The identification and characterization of umbilical cord blood-derived human basophils

Cross-linking allergen-specific immunoglobin E on human peripheral blood basophils results in the release of histamine and other inflammatory mediators that initiate allergy and asthma. The signaling pathways leading from IgE binding to mediator release have not been well established, mainly due to the difficulty in obtaining adequate numbers of highly purified basophils. It was the goal of this study to easily obtain Fc epsilonRI-positive human basophils in high yield and purity for studies of signal transduction pathways. We describe an in vitro culture system in which pulsing normal human cord blood leukocytes with interleukin-3 (IL-3) for 3-4 h followed by incubation in medium with fetal bovine serum generates a cell population that is predominately Fc epsilonRI positive between 14 and 28 days of culture. These cells resemble peripheral blood basophils when examined by light and electron microscopy. Like normal blood basophils, they express the integrins, CD11b, CD18, CD29, and CD49d. A majority of the IL-3-pulsed cells also express a marker recognized by the basophil-specific antibody, 2D7. Fc epsilonRI cross-linking results in a time and dose-dependent release of histamine. Fc epsilonRI cross-linking also stimulates protein-tyrosine phosphorylation, thought to be the first event leading to the IgE-mediated activation of peripheral blood basophils. These studies establish cord blood as an accessible source from which basophil-like cells can be developed to examine Fc epsilonRI-mediated signal transduction.     

Effects of hydroxyl radicals on purified angiotensin I converting enzyme

Somatic angiotensin-converting enzyme (ACE) is a protein which contains two similar domains (N and C), each possessing a functional active site. The relationship between ACE, its natural substrates and oxygen free radicals is starting to be explored. On one hand, superoxide anions production is induced by angiotensin II and on the other hand, activated polynuclear neutrophils, through free radicals generation, alter endothelial ACE activity. In this study, we examined the impact of hydroxyl radicals (.OH) on purified ACE. .OH were produced using a generator: 2,2'-azo-bis 2-amidinopropane (GRH) provided by Lara-Spiral (Fr). GRH (3 mM), in a time-dependent fashion, inhibited ACE activity. When ACE was co-incubated for 4 h with GRH, its activity decreased by 70%. Addition of dimethylthiourea (DMTU: 0.03 to 1 mM) or mannitol + methionine (20/10 mM), two sets of .OH scavengers, produced a dose-dependent protection on ACE activity. To examine whether oxidation of thiol groups in the ACE molecule could be involved in the action of GRH, the effects of thiol reducing agents: mercaptoethanol and dithiotreitol (DTT) were investigated. These compounds produced a dose-dependent and significant protection; with 100% protection at 0.2 and 0.3 mM for mercaptoethanol and at 0.1 mM for DTT. The hydrolysis of two natural and domain-specific substrates were also explored. The hydrolysis of angiotensin I preferentially cleaved by the C domain was significantly (p < 0.01) inhibited by 57, 58 and 69% in contact with 0.3, 1 and 3 mM GRH [in nmol angio II formed/min/nmol of ACE, n = 4; 35.9 +/- 0.6 (control), 15.5 +/- 2.8 (GRH : 0.3 mM), 15.1 +/- 0.5 (1), 10.9 +/- 0.6 (3)]. The hydrolysis of the hemoregulatory peptide (hp), preferential substrate for the N domain was not affected by GRH at 0.3 mM and inhibited by 28% (not significant) by 1 mM GRH [in nmol ph hydrolized/min/nmol ACE, n = 4; 12.6 +/- 1.9 (control), 14.9 (GRH : 0.3 mM), 8.3 +/- 4.0 (1). These results demonstrated that .OH affect ACE activity and could suggest a privileged impact of GRH on the C domain. The precise sites of action of .OH remain unknown. The Cys residues near the active centers, by forming disulphide bridges during the oxidation could be of critical importance. Further studies will be needed to determine whether oxidative stress again ACE can be involved in the genesis of inflammatory vascular pathologies.     

Mortality in multiple trauma patients with fractures

A multicentered study was performed to determine the mortality rate of patients with multiple injuries with major pelvic and long bone fractures who have early total care of their injuries. A 2-year review of patients with ISSs > or = 18 with major fractures treated at the trauma centers in Buffalo, New York, Camden, New Jersey, Nashville, Tennessee, Baltimore, Maryland, Tampa, Florida, and Seattle, Washington was performed. This group of 676 patients was compared with a similar group of 906 patients from the American College of Surgeons' Multiple Trauma Outcome Study. Mortality was significantly reduced in the patients who had early total care of all their injuries including fracture stabilization for patients less than 50 years of age and those 50 years and older. In a subgroup of patients less than 50 years of age and an ISS of 18-34 and 35-45 there was a mortality reduction from 11.8% to 5.1% and from 25.8% to 11.5%, respectively, when the fractures were managed acutely. Similar reductions in mortality were found in the patients 50 years of age and older with early fracture stabilization with a reduction from 26.4% to 8% in patients with ISSs of 18-24 and a reduction from 42.3% to 18.4% in the patients with ISSs of 35-45. This study clearly shows the additional benefit of early fracture stabilization in reducing mortality rates in the patient with multiple injuries.     

Brain tumors in children under 2 years: treatment, survival and long-term prognosis

Survival for children with brain tumors less than 2 years of age at diagnosis is dismal, and the quality of life of long-term survivors poor. Between 1975 and 1987, 78 (13%) of 579 patients with brain tumors treated at Children's Hospital of Philadelphia were under 2 years of age. Tumor site was posterior fossa in 31 (40%) and supratentorial in 47 (60%). Nine of 37 patients (24%) with malignant tumors, and 30 of 41 (73%) patients with benign tumors are alive with a mean follow-up of 116 months. Long-term survival after treatment with chemotherapy alone occurred in 10 patients, including 3 with malignant tumors. In 5 additional patients, chemotherapy delayed the need for irradiation a mean of 30 months. Of the 29 patients who relapsed after initial therapy, 12 are alive without progressive disease (2 patients with malignant tumors and 10 with benign tumors) a mean of 80 months after relapse, 2 children are alive with progressive disease, and 14 died a median of 48 months (range 9-115 months) after relapse. Twenty-one of the 39 survivors have minimal or no neurological or intellectual dysfunction. Surviving patients treated with surgery and chemotherapy have better intellectual function than patients treated with surgery and radiation (with or without chemotherapy) in that 8 of 10 children treated with surgery and chemotherapy have normal or above normal intelligence compared with 5 of 12 children receiving irradiation before their second birthday.     

Bioenergetics of contracting skeletal muscle after partial reduction of blood flow

The purpose of this study was to examine the bioenergetics and regulation of O2 uptake (VO2) and force production in contracting muscle when blood flow was moderately reduced during a steady-state contractile period. Canine gastrocnemius muscle (n = 5) was isolated, and 3-min stimulation periods of isometric, tetanic contractions were elicited sequentially at rates of 0.25, 0.33, and 0.5 contractions/s (Hz) immediately followed by a reduction of blood flow [ischemic (I) condition] to 46 +/- 3% of the value obtained at 0.5 Hz with normal blood flow. The VO2 of the contracting muscle was significantly (P < 0.05) reduced during the I condition [6.5 +/- 0.8 (SE) ml . 100 g-1 . min-1] compared with the same stimulation frequency with normal flow (11.2 +/- 1.5 ml . 100 g-1 . min-1), as was the tension-time index (79 +/- 12 vs. 123 +/- 22 N . g-1 . min-1, respectively). The ratio of VO2 to tension-time index remained constant throughout all contraction periods. Muscle phosphocreatine concentration, ATP concentration, and lactate efflux were not significantly different during the I condition compared with the 0. 5-Hz condition with normal blood flow. However, at comparable rates of VO2 and tension-time index, muscle phosphocreatine concentration and ATP concentration were significantly less during the I condition compared with normal-flow conditions. These results demonstrate that, in this highly oxidative muscle, the normal balance of O2 supply to force output was maintained during moderate ischemia by downregulation of force production. In addition, 1) the minimal disruption in intracellular homeostasis after the initiation of ischemia was likely a result of steady-state metabolic conditions having already been activated, and 2) the difference in intracellular conditions at comparable rates of VO2 and tension-time index between the normal flow and I condition may have been due to altered intracellular O2 tension.