a membrane-bound repressor protein) and LiaS (a sensor kinase) reside within the GAS membrane microdomain (ExPortal). Cell envelope stress induced by antimicrobials resulted in ExPortal disruption and activation of the LiaFSR system. The only human antimicrobial peptide whose presence resulted in ExPortal disruption and LiaFSR activation was the alpha-defensin human neutrophil peptide 1 (hNP-1). Elimination of membrane cardiolipin through targeted gene deletion resulted in loss of LiaS colocalization with the GAS ExPortal and activation of LiaFSR, whereas LiaF membrane localization was unaffected. Isogenic mutants lacking either LiaF or LiaS revealed a critical role of LiaF in ExPortal integrity. Thus, LiaF and LiaS colocalize with the GAS ExPortal by distinct mechanisms, further supporting codependence. These are the first data identifying a multicomponent signal system within the ExPortal, thereby providing new insight into bacterial intramembrane signaling in GAS that may serve as a paradigm for Gram-positive bacteria. IMPORTANCE Bacterial two-component systems sense and induce transcriptional changes in response to environmental stressors, including antimicrobials and human antimicrobial peptides. Since the stresses imposed by the host’s defensive responses may act as markers of specific temporal stages of disease progression or host compartments, pathogens often coordinately regulate stress response programs with virulence factor expression. The mechanism by which bacteria recognize these stresses and subsequently induce transcriptional responses remains not well understood. In this study, we showed that LiaFSR senses cell envelope stress through colocalization of LiaF and LiaS with the group A Streptococcus (GAS) ExPortal and is activated in direct response to ExPortal disruption by antimicrobials or human antimicrobial peptides. Our studies shed new light on the sensing of cell envelope stress in Gram-positive bacteria and may contribute to the development of therapies targeting these processes.

Subtle differences were noted at the bones' microstructural level, however these are likely the result of random effects that do not translate into changes that are biologically relevant. Similarly, differences were not seen at the mechanical level, nor were they reflected in blood-based biomarkers of bone metabolism. Altogether, dietary consumption of PUFA do not seem to affect bone structure or metabolism in a healthy model of growing mice.