Endochondral fracture healing with external fixation in the Sost knockout mouse results in earlier fibrocartilage callus removal and increased bone volume fraction and strength.

Sclerostin deficiency, via genetic knockout or anti-Sclerostin antibody treatment, has been shown to cause increased bone volume, density and strength of calluses following endochondral bone healing. However, there is limited data on the effect of Sclerostin deficiency on the formative early stage of fibrocartilage (non-bony tissue) formation and removal. In this study we extensively investigate the early fibrocartilage callus. Closed tibial fractures were performed on Sost(-/-) mice and age-matched wild type (C57Bl/6J) controls and assessed at multiple early time points (7, 10 and 14days), as well as at 28days post-fracture after bony union. External fixation was utilized, avoiding internal pinning and minimizing differences in stability stiffness, a variable that has confounded previous research in this area. Normal endochondral ossification progressed in wild type and Sost(-/-) mice with equivalent volumes of fibrocartilage formed at early day 7 and day 10 time points, and bony union in both genotypes by day 28. There were no significant differences in rate of bony union; however there were significant increases in fibrocartilage removal from the Sost(-/-) fracture calluses at day 14 suggesting earlier progression of endochondral healing. Earlier bone formation was seen in Sost(-/-) calluses over wild type with greater bone volume at day 10 (221%, p<0.01). The resultant Sost(-/-) united bony calluses at day 28 had increased bone volume fraction compared to wild type calluses (24%, p<0.05), and the strength of the fractured Sost(-/-) tibiae was greater than that that of wild type fractured tibiae. In summary, bony union was not altered by Sclerostin deficiency in externally-fixed closed tibial fractures, but fibrocartilage removal was enhanced and the resultant united bony calluses had increased bone fraction and increased strength.

Mechanical load increases in bone formation via a sclerostin-independent pathway.

Sclerostin, encoded by the Sost gene, is an important negative regulator of bone formation that has been proposed to have a key role in regulating the response to mechanical loading. To investigate the effect of long-term Sclerostin deficiency on mechanotransduction in bone, we performed experiments on unloaded or loaded tibiae of 10 week old female Sost-/- and wild type mice. Unloading was induced via 0.5U botulinum toxin (BTX) injections into the right quadriceps and calf muscles, causing muscle paralysis and limb disuse. On a separate group of mice, increased loading was performed on the left tibiae through unilateral cyclic axial compression of equivalent strains (+1200 µe) at 1200 cycles/day, 5 days/week. Another cohort of mice receiving equivalent loads (-9.0 N) also were assessed. Contralateral tibiae served as normal load controls. Loaded/unloaded and normal load tibiae were assessed at day 14 for bone volume (BV) and formation changes. Loss of BV was seen in the unloaded tibiae of wild type mice, but BV was not different between normal load and unloaded Sost-/- tibiae. An increase in BV was seen in the loaded tibiae of wild type and Sost-/- mice over their normal load controls. The increased BV was associated with significantly increased mid-shaft periosteal mineralizing surface/bone surface (MS/BS), mineral apposition rate (MAR), and bone formation rate/bone surface (BFR/BS), and endosteal MAR and BFR/BS. Notably, loading induced a greater increase in periosteal MAR and BFR/BS in Sost-/- mice than in wild type controls. Thus, long-term Sclerostin deficiency inhibits the bone loss normally induced with decreased mechanical load, but it can augment the increase in bone formation with increased load.

Modulation of endochondral ossification by MEK inhibitors PD0325901 and AZD6244 (Selumetinib).

MEK inhibitors (MEKi) PD0325901 and AZD6244 (Selumetinib) are drugs currently under clinical investigation for cancer treatment, however the Ras-MAPK pathway is also an important mediator of normal bone cell differentiation and function. In this study we examined the effects of these compounds on endochondral processes using both in vitro and in vivo models. Treatment with PD0325901 or AZD6244 significantly increased Runx2 and Alkaline phosphate gene expression in calvarial osteoblasts and decreased TRAP+ cells in induced osteoclast cultures. To test the effects of these drugs on bone healing, C57/Bl6 mice underwent a closed tibial fracture and were treated with PD0325901 or AZD6244 at 10mg/kg/day. Animals were culled at day 10 and at day 21 post-fracture for analysis of the fracture callus and the femoral growth plate in the contralateral leg. MEKi treatment markedly increased cartilage volume in the soft callus at day 10 post-fracture (+60% PD0325901, +20% AZD6244) and continued treatment led to a delay in cartilage remodeling. At the growth plate, we observed an increase in the height of the hypertrophic zone relative to the proliferative zone of +78% in PD0325901 treated mice. Osteoclast surface was significantly decreased both at the terminal end of the growth plate and within the fracture calluses of MEKi treated animals. The mechanistic effects of MEKi on genes encoding cartilage matrix proteins and catabolic enzymes were examined in articular chondrocyte cultures. PD0325901 or AZD6244 led to increased matrix protein expression (Col2a1 and Acan) and decreased expression of catabolic factors (Mmp13 and Adamts-5). Taken together, these data support the hypothesis that MEKi treatment can impact chondrocyte hypertrophy, matrix resorption, and fracture healing. These compounds can also affect bone architecture by expanding the hypertrophic zone of the growth plate and reducing osteoclast surface systemically.

Postmarketing surveillance: strengths and limitations. The flucloxacillin-dicloxacillin story.

Spontaneous reporting of adverse drug reactions continues to be the principal method used for monitoring the safety of marketed drugs. Despite the many successes attributed to these schemes, they can reliably detect only a small fraction of the range of possible drug-related events and provide virtually no useful quantitative data. Some of the limitations of spontaneous reporting were demonstrated recently in relation to flucloxacillin. Reports in Australia suggested the likelihood of an unacceptable risk of flucloxacillin-associated jaundice, but the data from spontaneous reporting in countries with apparently similar use of the drug, such as New Zealand and the UK, were insufficient to confirm or refute this proposition. Spontaneous monitoring should be supplemented by the systematic monitoring of cohorts of users of new drugs, using record-linkage to track their subsequent health. Although several impediments exist to the introduction of such a scheme in Australia, consideration should be given to addressing how such a system might be implemented.

Loss of bactericidal activity from contact lens storage solutions.

PURPOSE: The purpose of this study was to evaluate the effect of lens storage on the bactericidal activities of disinfecting solutions. METHODS: HYDROCURVEII (55% water content) soft contact lenses were soaked in OPTI-FREE Rinsing, Disinfecting, and Storage Solution (preserved with polyquaternium-1 [PQ-1]) or ReNu Multi-Purpose Solution (polyaminopropyl biguanide [PHMB]). After the lenses were removed, solutions were challenged with Pseudomonas aeruginosa and Staphylococcus aureus. Uptake of the antimicrobial agents by the lenses was evaluated by placing the lenses on plates seeded with S. aureus and observing for zones of growth inhibition. RESULTS: The bactericidal activity of the PHMB preserved solution decreased after 4 hours of storage time and showed no activity after storage for 3 days. Lenses soaked in the PHMB preserved solution produced zones of inhibition. The results suggest that the PHMB in the solution accumulates in the lens. This decreases the levels of preservative in the solution that is no longer available during lens storage. Neither decrease in bactericidal activity nor accumulation of the PQ-1 preservative in the lens occurred during storage with the PQ-1 solution. CONCLUSION: These results demonstrate that PQ-1 preserved solutions maintain bactericidal activity overnight and during prolonged storage without detectable uptake by lenses.

Hydronephrosis in mice exposed to TCDD-contaminated breast milk: identification of the peak period of sensitivity and assessment of potential recovery.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent inducer of hydronephrosis in both fetal and neonatal mice. A critical period of sensitivity to TCDD could not be identified for prenatally induced hydronephrosis since the urinary tract appeared equally sensitive throughout organogenesis. To identify the critical period of susceptibility for development of lactationally induced hydronephrosis in neonatal mice, as well as to characterize the potential for recovery from this renal lesion, dose-response and time-course studies were conducted in the postnatal period. Pregnant C57BL/6N mice were allowed natural delivery. In the dose-response phase of this investigation, mothers were administered 0, 3, 6, or 12 micrograms TCDD/kg once by gavage on Postnatal Day (PND) 1, 4, 8, or 14, and dams and pups were euthanized on PND 26. The kidneys were examined, and hydronephrotic severity was scored. The incidence and severity of hydronephrosis were significantly increased above controls only following treatment on PND 1 or 4, while on PND 8 the increase was marginal and pairwise tests were nonsignificant. Following treatment of dams on PND 1, the hydronephrotic response detected in 26-day-old pups was significantly greater than that for all later exposure days. In the time-course study, dams were given a single oral dose of 0 or 9 micrograms TCDD/kg on PND 1, and mothers and litters were subsequently euthanized on PND 7, 13, 19, or 26. Both hydronephrotic incidence and severity increased with time to euthanization following treatment on PND 1. Thus with the dosing regimen used in this study, recovery does not appear to occur between PNDs 7 and 26. Sex-related differences were observed, as the hydronephrotic response in males was generally greater than in females. In conclusion, the postnatal window of sensitivity during which TCDD can induce hydronephrosis is very narrow. Nonetheless, the hydronephrotic response induced during this early postnatal time is dramatic. Finally, PND 1 is the peak postnatal period of susceptibility for development of TCDD-induced hydronephrosis.

Differential toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in C57BL/6J mice congenic at the Ah Locus.

The acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was examined in male C57BL/6J mice differing only at the Ah locus. Wild type mice (Ahb/b, "b/b") were treated once with 0, 50, 100, 200, 300, and 400 micrograms TCDD/kg po while congenic mice (Ahd/d, "d/d") received a single dose of 0, 400, 800, 1600, 2400, and 3200 micrograms TCDD/kg. Mice were checked daily, weighed twice a week, and those that survived, killed 35 days post-treatment. The LD50 values were 159 and 3351 micrograms/kg for b/b and d/d mice, respectively. Mean time to death was 22 days and was independent of dose and genotype. Decrease in body weight gain was noted in both strains 5 days after treatment and occurred at doses greater than or equal to 100 micrograms/kg in b/b mice and 1600 micrograms/kg in d/d mice. Dose-related increases in liver weight (both absolute and relative to body weight) and decreases in thymus, spleen, testes, and epididymal fat pad weights were observed at 8-24-fold higher doses in d/d than in b/b mice. A dose-related increase in segmented neutrophils was observed in both strains. Serum chemistry values indicated that 8-24X greater doses of TCDD were needed to elevate sorbitol dehydrogenase, alanine aminotransferase, and 5'-nucleotidase and to decrease total and esterified cholesterol in d/d than in b/b mice. Few effects were seen on total bile acids, serum triglycerides, glucose, or nonesterified cholesterol. In the liver, hepatocellular cytomegaly, fatty change, and bile duct hyperplasia occurred in both strains in a dose-related manner, as did thymic and splenic atrophy. Necrosis of germinal epithelium in the testes and edema in the stomach submucosa occurred at acutely toxic doses. These lesions also occurred at doses 8-24X greater in d/d than in b/b mice. Thus, the spectrum of toxicity is independent of the allele at the Ah locus, but the relative dose needed to bring about various acute responses is approximately 8-24X greater in congenic mice homozygous for the "d" allele than for the wild type animals carrying two copies of the "b" gene.

Hepatocytes in the mouse stomach.

Hepatocytes occurred in the stomach as incidental findings in 4 110-112-week-old mice (3 B6C3F1 and 1 Crl:COBS-CD1) sacrificed at termination of 2-yr toxicity/carcinogenicity bioassays of unrelated chemicals. Both sexes, and control and treated animals, were affected. Grossly, 2 mice only had 1.0-5.0 mm, smooth, cream-colored nodules protruding from the glandular stomach mucosa. Histologically, the glandular stomach submucosa and lamina propria adjacent to the limiting ridge, and in one case, the forestomach submucosa had circumscribed accumulations of well-differentiated hepatocytes with abundant eosinophilic cytoplasm and round central nuclei. Adjacent gastric glands sometimes exhibited dilation, epithelial hyperplasia, mineralization and/or microherniation into the submucosa. Ultrastructurally, the hepatocytes were polygonal cells with abundant mitochondria and rough endoplasmic reticulum; intercellular bile canaliculus-like structures exhibiting intraluminal microvilli and bounded by desmosomes were also present. No evidence of hepatocellular carcinoma or primary gastric neoplasms was found. No definitive conclusions concerning cell of origin or pathogenesis of these hepatocytes could be made, but hypotheses include congenital anomaly or post-natal transdifferentiation (metaplasia).

Pancreatic hepatocytes associated with chronic 2,6-dichloro-p-phenylenediamine administration in Fischer 344 rats.

Pancreatic tissue (original and recut sections) from Fischer 344 rats fed 2,6-dichloro-p-phenylenediamine in a chronic (2-year) carcinogenesis bioassay was evaluated for presence of pancreatic hepatocytes (PH) by light microscopy. PH were found in dose groups as follows: males--0 ppm (controls)--0/50 (0%), 1,000 ppm--4/50 (8%), 2,000 ppm--9/50 (18%); females--0 ppm (controls)--1/50 (2%), 2,000 ppm-15/50 (30%), 6,000 ppm--15/49 (31%). This represented a significant dose-related increased incidence of PH in 2,000-ppm males, and 2,000- and 6,000-ppm females. A statistically significant increase (p less than 0.01) in pancreatic acinar atrophy and fibrosis was also seen in treated female rats, but the relationship of these lesions to the PH is unclear.

Egg yolk serositis in an American alligator (Alligator mississippiensis).

An adult female American alligator (Alligator mississippiensis) had diffuse, yellow, granular serosal thickening at necropsy. Light microscopic examination of affected stomach, small intestine and spleen revealed a chronic proliferative serositis associated with 3 to 15 micron eosinophilic extracellular globules identified histochemically and morphologically as egg yolk. The intracoelomic egg yolk was considered to be the cause of the serosal reaction.

Role of coagulopathy in newborn intracranial hemorrhage.

Fifty newborn infants of less than 33 weeks' gestation were followed prospectively from birth with serial coagulation and real-time ultrasound studies. A significant association of hypocoagulability in the first four hours of life with subsequent onset or progression of intraventricular or other clinical hemorrhages was documented. Abnormalities included lower values for fibrinogen, platelet count, antithrombin III, and factor VIII with higher values for fibrin monomer and longer Laidlaw whole blood clotting times. These abnormalities tended to correct spontaneously in surviving infants. An association between gestational complications and incidence of hypocoagulability and intracranial hemorrhage (ICH) was noted. Babies of preeclamptic mothers had fewer abnormalities and babies born to mothers with premature rupture of membranes and suspected amnionitis manifested more hypocoagulability and more severe intracranial hemorrhages.

Timing and antecedents of intracranial hemorrhage in the newborn.

Fifty newborn infants of less than 33 weeks' gestation were followed prospectively from birth to evaluate the temporal relationships of various clinical factors to the onset and progression of intracranial hemorrhage (ICH) in an inborn population given maximal support. ICH was diagnosed and followed with bedside ultrasound every eight hours. The incidence of intraventricular hemorrhage was 30% and of any ICH was 40% with onset from less than 2 hours to 8 days of age. Grades 2, 3, and 4 ICH correlated with Apgar scores of less than 5 at five minutes, vaginal delivery, longer labors, and intrapartum hemorrhage. There was a significant correlation between ICH and both blood pressure fluctuations of greater than 100% and rapid colloid infusions. Slow transfusions of packed red cells did not appear to precipitate episodes of ICH. In a setting of optimal care, ICH appears to be more related to prenatal stresses than to specific postnatal complications.

Anticoagulant therapy by continuous heparinization in newborn and older infants.

In order to evaluate the metabolism and anticoagulant effect of heparin in the newborn infant and young child, 15 babies were monitored during continuous intravenous heparinization for documented large vessel thromboses. Infants with the most significant thromboses had the highest clearance rates for heparin. Plasma heparin levels in the therapeutic range (0.3 to 0.5 U/ml) and clinical resolution of the thrombi were associated with heparin doses of 16 to 35 U/kg/hour (mean = 27 U/kg/hour). A micro whole blood clotting time was evaluated and shown to be a useful guide to heparin effect in these infants. With the exception of excessive oozing from puncture sites in one infant, no complications of heparin therapy were noted. The newborn infant appears to require a larger amount of heparin than adults in order to achieve adequate heparinization.

Biochemical and functional study of antithrombin III in newborn infants.

Antithrombin III (AT-III) was isolated by heparin affinity chromatography from adult venous and newborn term and preterm umbilical cord blood. The purified proteins were compared by SDS-PAGE, rocket immuno-electrophoresis, protein concentration by microbiuret relative to optical density at 280 nm, heparin cofactor specific activity, progressive neutralization of thrombin and factor Xa at 37 degree C and pH related antithrombin kinetics. The structural evaluations revealed a fetal AT-III of molecular weight, charge and electrophoretic migration indistinguishable from adult AT-III. The functional studies showed that, on an equimolar basis, the rates of thrombin and Xa interactions with fetal AT-III were as rapid as those with adult AT-III. The catalytic rates of various concentrations of heparin were also equal. The newborn infant, therefore, displays a quantitative but not quantitative deficiency of AT-III.

Heparin clearance in the newborn.

Twenty-five preterm newborns were given bolus infusions of sodium heparin (100 units/kg) and plasma heparin levels were assayed at 5, 20, 40, 90, and 150 min. Eight normal adults received a 75 units/kg bolus of heparin and levels were assayed at 5, 30, 60, 120, 180, and 240 min. In comparison with the adult data (mean adult plasma heparin half life (T1/2) = 63.3 min), the newborn infants demonstrated a significantly shorter plasma heparin half life (T1/2 = 35.5 min in group I, 33 to 36 wk gestation; 35.5 min in group II, 29 to 32 wk gestation; and 41.6 min in group III, 25 to 28 wk gestation), as demonstrated by a chromogenic heparin assay. The newborn groups, had a larger volume of distribution (Vd) of heparin which varied inversely with gestational age (Vd =36.6 ml/kg in the adults, 57.8 ml/kg in group I, 73.3 ml/kg in group II, and 81.0 ml/kg in group III babies. Heparin clearance (CI) was significantly greater in all newborn groups when compared with the adult (CIadult = 0.43 ml/kg/min; ClGrIII = 1.49 ml/kg/min). A one-stage clotting assay for heparin generated similar results, although infant plasma heparin levels were slightly higher by this assay. Before heparin administration, the mean antithrombin III antigen (AT-III Ag) of the babies was 26.5% of normal adult and was not furthur decreased 90 min after the heparin bolus.

CT detection and course of intracranial hemorrhage in premature infants.

Twenty neonates with a suspected intracranial hemorrhage were studied by computed tomography (CT). The exact site and extent of the hemorrhage in all infants were clearly demonstrated on serial CT scans. In intraventricular hemorrhage, a dense subependymal halo lined the ventricular system and could be recognized for up to 2 weeks. Discrete hemorrhage adjacent to the ventricular system also appeared as discrete nodules rather than as a diffuse hemorrhage. Blood in the ventricular system could be recognized up to 2 weeks when there were blood-cerebrospinal fluid levels. Hydrocephalus was a common sequela and was readily detectable before a measurable change in head size.