Distinct patterns of cytokines, chemokines, and growth factors in synovial fluid after ACL injury in comparison to osteoarthritis.

This study analyzed knee synovial fluid after anterior cruciate ligament (ACL) tear and in osteoarthritis (OA) to test the hypotheses that concentrations of cytokines, chemokines, and growth factors differ (a) by diagnosis and (b) after ACL tear by time from injury and presence/absence of concomitant meniscus tear. Synovial fluid samples were collected from two groups, ACL tears (with or without meniscus tear) (N = 13) and Kellgren-Lawrence grade 3 and 4 OA (N = 16), undergoing clinically indicated aspiration of the knee joint. Multiple cytokines, chemokines, and growth factors were assessed using a multiplexed 45-protein panel. Comparisons were made for the concentrations of all molecules between ACL tear and OA patients, isolated versus combined ACL and meniscus tears, and categorized by time from injury: acute or early subacute (<15 days, N = 8) versus late subacute or chronic (>15 days and <3 months, N = 5). ACL tear patients have higher levels of six molecules (IL-4, IL-5, IL-13, PlGF-1, bNGF, TNF-α) in knee synovial fluid compared to OA patients. Isolated ACL tears express higher levels of IL-4, IL-13 and IFN-γ and lower levels of IL-7 than ACL tears with a concomitant meniscus tear. SDF-1α, PlGF-1, IL-1RA, HGF, bNGF, and BDNF levels are elevated immediately after injury and drop off significantly in the late subacute phase (after 15 days). Synovial fluid from knees with ACL tears have elevated metabolic activity compared to knees with OA. The cytokine profiles after ACL tears are influenced by the time from injury and the presence of meniscus tears. These findings offer valuable insights into the levels of cytokines, chemokines, and growth factors in the knee after ACL injury, information which may have important implications for the diagnosis, prognosis and treatment of this common pathology.

Molecular Magnetic Resonance Imaging of Aneurysmal Inflammation Using a Redox Active Iron Complex.

OBJECTIVES: Inflammation plays a key role in driving brain aneurysmal instability and rupture, but clinical tools to noninvasively differentiate between inflamed and stable aneurysms are lacking. We hypothesize that imaging oxidative changes in the aneurysmal microenvironment driven by myeloid inflammatory cells may represent a noninvasive biomarker to evaluate rupture risk. In this study, we performed initial evaluation of the oxidatively activated probe Fe-PyC3A as a tool for magnetic resonance imaging (MRI) of inflammation in a rabbit model of saccular aneurysm. MATERIALS AND METHODS: The difference in longitudinal relaxivity ( r1 ) in reduced and oxidized states of Fe-PyC3A was measured in water and blood plasma phantoms at 3 T. A rabbit saccular aneurysm model was created by endovascular intervention/elastinolysis with subsequent decellularization in situ. Rabbits were imaged at 4 weeks (n = 4) or 12 weeks (n = 4) after aneurysmal induction, when luminal levels of inflammation reflected by the presence of myeloperoxidase positive cells are relatively high and low, respectively, using a 3 T clinical scanner. Both groups were imaged dynamically using a 2-dimensional T1-weighted fast field echo pulse MRI sequence before and up to 4 minutes postinjection of Fe-PyC3A. Dynamic imaging was then repeated after an injection of gadobutrol (0.1 mmol/kg) as negative control probe. Rabbits from the 12-week aneurysm group were also imaged before and 20 minutes and 3 hours after injection of Fe-PyC3A using an axial respiratory gated turbo-spin echo (TSE) pulse sequence with motion-sensitized driven equilibrium (MSDE) preparation. The MSDE/TSE imaging was repeated before, immediately after dynamic acquisition (20 minutes postinjection), and 3 hours after injection of gadobutrol. Aneurysmal enhancement ratios (ERs) were calculated by dividing the postinjection aneurysm versus skeletal muscle contrast ratio by the preinjection contrast ratio. After imaging, the aneurysms were excised and inflammatory infiltrate was characterized by fluorometric detection of myeloperoxidase activity and calprotectin immunostaining, respectively. RESULTS: In vitro relaxometry showed that oxidation of Fe-PyC3A by hydrogen peroxide resulted in a 15-fold increase of r1 at 3 T. Relaxometry in the presence of blood plasma showed no more than a 10% increase of r1 , indicating the absence of strong interaction of Fe-PyC3A with plasma proteins. Dynamic imaging with Fe-PyC3A generated little signal enhancement within the blood pool or adjacent muscle but did generate a transient increase in aneurysmal ER that was significantly greater 4 weeks versus 12 weeks after aneurysm induction (1.6 ± 0.30 vs 1.2 ± 0.03, P < 0.05). Dynamic imaging with gadobutrol generated strong aneurysmal enhancement, but also strong enhancement of the blood and muscle resulting in smaller relative ER change. In the 12-week group of rabbits, MSDE/TSE imaging showed that ER values measured immediately after dynamic MRI (20 minutes postinjection) were significantly higher ( P < 0.05) in the case of Fe-PyC3A (1.25 ± 0.06) than for gadobutrol injection (1.03 ± 0.03). Immunohistochemical corroboration using anticalprotectin antibody showed that leukocyte infiltration into the vessel walls and luminal thrombi was significantly higher in the 4-week group versus 12-week aneurysms (123 ± 37 vs 18 ± 7 cells/mm 2 , P < 0.05). CONCLUSIONS: Magnetic resonance imaging using Fe-PyC3A injection in dynamic or delayed acquisition modes was shown to generate a higher magnetic resonance signal enhancement in aneurysms that exhibit higher degree of inflammation. The results of our pilot experiments support further evaluation of MRI using Fe-PyC3A as a noninvasive marker of aneurysmal inflammation.

ALS-linked PFN1 variants exhibit loss and gain of functions in the context of formin-induced actin polymerization.

Profilin-1 (PFN1) plays important roles in modulating actin dynamics through binding both monomeric actin and proteins enriched with polyproline motifs. Mutations in PFN1 have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, whether ALS-linked mutations affect PFN1 function has remained unclear. To address this question, we employed an unbiased proteomics analysis in mammalian cells to identify proteins that differentially interact with mutant and wild-type (WT) PFN1. These studies uncovered differential binding between two ALS-linked PFN1 variants, G118V and M114T, and select formin proteins. Furthermore, both variants augmented formin-mediated actin assembly relative to PFN1 WT. Molecular dynamics simulations revealed mutation-induced changes in the internal dynamic couplings within an alpha helix of PFN1 that directly contacts both actin and polyproline, as well as structural fluctuations within the actin- and polyproline-binding regions of PFN1. These data indicate that ALS-PFN1 variants have the potential for heightened flexibility in the context of the ternary actin-PFN1-polyproline complex during actin assembly. Conversely, PFN1 C71G was more severely destabilized than the other PFN1 variants, resulting in reduced protein expression in both transfected and ALS patient lymphoblast cell lines. Moreover, this variant exhibited loss-of-function phenotypes in the context of actin assembly. Perturbations in actin dynamics and assembly can therefore result from ALS-linked mutations in PFN1. However, ALS-PFN1 variants may dysregulate actin polymerization through different mechanisms that depend upon the solubility and stability of the mutant protein.

Gene Expression in Meniscal Tears at the Time of Arthroscopic Partial Meniscectomy Predicts the Progression of Osteoarthritis Within 6 Years of Surgery.

BACKGROUND: While knees with meniscal tears are associated with a heightened risk of developing osteoarthritis (OA), it is difficult to predict which patients are at the greatest risk for OA. Gene signatures in menisci that are resected during arthroscopic partial meniscectomy (APM) may provide insight into the risk of OA progression. HYPOTHESIS: Meniscal gene signatures at the time of APM will predict radiographic OA progression. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Meniscal fragments were collected from 38 patients without OA during clinically indicated APM of the medial meniscus. The expression of 28 candidate genes with known roles in cartilage homeostasis, OA, extracellular matrix degradation, and obesity was assessed by quantitative real-time polymerase chain reaction. Weightbearing radiographs obtained before surgery and at final follow-up were graded by a musculoskeletal radiologist using the Kellgren-Lawrence classification of OA. The association of meniscal gene expression at baseline with the progression of radiographic OA was determined. RESULTS: Gene expression and baseline and follow-up radiographic data were available from 31 patients (81.6%) at a mean follow-up of 6.2 ± 1.3 years. Patients without OA progression had significantly higher expression of 7 genes: MMP9 (5.1-fold; P = .002), IL8 (2.9-fold; P = .016), CCL3 (3.7-fold; P = .032), CCL3L1 (4.5-fold; P = .008), CXCL6 (6.2-fold; P = .010), LEP (5.2-fold; P = .004), and RETN (46-fold; P = .008). CONCLUSION: Gene expression in the meniscus at the time of APM may be associated with the risk for progression of OA after surgery. Elevated expression of the aforementioned genes may reflect a chondroprotective response. Stratifying the risk for OA progression after APM could facilitate targeted interventions to delay or prevent the development of OA. Further studies in a larger cohort with an extended follow-up, and inclusion of additional genes, are warranted to better characterize this association.

Duration of symptoms prior to partial meniscectomy is not associated with the expression of osteoarthritis genes in the injured meniscus.

While there is emerging data on how duration of symptoms prior to surgery relates to outcomes of patients undergoing arthroscopic partial meniscectomy, little is known about how duration of symptoms relates to the biology of the knee in these patients. The purpose of this study was to test the hypothesis that duration of symptoms prior to arthroscopic partial meniscectomy is associated with expression of osteoarthritis (OA)-related genes in the meniscus. We collected resected meniscus from patients (N = 76) undergoing clinically indicated arthroscopic partial meniscectomy from knees without advanced degenerative changes. RNA from 64 patients was analyzed for 28 candidate OA transcripts by real-time polymerase chain reaction (PCR). RNA was also probed for identification of novel genes by RNA microarray in 12 patients followed by validation of selected candidates by real-time PCR. The association of gene expression with duration of symptoms prior to surgery was tested. Additional screening was performed with known OA genetic risk alleles assembled from published literature and with gene transcripts differentially expressed between non-OA and OA cartilage and menisci. Our data revealed that duration of symptoms did not predict expression of OA genes in the meniscus, other than limited association with CXCL3, BMP2, and HLA-DQA1. Microarray identified new genes and pathways with unknown role(s) in meniscus injury and OA and validation of a subset of genes by real-time PCR showed expression pattern highly concordant with the microarray data. While duration of symptoms prior to arthroscopic partial meniscectomy does not significantly alter the expression of OA related genes, the association with novel genes and pathways deserves further investigation.

Contact Lens Versus Non-Contact Lens-Related Corneal Ulcers at an Academic Center.

PURPOSE: To compare the infectious contact lens-related corneal ulcer (CLRU) and non-CLRU cases at Saint Louis University. METHODS: Retrospective review of corneal ulcer cases identified by search of the ophthalmology and microbiology department databases between 1999 and 2016. RESULTS: Six hundred seventy-seven cases of corneal ulcers were identified, of which 46% were CLRU. CLRU cases were seen more commonly in younger patients (P<0.001) and women (P=0.03) than non-CLRU cases. Many of the infections were vision-threatening as defined by central/paracentral location (73% CLRU and 71% non-CLRU [P=0.60]) and large size of ulcer >2 mm in 36% CLRU and 51% non-CLRU (P=0.002). Causative pathogen in cultured CLRU was predominately Pseudomonas species (44%, P<0.001 vs. the non-CLRU group), other gram-negative (6%), gram-positive (33%), fungi (13%), and Acanthamoeba (5%). Comparatively, cultured non-CLRU was predominately gram-positive (64%, P<0.001 vs. the CLRU group), gram-negative (26%), and fungi (11%). The combined oxacillin-resistant Staphylococcus aureus and coagulase-negative Staphylococcus isolates were 35% and 34%, respectively. Despite the progressive increase in the number of corneal ulcers seen, the annual trend for any one particular organism for either CLRU cases or non-CLRU cases did not change significantly. CONCLUSIONS: Most of the cases were non-CLRU. CLRU was disproportionately associated with Pseudomonas species and non-CLRU with Staphylococcal species. Fungal infections were predominately caused by filamentous organisms in both groups. Acanthamoeba keratitis was exclusively associated with CL use.

Nonlinear gene expression-phenotype relationships contribute to variation and clefting in the A/WySn mouse.

BACKGROUND: Cleft lip and palate is one of the most common human birth defects, but the underlying etiology is poorly understood. The A/WySn mouse is a spontaneously occurring model of multigenic clefting in which 20% to 30% of individuals develop an orofacial cleft. Recent work has shown altered methylation at a specific retrotransposon insertion downstream of the Wnt9b locus in clefting animals, which results in decreased Wnt9b expression. RESULTS: Using a newly developed protocol that allows us to measure morphology, gene expression, and DNA methylation in the same embryo, we relate gene expression in an individual embryo directly to its three-dimensional morphology for the first time. We find that methylation at the retrotransposon relates to Wnt9b expression and morphology. IAP methylation relates to shape of the nasal process in a manner consistent with clefting. Embryos with low IAP methylation exhibit increased among-individual variance in facial shape. CONCLUSIONS: Methylation and gene expression relate nonlinearly to nasal process morphology. Individuals at one end of a continuum of phenotypic states display a clinical phenotype and increased phenotypic variation. Variable penetrance and expressivity in this model is likely determined both by among-individual variation in methylation and changes in phenotypic robustness along the underlying liability distribution for orofacial clefting.

Laboratory Results, Epidemiologic Features, and Outcome Analyses of Microbial Keratitis: A 15-Year Review From St. Louis.

PURPOSE: To evaluate the laboratory results and prognostic factors of poor clinical outcomes in microbial keratitis cases over 15 years at Saint Louis University. DESIGN: Retrospective cohort and trend study. METHODS: Microbiological and clinical information from culture-positive cases seen at Saint Louis University from 1999 to 2013 were reviewed retrospectively. Statistical analyses were used to determine microbiological and antibiotic susceptibility trends. Prognostic factors of poor clinical outcome from the literature were used to create multivariate regression models to describe our cohort. RESULTS: Gram-positive organisms predominated (48%), followed by gram-negative organisms (34%) and fungi (16%). The most commonly isolated organism was Pseudomonas aeruginosa (21%). Oxacillin-resistant rates of Staphylococcus aureus and coagulase-negative staphylococci were 45% and 43%, respectively. Only the proportion of Pseudomonas changed significantly over time (P = .02). The only antibiotic found to lose efficacy over time was gentamicin for gram-positive organisms (P = .005). Multivariate logistic regression analyses revealed that major complications were associated with large ulcers (P < .006), fungal cases (P < .001), and comorbid ophthalmic conditions (P < .001). Poor healing was associated with large ulcers (P < .001) and fungal cases (P < .001). Lastly, poor visual outcome was associated with large ulcers (P < .01) and age ≥ 60 years (P < .02). CONCLUSIONS: In the St Louis area, oxacillin-resistant organisms, Pseudomonas aeruginosa, and fungi are commonly recovered from microbial keratitis cases with a disproportionally high incidence. Hence, empiric antibiotic choice should reflect these trends. Special care needs to be taken for patients with large ulcers and fungal infections, as well as elderly patients with comorbid ophthalmic conditions, as these patients have worse clinical outcomes.

Evidence for Genetic Contribution to Variation in Posttraumatic Osteoarthritis in Mice.

OBJECTIVE: Recombinant inbred mouse strains generated from an LG/J and SM/J intercross offer a unique resource to study complex genetic traits such as osteoarthritis (OA). We undertook this study to determine the susceptibility of 14 strains to various phenotypes characteristic of posttraumatic OA. We hypothesized that phenotypic variability is associated with genetic variability. METHODS: Ten-week-old male mice underwent surgical destabilization of the medial meniscus (DMM) to induce posttraumatic OA. Mice were killed 8 weeks after surgery, and knee joints were processed for histology to score cartilage degeneration and synovitis. Micro-computed tomography was used to analyze trabecular bone parameters including subchondral bone plate thickness and synovial ectopic calcifications. Gene expression in the knees was assessed using a QuantiGene Plex assay. RESULTS: Broad-sense heritability ranged from 0.18 to 0.58, which suggested that the responses to surgery were moderately heritable. The LGXSM-33, LGXSM-5, LGXSM-46, and SM/J strains were highly susceptible to OA, while the LGXSM-131b, LGXSM-163, LGXSM-35, LGXSM-128a, LGXSM-6, and LG/J strains were relatively OA resistant. This study was the first to accomplish measurement of genetic correlations of phenotypes that are characteristic of posttraumatic OA. Cartilage degeneration was significantly positively associated with synovitis (r = 0.83-0.92), and subchondral bone plate thickness was negatively correlated with ectopic calcifications (r = -0.59). Moreover, we showed that 40 of the 78 genes tested were significantly correlated with various OA phenotypes. However, unlike the OA phenotypes, there was no evidence for genetic variation in differences in gene expression levels between DMM-operated and sham-operated knees. CONCLUSION: For these mouse strains, various characteristics of posttraumatic OA varied with genetic composition, which demonstrated a genetic basis for susceptibility to posttraumatic OA. The heritability of posttraumatic OA was established. Phenotypes exhibited various degrees of correlations; cartilage degeneration was positively correlated with synovitis, but not with the formation of ectopic calcifications. Further investigation of the genome regions that contain genes implicated in OA, as well as further investigation of gene expression data, will be useful for studying mechanisms of OA and identifying therapeutic targets.

An In Vitro Model of Charcot-Marie-Tooth Disease Type 4B2 Provides Insight Into the Roles of MTMR13 and MTMR2 in Schwann Cell Myelination.

Charcot-Marie-Tooth Disorder Type 4B (CMT4B) is a demyelinating peripheral neuropathy caused by mutations in myotubularin-related (MTMR) proteins 2, 13, or 5 (CMT4B1/2/3), which regulate phosphoinositide turnover and endosomal trafficking. Although mouse models of CMT4B2 exist, an in vitro model would make possible pharmacological and reverse genetic experiments needed to clarify the role of MTMR13 in myelination. We have generated such a model using Schwann cell-dorsal root ganglion (SC-DRG) explants from Mtmr13-/- mice. Myelin sheaths in mutant cultures contain outfoldings highly reminiscent of those observed in the nerves of Mtmr13-/- mice and CMT4B2 patients. Mtmr13-/- SC-DRG explants also contain reduced Mtmr2, further supporting a role of Mtmr13 in stabilizing Mtmr2. Elevated PI(3,5)P2 has been implicated as a cause of myelin outfoldings in Mtmr2-/- models. In contrast, the role of elevated PI3P or PI(3,5)P2 in promoting outfoldings in Mtmr13-/- models is unclear. We found that over-expression of MTMR2 in Mtmr13-/- SC-DRGs moderately reduced the prevalence of myelin outfoldings. Thus, a manipulation predicted to lower PI3P and PI(3,5)P2 partially suppressed the phenotype caused by Mtmr13 deficiency. We also explored the relationship between CMT4B2-like myelin outfoldings and kinases that produce PI3P and PI(3,5)P2 by analyzing nerve pathology in mice lacking both Mtmr13 and one of two specific PI 3-kinases. Intriguingly, the loss of vacuolar protein sorting 34 or PI3K-C2ß in Mtmr13-/- mice had no impact on the prevalence of myelin outfoldings. In aggregate, our findings suggest that the MTMR13 scaffold protein likely has critical functions other than stabilizing MTMR2 to achieve an adequate level of PI 3-phosphatase activity.

Post-Traumatic Osteoarthritis in Mice Following Mechanical Injury to the Synovial Joint.

We investigated the spectrum of lesions characteristic of post-traumatic osteoarthritis (PTOA) across the knee joint in response to mechanical injury. We hypothesized that alteration in knee joint stability in mice reproduces molecular and structural features of PTOA that would suggest potential therapeutic targets in humans. The right knees of eight-week old male mice from two recombinant inbred lines (LGXSM-6 and LGXSM-33) were subjected to axial tibial compression. Three separate loading magnitudes were applied: 6N, 9N, and 12N. Left knees served as non-loaded controls. Mice were sacrificed at 5, 9, 14, 28, and 56 days post-loading and whole knee joint changes were assessed by histology, immunostaining, micro-CT, and magnetic resonance imaging. We observed that tibial compression disrupted joint stability by rupturing the anterior cruciate ligament (except for 6N) and instigated a cascade of temporal and topographical features of PTOA. These features included cartilage extracellular matrix loss without proteoglycan replacement, chondrocyte apoptosis at day 5, synovitis present at day 14, osteophytes, ectopic calcification, and meniscus pathology. These findings provide a plausible model and a whole-joint approach for how joint injury in humans leads to PTOA. Chondrocyte apoptosis, synovitis, and ectopic calcification appear to be targets for potential therapeutic intervention.

Corneal Toxicity Associated With Aquarium Coral Palytoxin.

PURPOSE: To report a series of patients who developed corneal toxicity after exposure to aquarium coral palytoxin. DESIGN: Multicenter retrospective case series. METHODS: Retrospective review. RESULTS: Seven patients presented with corneal findings ranging from superficial punctate epitheliopathy to bilateral corneal melt with subsequent perforation. Among those with mild corneal findings, resolution was achieved with topical steroids and lubrication, whereas some patients who developed progressive corneal melt required therapeutic penetrating keratoplasty. The history in all patients revealed exposure to aquarium zoanthid corals shortly before disease onset. A review of the literature revealed that there are few prior reports of coral-associated corneal toxicity and that some species of coral secrete a substance known as palytoxin, a potent vasoconstrictor that inhibits the membranous sodium-potassium ATPase pump across cell types and can cause rapid death if inhaled or ingested. CONCLUSIONS: This is the largest case series to date demonstrating patients with aquarium coral palytoxin-associated corneal toxicity, and is the first to provide details of related histopathologic findings. Similar to other forms of toxic keratoconjunctivitis, a detailed history and careful clinical assessment are required, as well as timely removal of the offending agent from the patients' ocular milieu and environment. Mild ocular surface and corneal disease may be treated effectively with aggressive topical steroid therapy and lubrication. Given the potential severity of ocular as well as systemic adverse effects, there should be increased awareness of this entity among eye care professionals, aquarium enthusiasts, and the general public.

Genetic loci that regulate ectopic calcification in response to knee trauma in LG/J by SM/J advanced intercross mice.

This study reports on genetic susceptibility to ectopic calcification in the LG/J and SM/J advanced intercross mice. Using 347 mice in 98 full-sibships, destabilization of medial meniscus (DMM) was performed to induce joint injury. We found that joint destabilization instigated ectopic calcifications as detected and quantified by micro-CT. We performed quantitative trait locus (QTL) analysis to map ectopic calcification phenotypes to discrete genomic locations. To validate the functional significance of the selected QTL candidate genes, we compared mRNA expression between parental LG/J and SM/J inbred strains. Overall, we detected 20 QTLs affecting synovial and meniscus calcification phenotypes with 11 QTLs linked to synovial calcification. Functional and bioinformatic analyses of single nucleotide polymorphism (SNP) identified functional classifications relevant to angiogenesis (Myo1e, Kif26b, Nprl3, Stab2, Fam105b), bone metabolism/calcification (Tle3, Tgfb2, Lipc, Nfe2l1, Ank, Fam105b), arthritis (Stab2, Tbx21, Map4k4, Hoxb9, Larp6, Col1a2, Adam10, Timp3, Nfe2l1, Trpm3), and ankylosing-spondylitis (Ank, Pon1, Il1r2, Tbkbp1) indicating that ectopic calcification involves multiple mechanisms. Furthermore, the expression of 11 out of 78 candidate genes was significantly different between LG/J and SM/J. Correlation analysis showed that Aff3, Fam81a, Syn3, and Ank were correlated with synovial calcification. Taken together, our findings of multiple genetic loci suggest the involvement of multiple genes contributing to ectopic calcification.

Molecular insight into the association between cartilage regeneration and ear wound healing in genetic mouse models: targeting new genes in regeneration.

Tissue regeneration is a complex trait with few genetic models available. Mouse strains LG/J and MRL are exceptional healers. Using recombinant inbred strains from a large (LG/J, healer) and small (SM/J, nonhealer) intercross, we have previously shown a positive genetic correlation between ear wound healing, knee cartilage regeneration, and protection from osteoarthritis. We hypothesize that a common set of genes operates in tissue healing and articular cartilage regeneration. Taking advantage of archived histological sections from recombinant inbred strains, we analyzed expression of candidate genes through branched-chain DNA technology directly from tissue lysates. We determined broad-sense heritability of candidates, Pearson correlation of candidates with healing phenotypes, and Ward minimum variance cluster analysis for strains. A bioinformatic assessment of allelic polymorphisms within and near candidate genes was also performed. The expression of several candidates was significantly heritable among strains. Although several genes correlated with both ear wound healing and cartilage healing at a marginal level, the expression of four genes representing DNA repair (Xrcc2, Pcna) and Wnt signaling (Axin2, Wnt16) pathways was significantly positively correlated with both phenotypes. Cluster analysis accurately classified healers and nonhealers for seven out of eight strains based on gene expression. Specific sequence differences between LG/J and SM/J were identified as potential causal polymorphisms. Our study suggests a common genetic basis between tissue healing and osteoarthritis susceptibility. Mapping genetic variations causing differences in diverse healing responses in multiple tissues may reveal generic healing processes in pursuit of new therapeutic targets designed to induce or enhance regeneration and, potentially, protection from osteoarthritis.

The CMT4B disease-causing phosphatases Mtmr2 and Mtmr13 localize to the Schwann cell cytoplasm and endomembrane compartments, where they depend upon each other to achieve wild-type levels of protein expression.

The demyelinating peripheral neuropathy Charcot-Marie-Tooth type 4B (CMT4B) is characterized by axonal degeneration and myelin outfoldings. CMT4B results from mutations in either myotubularin-related protein 2 (MTMR2; CMT4B1) or MTMR13 (CMT4B2), phosphoinositide (PI) 3-phosphatases that dephosphorylate phosphatidylinositol 3-phosphate (PtdIns3P) and PtdIns(3,5)P2, lipids which regulate endo-lysosomal membrane traffic. The catalytically active MTMR2 and catalytically inactive MTMR13 physically associate, although the significance of this association is not well understood. Here we show that Mtmr13 loss leads to axonal degeneration in sciatic nerves of older mice. In addition, CMT4B2-like myelin outfoldings are present in Mtmr13(-/-) nerves at postnatal day 3. Thus, Mtmr13(-/-) mice show both the initial dysmyelination and later degenerative pathology of CMT4B2. Given the key role of PI 3-kinase-Akt signaling in myelination, we investigated the state of the pathway in nerves of CMT4B models. We found that Akt activation is unaltered in Mtmr13(-/-) and Mtmr2(-/-) mice. Mtmr2 and Mtmr13 are found within the Schwann cell cytoplasm, where the proteins are partially localized to punctate compartments, suggesting that Mtmr2-Mtmr13 may dephosphorylate their substrates on specific intracellular compartments. Mtmr2-Mtmr13 substrates play essential roles in endo-lysosomal membrane traffic. However, endosomes and lysosomes of Mtmr13(-/-) and Mtmr2(-/-) Schwann cells are morphologically indistinguishable from those of controls, indicating that loss of these proteins does not cause wholesale dysregulation of the endo-lysosomal system. Notably, Mtmr2 and Mtmr13 depend upon each other to achieve wild-type levels of protein expression. Mtmr2 stabilizes Mtmr13 on membranes, indicating that the Mtmr13 pseudophosphatase is regulated by its catalytically active binding partner.

Sbf/MTMR13 coordinates PI(3)P and Rab21 regulation in endocytic control of cellular remodeling.

Cells rely on the coordinated regulation of lipid phosphoinositides and Rab GTPases to define membrane compartment fates along distinct trafficking routes. The family of disease-related myotubularin (MTM) phosphoinositide phosphatases includes catalytically inactive members, or pseudophosphatases, with poorly understood functions. We found that Drosophila MTM pseudophosphatase Sbf coordinates both phosphatidylinositol 3-phosphate (PI(3)P) turnover and Rab21 GTPase activation in an endosomal pathway that controls macrophage remodeling. Sbf dynamically interacts with class II phosphatidylinositol 3-kinase and stably recruits Mtm to promote turnover of a PI(3)P subpool essential for endosomal trafficking. Sbf also functions as a guanine nucleotide exchange factor that promotes Rab21 GTPase activation associated with PI(3)P endosomes. Of importance, Sbf, Mtm, and Rab21 function together, along with Rab11-mediated endosomal trafficking, to control macrophage protrusion formation. This identifies Sbf as a critical coordinator of PI(3)P and Rab21 regulation, which specifies an endosomal pathway and cortical control.

Epigenetic integration of the developing brain and face.

The integration of the brain and face and to what extent this relationship constrains or enables evolutionary change in the craniofacial complex is an issue of long-standing interest in vertebrate evolution. To investigate brain-face integration, we studied the covariation between the forebrain and midface at gestational days 10-10.5 in four strains of laboratory mice. We found that phenotypic variation in the forebrain is highly correlated with that of the face during face formation such that variation in the size of the forebrain correlates with the degree of prognathism and orientation of the facial prominences. This suggests strongly that the integration of the brain and face is relevant to the etiology of midfacial malformations such as orofacial clefts. This axis of integration also has important implications for the evolutionary developmental biology of the mammalian craniofacial complex.

Estimating cell count and distribution in labeled histological samples using incremental cell search.

Cell proliferation is critical to the outgrowth of biological structures including the face and limbs. This cellular process has traditionally been studied via sequential histological sampling of these tissues. The length and tedium of traditional sampling is a major impediment to analyzing the large datasets required to accurately model cellular processes. Computerized cell localization and quantification is critical for high-throughput morphometric analysis of developing embryonic tissues. We have developed the Incremental Cell Search (ICS), a novel software tool that expedites the analysis of relationships between morphological outgrowth and cell proliferation in embryonic tissues. Based on an estimated average cell size and stain color, ICS rapidly indicates the approximate location and amount of cells in histological images of labeled embryonic tissue and provides estimates of cell counts in regions with saturated fluorescence and blurred cell boundaries. This capacity opens the door to high-throughput 3D and 4D quantitative analyses of developmental patterns.

Engaging physicians in risk factor reduction.

OptumHealth tested the feasibility of physician-directed population management in 3 primary care practices and with 546 continuously insured patients who exhibited claims markers for coronary artery disease, diabetes, and/or hypertension. During the intervention portion of the study, we asked physicians to improve the following health measurements: blood pressure, body mass index, cholesterol, hemoglobin A1c, and smoking status. We offered a modest pay-for-outcomes incentive for each risk factor improvement achieved. Additionally, on an eligible subset of these patients, we asked physicians to actively refer to population management programs those patients they determined could benefit from nurse or health coach interventions, advising us as to which components of their treatment plan they wished us to address. The 6-month intervention period exhibited a 10-fold improvement in the trend rate of risk factor management success when compared to the prior 6-month period for the same patients. A net of 96 distinct risk factor improvements were achieved by the 546 patients during the intervention period, whereas 9 net risk factor improvements occurred in the comparison period. This difference in improvement trends was statistically significant at P < 0.01. Of the 546 study participants, a subset of 187 members was eligible for participation in OptumHealth care management programs. Physicians identified 80 of these 187 eligible members as appropriate targets for program intervention. Representing ourselves as "calling on behalf" of the physician practices, we established contact with 50 referred members; 43 members (86%) actively enrolled in our programs. This enrollment rate is 2 to 3 times the rate of enrollment through our standard program outreach methods. We conclude that physician-directed population management with aligned incentives offers promise as a method of achieving important health and wellness goals.