[Evaluation of coagulopathies and fibrinolytic abnormalities in central retinal vein occlusion in patients under 60 years of age]. / Apport du bilan des anomalies de la coagulation et de la fibrinolyse dans les occlusions de la veine centrale de la rétine chez les sujets de moins de 60ans.

PURPOSE: To investigate the association of thrombophilic and fibrinolytic factors with central retinal vein occlusion (CRVO) in patients under 60 years of age. MATERIALS AND METHODS: A prospective, observational study of 21 patients with CRVO compared with an age- and sex-matched control group of 23 volunteers was performed. All participants were tested for: cholesterol, hypertension, factors VIII, IX, and XI, homocysteine, antiphospholipid antibodies, antithrombin III, proteins C and S, protein Z and protein Z antibodies, resistance to activated protein C, factor V Leiden mutation, prothrombin mutation, MTHFR genotypes, plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (t-PA) polymorphisms. RESULTS: There was a significantly higher rate of hyperhomocysteinemia (23.8% versus 0%, P=0.018) in CRVO patients. Increased level of factor VIII was more common in the CRVO group at diagnosis (23.8% versus 0%, P=0.018) but no significant difference was observed after one month (P=0.1). Hypercholesterolemia was more common in the CRVO group (42.8% versus 17.4%, P=0.09). Thirty-eight percent of patients with CRVO were hypertensive. Frequencies of other hypercoagulable states were rare. No significant differences were observed for hereditary fibrinolytic abnormalities. DISCUSSION AND CONCLUSION: Among the coagulopathies studied, hyperhomocysteinemia appears to be a risk factor for central retinal vein occlusion in patients under 60 years of age. Conversely, polymorphisms of fibrinolytic factors do not appear to play a role in this population.

Use of recombinant factor VIIa (NovoSeven(®)) in 8 patients with ongoing life-threatening bleeding treated with fondaparinux.

BACKGROUND: Fondaparinux has a favourable efficacy-safety profile but if major bleeding occurs, reversal of antithrombotic treatment is challenging. We present clinical and biological observations from patients treated with rFVIIa for bleeding under fondaparinux. METHODS: Fondaparinux-treated patients with bleeding (>10% haematocrit decrease) and cardiovascular collapse were eligible. Patients received a single 90 µg/kg bolus rFVIIa. Clinical success was defined as clinical bleeding control without thrombotic complication. A biological criterion of successful antagonization was defined as a >100% increase in peak thrombin generation (C(max)). RESULTS: 8 patients were treated (5 ACS, 3 VTE). Patients received aspirin and clopidogrel (n = 5), eptifibatide (n = 2), fluindione (n = 5). In addition to standard haemostatic methods, all patients received rFVIIa and transfusion. Clinical progression was favourable in 4, with bleeding clinically controlled in <6 h. 1 patient died. Biological success was observed in 4 patients with lowest baseline anti-Xa (0.67-0.92 U/L); ¾ had clinical success. In patients with baseline anti-Xa >1.0 U/L (1.14-1.62 U/L), increase in C(max) was low; ¾ had no clinical bleeding control. CONCLUSION: This series is the largest describing rFVIIa use to control bleeding in patients under fondaparinux. rVFIIa was considered efficient in 50%, suggesting inefficacy in the context of elevated anti-Xa.

Cord blood volume reduction using an automated system (Sepax) vs. a semi-automated system (Optipress II) and a manual method (hydroxyethyl starch sedimentation) for routine cord blood banking: a comparative study.

Background With the development of cord blood banking, solutions have to be found to solve the storage space problem, by reducing the volume of cord blood units (CBU). Methods We compared total nucleated cell (TNC) and CD34(+) cell counts before and after processing with three different CBU volume reduction methods used consecutively in our bank: a manual method based on hydroxyethyl starch sedimentation (HES) (n=447), a top-and-bottom (TB) semi-automated method (n=181) using Optipress II, and the Sepax automated method (n=213). Statistical analysis was done using t-tests, linear regression and Spearman correlation coefficients. Adjusted variables included TNC, CD34(+) cell counts, CD34(+) cell percentage and CB volume before processing. Results TNC recovery was higher with Sepax (80.3+/-7.7%) than with HES (76.8+/-9.1%) and TB (60.7+/-13.5%) (P<0.0001, both). It was higher with HES than with TB (P<0.0001). CD34(+) cell recovery was higher with Sepax (86+/-11.6%) than with HES (81.5+/-12.5%) and TB (82.0+/-17.7%) (P<0.008 and <0.0001, respectively) and results with HES and TB were not significantly different (P=0.7). Interestingly, with Sepax, TNC and CD34(+) cell recoveries were not correlated with pre-processing values (P=0.8 and 0.4, respectively). Discussion In conclusion, the Sepax volume reduction method allows higher TNC and CD34(+) cell recoveries.

Induction of tissue factor expression on human umbilical vein endothelial cells by cell-specific HLA class I antibody: preliminary data.

Donor-specific antibodies may play an important role in the development of chronic allograft rejection process. However, the mechanisms leading to intimal vascular proliferation and fibrosis remain poorly understood. The aim of this study was to examine whether donor-specific HLA antibodies induce overexpression of tissue factor (TF) by endothelial cells. HLA typed human umbilical vein endothelial cells (HUVEC) were incubated for 1 to 12 hours with LPS (10 microg/mL), and increasing concentrations (1 to 500 microg/mL) of anti-HLA A1 antibody specific for an antigen expressed by HUVEC and of an anti-HLA A2 antibody for which A2 was not expressed by the HUVEC. Expression of TF mRNA transcripts was quantified using real time Q-RT PCR and TF activity was tested in cell lysates of cultured HUVEC using a chromogenic TF activity assay. HUVEC-specific anti-HLA A1 antibody at low concentrations (10 microg/mL) induced both a significant increase of TF mRNA transcripts after 1 hour of incubation and TF activity after 3 hours incubation compared to incubation with medium alone or with the nonspecific anti-HLA A2 antibody (n = 4 for all experiments, P < .05). These data show for the first time that specific anti-HLA antibody can induce overexpression of TF on endothelial cells. TF, a transmembrane glycoprotein involved not only in the onset of the coagulation cascade, but also in cell proliferation and anti-apoptotic processes, may play a role in the development of alloantibody-induced chronic rejection.

CD34+ cells and CD34+CD38- subset from mobilized blood show different patterns of adhesion molecules compared to those from steady-state blood, bone marrow, and cord blood.

As suggested previously, a down-regulation of some cellular adhesion molecules (CAMs) on CD34(+) hematopoietic progenitor cells (HPC) may contribute to their egress from bone marrow (BM) to peripheral blood (PB) by decreasing their adhesion to BM stromal cells. Besides counting the percentage of CAM-positive cells, we decided to define clearly the antigen density (AgD) of the CAM on mobilized- and steady-state CD34(+) HPC using QIFIKIT calibration beads. Five sources of cells were compared: PB and BM from normal donors (nPB, nBM) cord blood (CB), mobilized PB obtained from leukapheresis products (LKP), and mobilized BM (mBM) samples. In our study the CAM-AgD was the lowest on CD34(+) cells in LKP which, on the contrary, contained the highest percentage of CD117(+), CD54(+), CD58(+) cell subsets. As for CB, a greater proportion of CD44(+) and CD62L(+) cells was observed in LKP than in other products. The LKP-CD34(+) cell population contained a greater percentage of CD11a(+) cells when compared to mBM, but the lowest percentage of CD49d(+) and CD49e(+) cells when compared to all products. The proportion of the CD34(+)CD38(-) immature subset expressing CD11a, CD44, CD54, or CD62L was greater in LKP than in mBM; the CD62L-AgD was higher in LKP than in mBM. This quantitative analysis clearly showed a downregulation of all CAM on LKP-CD34(+). The CD44, CD62L, CD11a, and CD54 AgD decrease appears to be specifically involved in the egress of the CD34(+) subsets into PB. The control of antigen density of these adhesion molecules is likely to be clinically important for effective mobilization of HPC as well as for rapid engraftment following HPC transplant.

[Relative value of different antiphospholipid antibodies detected in a department of internal medicine: retrospective study of 124 patients]. / Valeur relative des différents anticorps antiphospholipides détectés dans un service de médecine interne: étude rétrospective chez 124 patients.

PURPOSE: The value of antiphospholipid antibodies (aPL) detected in the sera of the patients of an Internal Medicine department is not univocal and is still much debated. To test the contribution of such new markers, we reviewed the records of patients having antiphospholipid antibodies detected between 1996 and 1997. METHODS: One hundred and twenty four patients, having at least one of these two aPL: lupus anticoagulant (LA), anticardiolipin antibodies (aCL), or one of these two anti-proteins: anti-beta 2glycoprotéin I antibodies (anti-beta 2GPI) or anti-prothrombin antibodies (aPT), were studied. LA was detected by a PTT-LA technique and aCL, anti-beta 2GPI and aPT by ELISA-sandwich techniques. For each patient we recorded sex, age, personal and familial history of thrombosis, fetal losses and systemic disease, the reason of aPL detection, the final diagnosis, activated partial thromboplastin time (aPTT), platelets count and type of aPL. RESULTS: The population was composed of 77 women (62%) and 47 men (38%) with a mean age of 54 years [12-92 years]. A thrombocytopenia was strongly correlated to aCL presence (OR = 6.15 et p = 0.03). The reason of aPL detection was venous thrombosis, recurrent fetal losses, systemic disease, infectious disease or fortuitous discovery of a prolonged aPTT. The final diagnosis was a systemic disease in 57% of cases, an infectious disease in 14.5%, a thrombosis in 4.5% and a neoplasia in 3%. LA was detected in 54% of patients, aCL in 39.5%, anti-beta 2GPI in 23% and aPT in 31%. No relationship between the aPTT value and the type of aPL could be established. CONCLUSION: Our study shows that familial histories of venous thrombosis or systemic disease are useful to enhance antiphospholipid antibodies detection; that LA is mostly associated to systemic and infectious diseases; that aCL and anti-beta 2GPI are predominant in case of venous thrombosis and that thrombocytopenia has to enhance aCL detection and the discussion about a possible APS.

[Liveoid vasculopathy with combined thrombophilia: efficacy of iloprost]. / Vasculopathie livédoïde et thrombophilie combinée: efficacité de l'iloprost.

INTRODUCTION: Livedoid vasculopathy is characterized by early, focal painful purpuric lesions of the lower skin extremities without histologic finding of small vessel vasculitis. EXEGESIS: A 38-year-old man was seen in our unit for painful purpuric lesions of both feet localized on toes and external sides. Skin biopsy showed dermic vessel thrombosis and endothelial cell proliferation. Lupus anticogulant antibody was positive in association with a heterozygous factor V (Leiden) gene mutation (G1691A). Anticoagulation failed to relieve pain and cutaneous lesions. Intravenous iloprost, a prostacylcin analogous (Ilomedine) was dramatically and rapidly effective in our patient. CONCLUSION: Livedoid vasculopathy is a cutaneous affection related to vascular thrombotic events in which thrombophilia plays a central role. Iloprost might be an interesting alternative treatment of painful purpuric lesions when anticoagulant treatments are ineffective.

Cost-effectiveness of CD34+ dose in peripheral blood progenitor cell transplantation for non-Hodgkin's lymphoma patients: a single centre study.

Intensive high-dose chemotherapy with peripheral blood progenitor cell (PBPC) transplantation is a common strategy for aggressive non-Hodgkin's lymphomas (NHL). A retrospective cost-effectiveness analysis of CD34+ cell dose was carried out. Between 1994 and 1998, 28 patients were included. Efficacy was measured by the length of aplasia. Data collection concerned the period from graft day until discharge from hospital, and the post-graft period until graft day +100. Patients transplanted using a cell dose greater than 5 x 106/kg were found to have a faster hematological recovery. Average length of post-graft hospitalization was shorter and fewer blood products were required for patients with more than 5 x 106/kg CD34+ cells transplanted. Hospitalization was the major cost driver. A large reduction in procedure cost was obtained with a CD34+ cell count higher than 5 x 106/kg (-US$2740, -11%). This difference was directly related to hospitalization (-US$860) and platelet units transfused (-US$1,340). A sensitivity analysis showed the robustness of results. Our findings indicated that a CD34+ cell dose higher than 5 x 106/kg was more cost-effective than a lower dose in NHL patients. The collection of 5 x 106/kg CD34+ cells appeared necessary to optimize the PBPC procedure.

T-cell immune defect and B-cell activation in renal transplant recipients with monoclonal gammopathies.

Monoclonal immunoglobulins (molg) have repeatedly been described in organ and bone marrow transplantation. Although their exact significance is not known, their occurrence is often associated with intensive immunosuppression. We investigated whether molg reflect T-cell immune defect and B-cell activation in renal transplant recipients. Immunofixations and lymphocyte subset analysis (CD4, CD8, CD19) were performed in 182 renal transplant recipients. Soluble CD23 concentrations were measured in patients with molg and in control transplant patients without molg. Monoclonal immunoglobulins were identified in 54 patients (29.6 %). Transplant endurance was shorter (62 +/- 53 months vs 81 +/- 47 months; P < 0.02) and age was older (53 +/- 13 years vs 46 +/- 13 years; P < 0.005) in patients with molg. Maintenance immunosuppression did not differ between patients with and without molg. Mean CD4-cell count was significantly lower in patients with molg (387 +/- 286/mm(3) vs 538 +/- 341/mm(3); P < 0.005). Both CD8- and CD19-cell counts were similar for the 2 groups. Soluble CD23 concentrations were higher in patients with abnormal immunoglobulin values than in patients with normal immunofixation (12.8 +/- 8 vs 1.9 +/- 1.8 microg/l; P < 0. 005). Our study provides new evidence that molg reflect T-cell immune defect in renal transplant recipients. Further studies are required to determine whether CD4-cell count and sCD23 may help to predict the risk of lymphoma in transplant patients with molg.

Interferon gamma, IL2, IL4, IL10 and TNFalpha secretions in multiple sclerosis patients treated with an anti-CD4 monoclonal antibody.

In order to better understand the mechanisms of action of a monoclonal anti-CD4/BF5 antibody(mAb), cytokine secretions were studied in 14 multiple sclerosis (MS) patients treated in a phase 1 trial. Secretion patterns of IFNgamma, IL2, IL4, IL10 and TNFalpha by peripheral blood mononuclear cells were studied before (DO) and after (D30) the treatment. We decided to undertake this study because in a previous one we observed no variations in serum levels of TNFalpha, IFNgamma, IL1, IL6. Results showed significant reductions in IFNgamma, IL2 and TNFalpha secretions after treatment. The anti-CD4 mAb seemed to act on both Th1- and Th2-cells but with preferential action on Th1-cells. Results on Th2-cells were less obvious even though a significant decrease in IL10 was observed. There was no correlation between any of the immunological markers studied and disease activity. This study demonstrates that pharmacological modifications of the CD4 receptor can induce variations in several cytokine secretion levels. It also stresses the role played by Th1- and Th2-cells in the etiopathogenesis of MS.

Low dose T-cell lymphocyte infusion combined with marrow T-cell depletion as prophylaxis of acute graft vs host disease for HLA identical sibling bone marrow transplantation.

T-cell depletion (TCD) of the bone marrow graft remains the most effective method to prevent severe graft versus host disease after allogeneic bone marrow transplantation. Early studies of HLA-identical sibling transplants showed that although T-cell depletion decreased GVHD, T-cell depleted transplants had higher risks of graft failure and leukemia relapse, leukemia free survival (LFS) was not improved compared to non-T-cell depleted transplants. In order to avoid graft failure and increased risk of relapse associated with this approach, we initiated a pilot study of T-cell depletion of the marrow graft combined with reinfusion of a fixed quantity of CD2+ peripheral blood T-cells. Depletion technique consisted in negative purging using CD2 and CD7 monoclonal antibodies (MoAbs) followed by rabbit complement cytolysis. This approach was associated with an intensified conditioning regimen using total body irradiation, high-dose cytosine arabinoside and melphalan (TAM) for all but one patient. Twenty-one patients were included with a mean age of 40 years. Only one acute severe Graft Versus Host Disease (GVHD) was observed and all patients engrafted. At 63 months, probability of survival is 42.86% with a relapse risk of 19.89%, two patients died from B-cell lymphoproliferative disease, seven other died from the procedure partially because of the use of the TAM as pretransplant regimen. This approach is being pursued by a gene therapy trial using herpes-simplex - 1 thymidine kinase gene expressing peripheral donor T-cells.

Prevalence and clinical significance of antiphospholipid antibodies in renal transplant recipients.

BACKGROUND: The prevalence and clinical significance of antiphospholipid antibodies (APAs) have not been extensively studied in non-systemic lupus erythematosus (non-SLE) renal transplant recipients. METHODS: To further define the prevalence and clinical significance of APAs in non-SLE renal transplant recipients and the appearance of dialysis-related APAs after renal transplantation, we conducted a retrospective study on 178 renal transplant recipients. Documentation of anticardiolipin antibodies (ACAs) and lupus anticoagulant in non-SLE renal transplant recipients, retrospective documentation of ACAs on pretransplant frozen plasma and standardized collection of demographic characteristics and posttransplant history of thrombosis were assessed. RESULTS: Fifty of 178 patients (28.1%) had APAs. Transplant duration was shorter and hemodialysis duration was longer in patients with APAs. A posttransplant history of both venous and arterial thrombosis was more frequent in patients with posttransplant APAs (respectively, 18% vs. 6.2% [P<0.001] and 8% vs. 2.3% [P<0.001]). Pretransplant sera were available from 55 patients. Most of patients with posttransplant ACAs had ACAs in the pretransplant period (85%). Pretransplant ACAs were associated with a posttransplant history of venous thrombosis (P<0.001). CONCLUSIONS: Our study demonstrates a high prevalence of APAs in non-SLE renal transplant recipients. Most of them have been acquired in the pretransplant period. Both pretransplant ACAs and posttransplant APAs are associated with posttransplant episodes of thrombosis. Further studies are required to determine the interest of prophylactic measures.

[Antigenicity of fresh, cryopreserved, or liquid-medium-preserved human parathyroid adenomas]. / Antigénicité des adénomes parathyroïdiens humains frais, cryopréservés et conservés en milieu liquide.

We have studied the ability of the cryopreservation and culture techniques to reduce the antigenicity of human parathyroid tissue by suppressing HLA DR bearing cells. Antigenicity was studied with an immunoperoxidase technique applied on frozen sections. Antibody against HLA DR, CD1a, CD3, CD22, CD45RA, CD68 and H et Y antigens were used. In fresh parathyroid tissue, endothelial cells, histiocytes and interstitial dendritic cells expressed HLA DR antigens. Antigenicity of cryopreserved tissue were not altered. In cultured tissue, interstitial HLA DR bearing cells have disappeared but antigenicity of endothelial cells were not modified.

A randomized, double blind, placebo controlled multicenter trial of murine anti-CD4 monoclonal antibody therapy in rheumatoid arthritis.

OBJECTIVE: To assess safety and efficacy of a murine anti-CD4 monoclonal antibody (Mab) in a population of patients with rheumatoid arthritis (RA) compared to treatment with placebo. METHODS: Fifty-eight patients with defined RA were included in this placebo controlled, randomized, double blind, multicenter study. Of the 48 women and 10 men (mean age 54.5 years), 25 were functional class II and 31 were class III, with 9 years' disease duration; the mean of previous disease modifying antirheumatic drugs was 4; 49 were taking steroids (mean dosage 11 mg/day of prednisone). Eighty percent were rheumatoid factor positive. All were in an active state of the disease with: pain > 4 (mean at inclusion 6.6), tender joints > 4 (mean 12), swollen joint count > 3 (mean 9), morning stiffness > 45 min (mean 185), erythrocyte sedimentation rate > 30 mm (mean 59) or C-reactive protein (CRP) > 30 mg/l (mean 63). Treatment was randomized between murine anti-CD4 Mab (B-F5, Diaclone, 20 mg/day) or placebo intravenously for 10 consecutive days. Efficacy was assessed with a composite index (Paulus), with evaluation of number of patients with 20 or 50% improvement in each group. Changes in measures of single clinical or biological variables were also evaluated. RESULTS: The 2 groups were comparable at inclusion. Treatment was well tolerated. Mild side effects (chills, fever, rash) were seen in both groups. Percentage of patients with global 20 or 50% response did not differ between placebo and Mab groups at Day 10 or at Day 30. Evaluation of single variables showed reduced CRP, swollen joint count, and Ritchie index in some B-F5 patients at Day 10, although in the B-F5 group as a whole only CRP was significant. CONCLUSION: No significant improvement in RA after murine anti-CD4 Mab was observed.

Soluble intercellular adhesion molecule 1 in spondylarthropathies.

The aim of the study was to evaluate the soluble form of intercellular adhesion molecule 1 (sICAM-1), a ligand of LFA-1, in the serum of patients with spondylarthropathies (SpA) and to look for a correlation with several inflammatory parameters. sICAM-1 levels were measured by ELISA in 25 SpA patients, 20 healthy controls and 20 patients with rheumatoid arthritis (RA). Results showed that sICAM-1 levels were increased (but not significantly) in SpA patients compared with controls; high levels (> 400 ng/ml) where found in 28% of SpA patients but in none of the RA or control groups. In SpA, correlations were found between sICAM-1 and erythrocyte sedimentation rate, C-reactive protein and interleukin 6, but not with tumour necrosis factor alapha or IgA. These correlations were absent in RA. In conclusion, these results suggest that sICAM-1 levels in SpA may reflect the acute phase of inflammation.

Comparative analysis of class I, II and III epitope-detecting CD34 monoclonal antibodies by quantitative flow cytometry.

UNLABELLED: The aim of this study was to compare different CD34 monoclonal antibodies (MAbs) belonging to three different classes: MY10 class I, QBend10 class II, a mixture of three selected MAbs class I and II designated as CD34 Pool, and 8G12 class III. Bone marrow (BM) samples from 13 healthy donors were analyzed for: 1) percentage of CD34+ cells, 2) quantitative expression of CD34 epitopes (antigen's density - AgD) using a quantitative indirect immunofluorescence (QIFI) test, 3) study of CD34+ cell subsets defined by CD34 and CD38 coexpression. 8G12 MAb showed the highest reactivity with regard to the percentage of detected CD34+ cells and AgD on these cells. A nearly identical percentage of CD34+ cells was detected with CD34 Pool, but with a lower AgD. With QBend10, the percentage of CD34 expressing cells was insignificantly decreased and the AgD was slightly lower. The expression of the MY10 epitope was the lowest and was detected on the lowest number of CD34+ cells. Concerning CD34 and CD38 coexpressing subset, we observed that 8G12 class III MAb detected CD34loCD38dim cells with comparable efficiency with MY10 class I MAb, but with significantly higher level than QBend10 class II and CD34 Pool class I+II MAbs. The CD34hiCD38dim subset was detected with the same efficiency by QBend10, CD34 Pool or 8G12 MAbs but with significantly higher frequency than MY10 MAb. IN CONCLUSION: class II and III MAbs appear preferable for flow cytometric quantification of CD34+ cells; for CD34+ cell subsets determination class III MAbs should be suitable.