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.

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.

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.

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.

Anti-tumoral effects of miR-3189-3p in glioblastoma.

Glioblastoma is one of the most aggressive brain tumors. We have previously found up-regulation of growth differentiation factor 15 (GDF15) in glioblastoma cells treated with the anticancer agent fenofibrate. Sequence analysis of GDF15 revealed the presence of a microRNA, miR-3189, in the single intron. We then asked whether miR-3189 was expressed in clinical samples and whether it was functional in glioblastoma cells. We found that expression of miR-3189-3p was down-regulated in astrocytoma and glioblastoma clinical samples compared with control brain tissue. In vitro, the functionality of miR-3189-3p was tested by RNA-binding protein immunoprecipitation, and miR-3189-3p coimmunoprecipitated with Argonaute 2 together with two of its major predicted gene targets, the SF3B2 splicing factor and the guanine nucleotide exchange factor p63RhoGEF. Overexpression of miR-3189-3p resulted in a significant inhibition of cell proliferation and migration through direct targeting of SF3B2 and p63RhoGEF, respectively. Interestingly, miR-3189-3p levels were increased by treatment of glioblastoma cells with fenofibrate, a lipid-lowering drug with multiple anticancer activities. The attenuated expression of miR-3189-3p in clinical samples paralleled the elevated expression of SF3B2, which could contribute to the activation of SF3B2 growth-promoting pathways in these tumors. Finally, miR-3189-3p-mediated inhibition of tumor growth in vivo further supported the function of this microRNA as a tumor suppressor.

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.

SDF-1α/CXCR4 signaling mediates digit tip regeneration promoted by BMP-2.

Previously we demonstrated that BMP signaling is required for endogenous digit tip regeneration, and that treatment with BMP-2 or -7 induces a regenerative response following amputation at regeneration-incompetent levels (Yu et al., 2010, 2012). Both endogenous regeneration and BMP-induced regeneration are associated with the transient formation of a blastema, however the formation of a regeneration blastema in mammals is poorly understood. In this study, we focus on how blastema cells respond to BMP signaling during neonatal digit regeneration in mice. First, we show that blastema cells retain regenerative properties after expansion in vitro, and when re-introduced into the amputated digit, these cells display directed migration in response to BMP-2. However, in vitro studies demonstrate that BMP-2 alone does not influence blastema cell migration, suggesting a requirement of another pivotal downstream factor for cell recruitment. We show that blastema cell migration is stimulated by the cytokine, SDF-1α, and that SDF-1α is expressed by the wound epidermis as well as endothelial cells of the blastema. Blastema cells express both SDF-1α receptors, CXCR4 and CXCR7, although the migration response is inhibited by the CXCR4-specific antagonist, AMD3100. Mice treated with AMD3100 display a partial inhibition of skeletal regrowth associated with the regeneration response. We provide evidence that BMP-2 regulates Sdf-1α expression in endothelial cells but not cells of the wound epidermis. Finally, we show that SDF-1α-expressing COS1 cells engrafted into a regeneration-incompetent digit amputation wound resulted in a locally enhanced population of CXCR4 positive cells, and induced a partial regenerative response. Taken together, this study provides evidence that one downstream mechanism of BMP signaling during mammalian digit regeneration involves activation of SDF-1α/CXCR4 signaling by endothelial cells to recruit blastema cells.

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.

Trp53 negatively regulates autoimmunity via the STAT3-Th17 axis.

Emerging evidence suggests that the tumor suppressor p53 is also a crucial regulator for many physiological processes. Previous observations indicate that p53 suppresses inflammation by inhibiting inflammatory antigen-presenting cells. To investigate the potential role of p53 in autoimmune effector T cells, we generated p53(null)CD45.1 mice by crossing p53(null)CD45.2 and CD45.1 mice. We demonstrate that p53(null)CD45.1 mice spontaneously developed autoimmunity, with a significant increase in IL-17-producing Th17 effectors in their lymph nodes (4.7 ± 1.0%) compared to the age-matched counterparts (1.9 ± 0.8% for p53(null)CD45.2, 1.1 ± 0.2% for CD45.1, and 0.5 ± 0.1% for CD45.2 mice). Likewise, p53(null)CD45.1 mice possess highly elevated serum levels of inflammatory cytokines IL-17 and IL-6. This enhanced Th17 response results largely from an increased sensitivity of p53(null)CD45.1 T cells to IL-6-induced STAT3 phosphorylation. Administration of STAT3 inhibitor S31-201 (IC50 of 38.0 ± 7.2 µM for IL-6-induced STAT3 phosphorylation), but not PBS control, to p53(null)CD45.1 mice suppressed Th17 effectors and alleviated autoimmune pathology. This is the first report revealing that p53 activity in T cells suppresses autoimmunity by controlling Th17 effectors. This study suggests that p53 serves as a guardian of immunological functions and that the p53-STAT3-Th17 axis might be a therapeutic target for autoimmunity.

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.

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.

Expression of doublecortin reveals articular chondrocyte lineage in mouse embryonic limbs.

The doublecortin (Dcx) gene encodes a microtubule-binding protein that was originally found in immature neurons. In this study, we used two mouse strains that express reporter genes (LacZ and enhanced green fluorescence protein, respectively) driven by the endogenous Dcx promoter. We found that Dcx was expressed in the mesenchymal cells in the mouse embryonic limb buds. A population of the mesenchymal cells continued Dcx expression after they differentiated into joint interzone cells and then articular chondrocytes. In contrast, the endochondral chondrocytes lost Dcx expression when the mesenchymal cells differentiated into endochondral chondrocytes. These data support a concept that the articular and endochondral chondrocytes originate from the same mesenchymal cells that express Dcx. In contrast to the notion that articular chondrocytes are derived from de-differentiated endochondral chondrocytes, our findings demonstrate that the lineages of articular and endochondral chondrocytes bifurcate at the stage of endochondral chondrogenesis.

Long-term follow-up of arthroscopic rotator cuff repair.

PURPOSE: The purpose of this study was to perform a case series assessing the clinical outcomes of patients with at least 9 years of follow-up after an all-arthroscopic rotator cuff repair. METHOD: We performed a review of all of the arthroscopic rotator cuff repairs done by the senior author from 1991 to 2001. Study patients identified were contacted and evaluated by the first author and the senior author. A thorough in-office shoulder examination was completed and a current University of California, Los Angeles shoulder score was obtained during the evaluation. RESULTS: Seven hundred seventy-two patients were in the initial database. Forty-eight patients were identified from the database after inclusion and exclusion criteria were applied. Follow-up ranged from 110 to 223 months, averaging 151.7 months. All repairs were single row and received an arthroscopic subacromial decompression. We identified 33 all-arthroscopic rotator cuff repairs for follow-up in 24 patients included in the study. The mean University of California, Los Angeles score at follow-up was 31.8, with 87.7% of patients having excellent and good outcomes. Of the patients, 18 showed excellent results, 11 good, 2 fair, and 2 poor. All the patients presented with no loss of motion. CONCLUSIONS: Our data suggest that patients maintain good outcomes 10 years after the index surgery. These findings are comparable to the outcomes reported in short-term and midterm follow-up studies. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

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.

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.

Alveolar macrophage-mediated killing of Pneumocystis carinii f. sp. muris involves molecular recognition by the Dectin-1 beta-glucan receptor.

Innate immune mechanisms against Pneumocystis carinii, a frequent cause of pneumonia in immunocompromised individuals, are not well understood. Using both real time polymerase chain reaction as a measure of organism viability and fluorescent deconvolution microscopy, we show that nonopsonic phagocytosis of P. carinii by alveolar macrophages is mediated by the Dectin-1 beta-glucan receptor and that the subsequent generation of hydrogen peroxide is involved in alveolar macrophage-mediated killing of P. carinii. The macrophage Dectin-1 beta-glucan receptor colocalized with the P. carinii cyst wall. However, blockage of Dectin-1 with high concentrations of anti-Dectin-1 antibody inhibited binding and concomitant killing of P. carinii by alveolar macrophages. Furthermore, RAW 264.7 macrophages overexpressing Dectin-1 bound P. carinii at a higher level than control RAW cells. In the presence of Dectin-1 blockage, killing of opsonized P. carinii could be restored through FcgammaRII/III receptors. Opsonized P. carinii could also be efficiently killed in the presence of FcgammaRII/III receptor blockage through Dectin-1-mediated phagocytosis. We further show that Dectin-1 is required for P. carinii-induced macrophage inflammatory protein 2 production by alveolar macrophages. Taken together, these results show that nonopsonic phagocytosis and subsequent killing of P. carinii by alveolar macrophages is dependent upon recognition by the Dectin-1 beta-glucan receptor.

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.