[Congenital piriform aperture stenosis and odontogenic disorders]. / Sténose congénitale de l'orifice piriforme et troubles de l'odontogenèse.

PURPOSE: We had for objective to assess odontogenic disorders associated to a congenital piriform aperture stenosis and to study their various presentations. METHODS: Twelve patients presenting with a congenital piriform aperture stenosis, 1 week to 3 months of age, were retrospectively included from 1998 to 2008. All patients underwent an initial CT scan to evaluate the temporary dental germs. RESULTS: Deciduous dental germs were abnormal in 75% of the cases. Thirty-three percent had a single median maxillary central incisor. DISCUSSION: The concept of solitary median maxillary central incisor syndrome makes for a more pathophysiological approach of this type of disease, with various clinical presentations, corresponding to various levels of severity of a same pathological process.

[Palatal necrosis due to cocaine abuse]. / Nécrose palatine par consommation de cocaïne.

The easier access to cocaine allows chronic and intensive consumption. Nasally inhaled cocaine causes important midfacial lesions called: Cocaine Induced Midline Destructive Lesions. These lesions are due to several factors, the anesthetic, vasoconstrictive, locally prothrombotic properties of cocaine and its components combined with cytotoxic effects and traumatic nasal injuries related to consumption mode. Functional signs are: nasal regurgitation, rhinolalia, rhinorrhea, and midfacial pain. The morphological modifications of the nasal pyramid feature the destruction of bone and cartilage structures. Endo-buccal examination, anterior rhinoscopy, and TDM reveal palatine necrosis of variable extension. Nasal fossae superinfection is always present. Sinus superinfection is frequent. Management is multidisciplinary. Weaning must be achieved before surgery. It is necessary to rehabilitate speech and swallowing functions by the means of various local or free flaps.

[Cord blood: from bench to bedside]. / Le sang de cordon: du laboratoire au lit du malade.

Since 1974, umbilical cord blood (CB) has been shown to contain haematopoietic stem cells similar to stem cells from the bone marrow. In 1988, E. Gluckman and her colleagues performed - successfully - the first familial CB transplantation and cured a 5 years old child suffering from Fanconi's anemia. Rapidly, CB banks were organised throughout in the world and thanks to this novel source of haematopoietic stem cells, we can now find a donor for 75 % of the patients requiring a "bone marrow" transplantation. The major benefit of CB as a source of hematopoietic stem cells is its easy access. CB also allows a more significant degree of HLA incompatibility and thus offers an opportunity of transplantation to ethnic minorities for whom no HLA identical donors are available. However, several studies have shown that the number of cells harvested in a CB was closely correlated with the engraftment post transplantation and today, a minimum of 3.7 x 10(7) mononucleated cells/kg is recommended. This required amount of cells is not always reached due to the small volume often harvested from a CB. Therefore, to apply CB transplantations to adults, different approaches are currently being investigated : coinfusion of haploidentical cells, mesenchymal cells, a second CB, or the addition of CB expanded ex-vivo. Among these approaches, double CB transplantation seems nowadays the most promising alternative and ongoing studies should soon inform us whether the duration of aplasia will be improved.

Ex vivo expansion of neutrophil precursor cells from mobilized peripheral blood cells: similar results in cancer patients and normal donors.

BACKGROUND: Infusion of ex vivo differentiated myeloid progenitors may reduce or abrogate severe neutropenia following mobilized peripheral blood transplantation. We compared the ex vivo expansion of myeloid progenitor cells starting from cancer patients (CP) and from normal donors (ND) and evaluated the influence of the CD34(+) cell mobilization on the capacities of cells to be expanded. METHODS: The ex vivo-expanded cells were evaluated for their phenotype, the presence of primary and secondary granules and their functional capacities (oxidative burst activity and phagocytosis). RESULTS: We did not observe significant differences between ND and CP for the total leukocyte and CD34(+) cell expansions nor for the myeloid progenitor production. In CP as well as in ND, the expanded cells were functionally competent. DISCUSSION: This suggests that the capacities of CD34(+) cells to proliferate and differentiate ex vivo are not impaired by prior chemotherapy and/or disease status. On the other hand, we did not observe any significant correlation between the number of mobilized CD34(+) cells before apheresis and the cell expansion. In conclusion, the ex vivo expansion of CP and ND cells is comparable and achievable even with a low CD34(+) cell number in mobilized peripheral blood.

Ex vivo expansion of megakaryocyte progenitor cells: cord blood versus mobilized peripheral blood.

Thrombocytopenia is a problematic and potentially fatal occurrence after transplantation of cord blood stem cells. This problem may be alleviated by infusion of megakaryocyte progenitor cells. Here, we compared the ability of hematopoietic progenitor cells obtained from cord blood and expanded in culture to that of mobilized peripheral blood cells. The CD34(+) cells were plated for 10 days in presence of thrombopoietin (TPO) alone and combined with stem cell factor (SCF), Flt3-ligand (FL), interleukin-3 (IL-3), IL-6, and IL-11. Cells were analyzed for the CD41 and CD42b expression and for their ploidy status. Ex vivo produced platelets were enumerated. We show that (1) TPO alone was able to induce differentiation of CD34(+) cells into CD41(+) cells, with limited total leucocyte expansion; (2) the addition of SCF to TPO decreased significantly CD41(+) cell percentage in CB, but not in MPB; and (3) in CB, the addition of FL, IL-6, and IL-11 to TPO increased the leukocyte expansion with differentiation and terminal maturation into MK lineage. In these conditions, high numbers of immature CD34(+)CD41(+) MK progenitor cells were produced. Our results thereby demonstrate a different sensitivity of CB and MPB cells to SCF, with limited CB MK differentiation. This different sensitivity to SCF (produced constitutively by BM stromal cells) could explain the longer delay of platelet recovery after CB transplant. Nevertheless, in CB, the combination of TPO with FL, IL-6, and IL-11 allows generation of a suitable number of immature MK progenitor cells expressing both CD34 and CD41 antigens, which are supposed to be responsible for the platelet recovery after transplantation.

Growth factors and DLI in adult haploidentical transplant: a three-step pilot study towards patient and disease status adjusted management.

Haploidentical transplant is now established as a procedure of choice for patients who lack a compatible donor. However, they are still referred too late, heavily pretreated, at very advanced stages. We initiated a three-step phase I study trying improve transplant-related mortality, relapse rate, and immunity: G-CSF + DLI, GM-CSF + DLI, patient- and disease-adapted strategy. Thirty-three consecutive leukemia patients, aged 18-55, were investigated (20 very poor risk, 11 poor risk, and 2 better risk). GvH type NK alloreactivity was chosen when possible (18/33) and balanced across the three groups. In the first nine patients, G-CSF was used and escalated prophylactic DLI started at month 1. Thus, G-CSF and 1-3 DLI (10(4) CD3/kg) is safe. It results in faster CD4 recovery and a low rate of infections. However, it was insufficient to induce a GVL effect. In the next 12 patients, GM-CSF was used plus 1 DLI (10(4) CD3/kg) at day 30 unless aGVHD (3 patients). The comparison between the two first groups can be summarized as follows: G-CSF + DLI: TRM at day 100: 0, RR: 6/9, severe aGVHD: 0. GM-CSF + 1 DLI group: RR: 1/12, TRM at day 100: 3, aGVHD > 1: 9/12, price to pay: GVHD resulting in five deaths in total. Step 3 (13 patients) consists of a patient-adapted strategy: no more aspecific DLI (selected anti-CMV and aspergillus DLI planned in all patients); in myeloid disorders with NK alloreactivity: no GF. In the other cases, GM-CSF (at a reduced total dose of 500 mug) is given the follow-up of these 13 patients, although promising is currently short (median 5 months). Overall, TRM at day 100 is 3/29, reflecting the good tolerance of the conditioning in a heavily pretreated population (median age: 43). NRR mortality (8/26) at 1 year is greater in the GM-CSF + DLI group, reflecting the impact of severe aGVHD. We conclude that the third strategy might improve the outcome without exposing patients to unnecessary severe GVHD.

Isolation of BM mesenchymal stem cells by plastic adhesion or negative selection: phenotype, proliferation kinetics and differentiation potential.

BACKGROUND: BM mesenchymal stem cells (MSC) have the capacity for renewal and the potential to differentiate into multiple tissues. In this study, we compared different enrichment methods to obtain MSC from BM. METHODS: Three different methods were compared with a view to obtaining MSC more rapidly from BM: negative selection (RosetteSep and MACS) and plastic adhesion. The three cell fractions were grown in complete alpha-minimum essential medium in order to evaluate their proliferative capacity, their phenotype during culture and their potential to differentiate into adipocytes, osteocytes and chondrocytes. Identification of MSC was performed by immunofluorescence with putative mesenchymal markers SH2 and SH3 but also with hematopoietic markers. RESULTS: After negative selection, only 1+/-0.2% and 2.9+/-0.8% of cells were recovered from BM with the RosetteSep and MACS methods, respectively. However, negative depletion permitted a homogeneous population of MSC, with more than 90% SH2+ and SH3+ cells, to be obtained rapidly and in large quantities after 10 days of culture. Similar homogeneity was observed after three passages if the plastic adhesion was used as selection method and after an average of 25-30 days of culture. Different levels of MSC maturity were also suggested by the variable level expression of Stro-1. DISCUSSION: Depleting selection by RosetteSep may represent an easy method of obtaining MSC rapidly from BM with the aim of potential therapeutic use.

Ex vivo myeloid differentiation of cord blood CD34+ cells: comparison of four serum-free media containing bovine or human albumin.

BACKGROUND: Infusion of ex vivo generated myeloid post-progenitor cells associated with unmanipulated cells appears to be a promising approach to reduce neutropenia following cord blood (CB) transplant. We compared four commercially available serum-free media, two containing BSA and two containing human albumin, on the in vitro differentiation of CB CD34+ cells into post-myeloid progenitor cells. METHODS: CB CD34+ cells were cultured for 7 days in CTM-H00 (Mabio-International), StemSpanH2000 (StemCell Technologies), RM-B00 (Mabio-International) and Stem(alpha)A (Stem Alpha). The cells were stimulated by SCF, G-SCF, IL3 and Flt3-ligand (FL) added once at Day 0. Expansion was evaluated as the increase of leucocytes, CD34+ cells and CD13+ cells. Maturation of cells into the myeloid lineage was evaluated by expression of CD15, CD11b and CD16 Ags and by the presence of primary (myeloperoxydase, MPO) and secondary granules (lacoferrin, LF). The capacity of cells to phagocyte latex beads was evaluated to assess their functionality. RESULTS: We observed that: a) the mononuclear cell and CD34+ cell expansions were significantly different according to the medium tested (respectively 61.5 +/- 7.7 and 15.5 +/- 3.4 for RM-B00, 37.3 +/- 5.4 and 10.3 +/- 1.6 for CTM-H00, 23.2 +/- 6.5 and 5.8 +/- 0.9 for StemSpanH2000 and 16.6 +/- 2.4 and 3.9 +/- 0.7 for Stem(alpha)A; b) the expansion of myeloid precursors is higher with RM-B00, similar with CTM-H00 and StemSpanH2000, and lower with Stem(alpha)A. This difference is essentially due to total leucocyte expansion, rather than to a selective expansion of myeloid cells, except for Stem(alpha)A, for which the percentages of neutrophil precursor cells [promyelocytes (CD15+ CD11b+), myelocytes (CD11b+ CD16-) and mature cells (CD11b+ CD16+)] are significantly decreased. DISCUSSION: It appears that during ex vivo differentiation into myeloid lineages, the medium is critical and should be systematically screened before use in preclinical protocols. The use of human rather than bovine albumin, seems to have neither a negative, nor positive impact on the effectiveness of the medium.

Ex vivo expansion of neutrophil precursor cells from fresh and cryopreserved cord blood cells.

BACKGROUND: Neutropenia following cord blood (CB) transplantation may be abrogated by infusion of granulopoietic progenitor cells. The purpose of this study was to determine whether myeloid progenitors can be obtained by ex vivo expansion of cryopreserved cord blood aliquots, and whether these progenitors present the morphologic, biologic and functional properties of myeloid progenitors at various stages of differentiation. METHODS: The cells, plated for 7 days in serum-free medium with SCF, IL-3, G-CSF, Flt3-ligand and thrombopoietin in various combinations were assessed for the expression of CD34, CD38 and CD13. Maturation of cells into the myeloid lineage was evaluated by the expression of CD15, CD11b and CD16 and by the presence of primary (myeloperoxidase) and secondary granules (lactoferrin). The capacity of cells to phagocyte latex beads was evaluated to assess their functionality. RESULTS: We have shown that a). CD34+ cells isolated from thawed samples were able to produce expansions similar to fresh samples. b). The best combination for the expansion of neutrophil precursor cells was S3FG; c). in these conditions, all stages of myeloid progenitors were represented, but few mature cells were observed. d). However, when the cells were plated on a BM stroma to try to reproduce conditions occurring during transplant, they acquired rapidly the characteristics of mature segmented cells. e). The ex vivo generated granulocytes were able to phagocyte latex beads. DISCUSSION: In conclusion, it seems reasonable to systematically aliquot CB samples before cryopreservation. Some aliquots can then be thawed, enriched in CD34+ cells and ex vivo differentiated into myeloid lineage, while the other aliquots are conserved to be infused without manipulation.

Donor lymphocyte infusions in adult haploidentical transplant: a dose finding study.

Haploidentical transplantation has become a clinical option for patients lacking a compatible donor. However, patients are still referred at advanced stages and are usually heavily pretreated. This results in a high risk of toxicity, relapses and infections. We therefore started a donor lymphocyte infusion (DLI) dose-finding protocol, to try to improve both relapse rate and immunity reconstitution. In all, 12 consecutive patients were investigated. All had a refractory, some progressive, disease. Conditioning consisted of TBI, melphalan, ATG, fludarabine and CSA pretransplant. In four rapidly progressive patients, Ara-C had to be given 1 week preconditioning. The graft was T- and B-cell depleted with a fixed reinfused CD3 dose of 5 x 10(4)/kg. All patients engrafted before day 20. G-CSF was given from day 5 post-transplant and replaced with GM-CSF in the last three patients. Nonrelapse related mortality was 0/12 at 1 year. DLI were started at day 28 (3 x 10(4) CD3/kg) in the two first patients. This resulted in acute graft-versus-host disease (aGVHD) and chronic graft-versus-host disease (cGVHD) in both, but they did not relapse. The next dose was 1 x 10(4)/kg monthly for 3 months. This was well tolerated with only one grade I GVHD. Given the high relapse rate, we escalated doses (1, 3 and 10 x 10(4)/kg). This produced GVHD in all. We next moved, to GM-CSF and 1 x 10(4) CD3/kg monthly. Overall, 6/12 patients relapsed and received therapeutic DLI, starting at 1 x 10(5) CD3/kg with escalation every 2 weeks. We conclude that prophylactic DLI are feasible in adult haploidentical transplantation, without GVHD at a monthly dose of 1 x 10(4) CD3/kg. They result in faster CD4 recovery and a low rate of infections. The impact of GM-CSF remains to be further investigated. This scheme seems ideal for patients transplanted early in the course of their disease. In very bad prognosis patients, it remains insufficient to rapidly induce a GVL effect. Escalated doses are feasible but the price is aGVHD. Therapeutic DLI can be given at higher doses, depending on the time post-transplant. Haploidentical transplantation with low-dose DLI is a safe procedure that should be considered in all patients needing a transplant, but lacking a matched donor, early in the course of the disease.

[Hematopoietic stem cells: source, indications and perspectives]. / Les cellules souches hématopoiétiques: sources, indications et perspectives.

The haematopoietic stem cell (HSC) has been first described in the mouse and now identify in human as well. Exposed to a cocktail of growth factor, this HSC can self re-new and/or differentiate into the three lineages we have in the peripheral blood. These HSC are of major importance in the clinics since they can be used for some marrow (or stem cell) transplantation, and lead to the cure of a number of malignant and non malignant hemopathies. We have today three sources of HSC: the bone marrow, the mobilized peripheral blood stem cell and the cord blood. Bone marrow used to be the classical source of HSC after harvesting by aspirations in the iliac crest. However, this approach is now supplanted by the recovery of HSC in peripheral blood using a cell separation after four days of G-CSF administration. These are several advantages of this technique, but the most important one is the more rapid hematopoietic recovery after transplantation, reducing the risk of infection and transfusion. A recent source of HSC is the umbilical cord blood. At the moment of delivery, the cord blood is extremely enriched in HSC due to the migration of these cells from the liver to the bone marrow stroma, where they will persist after birth. We have learned that the marrow stroma display a major role in the regulation of hematopoiesis and the pathogenesis of several malignant hemopathies can be explained by disturbance in the function of stromal cell. We have particularly studied the patho-genesis of chronic lymphocytic leukaemia. We have also observed that a subpopulation of stromal cells, the mesenchymal cells are of major importance in the microenvironment. In addition, the plasticity of these cells is demonstrated in vitro and we have currently a research program investigating its differentiation in neural cells. All these observations bring new promises in the treatment of hemopathies but also in some other neurological degenerative diseases.

Early induction of apoptosis in B-chronic lymphocytic leukaemia cells by hydroxychloroquine: activation of caspase-3 and no protection by survival factors.

We have investigated the effect of hydroxychloroquine (HCQ), an anti-rheumatic drug, on malignant B cells from 20 patients with B-chronic lymphocytic leukaemia (B-CLL). HCQ induced a decrease in cell viability in a dose- and time-dependent manner. The mean IC50 was 32 +/- 7 microg/ml (range, 10-75 microg/ml) for 24 h of exposure. This cytotoxic effect was owing to apoptosis, as demonstrated by morphological changes, annexin V binding capacity and DNA fragmentation (28 +/- 4% of apoptotic cells as early as 5 h post incubation, increasing to 82 +/- 4% at 18 h post treatment). The apoptosis was associated with caspase-3 activation because the cleavage and activity of caspase-3 were increased by HCQ. The amount of bcl-2 protein was reduced during apoptosis, evidenced using quantitative flow cytometry. As early as 1 h post-HCQ treatment, a reduction of the mitochondrial transmembrane potential was measured by 3,3'-dihexyloxacarbocyanine iodide. Interestingly, the HCQ effect was not affected by exposure to interleukin-4 or co-culture with bone marrow stromal cells. Our observations suggest that HCQ may offer a new therapeutic tool in the treatment of B-CLL patients.

Characterization of CD34+ subsets derived from bone marrow, umbilical cord blood and mobilized peripheral blood after stem cell factor and interleukin 3 stimulation.

We characterized CD34+ cells purified from bone marrow (BM), mobilized peripheral blood (PB) and cord blood (CB) and we tried to establish correlations between the cell cycle kinetics of the CD34+CD38- and CD34+CD38+ subpopulations, their sensitivity to SCF and IL-3 and their expression of receptors for these two CSFs. At day 0, significantly fewer immature CD34+CD38- cells from CB and mobilized PB are in S + G2M phases of the cell cycle (respectively 2.0 +/- 0.4 and 0.9 +/- 0.3%) than their BM counterpart (5.6 +/- 1.2%). A 48-h incubation with SCF + IL-3 allows a significant increase in the percentage of cycling CD34+CD38- cells in CB (19.2 +/- 2.2%, P < 0.0002) and PB (14.1 +/- 5.5%, P < 0.05) while the proliferative potential of BM CD34+CD38- progenitors remains constant (8.6 +/- 1.0%, NS). CD123 (IL-3 receptor) expression is similar in the three sources of hematopoietic cells at day 0 and after 48-h culture. CD117 (SCF receptor) expression, although very heterogeneous according to the subpopulations and the sources of progenitors evaluated, seems not to correlate with the difference of progenitor cell sensitivity to SCF nor with their proliferative capacity. Considering the importance of the c-kit/SCF complex in the adhesion of stem cells to the microenvironment, several observations are relevant. The density of CD117 antigen expression (expressed in terms of mean equivalent soluble fluorescence, MESF) is significantly lower on fresh PB cells than on their BM (P < 0.017) and CB (P < 0.004) counterparts, particularly in the immature CD34+CD38- population (560 +/- 131, 2121 +/- 416 and 1192 +/- 129 MESF respectively); moreover, when PB and BM CD34+CD38- cells are stimulated for 48 h with SCF + IL-3, the CD117 expression decreases by 1.5- and 1.66-fold, respectively. This reduction could modify the functional capacities of ex vivo PB and BM manipulated immature progenitor cells.

Adhesion to bone marrow stroma inhibits apoptosis of chronic lymphocytic leukemia cells.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of monoclonal long-lived B cells which are apparently resistant to normal apoptotic regulation. Since bone marrow stromal cells play an essential role in B lymphopoiesis, we have investigated whether stromal cells influence B-CLL cell survival. Our results indicate that intimate contact with stromal cells reduces B-CLL cell apoptosis and prevents the loss of bcl-2 protein expression. Binding of B-CLL cells to stromal cells requires simultaneous action of beta1 and beta2 integrins. The interaction between B-CLL cells and other cell types seems important for their survival and may represent an important mechanism underlying accumulation of malignant cells in B-CLL patients.

Circadian and seasonal variations of hematopoiesis in cord blood.

Cord blood hematopoietic progenitors undergo circadian and seasonal variations. The lowest values are obtained between 4:00 and 12:00, as well as between May and August. This represents the first observation of such rhythms before birth.

Myeloid and lymphoid recovery following allogeneic bone marrow transplantation: a comparative study between related, unrelated bone marrow and allogeneic peripheral stem cell transplantation.

We studied myeloid and lymphoid recovery during a period of 12 months following HLA matched allogeneic bone marrow transplantation (BMT) in 15 patients. Patients were divided into three groups. Each group contained 5 patients according to the source of hematopoietic stem cell transplantation (HST): 1) related bone marrow transplantation (BMT), 2) allogeneic peripheral blood stem cell transplantation (PBSCT) and 3) matched unrelated donor transplantation (MUD). The rate and pattern of recovery of granulocytes, lymphocytes (T-cell subsets, B-cells, NK cells, subsets of CD45) were studied by cell counting and flow cytometry. Our results suggest faster recovery of PMN after PBSCT. Higher CD4 cell counts observed in the PBSCT group may have an impact on a lower incidence of opportunistic infections. Chronic GvHD mediated GvL effect seems to be more important in blood stem cell transplanted patients and this may have an influence on disease free survival.

TGF-beta activity and expression of its receptors in B-cell chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in Western countries and results from the accumulation of B-lymphocytes which are functionally abnormal and predominantly non-cycling in vivo. Consequently, it is important to understand why B-CLL cells accumulate in GO phase. Since TGF-beta is an important negative regulator of the immune system, a loss of responsiveness to this factor might provide a selective advantage to B-CLL cells. Here we review data on the role of TGF-beta in B-CLL. We show that the B-CLL cell response to TGF-beta signals is abnormal in vitro (inhibition of proliferation and induction of apoptosis). This lack of response of B-CLL cells to TGF-beta inhibition appears to be accompanied by a decrease or a loss of TGF-beta receptor expression.

Chronic lymphocytic leukemic B cells but not normal B cells are rescued from apoptosis by contact with normal bone marrow stromal cells.

The leukemic B lymphocytes from chronic lymphocytic leukemic (CLL) patients have a long survival in vivo, although ex vivo they rapidly die by apoptosis. To further investigate the mechanism of this, we have studied the influence of bone marrow stromal cells from normal subjects on apoptosis of B-CLL cells and normal umbilical cord blood (UCB) B lymphocytes. After 48 hours of incubation in medium alone, leukemic and normal B cells showed, respectively, 22 +/- 3% and 31 +/- 5% of apoptosis. Cocultures with stromal cells reduced the percentage of leukemic cells undergoing apoptosis (8 +/- 2%, P < . 0005) and prevented the loss of bcl-2 protein expression. In contrast, stromal cells slightly increased normal B-cell apoptosis (37 +/- 6%). Direct contact between leukemic cells and stromal cells was found to be essential for inhibition of leukemic cell apoptosis; indeed, separation of leukemic cells from stromal cells by microporous membrane increased spontaneous apoptosis, and comparable results were obtained with stromal cell conditioned medium. The difference in behavior observed between normal and leukemic B cells plated on stromal cells can be explained by the fact that only a few normal B cells adhere to stromal cells in comparison with B-CLL cells. B-CLL cell adhesion to stromal cells is mediated by beta1 and beta2 integrins acting simultaneously. Contact between B-CLL cells and bone marrow stromal cells seems to play a major role in the accumulation and survival of B-CLL cells in the bone marrow.

Heterogenous response of B lymphocytes to transforming growth factor-beta in B-cell chronic lymphocytic leukaemia: correlation with the expression of TGF-beta receptors.

We investigated the potential role of transforming growth factor-beta (TGF-beta) on spontaneous and cytokine-induced proliferation of B-cell chronic lymphocytic leukaemia (B-CLL) cells in vitro. Purified B lymphocytes from 21 B-CLL patients were cultured for 5 d in the presence of medium alone, IL-2 and/or IL-10, in the presence or absence of TGF-beta, and proliferation was measured by 3H-thymidine incorporation. TGF-beta inhibited B-cell proliferation in the majority of patients (15/21) but no inhibition was detected in 6/21 patients whatever the type of stimulant used. Addition of neutralizing antibodies to TGF-beta increased spontaneous and cytokine-induced proliferation; this effect was dose dependent and specific because addition of an irrelevant chicken IgG had no effect on B-CLL proliferation. In resistant patients, neutralizing antibodies to TGF-beta did not increase the proliferation. The expression of TGF-beta receptors on B-CLL cells was significantly lower than the one observed on normal CD5+ B lymphocytes for which the sensitivity to TGF-beta inhibition was more marked than in CLL. In addition, we found a strong correlation between the response of leukaemic B cells to TGF-beta inhibitory action and the expression of TGF-beta receptors on these cells. In summary, TGF-beta appears to function in CLL as a negative regulator of B lymphocytes but loss of responsiveness to this factor accompanied by a decrease of TGF-beta receptor expression, might provide a selective advantage to B-CLL lymphocytes.