Stackable Guide and Immediate Full Arch Loading: A Retrospective Cohort Study of 43 Edentulous Arches-The STAGE Study.

PURPOSE: As the population gets older, the prevalence of complete or partial tooth loss is increasing, significantly impacting people's quality of life. Scientific research demonstrates that implant-fixed complete dentures offer high levels of satisfaction. In certain cases, tooth loss can lead to significant bone atrophy, necessitating pre-implant bone reconstruction. We conducted a retrospective cohort study involving 43 arches, including or not bone grafts, rehabilitated using a stackable guided approach, which included an immediate loading protocol. The primary outcome measure was the survival rate of the implant at 4 months. MATERIAL AND METHODS: The digital workflow helps the design of the provisional prothesis before the implant surgery, which will be loaded immediately after the implant's placement. The stacked guides integrate both surgical and prosthetic considerations into a digital workflow. RESULT: A total of 284 implants were placed. After a 4-month follow-up period, 10 implants (3.5%) exhibited no osseointegration and were subsequently replaced, resulting in an overall success rate of 96.5%. After 1 year of follow-up, a prosthetic success rate of 100% was observed, with all patients being able to progress to the stages for the permanent fixed dentures. CONCLUSION: Our findings support the use of this protocol for all patients, whether they require bone grafts or not. However, a long-term follow-up is essential for a comprehensive evaluation of these treatment outcomes.

European dermatology forum: Updated guidelines on the use of extracorporeal photopheresis 2020 - Part 2.

BACKGROUND: Following the first investigational study on the use of extracorporeal photopheresis for the treatment of cutaneous T-cell lymphoma published in 1983, this technology has received continued use and further recognition for additional earlier as well as refractory forms. After the publication of the first guidelines for this technology in the JEADV in 2014, this technology has maintained additional promise in the treatment of other severe and refractory conditions in a multidisciplinary setting. It has confirmed recognition in well-known documented conditions such as graft-vs.-host disease after allogeneic bone marrow transplantation, systemic sclerosis, solid organ transplant rejection including lung, heart and liver and to a lesser extent inflammatory bowel disease. MATERIALS AND METHODS: In order to further provide recognized expert practical guidelines for the use of this technology for all indications, the European Dermatology Forum (EDF) again proceeded to address these questions in the hands of the recognized experts within and outside the field of dermatology. This was done using the recognized and approved guidelines of EDF for this task. All authors had the opportunity to review each contribution as it was added. RESULTS AND CONCLUSION: These updated 2020 guidelines provide at present the most comprehensive available expert recommendations for the use of extracorporeal photopheresis based on the available published literature and expert consensus opinion. The guidelines were divided into two parts: PART I covers Cutaneous T-cell lymphoma, chronic graft-vs.-host disease and acute graft-vs.-host disease, while PART II will cover scleroderma, solid organ transplantation, Crohn's disease, use of ECP in paediatric patients, atopic dermatitis, type 1 diabetes, pemphigus, epidermolysis bullosa acquisita and erosive oral lichen planus.

European dermatology forum - updated guidelines on the use of extracorporeal photopheresis 2020 - part 1.

BACKGROUND: Following the first investigational study on the use of extracorporeal photopheresis for the treatment of cutaneous T-cell lymphoma published in 1983, this technology has received continued use and further recognition for additional earlier as well as refractory forms. After the publication of the first guidelines for this technology in the JEADV in 2014, this technology has maintained additional promise in the treatment of other severe and refractory conditions in a multi-disciplinary setting. It has confirmed recognition in well-known documented conditions such as graft-versus-host disease after allogeneic bone marrow transplantation, systemic sclerosis, solid organ transplant rejection including lung, heart and liver and to a lesser extent inflammatory bowel disease. MATERIALS AND METHODS: In order to further provide recognized expert practical guidelines for the use of this technology for all indications, the European Dermatology Forum (EDF) again proceeded to address these questions in the hands of the recognized experts within and outside the field of dermatology. This was done using the recognized and approved guidelines of EDF for this task. All authors had the opportunity to review each contribution as it was added. RESULTS AND CONCLUSION: These updated 2020 guidelines provide at present the most comprehensive available expert recommendations for the use of extracorporeal photopheresis based on the available published literature and expert consensus opinion. The guidelines are divided in two parts: PART I covers cutaneous T-cell lymphoma, chronic graft-versus-host disease and acute graft-versus-host disease while PART II will cover scleroderma, solid organ transplantation, Crohn's disease, use of ECP in paediatrics practice, atopic dermatitis, type 1 diabetes, pemphigus, epidermolysis bullosa acquisita and erosive oral lichen planus.

Characterization of efficacy and safety of pathogen inactivated and quarantine plasma in routine use for treatment of acquired immune thrombotic thrombocytopenic purpura.

BACKGROUND: Auto-immune thrombotic thrombocytopenic purpura (TTP) is a morbid multi-organ disorder. Cardiac involvement not recognized in initial disease descriptions is a major cause of morbidity. Therapeutic plasma exchange (TPE) requires exposure to multiple plasma donors with risk of transfusion-transmitted infection (TTI). Pathogen inactivation (PI) with amotosalen-UVA, the INTERCEPT Blood System for Plasma (IBSP) is licensed to reduce TTI risk. METHODS: An open-label, retrospective study evaluated the efficacy of quarantine plasma (QP) and IBSP in TTP and defined treatment emergent cardiac abnormalities. Medical record review of sequential patient cohorts treated with QP and IBSP characterized efficacy by remission at 30 and 60 days (d) of treatment, time to remission, and volume (L/kg) of plasma required. Safety outcomes focused on cardiac adverse events (AE), relapse rates, and mortality. RESULTS: Thirty-one patients (18 IBSP and 13 QP) met study criteria for auto-immune TTP. The proportions (%) of patients in remission at 30 d (IBSP = 61·1, QP = 46·2, P = 0·570) and 60 d (IBSP = 77·8, QP = 76·9, P = 1·00) were not different. Median days to remission were less for IBSP (15·0 vs. 24·0, P = 0·003). Relapse rates (%) 60 d after remission were not different between cohorts (IBSP = 7·1, QP = 40·0, P = 0·150). ECG abnormalities before and during TPE were frequent; however, cardiac AE and mortality were not different between treatment cohorts. CONCLUSIONS: Cardiac and a spectrum of ECG findings are common in TTP. In this study, IBSP and QP had similar therapeutic profiles for TPE.

Guidelines on the use of extracorporeal photopheresis.

BACKGROUND: After the first investigational study on the use of extracorporeal photopheresis for the treatment of cutaneous T-cell lymphoma was published in 1983 with its subsequent recognition by the FDA for its refractory forms, the technology has shown significant promise in the treatment of other severe and refractory conditions in a multi-disciplinary setting. Among the major studied conditions are graft versus host disease after allogeneic bone marrow transplantation, systemic sclerosis, solid organ transplant rejection and inflammatory bowel disease. MATERIALS AND METHODS: In order to provide recognized expert practical guidelines for the use of this technology for all indications the European Dermatology Forum (EDF) proceeded to address these questions in the hands of the recognized experts within and outside the field of dermatology. This was done using the recognized and approved guidelines of EDF for this task. RESULTS AND CONCLUSION: These guidelines provide at present the most comprehensive available expert recommendations for the use of extracorporeal photopheresis based on the available published literature and expert consensus opinion.

[Four cases of photopheresis treatment for cutaneous lupus erythematosus refractory to standard therapy]. / Traitement par photochimiothérapie extracorporelle de quatre cas de lupus érythémateux cutané réfractaires aux traitements conventionnels.

BACKGROUND: Photopheresis is a leucopheresis procedure in which cells are photoactivated by psoralen and then irradiated by ultraviolet A. We report four cases of women with refractory cutaneous lupus erythematosus (LE) who responded to this treatment. PATIENTS AND METHODS: We treated one patient with subacute LE having a contraindication to antimalarials and to thalidomide and three patients with chronic LE (lupus panniculitis, lupus tumidus and disseminated discoid LE) refractory to treatment with hydroxychloroquine, chloroquine, thalidomide and dapsone, and also, in some cases, to oral and intravenous corticosteroids, methotrexate, colchicine, acitretine, sulfasalazine, mycophenolate mofetil and intravenous immunoglobulin. Treatment consisted of two 4-hour sessions fortnightly. Only antimalarials were continued during photopheresis. RESULTS: Photopheresis had a positive effect on all four patients. We noticed complete remission in two patients and interruption of progression followed by partial remission in the other two after a mean delay of two to three months of treatment. All treatments other than antimalarials were stopped. DISCUSSION: Photopheresis appears to be an effective treatment option in patients with cutaneous LE. Due to its high cost, it should nevertheless remain an exceptional therapeutic option restricted to patients with cutaneous LE resistant to standard therapy.

Hepatitis C virus structural proteins do not prevent human dendritic cell maturation.

AIM: Infection with hepatitis C virus (HCV) results in chronic hepatitis in more than 70% of cases. Alterations in the maturation of dendritic cells (DC) might play a role in the immune system's inability to eliminate the virus, although viral factors that could be involved have not been identified. This study in vitro investigated whether HCV structural proteins affect maturation of monocyte-derived DC. METHODS: HCV proteins (core, E1, E2) were expressed by transduction with recombinant adenoviruses of immature DC. The ability of these transduced DC to respond to a maturation stimulus was evaluated by measuring cell surface markers, allogenic lymphocyte stimulation and interleukin (IL)-12 production. RESULTS: Expression of HCV structural proteins did not modify DC maturation in the presence of lipopolysaccharide, as determined by their phenotype and stimulatory functioning. IL-12 secretion was not affected by HCV protein expression in mature DC. CONCLUSION: Our results suggest that HCV structural proteins do not affect maturation of monocyte-derived DC by lipopolysaccharide. These findings are important for further studies to clarify the pathogenesis of chronic HCV infection and towards the rational design of cellular vaccine approaches for immunotherapy against hepatitis C.

Macrophage activation in type 1 diabetic patients with catheter obstruction during peritoneal insulin delivery with an implantable pump.

OBJECTIVE: The purpose of this study was to evaluate the activation of macrophages in type 1 diabetic patients during peritoneal insulin delivery with an implantable pump against two types of insulin: that which was collected from the pump reservoir and that which came straight fromn the bottle (i.e., vial insltlin). Macrophage activation was studied in patients with and without cathcter obstruction and compared with activation in healthy subjects. RESEARCH DESIGN AND METHODS: Human insulin (21 PH, 400 U/ml; Hoescht) was collected from the pump reservoir (Minimed) of diabetic patients with (n = 3) or without (n = 7) catheter obstruction, as assessed by histological examination of the catheter tip. Monocytes were obtained from venous blood samples from both kinds of diabetic patients and from healthy subjects (n = 5) and were differentiated into monocyte-derived macrophages in culture. Their chemotaxis and tumor necrosis factor-alpha (TNF-alpha) release were studied with respect to both types of insulin, as previously stated. Formyl-methionyl-leucyl-phenylalanine (fMLP) and lipopolysaccharide (LPS) were used as controls. RESULTS: Neither insulin recovered from the pump reservoir nor vial insulin proved chemotactic to macrophages from either healthy subjects or those diabetic patients with and without catheter obstruction. The migration toward fMLP of macrophages from patients presenting a catheter obstruction was significantly higher than that observed with macrophages from either diabetic patients without obstruction or healthy subjects, the chemotactic index (mean +/- SD) was 3.81 +/- 0.36 vs. 2.30 +/- 0.89 and 2.60 +/- 0.80, respectively (P < 0.05). LPS significantly stimulated the TNF-alpha secretion of macrophages from diabetic subjects with a catheter obstruction, whereas both native and reservoir-recovered insulin had no effect on this release (144.83 +/- 67.25 vs. 5.15 +/- 2.93 and 5.27 +/- 2.43 pg/ml, P < 0.001). CONCLUSIONS: The human insulin used in implantable pumps, regardless of how long it had remained in the pump reservoir, did not induce macrophage activation in diabetic patients treated through intraperitoneal insulin delivery. In some of these diabetic patients, catheter obstruction could be explained by their high capacity of macrophage chemotaxis.

Modulation of MHC class II transport and lysosome distribution by macrophage-colony stimulating factor in human dendritic cells derived from monocytes.

The macrophage-colony stimulating factor (M-CSF) has been already shown to affect the function of dendritic cells (DC). Therefore, the differentiation of dendritic cells into macrophages (M(PHI)) might represent a pathway which could inhibit the immune response initiated by DC. Because Major Histocompatibility Complex class II molecules (MHC-II) are crucial for DC function, we asked whether M-CSF may influence the intracellular transport of MHC-II in monocyte derived DC. We found that, at early stages, M-CSF induced first a rapid redistribution of MHC-II from the MHC-II containing compartments (MIIC) to the plasma membrane and second an increase in MHC-II synthesis as observed with LPS or TNF-(alpha). These processes were associated with the sorting of MHC-II from lysosomal membranes which underwent a drastic structural reorganization. However, in contrast to tumor necrosis factor (TNF)-(alpha) or lipopolysaccharide (LPS), M-CSF neither potentiated the allostimulatory function of DC nor allowed the stabilization of MHC-II at the cell surface, but rather increased MHC-II turnover. We conclude that the rapid modulation of MHC-II transport and distribution may participate in the inhibitory effect of M-CSF on DC function and differentiation.

Vaccinia virus-related events and phenotypic changes after infection of dendritic cells derived from human monocytes.

The in vitro interactions between vaccinia virus (VV) and monocyte-derived human dendritic cells (DC) have been studied to gain a better understanding of the mechanisms involved in the induction of an immune response by VV. This work showed that VV binds to DC less efficiently than to HeLa cells (HeLa). Capping of viral antigens on the DC surface and electron microscopic examinations suggested that VV enters into DC mainly by endocytosis instead of fusion as for HeLa. Early viral-encoded proteins were expressed in DC but late viral proteins and viral DNA synthesis did not occur. Nevertheless, when successfully infected, DC expressed a similar amount of a foreign, viral-encoded protein, as HeLa, if the early component of the p7.5 promoter was used. VV infection did not lead to DC maturation as determined by following the level of several cell surface markers associated with maturation, but an inhibition of the expression of the costimulatory molecule CD80 was noticed. The proliferation of allogeneic peripheral blood lymphocytes (PBL) was stimulated by VV-infected DC or inhibited depending on the particular donor lymphocytes employed. PBL from VV-vaccinated individuals with good memory responses to VV antigens proliferated in the presence of infected autologous DC. PBL from individuals with poor memory responses to VV and one unvaccinated individual also proliferated, albeit to a lower level, in the presence of infected autologous DC. These results suggest that VV-infected DC could both stimulate memory cells and prime naive cells in vitro.

The MultiScreen filtration system to measure chemoattractant-induced release of leukocyte granule enzymes by differentiated HL-60 cells or normal human monocytes.

A variety of chemoattractants initiate chemotaxis by selective binding to chemoattractant receptors (CARs), a subfamily of seven transmembranous G-protein coupled receptors (7TM-GPCRs) expressed in the leukocyte plasma membrane. Whatever the chemoattractant, signaling leading to chemotaxis involves several common biological steps which occur within seconds to minutes of CAR ligand binding. Though each step can be used to study the progress of the chemotaxis activation process. only certain biological events are suitable for monitoring chemotaxis signaling on large sample numbers as required for drug screening. An example of such is the release of granule enzymes by leukocytes in response to a CAR ligand. In this study, promyelocytic HL-60 cells were employed to set up a 96-well microplate methodology using filtration instead of centrifugation to collect the extracellular fluid together with the cell-released enzymes. Undifferentiated HL-60 cells were found not to respond to any of the CAR ligands. With various types of HL-60 cells which had differentiated along the neutrophilic or monocytic pathways, a large enzyme release was dose-dependently triggered by fMLF or C5a, but none of the tested CC or CXC chemokines. The highest responsiveness was found for neutrophilic HL-60 cells differentiated with dibutyryl cyclic AMP. With normal human monocytes (prepared from the blood of healthy donors by leukapheresis and elutriation), the granule enzyme release response was large to fMLF or C5a, substantial to MCP-1, low to RANTES or MIP-1alpha, but insignificant to Eotaxin, IL-8 and GROalpha. The method readily measures N-acetyl-beta-D-glucosaminidase, beta-D-glucuronidase and elastase activities, and requires approximately five times fewer cells than classical methods, a very important feature when normal human cells are to be used in screening assays. The method was also adapted to large scale screening of antagonists such as cyclosporins A and H for fMLF-mediated signaling using HL-60 cells and monocytes, and truncated (9-76) MCP-1 for MCP-1-mediated signaling using monocytes.

Adhesive and/or signaling functions of CD44 isoforms in human dendritic cells.

The regulation and function of the CD44 family of surface glycoproteins were investigated in human monocyte-derived dendritic cells (DCs). Variant CD44 isoform transcripts encoding exons v3, v6, and v9 are differently regulated during the differentiation of monocytes into DCs. TNF-alpha treatment, which induces the maturation of DCs, up-regulates the expression of all v3-, v6-, and v9-containing isoforms examined. CD44 molecules are involved in the adhesion of DCs to immobilized hyaluronate (HA), and v3- and v6-containing variants participate in this function, whereas anti-CD44v9 mAbs were unable to inhibit DC adhesion to HA. The consequences of ligand binding to CD44 were examined by culturing DCs on dishes coated with HA or various anti-CD44 mAbs. HA, the anti-pan CD44 mAb J173, and mAbs directed against v6- and v9-containing (but not v3-containing) isoforms provoked DC aggregation, phenotypic and functional maturation, and the secretion of IL-8, TNF-alpha, IL-1beta, and granulocyte-macrophage CSF. In addition, IL-6, IL-10, and IL-12 were released by DCs stimulated with either J173 or HA, although these cytokines were not detected or were found only at low levels in the culture supernatants of DCs treated with anti-CD44v6 or anti-CD44v9 mAbs. Our study points to distinct capacities of the v3-, v6-, and v9-containing isoforms expressed by human DCs to mediate cell adhesion to HA and/or a signal inducing DC maturation and the secretion of cytokines.

Intracellular pathway for the generation of functional MHC class II peptide complexes in immature human dendritic cells.

Binding of antigenic peptides to MHC class II (MHC-II) molecules occurs in the endocytic pathway. From previous studies in B lymphocytes, it is believed that most but not all of the newly synthesized MHC-II molecules are directly targeted from the trans-Golgi network to endosomal compartments. By using pulse-chase metabolic labeling followed by cell surface biotinylation, we show here that in contrast to an EBV-transformed B cell line and human monocytes, the majority of newly synthesized MHC-II molecules (at least 55 +/- 13%) are first routed to the plasma membrane of dendritic cells derived from human monocytes. They reach the cell surface in association with the invariant chain (Ii), a polypeptide known to target MHC-II to the endosomal/lysosomal system. Following rapid internalization and degradation of Ii, these alphabeta Ii complexes are converted into alphabeta-peptide complexes as shown by their SDS stability. These SDS-stable dimers appear as soon as 15 to 30 min after internalization of the alphabeta Ii complexes. More than 80% of alphabeta dimers originating from internalized alphabeta Ii complexes are progressively delivered to the cell surface within the next 2 h. Depolymerization of microtubules, which delays the transport to late endosomal compartments, did not affect the kinetics of conversion of surface alphbeta Ii into SDS-stable and -unstable alphabeta dimers. Altogether, these data suggest that newly liberated class II alphabeta heterodimers may bind peptides in different compartments along the endocytic pathway in dendritic cells derived from human monocytes.

Polyethylenimine-mediated transfection of human monocytes with the IFN-gamma gene: an approach for cancer adoptive immunotherapy.

Human monocytes (Mo) and monocyte-derived macrophages (MdM) are major effectors in host defense systems against cancer. Their antitumoral activity is dependent upon two processes: recruitment and activation. One of the most powerful activators for these cells is recombinant human IFN-gamma (rhIFN-gamma). However, when the potential of activated rhIFN-gamma was evaluated in clinical trials by ex vivo adoptive cellular immunotherapy protocols, the major problem was the short duration of ex vivo activation by rhIFN-gamma. Thus repeated injections were required to obtain a clinical response. To overcome this limitation we have developed a gene transfer protocol with IFN-gamma cDNA and polyethylenimine so as to obtain an efficient, long-lasting autocrine cytocidal activation in transfected human Mo/MdM. We show, by clonogenic assays, that efficient transfection and tumoricidal activity can be obtained by this method in human monocyte populations. Although the proposed model must be improved before clinical use, IFN-gamma producing monocytes have potential for adoptive immunotherapy.

Regulation of CD44 isoform expression and CD44-mediated signaling in human dendritic cells.

Dendritic cells (DCs) express CD44, a cell surface receptor for the extracellular matrix ligand hyaluronate, involved in cell-cell interactions and cell migration. Besides the "standard" form of CD44, a variety of splice variants contain an additional extracellular region encoded by 10 "variable" exons termed v1 to v10. The standard form of CD44 as well as variants containing exon v6 (CD44v6) are known to play important roles in the immune system, yet largely unexplored in the DC lineage. In this study, we examined the regulation of CD44 isoforms in human DCs derived from monocytes cultivated in the presence of GM-CSF and IL-4. We found that v3, v6 and v9 variants are all up-regulated upon TNF-alpha stimulation of DCs. In addition, we show that stimulation of DCs using anti-CD44 mAbs can induce DC agregation, up-regulation of accessory molecule expression and secretion of cytokines. A mAb directed against CD44v6 variants was shown to mediate some of these effects.

Soluble CD16/Fc gamma RIII induces maturation of dendritic cells and production of several cytokines including IL-12.

Fc gamma RIII (CD16), a low affinity FcR which binds IgG-containing immune-complexes, exists under membrane-associated forms and under a soluble form (sFc gamma RIII). The latter, present in biological fluids (serum, saliva), is generated by proteolytic cleavage of the two membrane-associated Fc gamma RIII isoforms, Fc gamma RIII-A (expressed by macrophages and NK cells) and Fc gamma RIII-B (expressed exclusively by neutrophils). Herein we demonstrate that dendritic cells (DCs), generated by culturing monocytes with GM-CSF and IL-4, bind biotinylated recombinant sFc gamma RIII. This binding is specific and involves the complement receptor CR3 (CD11b/CD18) and CR4 (CD11c/CD18). Indeed, preincubation of DCs with anti-CD11b and anti-CD11c mAbs decreased by 52% and 62% respectively the binding with sFc gamma RIII. Moreover, electron microscopy showed that binding of gold-labeled sFc gamma RIII to DCs maintained at 4 degrees C occurred within clathrin-coated pits. Once internalized, at 37 degrees C, sFc gamma RIII entered the endocytic pathway and reached the MHC class II compartments. Furthermore, DCs incubated for 48 h with multivalent sFc gamma RIII expressed increased levels of CD40, CD80, CD86, CD54, CD58, HLA class I and class II molecules and decreased levels of CD23 and CD32. These effects result in an increased capacity of DCs to trigger proliferative responses by CD4+ CD45RA+ allogeneic T cells. RT-PCR amplification demonstrated that incubation of DCs for 20 h in the presence of multivalent sFc gamma RIII induced the appearance of GM-CSF and IL-12 p40 mRNA. Among the cytokines constitutively expressed, IL-1 beta and IL-8 were strongly up-regulated whereas IL-6 and IL-12 p35 mRNA were increased to a lesser extent and the expression of MIP-1 alpha mRNA remained constant. Finally, ELISA tests demonstrated that DCs incubated with multivalent sFc gamma RIII secreted the cytokines IL-1 beta, IL-6, IL-8, GM-CSF and IL-12 p75. Thus, while becoming internalized sFc gamma RIII could affect the capacity of DCs to present antigens and, via the induction of accessory molecules and the release of the IL-12 p75 protein, could initiate Th1 type immune response.

Adhesion and Transendothelial Migration of Human Monocytes: Role of Cytokine (IL3, GM-CSF) Activated Endothelial Cells.

In this study we have investigated the effect of GM-CSF and IL3 on Human Umbilical Vein Endothelial Cells (HUVEC). We studied the adhesion properties of HUVEC for non stimulated human elutriated monocytes, as well as the transendothelial migration of these cells. We analysed the expression of adhesion molecules (VLA4/CDw49d, VCAM1/CD106, LFA1/CD11a, ICAM1/CD54, CD18, L-selectin/CD62L, PeCAMl/CD31, ELAM1/CD62E) induced in monocyte adhesion and transmigration. Optimal conditions of HUVEC stimulation with IL3 and GM-CSF were obtained with 100 U/ml of each cytokine. IL3 and GM-CSF were found to induce HUVEC proliferation, more than twofold at day 7 of the culture compared to controls. HUVEC proliferation was not stimulated by IL1α, a slight inhibitory effect was observed at 250 and 500 U/ml. We showed that GM-CSF, IL3 and their combination mimic on activation like status that on which is expressed by an enhancement of adhesion and migration of non stimulated monocytes to and across cytokines activated HUVEC monolayers. After 6 hours activation with IL3 or GM-CSF, more than 60% of the monocytes are adherent to HUVEC after a contact of 30 minutes (vs 30.8 ± 4.6% for untreated control HUVEC). This percentage increased to 80% after a 7 days culture period in presence of the same cytokines (vs 40 ± 5.1% for untreated control HUVEC). IL3 was very effective at inducing monocyte transendothelial migration. The potency of IL3 is seen to be 2 to 3 fold higher than GM-CSF in this system. GM-CSF and IL3 modulate on HUVEC the expression of adhesion molecules induced in monocyte adhesion and transendothelial migration processes. We showed that anti-ELAMl inhibit in part monocyte migration (8.5 ± 3% vs 46.33 ± 4.03% without MoAb; vs 5.1 ± 2% with ICAM1, ELAM1 and VCAMI MoAbs).

Positive selection of CD34+ cells from cryopreserved peripheral blood stem cells after thawing: technical aspects and clinical use.

Autologous stem cell transplantation has become an important therapy in lymphoma, multiple myeloma and solid tumors. The rationale for the selection of CD34+ cells from peripheral blood or bone marrow progenitor cell collections is based on the observation that contaminating tumor cells can be depleted approximately 3 to 6 logs. This procedure may be limited because of lack of sufficient numbers of progenitor cells in the leukapheresis concentrates. The use of frozen/thawed peripheral blood mononuclear cell (PBMC) samples makes it possible to pool two or even more stem cell harvests collected at different time points to increase the total number of CD34+ progenitor cells. We report in this work the feasibility of frozen/thawed peripheral blood CD34+ positive cell selection, using the large-scale (Ceprate SC) and the lab-scale avidin-biotin immunoadsorption system (Ceprate LC). This procedure consists of a washing step and a positive selection step. Our results show that frozen/thawed CD34+ cells were obtained with a purity of 86.68 +/- 3.62%, a viability of 97.94 +/- 0.97% and a recovery of 91.85 +/- 10.84% (range 80 to 112%). The CFU-GM assays were performed in a methylcellulose based medium; 89.13 +/- 19.63 colonies were obtained for 10(3) cells plated. Two patients were grafted with peripheral blood CD34+ cells selected after freezing. Our clinical data show that these cells are capable of rapidly reconstituting hematopoiesis after high-dose chemotherapy.

Peripheral blood CD34+ cells: method of purification and ex vivo expansion.

Peripheral blood stem cells (PBSC) are used increasingly for autotransplantation in the treatment of acute leukemia, lymphoma, multiple myeloma, solid tumors such as ovarian and breast carcinoma. They are collected by leukaphereses during rapid hematopoietic recovery, following cytotoxic chemotherapy with or without administration of hematopoietic growth factors. We studied the clonogenic and cytokine-mediated expansion potential of CD34+ cells from mobilized PBSC. Low density mononuclear cells were processed using the CEPRATE LC CD34 KIT (CellPro). CD34+ purified cells, were cultured in suspension with 6 combined hematopoietic growth factors (IL1beta, IL3, IL6 at 100 U/ml and G-CSF, GM-CSF and stem cell factor at 10 ng/ml of each) for up to four weeks. Every week, cells were counted and CFU-GM assay was performed in a methylcellulose based medium. We have analysed the percentage of cells bearing CD34, CD33, CD38, HLA-DR, CD45RA, CD45RO antigens. Our results showed, that CD34+ cells were obtained with a purity of 92 +/- 2.3% and a yield of 71 +/- 10.7%. The majority co-expressed CD33 (57.76 +/- 34.16%) and CD38 (62.2 +/- 34%) antigens. These culture conditions, are necessary to obtain a fold increase of nucleated cells (377 fold at week 4), of CFU-GM progenitors (41.2 fold at week 3) and of CD34+ cell absolute number (10 fold at week 1) with an important differentiation of progenitors in particular myeloid progenitors.