Pegloticase co-administered with high MW polyethylene glycol effectively reduces PEG-immunogenicity and restores prolonged circulation in mouse.

The interactions between polymers and the immune system remains poorly controlled. In some instances, the immune system can produce antibodies specific to polymer constituents. Indeed, roughly half of pegloticase patients without immunomodulation develop high titers of anti-PEG antibodies (APA) to the PEG polymers on pegloticase, which then quickly clear the drug from circulation and render the gout treatment ineffective. Here, using pegloticase as a model drug, we show that addition of high molecular weight (MW) free (unconjugated) PEG to pegloticase allows us to control the immunogenicity and mitigates APA induction in mice. Compared to pegloticase mixed with saline, mice repeatedly dosed with pegloticase containing different MW or amount of free PEG possessed 4- to 12- fold lower anti-PEG IgG, and 6- to 10- fold lower anti-PEG IgM, after 3 rounds of pegloticase dosed every 2 weeks. The markedly reduced APA levels, together with competitive inhibition by free PEG, restored the prolonged circulation of pegloticase to levels observed in APA-naïve animals. In contrast, mice with pegloticase-induced APA eliminated nearly all pegloticase from the circulation within just four hours post-injection. These results support the growing literature demonstrating free PEG may effectively suppress drug-induced APA, which in turn may offer sustained therapeutic benefits without requiring broad immunomodulation. We also showed free PEG effectively blocked the PEGylated protein from binding with cells expressing PEG-specific B cell receptors. It provides a template of how we may be able to tune the interactions and immunogenicity of other polymer-modified therapeutics. STATEMENT OF SIGNIFICANCE: A major challenge with engineering materials for drug delivery is their interactions with the immune system. For instance, our body can produce high levels of anti-PEG antibodies (APA). Unfortunately, the field currently lack tools to limit immunostimulation or overcome pre-existing anti-PEG antibodies, without using broad immunosuppression. Here, we showed that simply introducing free PEG into a clinical formulation of PEG-uricase can effectively limit induction of anti-PEG antibodies, and restore their prolonged circulation upon repeated dosing. Our work offers a readily translatable method to safely and effectively restore the use PEG-drugs in patients with PEG-immunity, and provides a template to use unconjugated polymers with low immunogenicity to regulate interactions with the immune system for other polymer-modified therapeutics.

In-vivo detection of white adipose tissue browning: a multimodality imaging approach.

Detection and differentiation of brown fat in humans poses several challenges, as this tissue is sparse and often mixed with white adipose tissue. Non-invasive detection of beige fat represents an even greater challenge as this tissue is structurally and functionally more like white fat than brown fat. Here we used positron emission tomography with 18F-fluorodeoxyglucose, computed tomography, xenon-enhanced computed tomography, and dynamic contrast-enhanced ultrasound, to non-invasively detect functional and structural changes associated with the browning process of inguinal white fat, induced in mice by chronic stimulation with the ß3-adrenergic receptor agonist CL-316243. These studies reveal a very heterogeneous increase in baseline tissue radiodensity and xenon-enhanced radiodensity, indicative of both an increase in adipocytes water and protein content as well as tissue perfusion, mostly in regions that showed enhanced norepinephrine-stimulated perfusion before CL-316243 treatment. No statistically significant increase in 18F-fluorodeoxyglucose uptake or norepinephrine-stimulated tissue perfusion were observed in the mice after the CL-316243 treatment. The increase in tissue-water content and perfusion, along with the negligible increase in the tissue glucose uptake and norepinephrine-stimulated perfusion deserve more attention, especially considering the potential metabolic role that this tissue may play in whole body metabolism.

Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia.

OBJECTIVE: NRF2 is a master transcription factor that regulates the stress response. NRF2 is frequently mutated and activated in human esophageal squamous cell carcinoma (ESCC), which drives resistance to chemotherapy and radiation therapy. Therefore, a great need exists for NRF2 inhibitors for targeted therapy of NRF2high ESCC. DESIGN: We performed high-throughput screening of two compound libraries from which hit compounds were further validated in human ESCC cells and a genetically modified mouse model. The mechanism of action of one compound was explored by biochemical assays. RESULTS: Using high-throughput screening of two small molecule compound libraries, we identified 11 hit compounds as potential NRF2 inhibitors with minimal cytotoxicity at specified concentrations. We then validated two of these compounds, pyrimethamine and mitoxantrone, by demonstrating their dose- and time-dependent inhibitory effects on the expression of NRF2 and its target genes in two NRF2Mut human ESCC cells (KYSE70 and KYSE180). RNAseq and qPCR confirmed the suppression of global NRF2 signaling by these two compounds. Mechanistically, pyrimethamine reduced NRF2 half-life by promoting NRF2 ubiquitination and degradation in KYSE70 and KYSE180 cells. Expression of an Nrf2E79Q allele in mouse esophageal epithelium (Sox2CreER;LSL-Nrf2E79Q/+) resulted in an NRF2high phenotype, which included squamous hyperplasia, hyperkeratinization, and hyperactive glycolysis. Treatment with pyrimethamine (30 mg/kg/day, p.o.) suppressed the NRF2high esophageal phenotype with no observed toxicity. CONCLUSION: We have identified and validated pyrimethamine as an NRF2 inhibitor that may be rapidly tested in the clinic for NRF2high ESCC.

Combination of Polymeric Micelle Formulation of TGFß Receptor Inhibitors and Paclitaxel Produce Consistent Response Across Different Mouse Models of TNBC.

Triple-negative breast cancer (TNBC) is notoriously difficult to treat due to the lack of targetable receptors and sometimes poor response to chemotherapy. The transforming growth factor-beta (TGFß) family of proteins and their receptors (TGFR) are highly expressed in TNBC and implicated in chemotherapy-induced cancer stemness. Here we evaluated combination treatments using experimental TGFR inhibitors (TGFßi), SB525334 (SB), and LY2109761 (LY) with Paclitaxel (PTX) chemotherapy. These TGFßi target TGFR-I (SB) or both TGFR-I&II (LY). Due to the poor water solubility of these drugs, we incorporated each of them in poly(2-oxazoline) (POx) high-capacity polymeric micelles (SB-POx and LY-POx). We assessed their anti-cancer effect as single agents and in combination with micellar Paclitaxel (PTX-POx) using multiple immunocompetent TNBC mouse models that mimic human subtypes (4T1, T11-Apobec and T11-UV). While either TGFßi or PTX showed a differential effect in each model as single agents, the combinations were consistently effective against all three models. Genetic profiling of the tumors revealed differences in the expression levels of genes associated with TGFß, EMT, TLR-4, and Bcl2 signaling, alluding to the susceptibility to specific gene signatures to the treatment. Taken together, our study suggests that TGFßi and PTX combination therapy using high-capacity POx micelle delivery provides a robust anti-tumor response in multiple TNBC subtype mouse models.

In vivo imaging of brown adipose tissue vasculature reactivity during adrenergic stimulation of non-shivering thermogenesis in mice.

Brown adipose tissue (BAT) is a fat tissue specialized in heat production (non-shivering thermogenesis) and used by mammals to defend core body temperature when exposed to cold. Several studies have shown that during non-shivering thermogenesis the increase in BAT oxygen demand is met by a local and specific increase in tissue's blood flow. While the vasculature of BAT has been extensively studied postmortem in rodents using histology, optical and CT imaging techniques, vasculature changes during stimulation of non-shivering thermogenesis have never been directly detected in vivo. Here, by using computed tomography (CT) angiography with gold nanoparticles we investigate, non-invasively, changes in BAT vasculature during adrenergic stimulation of non-shivering thermogenesis by norepinephrine, a vasoconstrictor known to mediate brown fat heat production, and by CL 316,243, a specific ß3-adrenergic agonist also known to elicit BAT thermogenesis in rodents. We found that while CL 316,243 causes local vasodilation in BAT, with little impact on the rest of the vasculature throughout the body, norepinephrine leads to local vasodilation in addition to peripheral vasoconstriction. As a result, a significantly greater relative increase in BAT perfusion is observed following the injection of NE compared to CL. This study demonstrates the use of in vivo CT angiography as an effective tool in assessing vascular reactivity in BAT both qualitatively and quantitatively in preclinical studies.

Clinical and neuroradiological correlates of sleep in myotonic dystrophy type 1.

Abnormalities of sleep are common in myotonic dystrophy type 1 (DM1), but few previous studies have combined polysomnography with detailed clinical measures and brain imaging. In the present study, domiciliary polysomnography, symptom questionnaires and cognitive evaluation were undertaken in 39 DM1-affected individuals. Structural brain MRI was completed in those without contra-indication (n = 32). Polysomnograms were adequate for analysis in 36 participants. Sleep efficiency was reduced, and sleep architecture altered in keeping with previous studies. Twenty participants (56%) had moderate or severe sleep-disordered breathing (apnoea-hypopnoea index [AHI] ≥ 15). In linear modelling, apnoeas were positively associated with increasing age and male sex. AHI ≥ 15 was further associated with greater daytime pCO2 and self-reported physical impairment, somnolence and fatigue. Percentage REM sleep was inversely associated with cerebral grey matter volume, stage 1 sleep was positively associated with occipital lobe volume and stage 2 sleep with amygdala volume. Hippocampus volume was positively correlated with self-reported fatigue and somnolence. Linear relationships were also observed between measures of sleep architecture and cognitive performance. Findings broadly support the hypothesis that changes in sleep architecture and excessive somnolence in DM1 reflect the primary disease process in the central nervous system.

High MW polyethylene glycol prolongs circulation of pegloticase in mice with anti-PEG antibodies.

Pegloticase is an enzyme used to reduce serum uric acid levels in patients with chronic, treatment-refractory gout. Clinically, about 40% of patients develop high titers of anti-PEG antibodies (APA) after initial treatment, which in turn quickly eliminate subsequent doses of pegloticase from the systemic circulation and render the treatment ineffective. We previously found that pre-infusion with high MW free PEG (40 kDa) can serve as a decoy to saturate circulating APA, preventing binding to a subsequently administered dose of PEG-liposomes and restoring their prolonged circulation in mice, without any detectible toxicity. Here, we investigated the use of 40 kDa free PEG to restore the circulation of radio-labeled pegloticase in mice using longitudinal Positron Emission Tomography (PET) imaging over 4 days. Mice injected with pegloticase developed appreciable APA titers by Day 9, which further increased through Day 14. Compared to naïve mice, mice with pegloticase-induced APA rapidly cleared 89Zr-labeled pegloticase, with ~75% lower pegloticase concentrations in the circulation at four hours after treatment. The 96-h AUC in APA+ mice was less than 30% of the AUC in naïve mice. In contrast, pre-infusion of free PEG into PEG-sensitized mice restored the AUC of pegloticase to ~80% of that seen in naïve mice, resulting in a similar biodistribution to pegloticase in naïve mice over time. These results suggest that pre-infusion of free PEG may be a promising strategy to enable the safe and efficacious use of pegloticase and other PEGylated drugs in patients that have previously failed therapy due to induced APA.

Microgravity-induced alterations of mouse bones are compartment- and site-specific and vary with age.

The age at which astronauts experience microgravity is a critical consideration for skeletal health and similarly has clinical relevance for musculoskeletal disuse on Earth. While astronauts are extensively studied for bone and other physiological changes, rodent studies enable direct evaluation of skeletal changes with microgravity. Yet, mouse spaceflight studies have predominately evaluated tissues from young, growing mice. We evaluated bone microarchitecture in tibiae and femurs from Young (9-week-old) and Mature (32-weeks-old) female, C57BL/6N mice flown in microgravity for ~2 and ~3 weeks, respectively. Microgravity-induced changes were both compartment- and site-specific. Changes were greater in trabecular versus cortical bone in Mature mice exposed to microgravity (-40.0% Tb. BV/TV vs -4.4% Ct. BV/TV), and bone loss was greater in the proximal tibia as compared to the distal femur. Trabecular thickness in Young mice increased by +25.0% on Earth and no significant difference following microgravity. In Mature mice exposed to microgravity, trabecular thickness rapidly decreased (-24.5%) while no change was detected in age-matched mice that were maintained on Earth. Mature mice exposed to microgravity experienced greater bone loss than Young mice with net skeletal growth. Moreover, machine learning classification models confirmed that microgravity exposure-driven decrements in trabecular microarchitecture and cortical structure occurred disproportionately in Mature than in Young mice. Our results suggest that age of disuse onset may have clinical implications in osteoporotic or other at-risk populations on Earth and may contribute to understanding bone loss patterns in astronauts.

Spaceflight and hind limb unloading induces an arthritic phenotype in knee articular cartilage and menisci of rodents.

Reduced knee weight-bearing from prescription or sedentary lifestyles are associated with cartilage degradation; effects on the meniscus are unclear. Rodents exposed to spaceflight or hind limb unloading (HLU) represent unique opportunities to evaluate this question. This study evaluated arthritic changes in the medial knee compartment that bears the highest loads across the knee after actual and simulated spaceflight, and recovery with subsequent full weight-bearing. Cartilage and meniscal degradation in mice were measured via microCT, histology, and proteomics and/or biochemically after: (1) ~ 35 days on the International Space Station (ISS); (2) 13-days aboard the Space Shuttle Atlantis; or (3) 30 days of HLU, followed by a 49-day weight-bearing readaptation with/without exercise. Cartilage degradation post-ISS and HLU occurred at similar spatial locations, the tibial-femoral cartilage-cartilage contact point, with meniscal volume decline. Cartilage and meniscal glycosaminoglycan content were decreased in unloaded mice, with elevated catabolic enzymes (e.g., matrix metalloproteinases), and elevated oxidative stress and catabolic molecular pathway responses in menisci. After the 13-day Shuttle flight, meniscal degradation was observed. During readaptation, recovery of cartilage volume and thickness occurred with exercise. Reduced weight-bearing from either spaceflight or HLU induced an arthritic phenotype in cartilage and menisci, and exercise promoted recovery.

Inhibition of myostatin prevents microgravity-induced loss of skeletal muscle mass and strength.

The microgravity conditions of prolonged spaceflight are known to result in skeletal muscle atrophy that leads to diminished functional performance. To assess if inhibition of the growth factor myostatin has potential to reverse these effects, mice were treated with a myostatin antibody while housed on the International Space Station. Grip strength of ground control mice increased 3.1% compared to baseline values over the 6 weeks of the study, whereas grip strength measured for the first time in space showed flight animals to be -7.8% decreased in strength compared to baseline values. Control mice in space exhibited, compared to ground-based controls, a smaller increase in DEXA-measured muscle mass (+3.9% vs +5.6% respectively) although the difference was not significant. All individual flight limb muscles analyzed (except for the EDL) weighed significantly less than their ground counterparts at the study end (range -4.4% to -28.4%). Treatment with myostatin antibody YN41 was able to prevent many of these space-induced muscle changes. YN41 was able to block the reduction in muscle grip strength caused by spaceflight and was able to significantly increase the weight of all muscles of flight mice (apart from the EDL). Muscles of YN41-treated flight mice weighed as much as muscles from Ground IgG mice, with the exception of the soleus, demonstrating the ability to prevent spaceflight-induced atrophy. Muscle gene expression analysis demonstrated significant effects of microgravity and myostatin inhibition on many genes. Gamt and Actc1 gene expression was modulated by microgravity and YN41 in opposing directions. Myostatin inhibition did not overcome the significant reduction of microgravity on femoral BMD nor did it increase femoral or vertebral BMD in ground control mice. In summary, myostatin inhibition may be an effective countermeasure to detrimental consequences of skeletal muscle under microgravity conditions.

Altered rodent gait characteristics after ~35 days in orbit aboard the International Space Station.

The long-term adaptations to microgravity and other spaceflight challenges within the confines of a spacecraft, and readaptations to weight-bearing upon reaching a destination, are unclear. While post-flight gait change in astronauts have been well documented and reflect multi-system deficits, no data from rodents have been collected. Thus, the purpose of this study was to evaluate gait changes in response to spaceflight. A prospective collection of gait data was collected on 3 groups of mice: those who spent~35 days in orbit (FLIGHT) aboard the International Space Station (ISS); a ground-based control with the same habitat conditions as ISS (Ground Control; GC); and a vivarium control with typical rodent housing conditions (VIV). Pre-flight and post-flight gait measurements were conducted utilizing an optimized and portable gait analysis system (DigiGait, Mouse Specifics, Inc). The total data acquisition time for gait patterns of FLIGHT and control mice was 1.5-5 min/mouse, allowing all 20 mice per group to be assessed in less than an hour. Patterns of longitudinal gait changes were observed in the hind limbs and the forelimbs of the FLIGHT mice after ~35 days in orbit; few differences were observed in gait characteristics within the GC and VIV controls from the initial to the final gait assessment, and between groups. For FLIGHT mice, 12 out of 18 of the evaluated gait characteristics in the hind limbs were significantly changed, including: stride width variability; stride length and variance; stride, swing, and stance duration; paw angle and area at peak stance; and step angle, among others. Gait characteristics that decreased included stride frequency, and others. Moreover, numerous forelimb gait characteristics in the FLIGHT mice were changed at post-flight measures relative to pre-flight. This rapid DigiGait gait measurement tool and customized spaceflight protocol is useful for providing preliminary insight into how spaceflight could affect multiple systems in rodents in which deficits are reflected by altered gait characteristics.

A Mouse Model for Skeletal Structure and Function Changes Caused by Radiation Therapy and Estrogen Deficiency.

Radiation therapy and estrogen deficiency can damage healthy bone and lead to an increased fracture risk. The goal of this study is to develop a mouse model for radiation therapy using a fractionated biologically equivalent dose for cervical cancer treatment in both pre- and postmenopausal women. Thirty-two female C57BL/6 mice 13 weeks of age were divided into four groups: Sham + non-irradiated (SHAM + NR), Sham + irradiated (SHAM + IRR), ovariectomy + non-irradiated (OVX + NR) and ovariectomy + irradiated (OVX + IRR). The irradiated mice received a 6 Gy dose of X-rays to the hindlimbs at Day 2, Day 4 and Day 7 (18 Gy total). Tissues were collected at Day 35. DEXA, microCT analysis and FEA were used to quantify structural and functional changes at the proximal tibia, midshaft femur, proximal femur and L1 vertebra. There was a significant (p < 0.05) decline in proximal tibia trabecular BV/TV from (1) IRR compared to NR mice within Sham (- 46%) and OVX (- 41%); (2) OVX versus Sham within NR mice (- 36%) and IRR mice (- 30%). With homogenous material properties applied to the proximal tibia mesh using FEA, there was (1) an increase in whole bone (trabecular + cortical) structural stiffness from IRR compared to NR mice within Sham (+ 10%) and OVX (+ 15%); (2) a decrease in stiffness from OVX versus Sham within NR mice (- 18%) and IRR mice (- 14%). Fractionated irradiation and ovariectomy both had a negative effect on skeletal microarchitecture. Ovariectomy had a systemic effect, while skeletal radiation damage was largely specific to trabecular bone within the X-ray field.

Hemophilia A and B mice, but not VWF-/-mice, display bone defects in congenital development and remodeling after injury.

While joint damage is the primary co-morbidity of hemophilia, osteoporosis and osteopenia are also observed. Coagulation factor VIII deficient (FVIII-/-) mice develop an osteoporotic phenotype in the absence of induced hemarthrosis that is exacerbated two weeks after an induced joint injury. Here we have compared comprehensively the bone health of clotting factor VIII, factor IX, and Von Willebrand Factor knockout (FVIII-/-, FIX-/-, and VWF-/- respectively) mice both in the absence of joint hemorrhage and following induced joint injury. We found FVIII-/- and FIX-/- mice, but not VWF-/- mice, developmentally have an osteoporotic phenotype. Unilateral induced hemarthrosis causes further bone damage in both FVIII-/- and FIX-/- mice, but has little effect on VWF-/- bone health, indicating that the FVIII.VWF complex is not required for normal bone remodeling in vivo. To further investigate the bone healing following hemarthrosis in hemophilia we examined a two week time course using microCT, serum chemistry, and histological analysis. Elevated ratio of osteoprotegerin (OPG)/receptor activator of nuclear factor-kappa B ligand (RANKL), increased osterix+ osteoblastic cells, and decreased smoothness of the cortical bone surface were evident within several days of injury, indicative of acute heterotopic mineralization along the cortical surface. This was closely followed by increased interleukin-6 (IL-6) levels, increased osteoclast numbers, and significant trabecular bone loss. Uncoupled and disorganized bone formation and resorption continued for the duration of the study resulting in significant deterioration of the joint. Further elucidation of the shared mechanisms underlying abnormal bone homeostasis in the absence of FVIII or FIX is needed to guide evidence-based approaches to the screening and treatment of the prevalent bone defects in hemophilia A and B.

Prophylactic administration of glycoPEGylated factor IX provides protection and joint outcome superior to recombinant factor IX after induced joint bleeding.

BACKGROUND: Following induced joint hemorrhage, hemophilia B results in the abnormal persistence of iron deposition, inflammation, and neovascularity of the synovial tissue, as well as deterioration of the bone articular surface and strength. Previously, we demonstrated that a factor IX (FIX) replacement protein with extended circulating FIX activity, glycoPEGylated FIX nonacog beta pegol (N9-GP), could improve synovial and osteochondral parameters in F9 knockout mice when administered after joint injury. OBJECTIVE: We explored the use of N9-GP prior to unilateral joint hemorrhage and compared to unmodified recombinant FIX (rFIX). METHODS: Pharmacodynamics, histology, and microcomputed tomography were used to assess the effects of prophylactic administration of glycoPEGylated FIX. RESULTS: In comparison to rFIX, N9-GP significantly improved soft tissue histological parameters, as well as bone outcome at 2 weeks post injury, while performing equally in reduction of blood present in the joint space assessed 1 day after injury. CONCLUSIONS: These results indicate that, in comparison to rFIX, the prophylactic use of extended half-life FIX provides superior protection from bleeding-induced joint damage, manifested by improved correction of histologic parameters.

Chronic kidney disease and aging differentially diminish bone material and microarchitecture in C57Bl/6 mice.

Chronic kidney disease (CKD) is a common disease of aging and increases fracture risk over advanced age alone. Aging and CKD differently impair bone turnover and mineralization. We thus hypothesize that the loss of bone quality would be greatest with the combination of advanced age and CKD. We evaluated bone from young adult (6 mo.), middle-age (18 mo.), and old (24 mo.) male C57Bl/6 mice three months following either 5/6th nephrectomy, to induce CKD, or Sham procedures. CKD exacerbated losses of cortical and trabecular microarchitecture associated with aging. Aging and CKD each resulted in thinner, more porous cortices and fewer and thinner trabeculae. Bone material quality was also reduced with CKD, and these changes to bone material were distinct from those due to age. Aging reduced whole-bone flexural strength and modulus, micrometer-scale nanoindentation modulus, and nanometer-scale tissue and collagen strain (small-angle x-ray scattering [SAXS]. By contrast, CKD reduced work to fracture and variation in bone tissue modulus and composition (Raman spectroscopy), and increased percent collagen strain. The increased collagen strain burden was associated with loss of toughness in CKD. In addition, osteocyte lacunae became smaller, sparser, and more disordered with age for Sham mice, yet these age-related changes were not clearly observed in CKD. However, for CKD, larger lacunae positively correlated with increased serum phosphate levels, suggesting that osteocytes play a role in systemic mineral homeostasis. This work demonstrates that CKD reduces bone quality, including microarchitecture and bone material properties, and that loss of bone quality with age is compounded by CKD. These findings may help reconcile why bone mass does not consistently predict fracture in the CKD population, as well as why older individuals with CKD are at high risk of fragility.

Methods for quantitative characterization of bone injury from computed-tomography images.

Computed tomography (CT) images can potentially provide insights into bone structure for diagnosis of disorders and diseases. However, evaluation of trabecular bone structure and whole bone shape is often qualitative or semi-quantitative. This limits inter-study comparisons and the ability to detect subtle bone quality variations during early disease onset or in response to new treatments. In this work, we enable quantitative characterization of bone diseases through bone morphometry, texture analysis, and shape analysis methods. The potential of our analysis methods to identify the impact of hemophilia is validated in a mouse femur wound model. In our results, shape localizes and characterizes the formation of spurious bone, and our texture and bone morphometry analysis results provide extra information about the composition of that bone. Some of our one-dimensional (1D) textural features were able to significantly differentiate our injured femurs from our healthy femurs, even with this small sample size demonstrating the potential of the proposed analysis framework. While trabecular bone morphometrics have been a pillar in 3D microCT bone research for decades, the proposed analysis framework augments how we define and understand phenotypical presentation of bone disease. The contributed open source software is exposed to the medical image analysis community through 3D Slicer extensions to ensure both robustness and reproducibility.

Impaired Annulus Fibrosus Development and Vertebral Fusion Cause Severe Scoliosis in Mice with Deficiency of c-Jun NH2-Terminal Kinases 1 and 2.

Mitogen-activated protein kinases, including c-Jun NH2-terminal kinase (JNK), play an important role in the development and function of a large variety of tissues. The skeletal phenotype of JNK1 and JNK2 double-knockout (dKO) mice (JNK1fl/flCol2-Cre/JNK2-/-) and control genotypes were analyzed at different embryonic and postnatal stages. JNK1/2 dKO mice displayed a severe scoliotic phenotype beginning during development that was grossly apparent around weaning age. Alcian blue staining at embryonic day 17.5 showed abnormal fusion of the posterior spinal elements. In adult mice, fusion of vertebral bodies and of spinous and transverse processes was noted by micro-computed tomography, Alcian blue/Alizarin red staining, and histology. The long bones developed normally, and histologic sections of growth plate and articular cartilage revealed no significant abnormalities. Histologic sections of the vertebral column at embryonic days 15.5 and 17.5 revealed an abnormal organization of the annulus fibrosus in the dKOs, with chondrocyte-like cells and fusion of dorsal processes. Spinal sections in 10-week-old dKO mice showed replacement of intervertebral disk structures (annulus fibrosus and nucleus pulposus) by cartilage and bone tissues, with cells staining for markers of hypertrophic chondrocytes, including collagen X and runt-related transcription factor 2. These findings demonstrate a requirement for both JNK1 and JNK2 in the normal development of the axial skeleton. Loss of JNK signaling results in abnormal endochondral bone formation and subsequent severe scoliosis.

Modeling Space Radiation Induced Bone Changes in Rat Femurs through Finite Element Analysis.

As the duration of manned missions outside of the Earth's protective shielding increase, astronauts are at risk for exposure to space radiation. Various organ systems may be damaged due to exposure. This study investigates the bone strength changes using finite element modeling of Long Evans rats (n=85) subjected to graded, head-only proton (0, 10, 25, and 100 cGy, 150 MeV/n) and 28silicon (0, 10, 25, and 50 cGy, 300 MeV/n) radiation. The strength of the femoral neck will be examined due its clinical relevance to hip fractures. It has been shown in previous studies that bone mineral density was not reduced at the site of fracture. These findings question whether measurements of bone mineral density may be used to assess risk of hip fracture. The mechanisms leading to the irregular relationship between bone density and strength are still uncertain within literature and investigated to greater extent in clinical applications. Finite element analysis within this study simulated physiological loading of the femoral neck. No significant changes in femoral neck strength were found across doses of proton or 28silicon head-only radiation. Future work includes performing mechanical testing of the bone samples. Moving from mouse to larger animal models may also provide the increased lifespan for assessing the long-term outcomes of radiation exposure.

Research synergy and drug development: Bright stars in neighboring constellations.

Drug discovery and subsequent availability of a new breakthrough therapeutic or 'cure' is a compelling example of societal benefit from research advances. These advances are invariably collaborative, involving the contributions of many scientists to a discovery network in which theory and experiment are built upon. To document and understand such scientific advances, data mining of public and commercial data sources coupled with network analysis can be used as a digital methodology to assemble and analyze component events in the history of a therapeutic. This methodology is extensible beyond the history of therapeutics and its use more generally supports (i) efficiency in exploring the scientific history of a research advance (ii) documenting and understanding collaboration (iii) portfolio analysis, planning and optimization (iv) communication of the societal value of research. Building upon prior art, we have conducted a case study of five anti-cancer therapeutics to identify the collaborations that resulted in the successful development of these therapeutics both within and across their respective networks. We have linked the work of over 235,000 authors in roughly 106,000 scientific publications that capture the research crucial for the development of these five therapeutics. Applying retrospective citation discovery, we have identified a core set of publications cited in the networks of all five therapeutics and additional intersections in combinations of networks. We have enriched the content of these networks by annotating them with information on research awards from the US National Institutes of Health (NIH). Lastly, we have mapped these awards to their cognate peer review panels, identifying another layer of collaborative scientific activity that influenced the research represented in these networks.

Abnormal joint and bone wound healing in hemophilia mice is improved by extending factor IX activity after hemarthrosis.

Wound healing requires interactions between coagulation, inflammation, angiogenesis, cellular migration, and proliferation. Healing in dermal wounds of hemophilia B mice is delayed when compared with hemostatically normal wild-type (WT) mice, with abnormal persistence of iron deposition, inflammation, and neovascularity. We observed healing following induced joint hemorrhage in WT and factor IX (FIX) knockout (FIX-/-) mice, examining also parameters previously studied in an excisional skin wound model. Hemostatically normal mice tolerated this joint bleeding challenge, cleared blood from the joint, and healed with minimal pathology, even if additional autologous blood was injected intra-articularly at the time of wounding. Following hemarthrosis, joint wound healing in hemophilia B mice was impaired and demonstrated similar abnormal histologic features as previously described in hemophilic dermal wounds. Therefore, studies of pathophysiology and therapy of hemophilic joint bleeding performed in hemostatically normal animals are not likely to accurately reflect the healing defect of hemophilia. We additionally explored the hypothesis that the use of a FIX replacement protein with extended circulating FIX activity could improve synovial and osteochondral wound healing in hemophilic mice, when compared with treatment with unmodified recombinant FIX (rFIX) in the established joint bleeding model. Significantly improved synovial wound healing and preservation of normal osteochondral architecture are achieved by extending FIX activity after hemarthrosis using glycoPEGylated FIX when compared with an equivalent dose of rFIX. These results suggest that treating joint bleeding only until hemostasis is achieved may not result in optimal joint healing, which is improved by extending factor activity.