Sickle erythrocyte-endothelial interactions in microcirculation: the role of von Willebrand factor and implications for vasoocclusion

To determine the role of von Willebrand factor (vWF) in adhesion of sickle (SS) erythrocytes in microvascular flow conditions, we have perfused the ex vivo mesocecum vasculature of the rat with desmopressin, an analogue of vasopressin that causes the release of endothelial vWF. Analysis of vWF in the venous effluent of the isolated vasculature showed mainly the presence of extra-large molecular weight forms characteristic of endothelial vWF, which in the presence of desmopressin showed an average increase of 54%. Also, desmopressin induced a significant increase in adhesion of washed oxygenated (oxy) unseparated SS erythrocytes, accompanied by a persistent microvascular obstruction and a pronounced increase in the peripheral resistance (PRU). In contrast, infusion of SS deformable discocytes (SS2) in desmopressin-perfused vasculature resulted in a significant adhesion but not in persistent vasoocclusion, showing that SS2 discocytes alone are not sufficient for microvascular obstruction. Furthermore, SS4 erythrocytes (dense discocytes and irreversibly sickled erythrocytes) caused a persistent microvascular blockage and a significantly higher PRU than SS2 discocytes. However, the increase in PRU for SS4 erythrocytes following desmopressin treatment was 50% less compared with a corresponding increase for SS2 discocytes over the control values, which showed a smaller effect of desmopressin on the hemodynamic behavior of SS4 dense erythrocytes. Incubation of desmopressin-treated vasculature with anti-vWF antibodies resulted in a pronounced decrease in adhesion and significantly improved hemodynamic behavior of SS cells. Also, in untreated vasculature, similarly incubated with anti-vWF antibodies, there was almost complete inhibition of adhesion. Under the described perfusion conditions, antibodies to fibronectin and thrombospondin, as well as incubation of SS erythrocytes with anti-vWF antibodies did not affect adhesion. These results are compatible with a model for SS vasoocclusion in which extra- large vWF-mediated adhesion of deformable SS erythrocytes is the first step followed by an accelerated entrapment of dense SS erythrocytes.     

Lung findings on high-resolution computed tomography in idiopathic ankylosing spondylitis--correlation with clinical findings, pulmonary function testing and plain radiography

Previous studies on the association of ankylosing spondylitis and abnormalities of the lung parenchyma have been based largely on plain radiography and pulmonary function testing. This study, although uncontrolled, is the first to use high-resolution computed tomography to examine the entire lung parenchyma in ankylosing spondylitis patients, and to correlate the findings with clinical assessment, plain radiography and pulmonary function testing. The study population comprised 26 patients meeting the New York criteria for idiopathic ankylosing spondylitis who attended the out-patient department at our institution. High-resolution computed tomography examination revealed abnormalities in 19 patients (70%): these included interstitial lung disease (n = 4), bronchiectasis (n = 6), emphysema (n = 4), apical fibrosis (n = 2), mycetoma (n = 1) and non-specific interstitial lung disease (n = 12). Plain radiography was abnormal in only four patients and failed to identify any patient with interstitial lung disease. All patients with interstitial lung disease on high-resolution computed tomography had respiratory symptoms and three of the four had evidence of a restrictive process on pulmonary function testing. This study raises, for the first time, the possible association between interstitial lung disease and ankylosing spondylitis, and highlights the use of high-resolution computed tomography in detecting such disease in ankylosing spondylitis patients.      

Interleukin 1 induces human marrow stromal cells in long-term culture to produce granulocyte colony-stimulating factor and macrophage colony- stimulating factor

Pure interleukin 1 (IL 1) was found to stimulate established human bone marrow stromal layers in long-term culture to produce colony- stimulating activity (CSA). Maximal concentrations in the culture medium were reached 24 hours after a single IL 1 pulse. The effect could be neutralized by a specific rabbit anti-IL 1 antiserum. Stromal layers, once stimulated by IL 1, continued to release CSA into the culture medium in the absence of exogenous IL 1. A second IL 1 pulse induced CSA release in an identical manner, as did the primary stimulation, indicating that the CSA released was actively produced. Using specific immunologic assays, both granulocyte colony-stimulating factor (G-CSF) and macrophage CSF (M-CSF) could be identified in the culture supernatants, and production of both factors was inducible by IL 1. Shortly after initiation of the long-term marrow cultures "spontaneous" G-CSF and M-CSF release occurred. The release of G-CSF diminished following addition of the anti-IL 1 antiserum, indicating that endogenous production of IL 1 by stromal cells had contributed to this effect. These results further support the role of IL 1 as an important modulator of CSF production by cells of the hematopoietic microenvironment.     

Hepatitis C virus infection and chronic liver disease in children with leukemia in long-term remission

Antibody to the recently identified hepatitis C virus (HCV) was investigated in sera of 50 leukemic children who had chronic liver disease (CLD), observed for 1 to 12.6 years after therapy withdrawal. All patients were tested for anti-HCV at regular intervals: Ortho- enzyme-linked immunosorbent assay (ELISA) test was performed in all cases. Reactive sera were also tested by recombinant immunoblotting assay to define the specificity of the results obtained by ELISA. Twelve cases (24%) were persistently positive (group A), 11 (22%) were transiently anti-HCV+ positive (group B), and 27 (54%) were negative. Mean SGPT peak during follow-up was significantly higher in group A (P = .014, A v B and P less than .00001, A v C). SGPT normalized off- therapy in 1 of 12 cases (group A), 10 of 11 (group B), and 19 of 27 (group C) (P = .0004, A v B and P = .012, A v C). Accordingly, liver histology, available in 37 patients, showed signs of chronic hepatitis in all patients in group A while most patients in group B and C had less severe liver lesions. These results indicate that HCV plays a significant role in the etiology of chronic hepatitis in leukemic patients and that persistent anti-HCV activity correlates with a more severe CLD, which could jeopardize the final prognosis of children cured of leukemia.     

The Effect of Body Weight and Body Surface Area Correction on the Distribution of the ACT Response to Bolus Doses of Heparin for PTCA

Considerable controversy exists as to the appropriate dosing of heparin for PTCA. We retrospectively reviewed records of 335 patients undergoing PTCA to determine: 1) the effects of correcting for weight and body surface area (BSA) on the heparin dose-response distribution; and 2) the average dose of heparin (standard, weight-based, and BSA-based) required to achieve an activated clotting time (ACT) of 300 seconds. For each patient, height, weight, BSA, baseline ACT (HemoTec), bolus heparin dose, and post-heparin ACT were recorded and the heparin response calculated. There were no significant differences in the distributions of standard (SD =.017 +/- 006 sec/U, 34% of mean), weight-based (SD = 1.41 +/- 0.46 sec/U/kg, 33% of mean), and BSA-based (SD = 0.033 +/- 0.011 sec/U/m2, 32% of mean) heparin response. There were slight, but significant correlations between heparin response and weight (r = 0.37) and heparin response and BSA (r = 0.36). The estimated doses of heparin to achieve a HemoTec ACT of 300 seconds were 10,650 +/- 1270 U, 130 +/- 15 U/kg, and 5390 +/- 640 U/m2. CONCLUSIONS: There are slight but significant correlations between heparin response and both weight and BSA. The distributions of weight- and BSA-corrected heparin response are similar to that of standard heparin dosing. Thus, weight adjusted heparin dosing would not appear to be likely to provide a more reliable ACT response to bolus doses of heparin.     

The value of flow cytometric analysis of platelet glycoprotein expression of CD34+ cells measured under conditions that prevent P- selectin-mediated binding of platelets

In the present study, we show by adhesion assays and ultrastructural studies that platelets can bind to CD34+ cells from human blood and bone marrow and that this interaction interferes with the accurate detection of endogenously expressed platelet glycoproteins (GPs). The interaction between these cells was found to be reversible, dependent on divalent cations, and mediated by P-selectin. Enzymatic characterization showed the involvement of sialic acid residues, protein(s). The demonstration of mRNA for the P-selectin glycoprotein ligand 1 (PSGL-1) in the CD34+ cells by polymerase chain reaction (PCR) analysis suggests that this molecule is present in these cells. Under conditions that prevent platelet adhesion, a small but distinct subpopulation of CD34+ cells diffusely expressed the platelet GPIIb/IIIa complex. These cells were visualized by immunochemical studies. Furthermore, synthesis of mRNA for GPIIb and GPIIIa by CD34+ cells was shown using PCR analysis. The semiquantitative PCR results show relatively higher amounts of GPIIb mRNA than of PF4 mRNA in CD34+CD41+ cells in comparison with this ratio in platelets. This finding is a strong indication that the PCR results are not caused by contaminating adhering platelets. MoAbs against GPIa GPIb alpha, GPV, P- selectin, and the alpha-chain of the vitronectin receptor did not react with CD34+ cells. The number of CD34+ cells expressing GPIIb/IIIa present in peripheral blood stem cell (PBSC) transplants was determined and was correlated with platelet recovery after intensive chemotherapy in 27 patients. The number of CD34+CD41+ cells correlated significantly better with the time of platelet recovery after PBSC transplantation (r = .83, P = .04) than did the total number of CD34+ cells (r = .55). Statistical analysis produced a threshold value for rapid platelet recovery of 0.34 x 10(6) CD34+CD41+ cells/kg. This study suggests that if performed in the presence of EDTA the flow cytometric measurement of GPIIb/IIIa on CD34+ cells provides the most accurate indication of the platelet reconstitutive capacity of the PBSC transplant.     

Collagen-induced arthritis in rhesus monkeys: evaluation of markers for inflammation and joint degradation

The objective of this study was to analyse parameters in rhesus monkey collagen-induced arthritis (CIA) with which the inflammation and destruction of the joints can be described in quantitative terms. CIA was induced in genetically susceptible and resistant monkeys, which can be distinguished on the basis of the dominant resistance marker Mamu- A26. The disease course was monitored daily using a semiquantitative scoring system. Plasma samples were collected once or twice weekly and analysed for C-reactive protein (CRP). Urines were collected overnight once a week and analysed for excretion rates of the collagen cross- links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP). The results show that periods of active CIA are characterized by substantial weight loss and increased plasma CRP levels, followed shortly thereafter by increased excretion rates of the collagen cross- links HP and LP. Remission of the disease can be recognized by a decline in plasma CRP levels and especially an increase in body weight. The highest CRP levels were found in the most severely arthritic monkeys, indicating a possible relationship of the absolute plasma CRP levels to the severity of inflammation. During periods of active arthritis, increased excretion rates of collagen cross-links HP and LP in the urine were found. In particular, the major collagen cross-link in articular cartilage, HP, showed a strong increase (9- to 15-fold). The excretion rates of LP, which is considered as a bone-specific degradation marker, only increased 4- to 6-fold, thus indicating predominant destruction of cartilage and less of bone. In conclusion, the severity of CIA can be monitored in a quantitative manner using plasma CRP levels, urinary excretion rates of HP and LP, and body weights, superimposed on semiquantitative clinical scores. The parameters also facilitate a more objective assessment of the effect of anti-arthritic drugs in the model than with the clinical scores alone.      

Effect of recombinant human interleukin-6 (rhIL-6) and rhIL-3 on hematopoietic regeneration as demonstrated in a nonhuman primate chemotherapy model

Using a recently developed hepsulfam-induced pancytopenia model in rhesus macaques, we have studied the effects of recombinant human interleukin-6 (rhIL-6) and rhIL-3 on marrow regeneration. Control animals were given hepsulfam (1.5 g/m2 by a single 30-minute intravenous [i.v.] injection, n = 4), while study animals received hepsulfam followed by rhIL-6, rhIL-3, or a combination of rhIL-6 and rhIL-3 (n = 3 per study group). Each cytokine was administered by once- daily subcutaneous (SC) injection (15 micrograms/kg/d) for 3 weeks beginning the day after chemotherapy (days 2 through 22). Mean platelet counts in control animals were < 100,000/microL on days 15 through 24, with 50% of the counts < 50,000/microL and two of four animals requiring platelet transfusion. In the rhIL-6- and rhIL-6/rhIL-3- treated groups, the nadir mean platelet counts were 164,000 +/- 58,700/microL and 162,300 +/- 23,800/microL, respectively, and occurred on day 15. Platelet counts in the rhIL-3-treated group were similar to those in controls. Mean absolute neutrophil counts (ANCs) < 1,000/microL occurred on days 10 through 29 in control animals, days 8 through 15 in rhIL-6-treated animals, and days 6 through 8 and 13 in rhIL-6/rhIL-3-treated animals. The frequency of ANCs < 500/microL was significantly less in the rhIL-6- and rhIL-6/rhIL-3-treated groups versus control groups (2.7 +/- 0.6 and 2.0 +/- 1.0 vs 7.0 +/- 1.4 occurrences, respectively; P < .05). rhIL-3-treated animals had ANCs similar to those in controls; one animal died with septicemia on day 21. Monkeys receiving rhIL-6 were significantly more anemic during the cytokine administration period; however, the anemia resolved by day 24. Coadministration of rhIL-3 and rhIL-6 partially corrected the anemia. The data indicate that rhIL-6 prevents significant thrombocytopenia and shortens the neutropenic period in this chemotherapy model.