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.     

低分子肝素在麻醉大鼠胃肠道的吸收

以血浆抗Ｘａ活性及体内抗静脉血栓形成为指标，考察了低分子肝素溶液及乳剂在麻醉大鼠胃肠道的吸收情况。     

Aspirin does not inhibit adenosine diphosphate-induced platelet alpha- granule release

The involvement of metabolites of arachidonic acid in platelet-dense granule secretion and secondary platelet-platelet interactions is well characterized. However, their role in heterotypic interactions dependent on alpha-granule secretion is less well understood. Using platelet-surface expression of P-selectin as a marker of alpha-granule secretion, we have shown that: (1) aspirin treatment of platelets at doses that block dense granule secretion does not inhibit alpha-granule secretion to adenosine diphosphate (ADP); (2) synergism between epinephrine and ADP in the induction of P-selectin expression is similarly unaffected by aspirin; and (3) the ability of P-selectin to mediate adhesion of activated platelets to monocytes and polymorphonuclear lymphocytes in whole blood is also unchanged by aspirin treatment. To further explore the mechanisms responsible for platelet alpha-granule secretion, we have shown that inhibition of Na+/H+ exchange by either acidification of the extracellular medium or amiloride treatment blocked ADP-induced P-selectin expression. In contrast, incubation with the platelet lipoxygenase inhibitor 5,8,11- eicosatrynoic acid, by itself and with aspirin, did not decrease ADP- induced P-selectin expression. We conclude that platelet alpha-granule secretion in response to ADP is dependent on intact Na+/H+ exchange but is independent of the lipoxygenase- and cyclooxygenase-dependent metabolites of arachidonic acid.     

Expression of apoptosis regulatory proteins p53 and Bcl-2 in skin of patients with chronic renal failure on maintenance haemodialysis

BACKGROUND: Chronic renal failure results in multi-organ system derangement including cardiovascular, gastrointestinal, neurological, endocrinal, blood and dermatological abnormalities. Maintenance of skin homeostasis requires a delicate balance between proliferation, differentiation and apoptosis. p53 and Bcl-2 proteins play a central role in the regulation of apoptosis. OBJECTIVE: Evaluation of the expression of apoptosis regulatory proteins p53 and Bcl-2 in apparently normal skin of patients, with chronic renal failure on maintenance haemodialysis, with respect to their role in the apoptotic process. METHODS: Biopsy specimens were obtained from 10 patients with chronic renal failure on maintenance haemodialysis, as well as seven age-matched control subjects. Computer-assisted image analysis was employed to measure epidermal thickness in H&E-stained sections. Immunoperoxidase technique was also used to demonstrate p53 and Bcl-2 proteins and the TUNEL technique for detection of apoptotic cells in these specimens. RESULTS: The mean epidermal thickness was significantly higher (P < 0.0001) in patients than controls. Meanwhile, no apoptotic cells were detected in the epidermis of patients. On the other hand, a statistically significant difference was observed in both p53 (P = 0.0001) and Bcl-2 expression (P = 0.0003) when comparing patients and controls. Expression of p53 (2.74 +/- 0.84) and Bcl-2 (3.45 +/- 1.35) proteins was higher in skin samples obtained from patients with chronic renal failure and on maintenance haemodialysis than those from control cases (0.5 +/- 0.96 and 0.8 +/- 0.6, respectively). Moreover, Bcl-2 expression in patients was observed in basal as well as squamous cell layers of skin, whereas in control subjects it was confined to the basal cell layer only. CONCLUSION: These findings suggest that an alteration in the proliferation/apoptosis balance may be present in the skin of such patients.     

Patient controlled analgesia (PCA) in paediatric surgery: a prospective study following laparoscopic and open appendicectomy

Patient controlled analgesia (PCA) has not yet gained universal acceptance for the management of postoperative pain in paediatric surgery. In a prospective study we evaluated feasibility and complications of PCA following 90 cases of laparoscopic or open appendicectomy. PCA proved to be a safe and feasible method with few complications (2% of medical complications, no abort of application, 17 technical checks in a total running time of 4125 h). Acceptance by patients was high and children of all age groups worked the system properly. Assessment of application protocols showed, that the consumption of analgesics was significantly reduced following laparoscopic appendicectomy (P < 0.05). PCA is a safe and feasible method for the management of postoperative pain in children and PCA recording provides an excellent insight into the consumptional behaviour of patients, enabling staff to evaluate postoperative pain for various procedures.