Membrane expression of platelet calpain

Platelet calpain has many platelet substrates, including external membrane proteins. We thus investigated whether platelet calpain II was associated with platelet membranes in unstimulated and thrombin- activated platelets. A monospecific, goat polyclonal antibody was reared to purified platelet calpain II. Sixteen whole platelet lysates were found to contain 4.5 +/- 0.7 micrograms calpain antigen II per 10(8) platelets (mean +/- SEM) as determined by a competitive enzyme- linked immunosorbent assay. Using the dipeptide fluorogenic substrate, Suc-Leu-Tyr-MCA, 17 human platelet lysates contained 3.6 +/- 0.4 micrograms calpain activity per 10(8) platelets. Platelet calpain II was associated with the Triton X-100 insoluble platelet cytoskeletons from both unstimulated and thrombin-activated platelets. When compared with the total cell content of platelet calpain II, calpain antigen (10% to 13%) and calpain activity (24% to 28%) was associated with platelet cytoskeletons in unstimulated and thrombin-activated platelets, respectively. On immunoblot, the heavy chain (80 Kd) of calpain II was detected in platelet cytoskeletons. Subcellular fractionation studies on both unstimulated and thrombin-activated platelets, revealed that half of the total platelet calpain II antigen was associated with cytosol, and the other half was associated with the membrane fraction. Platelet calpain II was not seen on the surface of unstimulated, paraformaldehyde fixed platelets by immunofluorescence. However, on thrombin-activated platelets, rim immunofluorescence was seen, indicating that activated platelets externalize their calpain. This observation was confirmed by the finding that about 2,000 molecules per platelet of an 125I-anti-calpain II Fab' specifically bound to thrombin-activated but not unstimulated platelets. Both dibucaine (1 mmol/L) and platelet activating factor (1.86 mumol/L) in the absence of external Ca++, but not collagen (5 micrograms/mL) or ionophore A23187 (2.5 mumol/L) in the absence of external Ca++, were also able to externalize platelet calpain II antigen, as indicated by a similar level of specific 125I-anti-calpain II Fab'-platelet binding. These combined studies indicate that platelet calpain II is a major protein, comprising 2% of total platelet protein, a substantial portion of which is membrane-associated. When platelets are activated by thrombin and platelet activating factor, calpain II antigen also becomes present on the external platelet surface.     

Proinflammatory mediator-induced reversal of CD4+,CD25+ regulatory T cell-mediated suppression in rheumatoid arthritis

OBJECTIVE: We previously demonstrated that CD4+,CD25+ regulatory T (Treg) cells are present in increased numbers in the synovial fluid (SF) of rheumatoid arthritis (RA) patients and display enhanced suppressive activity as compared with their peripheral blood (PB) counterparts. Despite the presence of these immunoregulatory cells in RA, chronic inflammation persists. The purpose of the present study was to investigate whether particular proinflammatory mediators that are associated with RA could abrogate CD4+,CD25+ Treg-mediated suppression. METHODS: Monocyte phenotype was determined by flow cytometry and cytokine levels by enzyme-linked immunosorbent assay. Magnetically sorted CD4+,CD25- and CD4+,CD25+ T cells derived from the PB and SF obtained from RA patients were stimulated alone or in coculture with anti-CD3 monoclonal antibody (mAb) and autologous antigen-presenting cells, in the absence or presence of anti-CD28 mAb or the proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), or IL-7. RESULTS: Monocytes from the SF of RA patients displayed increased expression of HLA class II molecules, CD80, CD86, and CD40 as compared with PB-derived monocytes, indicating their activated status. Mimicking this increased costimulatory potential, addition of anti-CD28 mAb to cocultures of CD4+,CD25- and CD4+,CD25+ T cells resulted in reduced CD4+,CD25+ Treg-mediated suppression in both PB and SF. Furthermore, IL-7 and, to a limited extent, TNFalpha, both of which are produced by activated monocytes and were detected in SF, abrogated the CD4+,CD25+ Treg-mediated suppression. In contrast, IL-6 did not influence Treg-mediated suppression. CONCLUSION: Our findings suggest that the interaction of CD4+,CD25+ Treg cells with activated monocytes in the joint might lead to diminished suppressive activity of CD4+,CD25+ Treg cells in vivo, thus contributing to the chronic inflammation in RA.     

Glucose production and oxidation in preterm infants during total parenteral nutrition

During total parenteral nutrition in preterm infants, glucose may be infused at high rates, but it is not known if the endogenous glucose production is fully suppressed under these circumstances. Eight preterm appropriate for gestational age (AGA) (birth wt: 1613 +/- 151 g, gestational age: 31.1 +/- 1.5 wk) and eight preterm small for gestational age (SGA) newborn infants (1185 +/- 241 g, 32.9 +/- 2.6 wk) receiving a glucose infusion rate of 7.55 +/- 0.56 and 8.16 +/- 0.65 mg/kg.min, respectively, were studied during continuous total parenteral nutrition at postnatal d 8. Glucose oxidation rate was determined with a primed constant infusion of [U-13C] glucose, measuring the 13CO2 production in breath gas by isotope ratio mass spectrometry and the glucose production rate in plasma by gas chromatography mass spectrometry. In breath gas of AGA and SGA infants, 60 and 65%, respectively, of the infused tracer appeared as 13CO2. The glucose production rates were 7.97 +/- 1.61 and 8.12 +/- 1.84 mg/kg.min in AGA and SGA infants, respectively, indicating that no significant endogenous glucose production occurred. The glucose oxidation calculated from the glucose production and 13CO2 production was 4.74 +/- 0.99 mg/kg.min in AGA infants and was significantly different from the carbohydrate oxidation rate of 6.62 +/- 1.23 mg/kg.min measured by simultaneous indirect calorimetry. In SGA infants, however, the glucose and carbohydrate oxidation rates were not significantly different at 5.33 +/- 1.56 and 6.16 +/- 2.45 mg/kg.min. It is concluded that 1-wk-old AGA or SGA preterm infants receiving total parenteral nutrition of 80 kcal/kg.d produce no endogenous glucose and their glucose oxidation rates are similar at 63-65% of the glucose infused. It is suggested that the significant difference between glucose and carbohydrate oxidation rates observed in AGA but not in SGA infants is due either to a higher rate of lipogenesis from carbohydrates, or, less likely, to a higher rate of glycogen oxidation.     

Immature articular cartilage is more susceptible to blood-induced damage than mature articular cartilage: an in vivo animal study

OBJECTIVE: Cartilage of young but skeletally mature dogs is more susceptible to blood-induced damage than that of old dogs. The aim of the present study was to investigate whether cartilage of skeletally immature individuals is even more adversely affected by exposure to blood than that of mature individuals, as suggested by clinical practice experience with humans. METHODS: Right knees of 3 groups of 6 beagle dogs (skeletally immature, young mature, and old animals) were injected with autologous blood on days 0 and 2. On day 4, cartilage matrix proteoglycan turnover (content, synthesis, and release), synovial inflammation, and cartilage-destructive properties of the synovial tissue were determined and compared with those of the left uninjected control knees. RESULTS: Subsequent to intraarticular bleeding, cartilage proteoglycan content decreased in an age-dependent manner, with the largest decrease occurring in cartilage of immature animals. Proteoglycan synthesis per cell also decreased in an age-dependent manner, with the largest decrease occurring in the immature animals. Cartilage proteoglycan release increased in all 3 groups, but the decrease was not age dependent. Interestingly, immature animals showed a large increase in cartilage DNA content upon exposure to blood, whereas mature animals did not. Histologic analysis showed a mild synovitis in animals of all ages, but catabolic inflammatory activity was found only in immature animals. CONCLUSION: Joints of skeletally immature dogs appeared to be more susceptible than joints of mature dogs to the adverse effects of a joint hemorrhage. These data suggest that for humans, specifically young children are at risk for joint damage after a joint hemorrhage.     

Predictive value of EEG in neonates with periventricular leukomalacia

The aim of this study was to evaluate whether EEG (i.e. positive Rolandic sharp waves) can be used to predict neurodevelopment in newborn infants with periventricular leukomalacia and compare the predictive value with that of MRI. A sequential cohort of neonates (n=45; 33 males, 12 females; mean gestational age 31.2 weeks, SD 2.7, range 27 to 37.8 weeks; mean birthweight 1592 g, SD 601 g) with periventricular hyperechogenicities on cranial ultrasound was recruited for this study. EEGs were analyzed for positive Rolandic sharp waves. Neurodevelopment was evaluated at the ages of 12 and 18 months. In the whole group the probability of a poor outcome was 24% and the probability of any impairment was 33%. If the number of positive Rolandic sharp waves was no more than 0.1 per minute, the probability of a poor outcome was reduced to 9% (95% confidence interval [95%CI] 2 to 27%) and the probability of any impairment was reduced to 13% (95%CI 4 to 32%). In all infants with more than 0.1 positive Rolandic sharp waves per minute the probability of a poor outcome was 41% (95%CI 23 to 61%) and of any impairment was 55% (95%CI 34 to 73%). In these infants MRI identified infants with a poor outcome with a sensitivity of 1.00 (95%CI 0.70 to 1.00) and a specificity of 0.92 (95%CI 0.67 to 0.99), and infants with any impairment with a sensitivity of 0.83 (95%CI 0.55 to 0.95) and a specificity of 1.00 (95%CI 0.72 to 1.00). Results suggest that if an EEG of an infant with periventricular leukomalacia contains no more than 0.1 positive Rolandic sharp waves per minute the probability of a normal or mildly delayed development is high (0.91, 95%CI 0.73 to 0.98). MRI enhances the accuracy of the outcome prediction slightly; however, owing to a wide confidence interval, this advantage is negligible. However, if the frequency of the positive Rolandic sharp waves exceeds 0.1per minute, MRI can significantly enhance the precision of the prediction of outcome.     

Neuromotor function and school performance in 7-year-old children born as high-risk preterm infants

Neuromotor behavior was studied in 63 children at a mean age of 7 years. They were born at a gestational age less than 32 weeks and/or birthweight under 1500 g and were categorized according to their medical history in conformance with the Neonatal Medical Index (from category I to V, from few to serious complications). We included only children considered at high risk as categorized in III to V. The neuromotor behavior study focuses on different subcategories, such as hand function, quality of walking, posture, passive muscle tone, coordination, and diadochokinesia. Hand preference and/or lateralization, the presence of associated movements, and/or asymmetry were noted, as was school performance. Then gender, gestational age, birthweight, and dysmaturity were investigated as confounding factors. The outcome at 7 years was correlated with the Neonatal Medical Index and the neonatal brain ultrasonography classification. None of the children scored 100% on the combined subcategories. Nineteen children (30%) had an overall score between 75 and 99%. Significant relationships between all different subcategories were found. Lack of hand preference, poor lateralization, and male gender were related to poor overall outcome. Poor motor control was correlated to special schooling and education below age level. The Neonatal Medical Index proved to have a significant influence on total outcome and the subcategories at the age of 7 years, with the worst outcome in children formerly classified in category V. Neuromotor behavior at 7 years of age was not related to birthweight, gestational age, dysmaturity, and neonatal brain ultrasonography classification only.     

Joint distraction as an alternative for the treatment of osteoarthritis

Irrespective of underlying mechanisms, the long-term efficacy of joint distraction in the treatment of severe ankle osteoarthritis at young age validates the concept of joint distraction in the treatment of osteoarthritis. Therefore, joint distraction in the case of severe ankle osteoarthritis may be a treatment of choice. This opens the possibility to study joint distraction as a treatment for other joints. Because knee osteoarthritis is much more common, it is a much greater social and economic problem. Beneficial effects of joint distraction in the case of ankle osteoarthritis, and specifically in the treatment of more common forms of osteoarthritis such as severe knee and hip osteoarthritis, may therefore have a great impact, especially in view of the increasing age of our population.