Influence of a calcium dependent protease inhibitor on platelet activation and secretion

Continuous proteolysis resulting in consumption of major cytoskeletal proteins may be essential for platelet activation and aggregation. In this study we evaluated the effect of a known protease inhibitor, Leupeptin, on agonist induced platelet aggregation and secretion. Platelets exposed to 10 ugs/ml of Leupeptin did not aggregate in response to the action of thrombin (0.2u/ml). However, a concentration of Leupeptin as high as 250 ugs/ml did not prevent arachidonate induced aggregation and secretion. Leupeptin (100 ugs/ml) effectively blocked thrombin (0.2 u/ml) induced elevation of cytosolic calcium, but did not affect arachidonate induced elevation of platelet intracellular calcium levels. At a concentration of 100 ug/ml, Leupeptin effectively blocked thrombin (0.5u/ml) induced clot formation of platelet poor plasma, suggesting that it can exert its effect on thrombin by preventing fibrinogen degradation. Effective K_i for the competitive inhibition of thrombin induced hydrolysis of a chromogenic substrate, S2238, by Leupeptin was 2.4 uM. Leupeptin inhibition of platelet function was reversible by washing platelets free of the polypeptide. Results of our study demonstrate that Leupeptin inhibits thrombin induced platelet activation, probably by interfering with its proteolytic activity on the platelet surface membrane. However, inhibition of platelet surface membrane associated proteases did not prevent activation of platelets by other agonists.     

Development of a high performance zinc-62/copper-62 radionuclide generator for positron emission tomography.

Clinical utilisation of positron emission tomography could be enhanced by the availability of short-lived radionuclides derived from generator systems. The zinc-62/copper-62 combination is one such system which could be used as a source for a number of copper-62 radiopharmaceuticals. We have developed and optimised a high activity (5.6 GBq, 150 mCi) zinc-62/copper-62 generator to provide 62Cu in a form that is suitable for direct labelling of pyruvaldehyde-bis-(N4-methylthiosemicarbazone)-copper(II), Cu(PTSM). The distribution coefficients of Zn(II) and Cu(II) between anion-exchange resin and various hydrochloric acid/organic solvent mixtures were measured. Based on these measurements a generator eluent of 0.3 M HCl/40% ethanol provided 62Cu in greater than 90% yield in a 3-ml volume. A very low 62Zn breakthrough of less than 3 x 10(-7)% was achieved. Copper-PTSM was successfully labelled with the no-carrier-added 62Cu eluent directly from the generator with 94% radiochemical yield.     

Influence of H-2 genes on growth of Mycobacterium tuberculosis in the lungs of chronically infected mice.

Mice infected by intraperitoneal injection of Mycobacterium tuberculosis were studied over a 23-week period. They showed progressive infection in the lung (with increasing microbial count and granuloma size) whereas viable bacillary counts remained largely stationary in the spleen and in the liver. The influence of H-2 genes on the progression of the lung infection was studied in four congenic strains of animals with B10 and three congenic strains of animals with BALB backgrounds. H-2k mice had significantly higher bacterial counts in the lung than H-2b mice on both B10 and BALB backgrounds, BALB. K (H-2k) mice were also more susceptible than BALB/c (H-2d) mice. Results with recombinant strains showed that bacillary counts and granulomatous infiltration were lower in the B10 (KbAbE-Db) compared with B10.A(3R) (KbAbEbDd) strain and in B10.A(4R) (KkAkE-Db) compared with B10.BR (KkAkEkDk) mice. This resistance to the late expansion of tuberculous infection in the lungs may be associated with the lack of an expressed I-E molecular or with the expression of the Db molecule.     

Use of abdominal ultrasonography to assess pediatric splenic trauma. Potential pitfalls in the diagnosis.

OBJECTIVE: The purpose of this study was to evaluate the accuracy of abdominal ultrasonography (US) for screening and grading pediatric splenic injury. SUMMARY BACKGROUND DATA: The use of abdominal US has increased rapidly as a method of evaluating organ damage after blunt abdominal trauma. Despite US's increasing use, little is known about its accuracy in children with splenic injury. METHODS: Children (N = 32) suffering blunt abdominal trauma who were diagnosed with splenic injury by computerized tomography (CT) scan prospectively were enlisted in this study. Degree of splenic injury was evaluated by both CT and US. The ultrasounds were evaluated by an initial reading as well as by a radiologist who was blinded as to the results of the CT. RESULTS: Twelve (38%) of the 32 splenic injuries found on CT were missed completely on the initial reading of the US. When the ultrasounds were graded in a blinded fashion, 10 (31%) of the splenic lacerations were missed and 17 (53%) were downgraded. Seven (22%) of the 32 splenic fractures were not associated with any free intraperitoneal fluid on the CT scan. CONCLUSIONS: This study has shown that US has a low level of sensitivity (62% to 78%) in detecting splenic injury and downgrades the degree of injury in the majority of cases. Reliance on free intraperitoneal fluid may be inaccurate because not all patients with splenic injury have free intra-abdominal fluid. Based on these findings, US may be of limited use in the initial assessment, management, and follow-up of pediatric splenic trauma.     

Genetic analysis of the interleukin‐1 receptor antagonist and its homologue IL‐1L1 in alopecia areata: strong severity association and possible gene interaction‡

Alopecia areata is an inflammatory hair loss disease with a major genetic component. The presence of focal inflammatory lesions with perifollicular T‐cell infiltrates reflects the importance of local cytokine production in the pathogenesis. In addition to its fundamental pro‐inflammatory role, the interleukin‐1 (IL‐1) system has major effects on hair growth regulation in vitro, with the inhibitory actions of IL‐1α and IL‐1β being opposed by the receptor antagonist IL‐1ra. The novel interleukin‐1 like molecule 1 (IL‐1L1) which has greatest gene sequence homology with IL1RN, the gene encoding IL‐1ra, is another potential IL‐1 antagonist. In view of previous studies suggesting a significant role for IL1RN polymorphisms in the pathogenesis of autoimmune/inflammatory disease, we have analysed polymorphisms of IL‐1ra (IL1RN+2018) and its homologue IL‐1L1 (IL1L1+4734) in a case–control association study on 165 patients and a large number of matched controls. Homozygosity for the rare allele of IL1RN (IL1RN*2) was significantly associated with alopecia areata [odds ratio (OR) = 1.89, 95% CI (1.09, 3.28); P = 0.02], confirming our previous findings of significant association with the IL1RN variable number tandem repeat (VNTR). The results also revealed a novel association involving a polymorphism of the interleukin‐1 receptor antagonist homologue IL1L1 at position + 4734, IL1RN+2018, and alopecia areata. The effect of a genotype combining three copies of the rare alleles at the IL1RN and IL1L1 loci conferred a more than additive increase in the risk of disease compared to IL1RN+2018 or IL1L1+4734 alone [OR 3.37 (1.60, 7.06); P = 0.002], suggesting possible synergy between the IL1RN and IL1L1 genes. This effect was stronger in patients with severe disease (alopecia totalis/universalis) [OR 4.62 (1.87, 11.40), P = 0.0022], and in those with early age at onset (< 20 years) [OR = 6.38 (2.64, 15.42), P = 0.0002]. Our results suggest that these polymorphisms within IL1RN and IL1L1 themselves or a gene in linkage disequilibrium with IL1RN and IL1L1 predispose to the more severe forms of alopecia areata.     

Ureteric jets: evaluation of normal flow dynamics with color Doppler sonography.

This study was designed to investigate a variety of sonographic features of ureteric jets in order to define patterns of flow and ranges of flow values in an asymptomatic population. The following features of ureteric jets were measured during a period of up to 30 min in a group of 15 asymptomatic volunteers after oral hydration (the mean value was calculated on each side): peak velocity (mean, 57 cm/sec); jet duration (mean, 4.6 sec); and number of peaks and subpeaks (mean, 2.2). Several flow patterns were observed, including discrete jets, ureteric streaming, and rest periods. For each patient the ratios of values obtained on the left and right sides were calculated for peak velocity (1.00-1.74; mean, 1.26); jet duration (1.00-4.69; mean, 1.83); and jet frequency (1.00-1.21; mean, 1.11). The interjet interval (period between jets) ranged from 2 to 150 sec. Bolus volume and jet frequency showed simultaneous moment-to-moment variation. The frequency and velocity rather than the duration ratios may be of greatest value in identifying patients with normal ureterodynamics. Our findings challenge two current concepts of renal pelvic and ureteral response to changes in urine output: (1) ureters have a fixed maximal discharge rate and (2) bolus volumes do not change until this rate is achieved. Asymmetric moment-to-moment fluctuations observed in jet frequency suggest that prolonged examination may be necessary to confirm normal symmetry of jet frequency in some patients with suspected ureteric obstruction.     

Calcium phosphate in catheter encrustation

Encrusted catheters from nine female patients were the source of samples of deposits which were examined by X-ray diffraction, atomic absorption spectroscopy, infra-red spectroscopy and extended X-ray absorption fine structure (EXAFS) spectroscopy. In eight samples the only crystalline phase which could be clearly distinguished by X-ray diffraction was ammonium magnesium orthophosphate hexahydrate, NH4MgPO4 X 6H2O, which occurs naturally as the mineral struvite. However, atomic absorption spectroscopy revealed an appreciable concentration of calcium in all samples. Calcium phosphates have previously been detected in catheter deposits. Infra-red and EXAFS spectra were consistent with the calcium phosphate being present as a poorly crystalline hydroxyapatite. Thus the deposits appear to consist of a mixture of crystalline struvite and a form of hydroxyapatite which is not fully crystalline.     

Hemodynamic consequences of right ventricular isolation: the contribution of the right ventricular free wall to cardiac performance

Surgical isolation of the right ventricular free wall was performed in 10 dogs to evaluate both the hemodynamic effects of the procedure and the postoperative contribution of right ventricular free wall contraction to overall cardiac performance. Following the procedure, there was no significant differences in peak right ventricular systolic pressure, right atrial pressure, right ventricular stroke volume, or cardiac index. Cardiac index remained at preoperative levels over a wide range of filling pressures. However, there was a significant decrease in right ventricular stroke work (6.0 +/- 1.3 gm-m/m2 to 5.1 +/- 0.5 gm-m/m2; p less than 0.05). Pacing the isolated right ventricular free wall resulted in marked hemodynamic improvement compared with an electrically silent right ventricular free wall. Cardiac index increased from 1.7 +/- 0.2 L/min/m2 to 2.6 +/- 0.2 L/min/m2 (p less than 0.0005), and right ventricular stroke work went from 3.0 +/- 0.6 gm-m/m2 to 6.4 +/- 0.9 gm-m/m2 (p less than 0.0005). Right ventricular performance was also significantly related to the timing of right ventricular free wall contraction. Thus, the right ventricular free wall played an important role in the maintenance of normal cardiac hemodynamics.