Chondrocytic and pharmacokinetic properties of Phlpp inhibitors.

Objective: The pleckstrin homology domain leucine-rich repeat protein phosphatases (Phlpp1/2) were recently identified as potential therapeutic targets for cartilage regeneration in osteoarthritic joints. Phlpp inhibitors NSC 117079 and NSC 45586 increase chondrocyte proliferation and matrix production, but the pharmacodynamics and pharmacokinetics of these compounds are not known. Design: Chondrocytic effects of Phlpp inhibitors, NSC 117079 and NSC 45586, were measured by western blotting of Phlpp substrates, glycosaminoglycan (GAG) assays, and transcriptomic assays. Liquid chromatography/mass spectroscopy assays were established to measure NSC 117079 and NSC 45586 in vitro and in vivo. The effects of NSC 117079 and NSC 45586 on articular cartilage structure in vivo after intra-articular injection were determined by histology. Results: The Phlpp inhibitors NSC 117079 and NSC 45586 were highly stable in vitro and stimulated GAG, Sox9, proteoglycan 4 and collagen 2 production in maturing but not more differentiated chondrocytes in vitro. Both molecules reduced Phlpp1/2 levels and suppressed matrix degradation to functionally extend their inhibitory effect on these phosphatases. In vivo, NSC 117079 was eliminated from the bloodstream within 4 â€‹h after intravenous injection, while NSC 45586 was eliminated in 8 â€‹h and had a higher volume distribution. Both molecules increased articular cartilage area on lateral and medial tibial plateaus and femoral condyles by 15% in C57Bl/6 mice between four and five weeks of age. Conclusion: These data advance our understanding of how Phlpp inhibitors promote and preserve cartilage formation and provide a basis for understanding their safety and activity in vivo.

Phlpp1 facilitates post-traumatic osteoarthritis and is induced by inflammation and promoter demethylation in human osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is the most common form of arthritis and a leading cause of disability. OA is characterized by articular chondrocyte deterioration, subchondral bone changes and debilitating pain. One strategy to promote cartilage regeneration and repair is to accelerate proliferation and matrix production of articular chondrocytes. We previously reported that the protein phosphatase Phlpp1 controls chondrocyte differentiation by regulating the activities of anabolic kinases. Here we examined the role of Phlpp1 in OA progression in a murine model. We also assessed PHLPP1 expression and promoter methylation. DESIGN: Knee joints of WT and Phlpp1(-/-) mice were surgically destabilized by transection of the medial meniscal ligament (DMM). Mice were assessed for signs of OA progression via radiographic and histological analyses, and pain assessment for mechanical hypersensitivity using the von Frey assay. Methylation of the PHLPP1 promoter and PHLPP1 expression were evaluated in human articular cartilage and chondrocyte cell lines. RESULTS: Following DMM surgeries, Phlpp1 deficient mice showed fewer signs of OA and cartilage degeneration. Mechanical allodynia associated with DMM surgeries was also attenuated in Phlpp1(-/-) mice. PHLPP1 was highly expressed in human articular cartilage from OA patients, but was undetectable in cartilage specimens from femoral neck fractures (FNFxs). Higher PHLPP1 levels correlated with less PHLPP1 promoter CpG methylation in cartilage from OA patients. Blocking cytosine methylation or treatment with inflammatory mediators enhanced PHLPP1 expression in human chondrocyte cell lines. CONCLUSION: Phlpp1 deficiency protects against OA progression while CpG demethylation and inflammatory cytokines promote PHLPP1 expression.

The consistency of students' self-assessments in short-essay subject matter examinations.

This longitudinal study compares the accuracy of self-assessments of 22 students across four examinations during their first 2 years of medical school. The four examinations used a similar short-essay format and covered many of the same basic science disciplines at similar levels of difficulty. Immediately after answering an average of 20 questions on each examination, students predicted their performance on those questions. After assigned subject matter experts had scored the questions, the differences between students' predictions and the experts' scores were calculated for each question. The degree to which students had over- and underestimated their performance across all questions was determined by separately averaging all positive and negative differences between students' and experts' assessments on each examination. The results of the study indicated that accuracy in self-assessment improved from examination 1 to examination 3 (with less overestimation) and dropped on examination 4 (with more underestimation). The results revealed no relationship between self-assessment estimations and actual scores received. Furthermore, the self-assessment estimations tended to be statistically correlated between contiguous examinations (i.e., examinations 1 and 2, 2 and 3, etc.) but not between non-contiguous ones (i.e., examinations 1 and 3, etc.). The results of the study are interpreted to suggest that the students in the study have a self-assessment tendency towards over- or underestimation that is somewhat stable but that gradually evolves over time with experience, maturity and self-assessment practice. The most frequent direction of change is towards decreased overestimation and increased underestimation. These results are consistent with the findings of other recent longitudinal self-assessment studies.

Morphologic effects of fast neutrons or photons on the canine kidney.

Thirty-nine adult male Beagles received either fast neutron or photon irradiation to the right thorax to determine the relative biological effectiveness of fast neutrons on normal pulmonary tissue. The right anterior abdomen, including the cranial half of the right kidney, was included in the field of irradiation. Twenty-four dogs (six/group) received fast neutrons with an average energy of 15 MeV to total doses of 1000, 1500, 2250, or 3375 cGy in four fractions per week for 6 weeks. Fifteen dogs received 3000, 4500, or 6750 cGy of photons (five/group) in an identical fractionation pattern. All 12 neutron irradiated dogs receiving 3375 and 2250 cGy and 1 of 6 receiving 1500 cGy, developed clinical and clinical pathologic signs of hepatic, pancreatic, and gastrointestinal disturbances, but no signs of renal injury were seen. These 13 dogs died or were euthanatized 47-367 days after irradiation. Only 1 of 5 dogs receiving 6750 cGy of photons developed similar signs and died 708 days post-irradiation. The remaining 11 neutron irradiated dogs and 14 photon irradiated dogs eventually died of other causes. All 39 dogs were necropsied and their kidneys were compared to each other and to control dogs. Radiation induced lesions included hemorrhages, necrosis and disappearance of tubular epithelia, glomerulosclerosis, atrophy and fibrosis. These lesions were associated with degenerative and occlusive vascular changes and were much more severe in the neutron irradiated dogs. The relative biologic effectiveness of fast neutrons for canine kidney assessed by gross and microscopic pathology is approximately 4.5 (6750/1500).

Radiobiological comparison of external beam irradiation and radioimmunotherapy in renal cell carcinoma xenografts.

Growth delay was measured in TK-82 renal cell carcinoma (RCC) xenografts implanted in nude mice receiving single fraction external beam irradiation (SF-XRT), multifraction external beam irradiation (MF-XRT), or radioimmunotherapy (RIT). Thermoluminescent dosimeter(s) (TLD) and autoradiography were used to ascertain the average absorbed dose delivered and the degree of heterogeneous uptake of radiolabeled antibody for the RIT irradiations. For intravenous administered activities of 100, 200, 400, and 600 microCi of I-131 labeled A6H antibody, volume doubling times (VDT) and TLD absorbed dose measurements for each administered activity were 7 days (341 cGy), 38 days (383 cGy), 85 days (886 cGy) and no regrowth (1034 cGy), respectively. For SF-XRT irradiations of 500, 1000, and 1500 cGy, VDT times were 11, 62, and 103 days, respectively. MF-XRT of 4 X 250 cGy over a 2-week period yielded a VDT of 25 days. Marked peripheral activity deposition was noted on most autoradiographs from multiple tumor samples. These data suggest that an equivalent to superior tumor growth delay is obtained for absorbed doses delivered by exponentially decaying low dose rate radioimmunotherapy RIT compared to similar doses of acute dose rate XRT as quantitated by the TLD method.

Pathologic effects of fractionated fast neutrons or photons on the pancreas, pylorus and duodenum of dogs.

Thirty-nine adult male Beagles received either fast neutron or photon irradiation to the right thorax to determine the relative biological effectiveness (RBE) of fast neutrons on normal pulmonary tissue. The right anterior abdomen was included in the field of radiation. Twenty-four dogs (six/group) received fast neutrons with an average energy of 15 MeV to total doses of 1000, 1500, 2250 or 3375 rad in four fractions per week for six weeks. Fifteen dogs received 3000, 4500 or 6750 rad of photons (five/group) in an identical fractionation pattern. All neutron irradiated dogs receiving 3375 and 2250 rad and one receiving 1500 rad developed clinical signs of pancreatic, hepatic and gastrointestinal disturbances. The liver enzymes of these dogs became elevated and they died or were euthanatized in extremis 47-367 days after irradiation. Only one 6750 rad photon dog developed similar signs and died 708 days post-irradiation. Five neutron and 10 photon exposed dogs died of other causes. Neutron-induced lesions in the stomach and duodenum included hemorrhages, erosions, ulcerations and fibrosis. Ulcers perforated the GI tract of five dogs. Pancreatic lesions included degranulation and necrosis of acinar cells, fibrosis ans atrophy. Islet cells were not obviously damaged. All lesions were associated with degenerative and occlusive vascular changes. The RBE of fast neutrons, assessed by clinical signs, gross and microscopic pathology, is approximately 3-4.5 for pancreas and about 4.5 for pylorus and duodenum.

Regional pulmonary function after irradiation of the canine lung: radionuclide evaluation.

The pulmonary effects of neutron and gamma irradiation were compared in a group of beagle dogs that were subjected to hemithorax irradiation with cobalt-60 gamma rays or 15-MeV neutrons. Integral cobalt-60 doses of 3000, 4500, or 6750 rad (30, 45, or 67.5 Gy) and neutron doses of 1000, 1500, or 2250 rad (10, 15, or 22.5 Gy) were given on a therapy-type schedule of four equal fractions per week for 6 weeks. Serial Tc-99m-macroaggregated albumin perfusion, Tc-99m-Sn-phytate aerosol, and xenon-133 ventilation studies were performed before irradiation and at 3, 6, 9, 12, 18, and 24 months postexposure. Pulmonary damage was more severe and persistent with neutron than with gamma radiation, but the changes were dose-dependent for both types of radiation. The perfusion and radioaerosol imaging studies provided the best scintigraphic evidence of lung damage. Abnormalities in the xenon-133 studies were relatively minor and were more apparent on the single-breath than on the equilibrium or clearance studies. The scintigraphic studies provided evidence of radiation-induced ventilation perfusion inequalities with both types of radiation, but required several times less neutron radiation than gamma radiation to produce similar alterations in ventilation-perfusion relationships.

Neoplasia in fast neutron-irradiated beagles.

One hundred fifty-one beagle dogs were irradiated with either photons or fast neutrons (15 MeV) to one of three dose-limiting normal tissues--spinal cord, lung, or brain. The radiation was given in four fractions per week for 5 weeks (spinal cord), 6 weeks (lung), or 7 weeks (brain) to total doses encompassing those given clinically for cancer management. To date, no nonirradiated dogs or photon-irradiated dogs have developed any neoplasms. Seven dogs receiving fast neutrons have developed 9 neoplasms within the irradiated field. Of the neutron-irradiated dogs at risk, the incidence of neoplasia was 15%. The latent period for radiation-induced cancers has varied from 1 to 4 1/2 years at this time in the study.

Anatomy of the beagle in cross-section: head and neck.

The transverse anatomy of the head and neck of the Beagle was studied. Cross-sectional preparations were photographed and compared with computerized tomographic scans, freshly prepared dissection specimens, and with skeletal preparations. Anatomic structures were identified by these means with the aid of anatomy texts. A series of labeled photographs were provided as a basis for interpretation of computerized tomography scans.

The pathologic effects of fractionated fast neutrons or photons on canine liver.

Thirty-nine adult male purebred beagles received either fast neutron or photon irradiation to the right thorax to determine the effects on pulmonary tissue. The right half of the liver was included in the field of radiation. Twenty-four dogs (six/group) received fast neutrons with a mean energy of 15 MeV to total doses of 1000, 1500, 2250, or 3375 rads in four fractions per week for 6 weeks. Fifteen dogs received 3000, 4500, or 6750 total rads of photons (five dogs/group) in an identical fractionation pattern. All neutron-irradiated dogs receiving 3375 and 2250 rads and one receiving 1500 rads developed clinical signs, hepatic enzyme, and bilirubin elevations, and the dogs died or were euthanized in extremis on postirradiation day 47-291. Signs of liver injury, other than enzyme changes, have not developed to date (1200-1300 days) in the remaining dogs, except in one 6750-rad photon dog that died of hepatic failure on postirradiation day 708. At necropsy, the irradiated right lobes of the liver were atrophic and the nonirradiated left lobes underwent compensatory hypertrophy. Hepatic arterioles and bile ducts were injured in every dog, but no obstructive lesions were observed in hepatic veins. Portal fibroplasia, bile retention, and proliferation of bile ductules was common; the latter two changes also occurred in the nonirradiated lobes. No qualitative differences were observed between hepatic lesions in neutron- versus photon-irradiated dogs. The relative biological effectiveness of fast neutrons for liver damage appears to be no less than 4.5.

Canine anatomy as assessed by computerized tomography.

Computerized tomographic studies of normal canine anatomy were obtained, using a whole body scanner. The regions of interest were head and neck, thorax, and abdomen and pelvis. Scans were compared with gross transverse sections from one euthanatized dog. Identification and labeling of anatomic structures were aided by reference to recognized texts of canine anatomy.