A Unique Case of Adult-Onset Still's Disease Not Responsive to Steroid Treatment.

A 53-year-old patient was admitted to the emergency department, presenting with fever, generalized weakness, and various myalgias and arthralgias lasting over seven days. Based upon the patient's worsening symptoms, elevated white blood cell count with neutrophilia and overall presentation, she was initially treated for an infectious cause and prescribed various antibiotics and antipyretic medications. As the patient's condition continued to worsen throughout the initial days of her intake, she was tested for a variety of infections, including coronavirus disease 2019 (COVID-19), Streptococcus, and influenza, and was administered a viral respiratory panel, all of which resulted negative. Upon the development of an evanescent rash on hospital day 9, as well as other symptoms including sore throat, arthritis, and an elevated fever present for over a week, a rheumatology consult now expressed concern for a possible case of Adult-Onset Still's Disease (AOSD). In line with the current treatment used for AOSD and the absence of all other infectious causes, the patient discontinued antibiotic treatment and was started on 125 milligrams of intravenous methylprednisolone every six hours. The patient showed minor improvements in symptoms over the next 24 hours but soon became refractory to treatment, resulting from multiorgan damage, and expired on hospital day 13.

Minoxidil weakens newly synthesized collagen in fibrotic synoviocytes from osteoarthritis patients.

PURPOSE: Synovial fibrosis (SFb) formation and turnover attributable to knee osteoarthritis (KOA) can impart painful stiffness and persist following arthroplasty. To supplement joint conditioning aimed at maximizing peri-operative function, we evaluated the antifibrotic effect of Minoxidil (MXD) on formation of pyridinoline (Pyd) cross-links catalyzed by Plod2-encoded lysyl hydroxylase (LH)2b that strengthen newly synthesized type-I collagen (COL1) in fibroblastic synovial cells (FSCs) from KOA patients. MXD was predicted to decrease Pyd without significant alterations to Col1a1 transcription by FSCs stimulated with transforming growth factor (TGF)ß1. METHODS: Synovium from 10 KOA patients grouped by SFb severity was preserved for picrosirius and LH2b histology or culture. Protein and RNA were purified from fibrotic FSCs after 8 days with or without 0.5 µM MXD and/or 4 ng/mL of TGFß1. COL1 and Pyd protein concentrations from ELISA and expression of Col1a1, Acta2, and Plod2 genes by qPCR were compared by parametric tests with α = 0.05. RESULTS: Histological LH2b expression corresponded to SFb severity. MXD attenuated COL1 output in KOA FSCs but only in the absence of TGFß1 and consistently decreased Pyd under all conditions with significant downregulation of Plod2 but minimal alterations to Col1a1 and Acta2 transcripts. CONCLUSIONS: MXD is an attractive candidate for local antifibrotic pharmacotherapy for SFb by compromising the integrity of newly formed fibrous deposits by FSCs during KOA and following arthroplasty. Targeted antifibrotic supplementation could improve physical therapy and arthroscopic lysis strategies aimed at breaking down joint scarring. However, the effect of MXD on other joint-specific TGFß1-mediated processes or non-fibrotic components requires further investigation.

Using the articularis genu to test peri-articular muscle health during knee osteoarthritis.

Knee osteoarthritis (OA) involves peri-articular sarcopenia. The infrapatellar articularis genu (AG) links to the quadriceps femoris (QF) and can be sampled from discarded tissue during arthroplasty. We predict disuse-mediated changes in AG myofiber type ratio and atrophy similar to reports on the QF during OA. OA AGs (n = 40) were preserved and grouped by poor (≤ 85°; n = 11), fair (90°-110°; n = 19), and good (≥ 115°; n = 10) range of motion (ROM). Immunolabeling of slow and fast myosin heavy chains in AG sections allowed comparing distribution and cross-sectional area (CSA) of type-I (T1) and type-II (T2) myofibers between groups and associating to ROM. T1/T2 ratios in fair and poor ROM groups was consistent with those published in OA QF. Increasing mean ± SD T2 percentages from good (43.31 ± 11.76), to fair (50.96 ± 5.85), and poor (60.02 ± 8.29) ROM groups was significant between poor versus fair (p = 0.018) and good (p < 0.0001) in association with ROM deficits (r = - 0.729; p < 0.0001). T1 and T2 CSA decreased with worsening ROM, which associates with lower symptom scores (r = 0.3198; p = 0.0472). In-depth evaluation of the OA AG as a surrogate for the OA QF relative to serum and/or synovial fluid biomarkers of sarcopenia could refine diagnostics of peri-articular muscle health to guide individualized strength rehabilitation after surgery.

Orphan Nuclear Receptor NR4A2 Is Constitutively Expressed in Cartilage and Upregulated in Inflamed Synovium From hTNF-Alpha Transgenic Mice.

Orphan nuclear receptor 4A2 (NR4A2/Nurr1) is a constitutively active transcription factor with potential roles in the onset and progression of inflammatory arthropathies. NR4A2 is overexpressed in synovium and cartilage from individuals with rheumatoid arthritis (RA), psoriatic arthritis, and osteoarthritis. This study documents the expression and tissue localization of NR4A2 and upstream regulator nuclear factor kappa B (NF-κB) in the human tumor necrosis factor-alpha (hTNF-α) transgenic mouse model of RA. Since TNF-α is a potent inducer of NR4A2 in vitro, we hypothesized that NR4A2 would also be upregulated and active during disease progression in this model. Expression levels of NR4A2, related receptors NR4A1 (Nur77) and 3 (NOR1), and NF-κB1 transcripts were quantified by RT-qPCR in hTNF-α and wild-type joints at three stages of disease. The protein distribution of NR4A2 and NF-κB subunit RelA (p65) was analyzed by quantitative immunohistochemistry. Global gene expression of 88 RA-related genes was also screened and compared between groups. Consistent with previous reports on the hTNF-α model, transgenic mice exhibited significant weight loss and severely swollen paws by 19 weeks of age compared to age-matched wild-type controls. NR4A1-3 and NF-κB1 were constitutively expressed at disease onset and in healthy joints. NF-κB1 transcript levels increased 2-fold in hTNF-α paws with established disease (12 weeks), followed by a 2-fold increase in NR4A2 at the late disease stage (19 weeks). NR4A2 and RelA proteins were overexpressed in inflamed synovium prior to symptoms of arthritis, suggesting that gene expression changes documented in whole paws were largely driven by elevated expression in diseased synovium. Broader screening of RA-related genes by RT-qPCR identified several differentially expressed genes in hTNF-α joints including those encoding inflammatory cytokines and chemokines, matrix-degrading enzymes and inhibitors, cell surface receptors, intracellular signaling proteins and transcription factors. Consensus binding sites for NR4A receptors and NF-κB1 were enriched in the promoters of differentially expressed genes suggesting central roles for these transcription factors in this model. This study is the first comprehensive analysis of NR4A2 in an animal model of RA and validates the hTNF-α model for testing of small molecules and genetic strategies targeting this transcription factor.

Differences in synovial fibrosis relative to range of motion in knee osteoarthritis patients.

This study tests if differences exist in the severity of synovial fibrosis between patients undergoing total knee arthroplasty (TKA) for osteoarthritis (OA) to help explain disparate deficits in pre- and postoperative range of motion (ROM) between patient groups. 117 knee OA patients were grouped by women (n = 74) and men (n = 43) or those who self-reported as Black (n = 48) or White (n = 69). ROM was measured pre- and post-TKA. Condyles and synovium collected during TKA were scored histologically for OA severity and synovitis. Fibrosis was measured from picrosirius-stained sections of the synovium. Data were analyzed using Mann-Whitney, parametric, and Spearman's rho tests with alpha at 0.05. We found no significant differences between patient age, BMI, radiographic scores, or deformity type when grouped by sex or race, or between metrics or OA severity when grouped by sex. Notably, higher synovitis was measured in women (p = .039) than men. White patients had greater ROM before (p = 0.46) and after surgery (p = .021) relative to Black patients. Fibrosis, but not OA severity and synovitis scores, for the total patient sample negatively correlated with preoperative (r s = -0.330; p = .0003) but not postoperative (rs = -0.032; p = .7627) ROM. Black patients manifested more fibrosis than White patients (p = <.0001), without significant differences between sexes. Statement of Clinical Significance: Coupled with histological scoring, measuring perioperative differences in synovial fibrosis against ROM may refine OA classification and justify the in-depth preoperative assessment of the knee as a whole. Such individualized analyses could guide personalized strategies to relieve symptomatic OA when TKA is not readily accessible and promote equitable TKA outcomes.

Combined Therapy With Avastin, a PAF Receptor Antagonist and a Lipid Mediator Inhibited Glioblastoma Tumor Growth.

Glioblastoma multiforme (GBM) is an aggressive, highly proliferative, invasive brain tumor with a poor prognosis and low survival rate. The current standard of care for GBM is chemotherapy combined with radiation following surgical intervention, altogether with limited efficacy, since survival averages 18 months. Improvement in treatment outcomes for patients with GBM requires a multifaceted approach due to the dysregulation of numerous signaling pathways. Recently emerging therapies to precisely modulate tumor angiogenesis, inflammation, and oxidative stress are gaining attention as potential options to combat GBM. Using a mouse model of GBM, this study aims to investigate Avastin (suppressor of vascular endothelial growth factor and anti-angiogenetic treatment), LAU-0901 (a platelet-activating factor receptor antagonist that blocks pro-inflammatory signaling), Elovanoid; ELV, a novel pro-homeostatic lipid mediator that protects neural cell integrity and their combination as an alternative treatment for GBM. Female athymic nude mice were anesthetized with ketamine/xylazine, and luciferase-modified U87MG tumor cells were stereotactically injected into the right striatum. On post-implantation day 13, mice received one of the following: LAU-0901, ELV, Avastin, and all three compounds in combination. Bioluminescent imaging (BLI) was performed on days 13, 20, and 30 post-implantation. Mice were perfused for ex vivo MRI on day 30. Bioluminescent intracranial tumor growth percentage was reduced by treatments with LAU-0901 (43%), Avastin (77%), or ELV (86%), individually, by day 30 compared to saline treatment. In combination, LAU-0901/Avastin, ELV/LAU-0901, or ELV/Avastin had a synergistic effect in decreasing tumor growth by 72, 92, and 96%, respectively. Additionally, tumor reduction was confirmed by MRI on day 30, which shows a decrease in tumor volume by treatments with LAU-0901 (37%), Avastin (67%), or ELV (81.5%), individually, by day 30 compared to saline treatment. In combination, LAU-0901/Avastin, ELV/LAU-0901, or ELV/Avastin had a synergistic effect in decreasing tumor growth by 69, 78.7, and 88.6%, respectively. We concluded that LAU-0901 and ELV combined with Avastin exert a better inhibitive effect in GBM progression than monotherapy. To our knowledge, this is the first study that demonstrates the efficacy of these novel therapeutic regimens in a model of GBM and may provide the basis for future therapeutics in GBM patients.

Quantifying Mediators of Racial Disparities in Knee Osteoarthritis Outcome Scores: A Cross-Sectional Analysis.

Studies on symptomatic osteoarthritis suggest that Black patients report worse pain and symptoms compared with White patients with osteoarthritis. In this study, we aimed to quantify the relationship among variables such as overall health and socioeconomic status that may contribute to disparities in patient-reported outcomes. METHODS: A total of 223 patients were enrolled. A mediation analysis was used to evaluate cross-sectional associations between race and the Knee injury and Osteoarthritis Outcome Score (KOOS) questionnaire, which was administered to patients prior to undergoing primary total knee arthroplasty. RESULTS: Black patients had worse KOOS pain, symptoms, and activities of daily living subscale scores than White patients. In our cohort, Black patients were younger, more likely to be female, and more likely to report lower educational status. We identified age, sex, Charlson Comorbidity Index, and education as partial mediators of racial disparities in KOOS subscale scores. Insurance status, deformity, radiographic (Kellgren-Lawrence) grade, C-reactive protein level, marital status, body mass index, and income did not show mediating effects. We found that, if age and sex were equal in both cohorts, the racial disparity in KOOS symptom scores would be reduced by 20.7% and 9.1%, respectively (95% confidence intervals [CIs], -5.1% to 47% and -5.5% to 26.3%). For KOOS pain scores, age and education level explained 18.9% and 5.1% of the racial disparity (95% CIs, -0.6% to 37% and -10.8% to 22.9%). Finally, for KOOS activities of daily living scores, education level explained 3.2% of the disparity (95% CI, -19.4% to 26.6%). CONCLUSIONS: No single factor in our study completely explained the racial disparity in KOOS scores, but our findings did suggest that several factors can combine to mediate this disparity in outcome scores. Quantification of variables that mediate racial disparity can help to build models for risk adjustment, pinpoint vulnerable populations, and identify primary points of intervention. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Proximal digit tip amputation initiates simultaneous blastema and transient fibrosis formation and results in partial regeneration.

Complete extremity regeneration in mammals is restricted to distal amputations of the digit tip, the terminal phalanx (P3). In mice, P3 regeneration is mediated via the formation of a blastema, a transient population of progenitor cells that form from the blending of periosteal and endosteal/marrow compartmentalized cells that undergo differentiation to restore the amputated structures. Compartmentalized blastema cells are formed independently, and periosteal compartment-derived cells are required for restoration of amputated skeletal length. P3 regenerative capacity is progressively attenuated at increasingly more proximal amputation levels, eventually resulting in regenerative failure. The continuum of regenerative capacity within the P3 wound milieu is a unique model to investigate mammalian blastema formation in response to distal amputation, as well as the healing response associated with regenerative failure at proximal amputation levels. We report that P3 proximal amputation healing, previously reported to result in regenerative failure, is not an example of complete regenerative failure, but instead is characterized by a limited bone regeneration response restricted to the endosteal/marrow compartment. The regeneration response is mediated by blastema formation within the endosteal/marrow compartment, and blastemal osteogenesis progresses through intramembranous ossification in a polarized proximal to distal sequence. Unlike bone regeneration following distal P3 amputation, osteogenesis within the periosteal compartment is not observed in response to proximal P3 amputation. We provide evidence that proximal P3 amputation initiates the formation of fibrotic tissue that isolates the endosteal/marrow compartment from the periosteal compartment and wound epidermis. While the fibrotic response is transient and later resolved, these studies demonstrate that blastema formation and fibrosis can occur in close proximity, with the regenerative response dominating the final outcome. Moreover, the results suggest that the attenuated proximal P3 regeneration response is associated with the absence of periosteal-compartment participation in blastema formation and bone regeneration.

Plasma flow distal to tourniquet placement provides a physiological mechanism for tissue salvage.

Military literature has demonstrated the utility and safety of tourniquets in preventing mortality for some time, paving the way for increased use of tourniquets in civilian settings, including perioperatively to provide a bloodless surgical field. However, tourniquet use is not without risk and the subsequent effects of tissue ischemia can impede downstream rehabilitative efforts to regenerate and salvage nerve, muscle, tissue and bone in the limb. Limb ischemia studies in both the mouse and pig models have indicated not only that there is residual flow past the tourniquet by means of microcirculation, but also that recovery from tissue ischemia is dependent upon this microcirculation. Here we expand upon these previous studies using portable Near-Infrared Imaging to quantify residual plasma flow distal to the tourniquet in mice, pigs, and humans and leverage this flow to show that plasma can be supersaturated with oxygen to reduce intracellular hypoxia and promote tissue salvage following tourniquet placement. Our findings provide a mechanism of delivery for the application of oxygen, tissue preservation solutions, and anti-microbial agents prior to tourniquet release to improve postoperative recovery. In the current environment of increased tourniquet use, techniques which promote distal tissue preservation and limb salvage rates are crucial.

Experimental Model of Zygomatic and Mandibular Defects to Support the Development of Custom Three-Dimensional--Printed Bone Scaffolds.

INTRODUCTION: Autologous reconstruction of segmental craniomaxillofacial bone defects is limited by insufficient graft material, donor site morbidity, and need for microsurgery. Reconstruction is challenging due to the complex three-dimensional (3D) structure of craniofacial skeleton. Customized 3D-printed patient-specific biologic scaffolds hold promise for reconstruction of the craniofacial skeleton without donor site morbidity. The authors report a porcine craniofacial defect model suitable for further evaluation of custom 3D-printed engineered bone scaffolds. METHODS: The authors created a 6 cm critical load-bearing defect in the left mandibular angle and a 1.5 cm noncritical, nonload bearing defect in the contralateral right zygomatic arch in 4 Yucatan minipigs. Defects were plated with patient-specific titanium hardware based on preoperative CT scans. Serial CT imaging was done immediately postoperatively, and at 3 and 6 months. Animals were clinically assessed for masticatory function, ambulation, and growth. At the 6-month study endpoint, animals were euthanized, and bony regeneration was evaluated through histological staining and micro-CT scanning compared to contralateral controls. RESULTS: All 4 animals reached study endpoint. Two mandibular plates fractured, but did not preclude study completion due to loss of masticatory function. One zygoma plate loosened while the site of another underwent heterotopic ossification. Gross examination of site defects revealed heterotopic ossification, confirmed by histological and micro-CT evaluation. Biomechanical testing was unavailable due to insufficient bony repair. CONCLUSIONS: The presented porcine zygoma and mandibular defect models are incapable of repair in the absence of bone scaffolds. Based on the authors' results, this model is appropriate for further study of custom 3D-printed engineered bone scaffolds.

Sirtuin 3 deficiency does not impede digit regeneration in mice.

The mitochondrial deacetylase sirtuin 3 (SIRT3) is thought to be one of the main contributors to metabolic flexibility-promoting mitochondrial energy production and maintaining homeostasis. In bone, metabolic profiles are tightly regulated and the loss of SIRT3 has deleterious effects on bone volume in vivo and on osteoblast differentiation in vitro. Despite the prominent role of this protein in bone stem cell proliferation, metabolic activity, and differentiation, the importance of SIRT3 for regeneration after bone injury has never been reported. We show here, using the mouse digit amputation model, that SIRT3 deficiency has no impact on the regenerative capacity and architecture of bone and soft tissue. Regeneration occurs in SIRT3 deficient mice in spite of the reduced oxidative metabolic profile of the periosteal cells. These data suggest that bone regeneration, in contrast to homeostatic bone turnover, is not reliant upon active SIRT3, and our results highlight the need to examine known roles of SIRT3 in the context of injury.

Molecular and Structural Traits of Insulin Receptor Substrate 1/LC3 Nuclear Structures and Their Role in Autophagy Control and Tumor Cell Survival.

Insulin receptor substrate 1 (IRS-1) is a common cytosolic adaptor molecule involved in signal transduction from insulin and insulin-like growth factor I (IGF-I) receptors. IRS-1 can also be found in the nucleus. We report here a new finding of unique IRS-1 nuclear structures, which we observed initially in glioblastoma biopsy specimens and glioblastoma xenografts. These nuclear structures can be reproduced in vitro by the ectopic expression of IRS-1 cDNA cloned in frame with the nuclear localization signal (NLS-IRS-1). In these structures, IRS-1 localizes at the periphery, while the center harbors a key autophagy protein, LC3. These new nuclear structures are highly dynamic, rapidly exchange IRS-1 molecules with the surrounding nucleoplasm, disassemble during mitosis, and require a growth stimulus for their reassembly and maintenance. In tumor cells engineered to express NLS-IRS-1, the IRS-1/LC3 nuclear structures repress autophagy induced by either amino acid starvation or rapamycin treatment. In this process, IRS-1 nuclear structures sequester LC3 inside the nucleus, possibly preventing its cytosolic translocation and the formation of new autophagosomes. This novel mechanism provides a quick and reversible way of inhibiting autophagy, which could counteract autophagy-induced cancer cell death under severe stress, including anticancer therapies.

The periosteal requirement and temporal dynamics of BMP2-induced middle phalanx regeneration in the adult mouse.

Regeneration of mammalian limbs is restricted to amputation of the distal digit tip, the terminal phalanx (P3). The adjacent skeletal element, the middle phalanx (P2), has emerged as a model system to investigate regenerative failure and as a site to test approaches aimed at enhancing regeneration. We report that exogenous application of bone morphogenetic protein 2 (BMP2) stimulates the formation of a transient cartilaginous callus distal to the amputation plane that mediates the regeneration of the amputated P2 bone. BMP2 initiates a significant regeneration response during the periosteal-derived cartilaginous healing phase of P2 bone repair, yet fails to induce regeneration in the absence of periosteal tissue, or after boney callus formation. We provide evidence that a temporal component exists in the induced regeneration of P2 that we define as the "regeneration window." In this window, cells are transiently responsive to BMP2 after the amputation injury. Simple re-injury of the healed P2 stump acts to reinitiate endogenous bone repair, complete with periosteal chondrogenesis, thus reopening the "regeneration window" and thereby recreating a regeneration-permissive environment that is responsive to exogenous BMP2 treatment.

Macrophages are required to coordinate mouse digit tip regeneration.

In mammals, macrophages are known to play a major role in tissue regeneration. They contribute to inflammation, histolysis, re-epithelialization, revascularization and cell proliferation. Macrophages have been shown to be essential for regeneration in salamanders and fish, but their role has not been elucidated in mammalian epimorphic regeneration. Here, using the regenerating mouse digit tip as a mammalian model, we demonstrate that macrophages are essential for the regeneration process. Using cell-depletion strategies, we show that regeneration is completely inhibited; bone histolysis does not occur, wound re-epithelialization is inhibited and the blastema does not form. Although rescue of epidermal wound closure in the absence of macrophages promotes blastema accumulation, it does not rescue cell differentiation, indicating that macrophages play a key role in the redifferentiation of the blastema. We provide additional evidence that although bone degradation is a component, it is not essential to the overall regenerative process. These findings show that macrophages play an essential role in coordinating the epimorphic regenerative response in mammals.

Fibroblast reticular cells engineer a blastema extracellular network during digit tip regeneration in mice.

The regeneration blastema which forms following amputation of the mouse digit tip is composed of undifferentiated cells bound together by an organized network of fibers. A monoclonal antibody (ER-TR7) that identifies extracellular matrix (ECM) fibers produced by fibroblast reticular cells during lymphoid organogenesis was used to characterize the ECM of the digit, the blastema, and the regenerate. Digit fibroblast reticular cells produce an ER-TR7+ ECM network associated with different tissues and represent a subset of loose connective tissue fibroblasts. During blastema formation there is an upregulation of matrix production that returns to its pre-existing level and anatomical pattern in the endpoint regenerate. Co-localization studies demonstrate a strong spatial correlation between the ER-TR7 antigen and collagen type III (COL3) in histological sections. ER-TR7 and COL3 are co-induced in cultured digit fibroblasts following treatment with tumor necrosis factor alpha and a lymphotoxin beta receptor agonist. These results provide an initial characterization of the ECM during digit regeneration and identify a subpopulation of fibroblasts involved in producing the blastema provisional matrix that is remodeled during the regeneration response.

Acute Ethanol Increases IGF-I-Induced Phosphorylation of ERKs by Enhancing Recruitment of p52-Shc to the Grb2/Shc Complex.

Ethanol plays a detrimental role in the development of the brain. Multiple studies have shown that ethanol inhibits insulin-like growth factor I receptor (IGF-IR) function. Because the IGF-IR contributes to brain development by supporting neural growth, survival, and differentiation, we sought to determine the molecular mechanism(s) involved in ethanol's effects on this membrane-associated tyrosine kinase. Using multiple neuronal cell types, we performed Western blot, immunoprecipitation, and GST-pulldowns following acute (1-24 h) or chronic (3 weeks) treatment with ethanol. Surprisingly, exposure of multiple neuronal cell types to acute (up to 24 h) ethanol (50 mM) enhanced IGF-I-induced phosphorylation of extracellular regulated kinases (ERKs), without affecting IGF-IR tyrosine phosphorylation itself, or Akt phosphorylation. This acute increase in ERKs phosphorylation was followed by the expected inhibition of the IGF-IR signaling following 3-week ethanol exposure. We then expressed a GFP-tagged IGF-IR construct in PC12 cells and used them to perform fluorescence recovery after photobleaching (FRAP) analysis. Using these fluorescently labeled cells, we determined that 50 mM ethanol decreased the half-time of the IGF-IR-associated FRAP, which implied that cell membrane-associated signaling events could be affected. Indeed, co-immunoprecipitation and GST-pulldown studies demonstrated that the acute ethanol exposure increased the recruitment of p52-Shc to the Grb2-Shc complex, which is known to engage the Ras-Raf-ERKs pathway following IGF-1 stimulation. These experiments indicate that even a short and low-dose exposure to ethanol may dysregulate function of the receptor, which plays a critical role in brain development. J. Cell. Physiol. 232: 1275-1286, 2017. © 2016 Wiley Periodicals, Inc.

Hyperbaric Oxygen Promotes Proximal Bone Regeneration and Organized Collagen Composition during Digit Regeneration.

Oxygen is critical for optimal bone regeneration. While axolotls and salamanders have retained the ability to regenerate whole limbs, mammalian regeneration is restricted to the distal tip of the digit (P3) in mice, primates, and humans. Our previous study revealed the oxygen microenvironment during regeneration is dynamic and temporally influential in building and degrading bone. Given that regeneration is dependent on a dynamic and changing oxygen environment, a better understanding of the effects of oxygen during wounding, scarring, and regeneration, and better ways to artificially generate both hypoxic and oxygen replete microenvironments are essential to promote regeneration beyond wounding or scarring. To explore the influence of increased oxygen on digit regeneration in vivo daily treatments of hyperbaric oxygen were administered to mice during all phases of the entire regenerative process. Micro-Computed Tomography (µCT) and histological analysis showed that the daily application of hyperbaric oxygen elicited the same enhanced bone degradation response as two individual pulses of oxygen applied during the blastema phase. We expand past these findings to show histologically that the continuous application of hyperbaric oxygen during digit regeneration results in delayed blastema formation at a much more proximal location after amputation, and the deposition of better organized collagen fibers during bone formation. The application of sustained hyperbaric oxygen also delays wound closure and enhances bone degradation after digit amputation. Thus, hyperbaric oxygen shows the potential for positive influential control on the various phases of an epimorphic regenerative response.

ERG2 and ERG24 Are Required for Normal Vacuolar Physiology as Well as Candida albicans Pathogenicity in a Murine Model of Disseminated but Not Vaginal Candidiasis.

Several important classes of antifungal agents, including the azoles, act by blocking ergosterol biosynthesis. It was recently reported that the azoles cause massive disruption of the fungal vacuole in the prevalent human pathogen Candida albicans. This is significant because normal vacuolar function is required to support C. albicans pathogenicity. This study examined the impact of the morpholine antifungals, which inhibit later steps of ergosterol biosynthesis, on C. albicans vacuolar integrity. It was found that overexpression of either the ERG2 or ERG24 gene, encoding C-8 sterol isomerase or C-14 sterol reductase, respectively, suppressed C. albicans sensitivity to the morpholines. In addition, both erg2Δ/Δ and erg24Δ/Δ mutants were hypersensitive to the morpholines. These data are consistent with the antifungal activity of the morpholines depending upon the simultaneous inhibition of both Erg2p and Erg24p. The vacuoles within both erg2Δ/Δ and erg24Δ/Δ C. albicans strains exhibited an aberrant morphology and accumulated large quantities of the weak base quinacrine, indicating enhanced vacuolar acidification compared with that of control strains. Both erg mutants exhibited significant defects in polarized hyphal growth and were avirulent in a mouse model of disseminated candidiasis. Surprisingly, in a mouse model of vaginal candidiasis, both mutants colonized mice at high levels and induced a pathogenic response similar to that with the controls. Thus, while targeting Erg2p or Erg24p alone could provide a potentially efficacious therapy for disseminated candidiasis, it may not be an effective strategy to treat vaginal infections. The potential value of drugs targeting these enzymes as adjunctive therapies is discussed.