The Journal of laboratory and clinical medicine最新文献

The effects of the administration of OKT3, a second immunoglobulin G2a (IgG2a) anti-CD3 monoclonal antibody (mAb), and its isotype switch variant IgA on granulocyte kinetics were compared for 5 hours after the first administration of the mAb. In addition, in vivo and in vitro studies were performed on alterations in expression of CD11b and CD62L induced by these mAbs. Within 15 minutes after administration OKT3 and IgG2a anti-CD3 mAbs induced a significant decrease in circulating granulocytes, whereas IgA anti-CD3 mAb did not. Apparently the initial decrease in circulating granulocytes depends on the heavy chain of the administered anti-CD3 mAb, resulting in immunocytoadherence and sequestration in the lungs. Increased adherence to pulmonary endothelium by altered expression of CD11b and CD62L plays a minor role in this first granulocytopenia, because each mAb exerted the same effects on these adhesion molecules in vitro. The second decrease in granulocyte counts occurred 60 minutes after administration of each mAb and correlated with a significant increase in expression of CD11b and CD62L in vivo and with upregulation of CD11b and down-regulation of CD62L in vitro. These alterations could be related to the presence of tumor necrosis factor-alpha both in vivo and in vitro. Thus granulocyte kinetics from 30 minutes after administration of each anti-CD3 mAb resemble neutrophil kinetics induced by TNF-alpha.

Leukoclastic vasculitis is thought to be initiated by deposition of immune complexes (ICs) in the vascular wall. To study the neutrophil response in a related in vitro model, we primed human umbilical vein endothelial cell (HUVEC) monolayers with antibodies against human fibronectin. The resulting respiratory burst to the immobilized ICs depended on the antibody concentration used to prime the monolayers and included a marked release of primary and secondary granule constituents. On IC-bearing HUVEC monolayers, but not on ICs directly bound to tissue culture dishes, blocking monoclonal antibodies (mAbs) to crystallizable fragment-gamma receptor II (Fc gamma RII) and Fc gamma RIII markedly inhibited the respiratory burst and the release of elastase. However, on both surfaces the neutrophil response was strongly inhibited by mAbs against CD18. Regardless of whether we used neutrophils from a patient with severe paroxysmal nocturnal hemoglobinuria (PNH) lacking the Fc gamma RIII, or whether the Fc gamma RII-mediated signal transduction was blocked by pertussis toxin, the respiratory burst to the IC-bearing HUVECs was essentially unchanged. With PNH neutrophils, the respiratory burst was predominantly blocked by an anti-Fc gamma RII mAb. In contrast, the response of pertussis toxin treated neutrophils was strongly inhibited by a mAb against Fc gamma RIII. Together these data indicate that the answer of neutrophils to ICs immobilized at the endothelial barrier depends on the cooperative function of both low-affinity Fc gamma Rs.

P-selectin is a granule membrane protein that is expressed on the surface of activated endothelial cells and platelets. Flow cytometry has been used as a means of detecting activated platelets in the circulation by using antibodies to P-selectin and other surface markers. In the study reported here, we developed a whole platelet ELISA for measuring P-selectin on platelets in platelet-rich plasma. Platelet-rich plasma samples for analysis were isolated from fresh blood by centrifugation, fixed with 1.0% paraformaldehyde, and used within 3 hours or after storage at -70 degrees C for up to 10 months. Paraformaldehyde-fixed, phorbol myristate acetate-activated or thrombin receptor peptide-activated platelets were used to construct a standard calibration curve. These platelets were stable after 10 months of storage at -70 degrees C. Interassay variability showed a high degree of correlation, with r = 0.98 +/- 0.03 (n = 12). The accuracy and specificity of the ELISA was verified by using fluorescence-activated flow cytometric analysis and is as sensitive (< or = 0.5%) as flow cytometry for detecting P-selectin expression on platelets. To assess the ability of the platelet ELISA to detect platelet activation in the systemic circulation, we examined 24 patients with unstable angina and 12 age-matched control subjects. Patients with unstable angina demonstrated significantly higher levels of circulating activated platelets than did age-matched control subjects. Although storage-dependent differences in absolute platelet activation levels were found, platelet ELISA results of samples evaluated within either 3 hours or after 10 months of storage were comparable to results obtained by fluorescence-activated flow cytometric analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

The aims of this study were to determine whether chronic ethanol consumption potentiates mitochondrial lipid peroxidation or impairment of mitochondrial oxidative metabolism in rats with chronic iron overload. Experimental iron overload was induced by feeding rats a chow diet supplemented with 2.5% carbonyl iron. After 8 to 12 weeks, half of the iron-loaded and control animals were changed to a liquid diet containing ethanol for 4 to 5 weeks. The remaining animals were fed an isocaloric amount of diet containing dextrin-maltose instead of ethanol for 4 to 5 weeks. Iron-supplemented animals had a 20-fold increase in hepatic iron concentration as compared with controls. Iron and ethanol independently increased plasma alanine aminotransferase (ALT) levels (p < 0.05) while the combination resulted in an additive increase in ALT levels (p < 0.01). Although iron overload increased the levels of mitochondrial conjugated dienes and significantly reduced the mitochondrial respiratory control ratio, ethanol administration did not affect these parameters in animals with or without iron overload. Livers from iron-loaded rats that received ethanol showed mild to moderate steatosis with scattered necroinflammatory foci. There was no significant increase in necroinflammatory foci in the livers of the iron plus ethanol group as compared with the iron group. In conclusion, we have demonstrated an additive increase in hepatocellular injury when ethanol is fed to iron-loaded rats, as evidenced by an increase in plasma ALT level. However, there were no additive or synergistic effects of iron and ethanol on either mitochondrial lipid peroxidation or mitochondrial oxidative metabolism.

The objective of this study was to assess the relationship between iron metabolism and pharmacokinetics of hemoglobin-based oxygen carrier-201 (HBOC-201), a polymerized hemoglobin product of bovine origin. A randomized, single-blind, single-dose study design was used. The study was performed at the Upjohn Research Clinics in Kalamazoo, Michigan. Four groups of healthy men and women (n = 24), who either received HBOC-201 (9 men, 9 women) or a control solution (Ringer's lactate) (3 men, 3 women) participated in the study. All subjects had phlebotomy (approximately 15% blood volume) followed by 3:1 hemodilution with Ringer's lactate and an intravenous infusion of HBOC-201 (up to 45 gm or 350 ml) or control solution (Ringer's lactate). Serial arterial blood gas samples with a radial artery catheter and simultaneous pulse oximetry were done during the first 24 hours. Serial samples for serum iron, ferritin, erythropoietin, and plasma HBOC-201 levels were taken over a 1-month period. In the HBOC-201-treated groups, serum iron and ferritin levels increased. Peak serum iron and ferritin levels occurred by hours 8 (up to 220 micrograms/dl) and 48 (up to 180 ng/ml), respectively. Serum iron levels paralleled HBOC-201 concentrations. Plasma half-life of HBOC-201 was about 20 hours. Serum erythropoietin increased by twofold to sixfold over baseline (p < 0.001) at 24 hours. No urinary hemoglobin was detected in the groups with HBOC-201-treated subjects. This study demonstrates that HBOC-201 produces increases in serum iron, ferritin, and erythropoietin that closely parallel plasma levels of HBOC-201 in men and women.

Although the effects of endothelin-1 (ET-1) on intact or perfused adult kidney are well understood, its effects in the fetus and the newborn have not been well studied. We examined the effects of infusions of 25, 50, and 100 ng/kg of ET-1 per minute on mean blood pressure (MBP), cardiac index (CI), renal blood flow (RBF), glomerular filtration rate (GFR), and urine volume (UV) in 7- to 10-day-old piglets (n = 24). In addition, the effects of pretreatment with a receptor antagonist (BQ-123) and with a cyclooxygenase inhibitor (indomethacin) were studied in 12 separate piglets. ET-1 produced a dose- and level-dependent decrease in CI (60%), RBF (50% to 75%), GFR (66% to 80%) and MBP 15% to 17%. These changes returned to 75% to 80% of baseline 60 minutes after discontinuation of ET-1. Low-dose infusion (25 ng/kg) did not result in any changes in systemic or renal hemodynamics. Plasma half-life of ET-1 in piglets was 2.1 +/- 0.4 minutes. Pretreatment with the specific ETA receptor antagonist BQ-123 completely blocked the ET-1-induced systemic and renal hemodynamic changes. Indomethacin blocked the ET-1-induced rise in MBP but failed to block any renal changes. In fact, indomethacin accentuated the changes induced by ET-1, especially the changes in RBF, RVR, and GFR. Studies of receptor binding in the renal cortex and medulla showed that, in the cortex, the Ki value for ET-1 was 6.32 +/- 1.57, and for ET-3 it was 20.05 +/- 4.38 (p < 0.05); in the medulla, the Ki values were similar for both ET-1 and ET-3. These results indicate that in piglets the renal vascular bed is highly sensitive to ET-1, and its effects are predominantly mediated through ETA receptors.

Cultured CD4+ cells are responsible for transfer of adoptive murine experimental hypersensitivity pneumonitis (EHP) (ARRD 1992; 146:1582-8). To characterize interactions that occur in vitro that result in cells able to transfer EHP, we added either antibody to IFN-gamma, antibody to IL-2, or 30 or 300 micrograms/ml IFN-gamma at the onset of 72-hour culture of C3H/HeJ spleen cells from either M. faeni or ovalbumin (control) sensitized donors with 30 micrograms/ml Micropolyspora faeni. We determined the phenotype of cells after culture and the amount of IL-2 or IFN-gamma in the culture supernatants, transferred cells to naive recipients, challenged the recipients intratracheally with M. faeni, and determined the extent of pulmonary inflammatory changes 4 days thereafter. Substantial amounts of IL-2 and IFN-gamma were detected in supernatants of cultures from M. faeni-sensitized animals, and lesser amounts were detected in culture supernatants from ovalbumin-sensitized donors. Treatment of cultures of M. faeni-sensitized cells with antibody to IL-2 or IFN-gamma blocked or reduced measurable IL-2 or IFN-gamma for the duration of culture. Treatment with IFN-gamma blocked increased levels of IL-2 at 48 and 72 hours of culture. Cultured M. faeni-sensitized cells adoptively transfer EHP. Cells from cultures depleted of either IL-2 or IFN-gamma or supplemented with IFN-gamma could transfer EHP equally well. We conclude that in vitro maturation of cells capable of adoptive EHP is not dependent on soluble IL-2 or IFN-gamma and is not altered by exogenous IFN-gamma.