Studies of circulating microparticle release in peripheral blood after pancreatic islet transplantation.

The loss of graft function after intraportal islet transplantation is likely multifactorial involving allogeneic rejection, recurrent autoimmunity, graft exhaustion due to a marginally implanted islet mass, immunosuppressant toxicity, and impaired ß-cell regeneration. Because early markers of the loss of ß-cell mass or function are lacking, monitoring of islet function remains a challenging issue. We have reported herein monitoring of membrane procoagulant microparticles (MPs) as markers of cell stress in the plasma of three recipients with various clinical histories. Early kinetics of C-peptide and MPs followed identical patterns during the first weeks after transplantation; a major increase probably reflected processes related to cell infusion and islet engraftment. Importantly in the case of rejection, MPs and C-peptide showed opposite patterns. A fall in C-peptide was associated with enhanced insulin needs. Our results suggested that a peak in MP levels might indicate rejection with prognotic value. Treatment of the loss of islet function by a new islet infusion or steroid therapy returned MP and C-peptide levels to their baselines with concomitant restoration of islet function. In the patient with suspected acute cellular rejection, MPs also appeared to be sensors of immunosuppressive steroid therapy.

Differentiation of antigen-presenting cells (dendritic cells and macrophages) for therapeutic application in patients with lymphoma.

The recent clinical trial in lymphoma using tumor antigen-loaded DCs (Hsu et al, Nature Med 1996; 2: 52) demonstrates the efficiency of the use of professional antigen presenting cells (APCs) for taking up, processing and presenting tumor protein in a vaccine strategy in cancer. However, the production of large quantities of clinical grade APCs remains to be resolved. Here, we describe that both dendritic cells (DCs) and macrophages (MOs) can be efficiently differentiated in large numbers from lymphoma patients in spite of their disease and previous therapy. These cells were produced using the VAC and MAK cell processors according to standard operating procedures. DCs and MOs were differentiated from circulating monocytes in gas permeable hydrophobic bags, with 2% autologous serum and in the presence of GM-CSF and IL-13 or GM-CSF alone, respectively. DCs and MOs were then purified by counter flow centrifugation. Phenotypic, morphological and functional analysis showed that cells differentiated from patients with lymphoma present quite similar features to DCs and MOs produced from monocytes of healthy donors. Moreover, we show that MOs, when combined with CD20 antibody (Rituximab), can efficiently engulf tumor cells and propose that a such combination could be used for initiating a clinical trial in lymphoma. Thus, the possibility of producing functional DC and MOs in large amounts in conditions compatible with therapeutic application will allow the development of new immune strategies to eradicate lymphoma.

[Evaluation of Cell-Dyn 3500 in extreme values]. / Etude du Cell-Dyn 3500 dans des valeurs extrêmes.

The quality tests made during the preparation of labile blood products permit to validate the product and to make sure the making procedures are applied properly. The labile blood products have to correspond to a number of standards set by a ministerial order. It is thus necessary to check the manufacturing unit of some products. On account of the original technology of Cell-Dyn 3500 which associates methods of impedance variation measure analysis and flow cytometric technique analysis on non-marked cells, and of its veterinary modulus permitting counting in extreme values, it seemed to be worth being evaluated in a blood bank quality control laboratory. Linearity was studied from a range of dilutions obtained from high value samples prepared by the centrifugation of blood bags and collection of the buffy coat by Optipress system. The study was performed on 22 samples. The linearity in the counting of red cells, white cells, platelets and haematocrit was measured. Intersample contamination was measured. It focused on the values of platelets and white cells. A sample of high cell concentration prepared in the condition mentioned above and a white cell-reduced blood concentrate were used. The study of repeatability was done by treating 20 times three samples covering a wide range of values of the different parameters of cell numeration. The linearity concerning parameters and the range of the values studied are good (example: from 10 to 3000 10(9)/l for platelets). Contamination is low for white cells and non appreciable for platelets. Repeatability shows excellent variation coefficients.