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PURPOSE: To evaluate the safety, pharmacokinetics, and efficacy of three different dose levels of pegylated granulocyte colony-stimulating factor (Ro 25-8315) on progenitor cell mobilization and hematologic recovery in cancer patients. PATIENTS AND METHODS: Breast cancer patients (n = 36) were randomly assigned to receive before (part I) and after (part II) chemotherapy either a single-dose injection of Ro 25-8315 (20 μg/kg, n = 9; 60 μg/kg, n = 9; 100 μg/kg, n = 10) or a standard daily dose of filgrastim (part I, 10 μg/kg/d; part II, 5 μg/kg/d) (control group, n = 8). RESULTS: Overall, Ro 25-8315 was well tolerated. In part I, more progenitor cell mobilization was observed with Ro 25-8315 100 μg/kg. The peak of circulating CD34+ cells was obtained at day +5 in the four groups, and the absolute neutrophil count (ANC) returned to less than 20 × 109/L by day +15. In part II, high levels of circulating CD34+ cells (> 20 cells/μL) were obtained in all four groups. The chemotherapy-induced neutropenia (< 1 × 109/L) was similar in the four groups. Ro 25-8315 100 μg/kg was more effective than filgrastim in reducing the number of patients with an ANC less than 0.5 × 109/L on day +12 after chemotherapy. CONCLUSION: A single injection of Ro 25-8315 100 μg/kg might be the optimal dose for steady-state peripheral-blood progenitor cell mobilization. A single injection of 20, 60, or 100 μg/kg could be as efficient as daily administration of filgrastim to correct chemotherapy-induced cytopenia. The optimal dose of Ro 25-8315 should be determined according to the planned chemotherapy regimen.

PURPOSE To determine whether recombinant human interleukin-3 (rhIL-3) reduces bone marrow depression and improves chemotherapeutic schedule adherence in ovarian cancer patients receiving first-line combination chemotherapy. PATIENTS AND METHODS In a randomized multicenter study, 185 patients received carboplatin (dose based on projected area under the concentration-time curve [AUC]=4) and cyclophosphamide (750 mg/m2) day 1, every 3 weeks for six cycles. Patients were randomized to receive rhIL-3 (5 microg/kg) or placebo once daily subcutaneously on days 3 to 12. RESULTS Adherence to chemotherapeutic regimen, mean chemotherapy cycle length, tumor response rate, and median survival at 24 months did not differ between groups. The number of side effects-primarily allergic reactions, flu-like symptoms and fever-were higher in the rhIL-3 group, which resulted in 21 discontinuations compared with one in the placebo group. Compared with placebo, the rhIL-3 group had higher platelet counts day 1 of cycles 2 to 6. The number of patients with World Health Organization (WHO) grade IV thrombocytopenia or number of platelet transfusions did not differ. Leukocyte counts differed only in cycles 1 and 2 between groups. The leukocyte nadir occurred earlier in the rhIL-3 (day 12) than in the placebo group (day 15, P=.006). Leukocytes and neutrophils were only higher in the rhIL-3 group day 1 of cycle 2. In cycles 4 and 5, more patients with WHO grade IV neutropenia received rhIL-3 (P < .005). Eosinophil counts were higher day 1 of cycles 2 to 6 in the rhIL-3 group (P < .0001). CONCLUSION rhIL-3 had stimulatory hematopoietic effects. This did not result either in reduction of platelet transfusions or in improvement of chemotherapeutic schedule adherence. There were more side effects in the rhIL-3 group than in the placebo group. rhIL-3 at 5 microg/kg/d is, therefore, not of clinical benefit in this chemotherapeutic regimen.

The authors describe, in a female with rheumatoid arthritis (RA) treated by methotrexate (MTX), the occurence during walking of a left femoral shaft fracture. This middle third femoral fracture needed early intramedullary locked nailing and was due to a malignant B follicular large cell lymphoma. Despite CHOP chemotherapy and remedial treatment (mitoxanthrone-holoxan-VP16), bone lymphoma spread and death occurred within six months. Our case (the 25th published) poses problems: what part, in RA, does MTX play in the occurrence of non-Hodgkin's malignant lymphoma (NHML)? There is debate about the role of MTX because NHML is frequent in RA. However, cases of regressive NHML after MTX therapy cessation are disturbing. Possibly, MTX facilitates proliferation of B lymphocytes infected by Epstein-Barr virus (EBV) whereas EBV is present in both lymphoma cells and in usual RA. Thus, clinical and laboratory survey is required in cases of RA treated with MTX.

New isoforms of CD44 with alternatively spliced exons have recently been described. Expression of exon v6 seems to be of particular interest. It has indeed been associated with poorer outcome of breast cancer patients with node invasion at diagnosis. However, no data were available for patients N0M0 (with neither metastasis nor node invasion at diagnosis). Moreover, previous statistical analyses were realized using immunohistochemical methods to detect CD44v6 expression although several variants with exon v6 have been described. We investigated expression of isoforms containing CD44v6 using an RT-PCR approach and a panel of 25 normal breast specimens, 10 mammary fibroadenomas, 8 cystic samples and 52 primary breast tumors (38 invasive N0M0). Normal breasts, fibroadenomas, and cysts all express the same variant, A (with exon v6 only), while several transcripts are amplified in tumors. Expression of variants other than A correlates with acquisition of a malignant phenotype. Invasive cancers also express additional variants in comparison with in situ carcinomas. Metastasis capacities seem to be associated with transcription of variants other than A but also with no transcription of some of them, variants D (with exons v6 and v10) and L (with exons v6 to v10). Expression of variants D and L correlates with higher percentages of disease-free survival and better outcome. Expression of CD44 splice variants with exon v6, as detected by RT-PCR, might be a useful prognostic factor for breast cancer. However, since the series size is small, our results need to be confirmed by later studies on a larger number of patients.

PURPOSE The usual therapy of osteosarcoma is neoadjuvant chemotherapy, followed by surgery, then by postoperative chemotherapy. There is no prognostic factor to predict, at diagnosis, the histologic response and final outcome. Inactivation of the retinoblastoma-susceptibility gene RB is associated with the pathogenesis of several human cancers. In primary osteosarcomas, loss of heterozygosity (LOH) at the RB locus has been found in greater than 60% of cases. The aim of this study was to determine the potential early prognostic value of LOH of RB gene on the biopsy material at diagnosis. PATIENTS AND METHODS Forty-seven patients with primary osteosarcoma, treated in four French institutions, were studied. LOH was studied by polymerase chain reaction (PCR) of an informative RB DNA polymorphism. RESULTS Assessment of LOH at the RB gene could be completed on 34 heterozygous patients only. LOH was found in 24 cases (70%). The event-free survival (EFS) rate at 60 months is 100% for patients without LOH, 43% for all patients with RB LOH, and 65% for nonmetastatic patients with RB LOH. The difference in EFS is highly significant at P = .008 and P = .024, respectively. Histologic response after preoperative chemotherapy did not show significant correlation with LOH status. CONCLUSION RB gene LOH appears to be an early predictive feature for osteosarcomas that indicates a potential unfavorable outcome. RB LOH study might shortly help to identify high-risk patients earlier. If this is verified, therapy could then be adapted earlier to the individual's real risk of relapse.

Expression of CD44 isoforms has been investigated on normal breasts, fibroadenomas, cysts and breast cancers. Carcinomas express additional variants in comparison with normal breasts while fibroadenomas and cysts do not. Invasive cancers also express more variants than in situ carcinomas. Recent studies tend to demonstrate that overexpression of CD44 is not a survival prognostic factor whereas expression of exon 2v and/or 3v could be. Results for exons 4v to 10v are presently conflicting. Further studies will then be necessary to clarify the role of CD44 isoforms in mammary carcinogenesis and metastasis. Metastatic capacity could be linked with expression of additional variants but also with no transcription of variants associating exon 6v with either exon 10v or both exons 9 and 10v.

Resume L'effet benefique de la decontamination digestive sur la reduction de l'incidence de la maladie du greffon contre l'hote (GvHD) est generalement admis. Nous rapportons ici notre experience portant sur 40 greffes allogeniques successives realisees a partir de donneurs apparentes HLA identiques. La decontamination totale a ete effectuee avec isolement en isolateur plastique ou en chambre a flux laminaire, alimentation sterilisee et antibiotiques oraux non-absorbables. Trente-huit patients sont evaluables (2 deces precoces). Seuls 4 d'entre eux restaient porteurs de bacteries au niveau intestinal alors que 24 etaient porteurs de levures. La plupart des patients etaient porteurs de germes au niveau nasal ou oropharynge (staphylocoques : 20 patients, bacilles a Gram negatif : 4 patients, levures : 16 patients). Vingt-huit patients (74 %) ont presente une GvHD aigue. Une relation significative a ete etablie entre colonisation bacterienne nasale ou oropharyngee et GvHD (p

The antiemetic efficacy and safety of granisetron (40 μg/kg), a selective and potent 5-hydroxytryptamine (serotonin) antagonist, was compared with that of metoclopramide (7 mg/kg) plus dexamethasone (12 mg) in patients receiving fractionated chemotherapy. Patients receiving cisplatin at doses of at least 15 mg/m2 or etoposide at least 120 mg/m2 or ifosfamide at least 1.2 g/m2 on each of 5 consecutive days were eligible. A total of 143 patients received granisetron and 141 received the comparator regimen. The 5-day complete response rate (no vomiting, no worse than mild nausea) for granisetron (46.8%) was equivalent to that for metoclopramide plus dexamethasone (43.9%). The overall 5-day response profile was superior for granisetron (P=0.013) because of fewer failures in this group. The overall incidence of adverse experiences was significantly lower in the granisetron group (60.8% versus 77.3%,P=0.003). Headache and constipation, more prevalent in the granisetron group, are recognized side-effects of serotonin antagonists. Extrapyramidal syndrome, not seen in any granisetron patients, occurred in 20.6% of comparator patients (P

Proteins of the complement system (C2, C3) are synthesized by human monocytes and macrophages, thus providing an important local source of these proteins in vivo which serve as a first-line host defense mechanism. In this study, we investigated the production of complement components C2, C4, and C9 by human monocytes/macrophages and by the pathologic cells of acute monocytic leukemia which represent a source of immature monocytic precursors. Human blood monocytes were collected and purified by cytapheresis and elutriation and leukemic cells by Ficoll gradient. Secretion of complement components was measured by a hemolytic assay. The evaluation of the mRNAs of the various complement components in the cells was performed by polymerase chain reaction (PCR) by adding 32P labeled deoxycytidinetriphosphate (dCTP) to the amplification step. Functional C2 was found to increase during in vitro maturation of macrophages up to the fourth week of culture. C2 mRNA was detected after amplification and increased during the maturation. Interferon-gamma (IFN-gamma) mediated a marked increase of the C2 mRNA. We found a decrease in synthesis of C4 mRNA during in vitro differentiation of human monocytes. The effect of IFN-gamma resulted in an increase in C4 mRNA. C9 mRNA was not detected although it was detected in the HepG2 hepatoma-derived cell line. Functional C2 was not detected by leukemic cells after 24 h of culture but little functional C4 was present in the cell supernatants. As they were by human monocytes and macrophages, C2 and C4 mRNAs were detected after amplification but C9 mRNAs were not detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Sixty-four patients with non metastatic high risk breast cancer were randomized in a double blind trial of adjuvant immunotherapy with sodium dithiocarb (DDC) versus placebo. All patients underwent prior surgery (mammectomy according to Patey) then adjuvant FAC chemotherapy +/- DDC. With a median follow-up of 5 years we observed 6 relapses and 5 deaths in DDC group; 13 relapses and 12 deaths in control group. At 6 years, overall survival is 81% in DDC group versus 55%. Disease free survival (DFS) is 76% in DDC group versus 55%. DDC associated to chemotherapy and locoregional treatment can improve survival and probably DFS in this high risk breast cancer subgroup.

Human blood monocytes (Mo) and monocyte-derived macrophages (M psi) possess cytotoxic effects against tumor cell lines when appropriately stimulated by various biological response modifiers, e.g., gamma interferon (gamma IFN) and muramyltripeptide (MTP). Activated Mo/M psi represent a new tool for the treatment of human malignancies, termed "adoptive cellular immunotherapy". Activated Mo/M psi express tissue factor procoagulant activity (PCA), which is a physiological trigger of blood coagulation. PCA was evaluated in vitro using a modification of the one-stage recalcification clotting time, and hemostatic changes were studied in vivo in cancer patients. Nine patients with peritoneal carcinomatosis were injected intraperitoneally with activated Mo and 11 patients with non-small cell lung carcinomas were infused intravenously with activated M psi. Hemostatic changes were followed using activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen level, antithrombin III (ATIII) and protein C (PC) activities. Fibrinolytic activity was estimated by euglobulin lysis time and assays for plasminogen and fibrin/fibrinogen degradation products (FDP). These assays were performed before and after each autologous infusion and on days 2 and 3. Activated Mo and M psi expressed potent PCA (85.5 +/- 7.5 U/ml for MTP activated Mo and 50 +/- 5.3 U/ml for gamma IFN activated M psi suspensions). In both groups of patients, APTT, PT, and TT underwent no significant variations. There was no significant consumption of ATIII or PC, and fibrinolysis was not activated during the study period. In the group injected intraperitoneally with MTP-activated Mo, fibrinogen showed a significant and progressive increase in relation to the development of an inflammatory reaction, reaching a maximum average value of 6.1 g/l at the end of the therapy with a concomitant increase in FDP levels. This increase was not observed after intravenous therapy with gamma IFN-activated M psi. No patient suffered from hemorrhagic or thrombotic events. In our experience, repeated injections of activated Mo or M psi expressing potent tissue factor PCA did not induce significant in vivo activation of the coagulation system in cancer patients.

Summary A total of 178 patients with meta.static renal cell cancer were randomized to receive interferon alfa-2a (rIFN alfa-2a) or interferon alfa-2a+vinblastine (VLB). IFN alfa 2a was injected intramuscularly at a dose of 18 MIU 3 times a week and VLB was given intravenously at a dose of 0.1 mg/kg once every 3 weeks. The response rate was 11% for patients on monotherapy and 24% for those on combination treatment. The 5-year survival for 145 eligible patients was 9%, independently from the treatment arm. The performance status was significantly related to long-term prognosis, and 13% of the patients with performance status 0 were alive at 5 years, as compared to 6% and 0% for patients with a WHO grade of 1 and 2, respectively. The most frequent adverse events in both treatment arms were flu-like symptoms (95%), fatigue (70%) and gastrointestinal disturbances (68%). Leukopenia was observed more frequently with combination treatment (53%) than with IFN alfa-2a alone (30%). In conclusion, rIFN alfa-2a monotherapy at this dose and schedule has modest antitumor activity in metastatic renal cell cancer. The combination of rIFN alfa-2a+VLB results in a doubling of the response rate, but this does not translate into prolonged survival. Toxicity (except leukopenia) and tolerance were similar in both treatment arms.

In vitro differentiation of human monocytes (Mo) provides large amounts of mature and functionally competent macrophages (M phi) which may be used as potentially powerful anticancer agents for adoptive immunotherapy. Granulocyte macrophage-colony stimulating factor (GM-CSF) was evaluated for its ability to influence long term cultures of Mo-derived M phi. Large quantities of Mo isolated by leukapheresis and elutriation were cultured in non-adherent cell culture bags or in plastic flasks with or without GM-CSF. At various stages of differentiation, GM-CSF treated M phi were recovered and assayed for survival, morphology, surface antigens, functional properties and proliferation in comparison with control M phi. In the present paper, we demonstrate that GM-CSF at a concentration of 50 U/ml (5 ng/ml) promotes better cell survival and the differentiation of Mo into M phi displaying certain morphological differences as compared to control M phi such as an increased expression of Max-1 antigen, CR3 and Fc gamma II receptors, higher phagocytic properties and increased capacities of cytotoxicity and TNF secretion when the cells are further activated by IFN-gamma. Furthermore, GM-CSF treated cells exhibit a low-grade proliferation although the nature of the proliferating cells has not been entirely elucidated. We conclude that the GM-CSF treated M phi would be particularly suitable for adoptive immunotherapy.

Human blood monocytes (Mo) and monocyte-derived macrophages (MØ) are known to be potent antitumor cytotoxic effector cells through activation with recombinant human interferon gamma (rIFN-y), bacterial muramyldipeptide or the synthetic derivative muramyltripeptide phosphatidylethanolamine entrapped in liposomes (L-MTP-PE). Large-scale generation of ex vivo activated Mo from the blood of cancer patients proved feasible. We report our experience with a fixed rotor speed counterflow centrifugation elutration (CEE) procedure using the newly available Beckman high capacity JE-5.0 rotor system that reproducibly isolates up to 1.0-1.5×109 Mo with > 90% purity, in suspension and functionally intact derived from peripheral blood mononuclear cell-enriched suspensions obtained by leukapheresis (LP) from healthy volunteers and cancer patients. The semiclosed, easy to handle CCE system, was adapted to a sterile technique that permitted clinical trials in adoptive monocyte immunotherapy. Freshly isolated Mo did not lose morphological or functional integrity and had no spontaneous activation. Their abilities to become activated to the cytotoxic state after 18-h stimulation with 500 U/ml rIFN-γ or 1 μg/ml L-MTP-PE and to differentiate into matured MØ in vitro were not altered. The system was therefore used to isolate large numbers of Mo for a phase I clinical trial of intraperitoneal immunotherapy with L-MTP-PE activated autologous Mo in nine patients with peritoneal carcinomatosis. Each patient received weekly Mo infusions (n=5) with an intrapatient dose escalation schedule (from 107 to 109 Mo). Toxicities were mild including fever, chills and abdominal pain. There was no treatment-induced thromboembolic event or capillary leak syndrome. The degree of Mo activation in vitro was shown by the release of procoagulant activity and monokines (IL-1, TN Fa and IL-6) in the culture supernatants and the antitumor activity against U937 cells and human ovarian carcinoma cells in vitro and in tumor-bearing nude mice in vivo. The in vivo immunomodulatory activity of i.p. infused Mo was demonstrated by an elevation of WBC and granulocyte counts, fibrinogen, C-reactive protein and neopterin in peripheral blood and generation of IL-1, TFNa and IL-6 in peritoneal fluid. Phase II clinical trials, now in progress, should further define the therapeutic potential of human adoptive cellular immunotherapy using Mo or MØ.

Les auteurs rapportent une observation de myopathie hypertrophique hypothyroïdienne consécutive à une radiothérapie en mantelet pour maladie de Hodgkin. L'élévation de la TSH est un phénomène fréquent après irradiation en mantelet et justifie un suivi prolongé de la fonction thyroïdienne de ces patients en raison du risque d'hypothyroïdie patente et peut-être de cancer. L'âge des patients, la lymphographie précédant l'irradiation, la chimiothérapie associée sont des facteurs influençant l'incidence des dysthyroïdies mais leur importance respective ne fait pas l'unanimité. Les myopathies hypertrophiques de l'hypothyroïdie sont rares; la présentation clinique spectaculaire contraste avec la quasi-normalité de l'histologie musculaire. Les altérations du métabolisme énergétique et les modifications des propriétés de la myosine consécutives au déficit hormonal rendent compte de la faiblesse musculaire de ces patients; par contre le mécanisme de l'hypertrophie reste discuté, l'hypothèse d'une augmentation du nombre des myotubes paraissant toutefois la plus probable. Après irradiation, en particulier pour maladie de Hodgkin, la fréquence de l'hypothyroïdie nécessite un suivi biologique régulier et systématique. Le traitement substitutif doit être instauré en cas d'élévation de la TSH basale, même si le taux de T4 est normal.

To evaluate the safety of on-line plasma perfusion over protein-A sepharose and the therapeutic advantage of combining plasma perfusion (PP) over protein-A sepharose with 5-fluorouracil (5-FU) chemotherapy in patients with metastatic colorectal carcinoma (MCRC), thirty patients were randomized after surgery of primary CRC to receive a combination of 5-FU and PP over protein-A sepharose (group A), or a combination of 5-FU and PP over sepharose (group B), or 5-FU alone (group C). Bi-weekly on-line PP over 200 ml protein-A sepharose gel (group A) or 200 ml sepharose gel (group B) were performed with a Cobe 2997 blood cell separator for a maximum of 19 treatments per patient. 5-FU was given at 1000 mg/m2/d on days 1-5 of a 4-weekly cycle until progression. PP was well tolerated and no severe or life-threatening toxicity was observed. Mild clinical side-effects consisted of fever and chills (36% in group A, 23% in group B). The most common biological effects of PP over protein-A sepharose were significant drops in IgG (66% of pre-PP values), CH50 and C3 (73% of pre-PP values) and a significant generation of C3a and C5a anaphylatoxins. Tumor response rates were 40% for group A, 0% for group B and 20% for group C. The median survival times tended to be longer in group A (17 months) than in group B (10 months) and in group C (9 months). This is the first randomized trial showing some therapeutic advantage in combining PP over protein-A sepharose with conventional chemotherapy in MCRC.

In order to analyze the kinetics of complement component synthesis by human monocytes/macrophages, we have developed a system of defined culture conditions in the absence of serum. Moreover, the use of the polymerase chain reaction (PCR) provides a high sensitivity for the detection of mRNAs and the study of the regulation of complement component synthesis by these cells. Human blood monocytes were collected and purified by cytapheresis and elutriation, and then cultured in nonadherent cell culture bags for up to 3 weeks. Cells were grown in Iscove's modified Dulbecco's medium supplemented with alpha-phosphatidylcholine, transferrin, insulin, glutamine and antibiotics. The phenotypic and functional properties of macrophages differentiated under these serum-free culture conditions have been previously analyzed [1]. Secretion of complement component was measured by a hemolytic assay. mRNA was prepared using guanidine isothiocyanate extraction followed by cesium gradient ultra-centrifugation. cDNA was obtained by reverse transcription, then amplified by PCR. Fresh monocytes did not display any secretion of C2 as measured by hemolytic assay. However, C2 secretion was detected on and after the 3rd day of serum-free cultured unstimulated monocytes. The rate of C2 production increased along with macrophage differentiation up to the 3rd week of culture. We were not able to detect any functional C4 secretion. In preliminary studies, C2 and C4 mRNAs were detected in macrophages and in unstimulated fresh monocytes. These preliminary studies show that the serum-free culture conditions we have developed allow very satisfactory survival and differentiation of human monocytes, and provide optimal conditions for the study of their secretory activity.

Resume Un malade presente une aplasie medullaire idiopathique, rebelle a tout traitement, et developpe, sous triple association d'antibiotiques, une pneumopathie infectieuse febrile non etiquetee. Un lavage broncho-alveolaire (LBA) est effectue et le patient est mis sous antifongiques. La culture du LBA permet le diagnostic de Cunninghamella bertholletiae des la 24eme heure. Malgre le diagnostic precoce et un traitement approprie le malade decede. L'examen histologique post-mortem confirmera l'envahissement mycosique de la trachee, des poumons, de la rate, de l'estomac et du pancreas. Cette observation est a notre connaissance la premiere dissemination generalisee consecutive a une primoinvasion pulmonaire par C. bertholletiae decrite en Europe. Le cas est compare aux 14 autres cas decrits dans le monde.

To elucidate the potential mechanisms of anemia induced by cisplatin (CDDP) we have evaluated hemolysis, dyserythropoiesis, ferrokinetics and cytotoxicity on erythroid progenitors in 12 patients treated by a CDDP-containing combination chemotherapy and in 6 patients treated by a similar combination but without CDDP. Eight patients, from the CDDP treated group, experienced a pronounced anemia. None had signs of hemolysis. Ferrokinetic study showed a very deep and protracted decrease of 59Fe incorporation during the chemotherapy cycle and the following 2 weeks. These results, along with a normal medullary erythroblastic cellularity, suggest that CDDP induces a deep but transient erythropoiesis alteration leading to anemia in some cases.

Resume L'incidence des infections a staphylocoques coagulase-negative est en forte augmentation dans les services d'hematologie depuis environ dix ans. La principale cause semble en etre l'usage de plus en plus frequent de voies veineuses centrales. Les autres facteurs exposant aux infections staphylococciques sont la neutropenie, l'hospitalisation souvent prolongee et surtout les lesions de mucite induite par la chimiotherapie et les infections herpetiques. Les infections a staphylocoques coagulase-negative sont cependant rarement fatales. Il ne parait pas indispensable de proposer l'introduction d'un glycopeptide anti-staphylococcique dans le traitement empirique des infections de tous les malades neutropeniques. Il est legitime de limiter cette attitude therapeutique aux patients les plus exposes au risque staphylococcique : les leucemiques et les transplantes medullaires.

To evaluate the advantage with regard to toxicity, response rate, time to progression and survival of combination chemoimmunotherapy over single-agent chemotherapy in patients with metastatic colorectal carcinoma (CRC), 30 patients were randomized to receive a combination of 5-fluorouracil (5-FU) by continuous i.v. infusion and plasma perfusion (PP) over protein A-Sepharose (group A), or a combination of 5-FU and PP over sepharose (group B) or 5-FU alone (group C). 5-FU was given at 1,000 mg/m2/d on days 1-5 of a 4-weekly cycle until progression. Patients of groups A and B received bi-weekly on-line PPs until disease progression or for a maximum of 19 treatments. PP was well tolerated and no severe or life-threatening toxicity was observed. The response rates were 10% for the group A (1 PR), 0% for the group B and 20% for the group C (1 CR + 1 PR). The times to tumor progression for patients in groups A and C were 22 months, 12 and 11 months, respectively and the median survival times were 17 months, 10 months and 9 months. Although the time to progression and survival tended to be higher in patients treated with protein A. PP, these differences were not statistically significant. This is the first report of a randomized trial showing some therapeutic advantage in combining protein A. PP with 5-FU in CRC patients. Further randomized studies are required to demonstrate the real true value of this chemoimmunotherapeutic approach.

Despite high response rates with platinum-based front-line chemotherapy, the prognosis for advanced ovarian carcinoma (AOC) is poor. Salvage chemotherapy for recurrent AOC was of little benefit before paclitaxel as single-agent therapy showed appreciable efficacy. Anthracyclines are effective, but are not often part of first-line therapy. In this pilot study, we investigated the feasibility of an anthracycline plus paclitaxel combination therapy for recurrent AOC. Twenty-four patients received 150 mg/m 2 paclitaxel on day 1, with either 50 mg/m 2 doxorubicin on day 1 or 75 mg/m 2 epirubicin on day 1 every 3 weeks. A 27% overall response rate was obtained. Myelosuppression was the major toxicity, but was manageable. No myocardiac toxicity was observed. We conclude that paclitaxel–anthracyclines is a promising salvage combination therapy in AOC that should be investigated further.

To evaluate the results, prognostic factors and especially side-effects of the treatment for subdiaphragmatic Hodgkin's disease (SHD) a retrospective study was conducted in the Haematology Departments and in the Cancer Centres of Nancy and Strasbourg between 1976 and 1990; 55 patients corresponding to the IA to IIB SHD stages were analysed. The median age was 45 years. In accordance with Ann Arbor classification, we observed 12 CS IA (21.3%), 2 CS IB (3.5%), 14 CS IIA (25.4%) and 27 CS IIB (49.7%). Twenty-five patients (45.4%) underwent laparotomy with spleen involvement in 10 cases. Fifteen patients (27.3%) had exclusive radiotherapy, 10 by inverted-Y field with or without splenic field, 5 by limited field to inguinal and homolateral iliac nodes. Forty patients had prior chemotherapy, 18 by MOPP protocol, 18 by hybrid MOPP/ABVD protocol and 4 by other schemes. The total dose delivered ranged from 26 to 45 Gy. With a median follow-up of 8 years, the overall and disease specific survival rates are respectively 61% and 83% at 10 years. Nine patients relapsed (16.4%), 4 among the 15 (26.6%) treated by exclusive irradiation and 5 among the 40 (12.5%) treated by combined therapy. We observed 8.3%, 21.4% and 18.5% of relapses respectively among the clinical stages IA, IIA and IIB. Eleven patients (20%) developed a second cancer. Twenty-six long-term complications were noted, nine of which concerned the digestive system. The only significant prognostic factor is age, with 10-year specific survival rates of 96% and 66% respectively for patients younger and older than 50 years (p=0.0003). Our data confirm that the most appropriate treatment for stage IA is exclusive radiotherapy and combined therapy for all other cases. With the use of CT-scan and eventually lymphography, the laparotomy is reserved only for cases with an uncertain diagnosis. Tobacco use is also clearly a risk factor in our series for late vascular complications and second cancers.

Among the different strategies which have been developed for immunotherapy of cancer, adoptive immunotherapy uses leucocytes activated in vitro and reinfused into the patients. Five leucocytes subsets can be employed for this immunotherapy with activated autologous cells. Blood monocytes can be isolated in high purity and large numbers and under special culture conditions differentiated into macrophages for adoptive transfer. Once activated ex vivo, these cells display very high antibody dependent and independent specific cytotoxicity for tumour cells, are capable of phagocytosis of cancer cells and, as antigen presenting, cells are able to involve CTL in the anticancer response. As the understanding of this activation to cytotoxicity is only recent, the present paper first provides a literature review of the main points in the field. Our own results are then discussed in relation to the development of a clinical protocol for adoptive transfer of MAK (macrophage activated killer) cells, a therapeutic strategy having a pivotal role in the immunosurveillance of cancer.

We report two patients suffering from carcinomatous meningitis who were treated by continuous intrathecal perfusion of methotrexate.

We analyzed the representation of CD44 isoforms with both exons v9 and v10 among CD44 total amount and also examined correlation between their expression, clinical parameters and survival. We used a semi-quantitative RT-PCR reaction and a panel of 25 normal breast specimens, 10 mammary fibroadenomas, 8 cystic samples and 52 primary breast tumors. CD44 expression level was statistically higher in malignant tumors than in normal breast tissues (p = 0.038) or in fibroadenomas (p = 0.047) and correlated with histological grading, p = 0.047. Ratios CD44 variants with both exons v9 and v10/ total CD44 were similar in normal breast tissues and fibroadenomas but lower in the cystic samples. In primary N(-)M(-) breast tumors, unfavourable outcome and relapse were correlated with low ratios.

Breast cancer is the most common female malignancy affecting approximately one woman in eight. Many attempts have been made to define markers which may have potential clinical applications in diagnosis as well as therapy. New isoforms of CD44 with alternative spliced exons have recently been described. We studied the expression of CD44 exon 6 using a semi-quantitative RT-PCR reaction on a panel of 25 normal breast specimens, 10 mammary fibroadenomas, eight cystic samples and 52 primary breast tumors. Significant correlation was found between CD44 exon 6 expression and the overall survival of the N-M-population, P = 0.032, (logrank test by Mantel's method). The same result was also observed for the disease-free survival, P = 0.000002 (logrank test by Mantel's method). CD44 exon 6 expression, as detected by our RT-PCR-based method, might be a useful prognostic indicator of metastasis in breast cancer. However, these preliminary results need to be confirmed by later retrospective and prospective studies on a larger number of patients.

Summary Background In 1989, S. Wadler reported very promising results (76% response rate) with a combination of 5-fluorouracil (5-FU) plus α-2a interferon (IFN) in the treatment of metastatic colorectal carcinoma (MCRC). in vitro, there are several potential explanations for synergism between the two agents. We therefore decided in 1989 to start a randomized study comparing 5-FU alone with 5-FU plus IFN. Patients and methods 105 non-pretreated patients with measurable metastatic colorectal carcinoma entered into this study. The patients were randomly allocated either in arm A (n = 49) with 5-FU: 750 mg/m2 i.v. CI d1-d5 followed by 750 mg/m2 iv. bolus once a week, or in arm B (n = 56) with 5-FU as in arm A plus IFN 9 × 106 IU sub-cutaneously three times a week. Results After two months of treatment we observed 1 CR and 2 PR in arm A (response rate 6.1%), 3 CR and 8 PR in arm B (response rate 19.6%), i.e., a significant difference (P = 0.05). Event-free survival was significantly higher in arm B (6 months) than in arm A (2 months) (P

Twenty three patients with relapsing (n = 11) or refractory (n = 12) non-Hodgkin's lymphoma (NHL) to one or two prior anthracycline based combination chemotherapy regimens were treated as second or third line regimen with 3 induction cycles of Idarubicin (IDA) (7 mg/m2/d i.v. d1-d3) and high dose cytarabine (HD Ara-C) (1 g/m2/12 h i.v. d1-d3), each cycle was repeated every 3 weeks. Responding patients received a maintenance therapy with monthly cycles of IDA: 15 mg/m2 d1-d3, Etoposide 100 mg/m2 d1-d3, both by oral route. Twenty two patients are evaluable and we observed 13 CR and 1 PR with an overall response rate of 61% (14/23: 95% Cl = 38.5% 80.3%). The median time to progression was 32 months (6.5 - 63 + m.). The response rate to IDA-HD Ara C was not different for patients with (n = 14) or without (n = 9) objective response to the last prior therapy. The main toxicity was hematological: all patients experienced grade 4 neutropenia and 22 patients had grade 4 thrombopenia, but there were no toxic deaths. IDA and HD-Ara-C combination is highly effective in refractory or relapsed. NHL. As hematological toxicity was the limiting factor for further escalation of dose-intensity, further studies might include hematopoietic growth factors support in the therapeutic scheme.

We report our experience of CBDCA-CPM combination chemotherapy as first line therapy in 101 ovarian cancers. The therapeutic scheme was: initial cytoreductive surgery followed by six chemotherapy cycles (CBDCA 400 mg/m2/d IV dl, CPM 600 mg/m2/d IV dl, dl = d21) and second-look laparotomy. The initial stages were four Ic, three IIa, four IIb, four IIc, 15 IIIa, 28 IIIb, 23 IIIc and 20 IV. After initial surgery, there were 39 macroscopic residual diseases superior to 2 cm, 26 macroscopic residual diseases inferior or equal to 2 cm, four microscopic diseases and no residual disease in 30 cases (unknown in two cases). The overall response rate to chemotherapy was 83% with 56% histologic complete response rate. The main toxicity was haematological with 60% of leucopenia grade III-IV, 52% of thrombopenia grade III-IV. Age at diagnosis, residual disease after first look surgery and length of CA 125 normalization were significant prognostic factors for survival in this series.

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.

E-cadherin (Epithelial-cadherin) is a subclass of the cadherin family that plays a major role in the maintenance of intercellular junctions in epithelial tissues. E-cadherin is also involved in the interactions between epithelial cells and T lymphocytes. In order to explore the relationship between E-cadherin expression, cancer invasion and metastases in vivo, we estimated its expression in normal breast specimens, fibroadenomas, cystic samples and primary breast carcinomas using a semiquantitative reverse transcription-polymerase chain reaction. The relationship between E-cadherin expression, survival and disease-free survival was also investigated. In comparison with normal breasts, 70% of the primary tumors showed reduced expression of E-cadherin suggesting that downregulation of this cell adhesion molecule is a common event in breast carcinoma. Significant correlation was found between E-cadherin expression and the histological classification. Most of the advanced tumors grades (10/13 tumors with grade III) presented decreased E-cadherin expression. No correlation was found between E-cadherin expression, estrogen and progesteron receptors, age and menopausal status at diagnosis. However, disease-free and overall survival was associated with E-cadherin expression. Patients showing poorly expressed E-cadherin in tumor tissue had a worse prognosis. The same results were observed for women without lymph node invasion or metastasis at diagnosis even when they were grouped according to their histological grade for statistical analysis. Therefore, E-cadherin mRNA expression in invasive breast carcinomas might be an early prognostic factor of metastasis.

Hodgkin's disease, non-Hodgkin lymphoma or leukemia occurring in a pregnant patient is a rare event. Diagnosis of hematologic malignancies in a pregnant woman usually poses a quandrum of psychological, ethical and medical considerations. All of these situations require a multidisciplinary team. Adequate staging and treatment of hematologic malignancies are complicated by the pregnancy. In the first trimester, termination of the pregnancy should be considered in order to provide the best outcome for the woman and fetus. In this review we have focused on the use of chemotherapy for the pregnant patient. This review reveals that the use of chemotherapy resulted in both objective disease remissions and the subsequent delivery of normal infant.

Hb Puttelange [beta 140(H18)Ala--Val] was found as a de novo mutation in two siblings of a French family suffering from polycythemia. Both parents were phenotypically normal and exclusion of paternity has been ruled out by the study of several polymorphic markers located on different chromosomes. The structural modification of Hb Puttelange was established by reversed-phase HPLC analysis of the tryptic digest of the abnormal chain. The amino acid composition of an abnormal beta T14 peptide revealed that one of the four residues of Ala was replaced by a Val. Tandem mass spectrometry demonstrated that the substitution concerned position beta 140 (H18). This hemoglobin displays an increased oxygen affinity that is responsible for the polycythemia. De novo mutations, as demonstrated again in the case of this variant, have the highest probabilities of detection when they lead to pathological manifestations. They may result either from a somatic mutation in a very early stage of the embryological development of the propositus or may have a parental origin with occurrence of a germline mosaicism. The study of the beta-globin gene indicated that this case of Hb Puttelange probably arose from a mutation affecting a part of the germline of the father, therefore leading to a true recurrence risk.

The efficacy of gastrointestinal decontamination in reducing the incidence of severe bacterial or fungal infections and of moderate to severe acute graft-versus-host disease (GVHD) has been suggested. We report here a retrospective study of 71 patients grafted consecutively in our institution with bone marrow from HLA genotypically identical siblings. Complete decontamination (plastic isolator or laminar airflow room, sterile nursing and oral antimicrobial drugs) was carried out in all patients. Sixty eight patients were evaluable. Only six patients had aerobic Gram negative rods or anaerobic bacteria in their faeces and 44 of 68 (65%) had yeasts in their faeces. Most patients had oropharyngeal and/or nasal colonisation with bacteria (Gram positive cocci: 39 patients (57%); Gram negative rods: 13 patients (19%)) or yeasts (29 patients (43%)). Thirty nine patients (57%) experienced severe gradeor = II acute GVHD (grade II-IV). A significant relation was found between bacterial oropharyngeal or nasal colonisation and GVHD (P0.01) but not between gastrointestinal microflora and GVHD, whatever microorganisms were considered (bacteria, yeasts).

Background: Extracorporeal photochemotherapy (EP) is a new immune-modulating therapy which combines cytapheresis and extracorporeal irradiation of leukocytes by UVA. Recently, patients with systemic sclerosis (SS) have been treated by this method, but the efficacy of EP is still controversial. Objective: Our purpose was to evaluate the efficacy and safety of EP in SS and in severe morphea. Methods: In an open study, 7 patients with SS and 2 patients with severe morphea were treated by EP and were evaluated after 6 months of treatment. No other treatments were allowed during this period. Results: Except one vasovagal reaction, EP was well tolerated in all patients. The surface of the cutaneous sclerosis was unchanged in 3 of the 7 patients with SS, whereas it was aggravated in the remaining 4 patients. There was no improvement of the visceral involvement of SS in these patients. Although the duration of SS was less than 4 years in 6 out of 7 patients, we did not observe any significant benefit from EP in SS patients. One patient with generalized morphea had stopped EP after 3 months because of loss of vascular access and could not be evaluated. In the last patient with linear morphea of the upper limbs, a reduction in the number of morphea plaques was observed, and the remaining lesions were less visible after 16 months of EP. Conclusions: The present results do not corroborate those previously published with regard to the efficiency of EP in SS of recent onset. The efficacy of ECP in morphea needs further confirmation.

PTL after solid organ transplantation are a serious complication occuring in 1–18% of patients. Between 1978 and february 1994, 1000 transplantations were performed and eleven secondary lymphoproliferative disorders were observed (1.1%). Seven of them were high-grade lymphoma and required chemotherapy.

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.

Multidrug Resistant (MDR) plays a major role in chemoresistance. Alpha Interferon (IFN) and all trans retinoic acid (ATRA) have antiproliferative effect and IFN regulates several genes, some of them implicated in the regulation of MDR gene expression. We have studied the modulations of adriamycin cytotoxicity by IFN and/or ATRA on K 562 (MDR-) and resistant to adriamycin K 562 adri (MDR+) cell lines. We observed an important increase of adriamycin cytotoxicity on both K 562 and K 562 adri by low dose of IFN and ATRA. Studies of MDR gene expression shows an increase in K 562 adri after exposure to IFN or ATRA. So the observed effect is not due to a down regulation of MDR gene expression but probably to the own antiproliferative effect of IFN and ATRA in combination with the cytotoxicity of adriamycin.

The tetrapeptide AcSDKP (Ser-Asp-Lys-Pro) is a reversible inhibitor of normal human hematopoietic progenitor growth. In this paper, we report that preincubation of bone-marrow mononuclear cells (MNC) with AcSDKP at 10(-10) M for 20 hours protects the granulocyte-macrophage colony-forming unit (CFU-GM) progenitors against photofrin II-mediated phototherapy. This protective effect was observed after short-term exposure to photofrin (2.5 micrograms/mL) and irradiation by high-energy doses at 514 nm. Nevertheless, AcSD-KP, which has no effect on leukemic cell proliferation, does not protect the HL-60 and K-562 leukemic cell lines against photosensitization.

The increasing interest in mononuclear phagocytes for adoptive cellular immunotherapy (ACI) trials in cancer patients led us to define a procedural approach to harvest reproducibly highly purified single-cell suspensions of large numbers of functional human circulating blood monocytes (Mo). A semiclosed counterflow centrifugal elutriation (CCE) system has been developed, using a new large capacity Beckman JE 5.0 rotor with one interchangeable 40 ml or 5 ml separation chamber, to purify Mo from mononuclear cell (MNC) concentrates of healthy donors and cancer patients obtained by continuous flow centrifugation leukapheresis (CFCL). This method does not require a Ficoll density gradient centrifugation step. A total of 115 leukapheresis procedures were carried out in 35 patients and in 30 healthy donors by either Cobe 2997 or Cobe Spectra, with a similar efficiency in MNC apheresis. The average yield per leukapheresis procedure was 5.6 x 10(9) MNC of purity 90-100% (25-45% Mo, 40-65% lymphocytes). The average yields per elutriation procedure (R/O fraction) were 1.1 x 10(9) cells (purity 93% Mo) using the 5 ml separation chamber, and 1.5 x 10(9) cells (purity 91%) using the 40 ml separation chamber, with a respective recovery of 82 +/- 7% and 78 +/- 8% Mo. In vitro analysis of the viability and function of the purified Mo shows that neither morphological integrity nor physiological activity was compromised by this two-step isolation procedure, which additionally provides highly purified human Mo suspensions, in a quantity suitable for ACl of cancer patients.

The objective of this phase II, multicenter, prospective study was to evaluate the safety and the response rate of paclitaxel administered to patients with ovarian cancer. Eligible patients had histologically proven ovarian carcinoma and measurable or evaluable disease and had received at least one platinum-containing regimen and no more than three prior chemotherapy regimens. Paclitaxel was given as a continuous intravenous (i.v.) infusion over 3 hours every 3 weeks. Dose was determined by the number of prior chemotherapy regimens. Patients with one or two prior chemotherapy regimens received 175 mg/m2 (group A) and patients with three prior chemotherapy regimens 135 mg/m2 paclitaxel (group B) after premedication. Treatment was repeated every 3 weeks. An interim safety analysis for the first 99 evaluable patients, 72 treated with 175 mg/m2 and 27 treated with 135 mg/m2 paclitaxel, is reported here. Median number of courses analyzed per patient was four (range, 1 to 6) in the two groups, for a total of 393 courses evaluable for toxicity (286 in group A, 107 in group B). World Health Organization (WHO) grade 3-4 neutropenia was observed in 29.6% of courses in group A and in 23.3% in group B. Two group A patients, 1 at courses 3 and 4 and 1 at course 6, experienced WHO grade 2 fever or infection associated with a WHO grade 4 neutropenia.(ABSTRACT TRUNCATED AT 250 WORDS)

Differentiation of the U937 cell line can be induced by various agents. We have investigated the differentiation abilities of gamma-interferon (IFN-gamma), interleukin-6 (IL-6), macrophage and granulocyte-macrophage colony-stimulating factors (M-CSF + GM-CSF) or the combinations IL-6 + M-CSF + GM-CSF and IFN-gamma + M-CSF + GM-CSF on the U937 cells by studying the morphology, cytochemical activity and several functional properties. The expression of the Leu-CAM proteins (CD11a, CD11b, CD11c, CD18) was also evaluated during the culture period. Our results show that the cytokines used in this study inhibit to a certain extent the proliferation of the tumor cells and drive the cells toward a differentiation phenotype that has several characteristics in common with mononuclear phagocytes, such as the expression of CD14, phagocytosis and release of superoxide anions. The adhesion molecules CD11b alpha chain and CD18 beta chain were strongly induced on the U937 cells with a maximal expression of the CD11b on the cells cultured with either M-CSF + GM-CSF or the combinations of IL-6 and IFN-gamma with M-CSF + GM-CSF. Conversely, for CD11a and CD11c alpha chains a rather low enhancement of the expression was noticed. In our culture system, cells incubated with the combination of M-CSF + GM-CSF exhibited differentiation characteristics which appeared to be largely potentialized when cytokines such as IL-6 or IFN-gamma were added.

We assessed the efficacy and tolerability of VIM (etoposide/ifosfamide/methotrexate) combination therapy in 24 patients who were failing the treatment protocol of the Lymphomes Non Hodgkiniens (LNH) 84 study. Eight patients were refractory to the LNH 84 induction cycles, but ten achieved a partial response (PR). The six remaining patients attained complete response (CR) after LNH 84 induction, but relapsed either during consolidation therapy or after completing the whole program. Twenty-three patients are evaluable for response. The VIM regimen provided a CR rate of 43% and a PR rate of 17%. Treatment failed in nine cases (39%). The CR rate was particularly high (67%) in the group of patients who had PR with LNH 84 induction treatment. Of the ten who had attained CR, five relapsed after 4 to 42 months and five are still alive with no evidence of disease after 29 to 62 months. VIM therapy was well tolerated. A total of 101 VIM courses were given. Myelotoxicity was the most common side effect. Grade 3 or 4 cytopenia was recorded after 11% of the cycles. Among eight infectious episodes recorded, one was fatal. This study demonstrates that CR and long disease-free survival are obtainable with the VIM regimen in a small number of patients failing a high-dose doxorubicin-containing first-line treatment.

In vivo, circulating blood monocytes (Mo) migrate into the various tissues where they undergo terminal maturation into macrophages (M phi) with morphological and sometimes functional properties that are characteristic for the tissue in which they reside. This tissue-specific M phi heterogeneity results from the immediate microenvironment, but may also originate from genetically distinct Mo subpopulations. The in vitro transformation of Mo to M phi is thought to reflect the events of the in vivo maturation and thus is widely used as a model to analyse M phi development. To study the heterogeneity within the mononuclear phagocyte system, we have investigated the phenotypic characterisation of mature tissue M phi, blood Mo and Mo-derived M phi cultured in medium with either GM-CSF, M-CSF or a combination of both cytokines. Tissue peritoneal and alveolar M phi showed different antigenic specificities, particularly concerning the transferrin receptor and CD68 and CD14 antigens. M-CSF-derived M phi when compared to the other M ø populations also exhibited a significantly increased expression of transferrin receptor and CD68 antigen. In contrast, GM-CSF treated cells which exhibited a better long term survival, showed notably more positivity for CD11b and CD32 antigens. These results show that the phenotypic heterogeneity of the different M phi populations is limited and appears to result from discrepancies in the differentiation and/or activation of the cells. The location of the CD68 antigen, which is generally considered to be an intracellular protein, was investigated at the ultrastructural level and found to be exclusively situated at the outer cell membrane.

Large quantities of human blood-derived monocytes have been cultured in suspension in nonadherent cell culture bags and maintained for up to 3 weeks in a serum-free medium. This serum-free medium contained Iscove's modified Dulbecco's medium (IMDM) supplemented with human albumin, alpha-phosphatidylcholine, transferrin, and insulin. Morphology, cell surface antigens, and functional properties of these in vitro maturing macrophages were studied in comparison with macrophages cultured in a standard medium containing 10% fetal calf serum. In this report we demonstrate that this serum-free medium allows a better yield of cell survival than the standard medium; it also allows the differentiation of blood monocytes into fully functional macrophagic cells that express the different antigens found in mature macrophages. The results indicate that the use of serum-free defined medium offers good conditions in which to culture large numbers of human monocytes and allows an accurate analysis of the effect of supplementation with growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) on the differentiation and survival of monocytes and macrophages. Serum-free cultures could also be helpful for the precise analysis of the cell secretion activity and for determining the factors that are responsible for monocyte maturation into macrophages.