One-year immunological evaluation of chronic hemodialysis in end-stage renal disease patients.

BACKGROUND: Much research has been devoted to the determination of acute leukocyte activation as well as acute cytokines production during and after blood hemodialysis membrane interaction. In contrast, few studies deal with chronic immunological evaluation of T-cell activation markers in hemodialysis. METHODS: We evaluated different immune parameters using a modified cellulose low-flux hemophan vs. synthetic high-flux polyamide membrane during 1 year in 35 stable chronic hemodialysis patients. Leukocyte counts, lymphocyte subpopulations, T-cell activation markers (CD69, CD25, HLA-DR, CD54, CD62L, CD45RO, CD11a, CD28), complement-activation products (C3a) and serum elastase were measured at 0, 3, 6 and 12 months in the two patient groups and compared to 13 healthy control subjects. RESULTS: Over dialysis time, all patients showed a significant level elevation of CD69/CD3 (p < 0.005) and CD25/ CD3 (p < 0.005) phenotypes. In contrast, HLA-DR and CD45RO remained unchanged suggesting a truncated pattern of activation. T lymphocyte subset analysis showed in both hemodialyzed groups a significant decrease in the expression of CD54 (ICAM-1) when compared to controls (p < 0.005). C3a and elastase measurements showed a significant upward trend with dialysis time in both hemodialyzed groups. CONCLUSION: Although the immunological changes seen in chronic hemodialyzed patients must be interpreted in conjunction with their basal uremic states and the membrane permeability properties, our study suggests that 1-year immunological evaluation of hemodialysis membranes biocompatibility is associated with changes in the pattern of chronic T-cell activation, which is in part related to the use of a particular membrane type. Moreover, some key molecules (CD54) are affected in patients with end-stage renal disease undergoing hemodialysis.

Characterization of human fibroleukin, a fibrinogen-like protein secreted by T lymphocytes.

We have recently cloned the human homologue of the murine pT49 cDNA (hpT49h), a transcript encoding a protein homologous to the beta- and gamma-chains of fibrinogen. Here, we report the identification of the hpT49h gene product using mAbs generated against a peptide corresponding to the carboxyl-terminal end of the deduced protein and a recombinant protein fragment expressed in Escherichia coli. mAbs 23A6, 7B12, and 3F4 specifically recognized a protein of 70 kDa in reducing SDS-PAGE in the culture supernatant of 293T cells transiently transfected with the full length hpT49h cDNA and freshly isolated PBMC. Under nonreducing conditions, the material migrated with a molecular mass of 250 to 300 kDa, indicating that the 70-kDa protein forms a disulfide bonded complex. Because of its homology with fibrinogen, we have termed this protein fibroleukin. Fibroleukin is spontaneously secreted in vitro by freshly isolated CD4+ and CD8+ T lymphocytes. RT-PCR analysis revealed preferential expression of fibroleukin mRNA in memory T lymphocytes (CD3+/CD45R0+) compared with naive T lymphocytes (CD3+/CD45RA+). Fibroleukin production by PBMC was rapidly lost in culture. Production could be partially maintained in the presence of IFN-gamma, while T lymphocyte activation had no effect. To demonstrate fibroleukin production in vivo, we analyzed colon mucosa by immunohistology. Fibroleukin staining was detected in the extracellular matrix of the T lymphocyte-rich upper portion of the lamina propria mucosa. While the exact function of fibroleukin remains to be defined, these data suggest that fibroleukin may play a role in physiologic lymphocyte functions at mucosal sites.

Post-remission therapy of adult acute myeloid leukaemia: one cycle of high-dose versus standard-dose cytarabine. Leukaemia Project Group of the Swiss Group for Clinical Cancer Research (SAKK).

BACKGROUND: Intensification of post-remission therapy improves the cure rate of acute myeloid leukemia (AML) but is often accompanied by unacceptable toxicity. From 1985 to 1992 the Swiss Group for Clinical Cancer Research (SAKK) performed a randomized phase III trial to evaluate the effectiveness of one single postremission course of high-dose cytarabine (HDAC) in terms of leukaemia-free and overall survival in adults with de novo AML. PATIENTS AND METHODS: Adult (15-65 years) AML patients in remission after two induction courses were randomly assigned to one consolidation course either with standard (SDAC: 100 mg/sqm 24 hours infusion over seven days) or with high-dose cytarabine (HDAC: 3000 mg/sqm every 12 hours as one-hour-infusion for six days). In addition, both arms included daunorubicin (45 mg/sqm daily on days 1 to 3). Thereafter, patients were observed without maintenance until relapse. RESULTS: After two induction courses 208/276 eligible patients achieved remission (CR: 169, 61%, PR: 39, 14%), 41 were resistant (15%) and 20 died early (7%). Seventy-one patients in remission were not randomized. One hundred thirty-seven were randomized in CR/PR (67 SDAC, 70 HDAC). 4/70 patients randomized to HDAC did not receive it. Treatment-related mortality in HDAC was 1.4% (1/66). WHO grade 3-4 toxicities occurred in 14/67 SDAC and in 38/66 HDAC patients (P < 0.0001). The median event free survival was 10.8 (SDAC) vs. 12.2 months (HDAC; P = 0.18). The median overall survival was 24.6 (SDAC) vs. 32.6 months (HDAC; P = 0.07). Although statistically uncertain, HDAC reduced the hazard of progression (hazard ratio: 0.69, P = 0.08) and of death (hazard ratio: 0.70, P = 0.13). For 112 patients stratified as CR the estimated four-year disease-free survival was 25% (+/-6%) with SDAC and 37% (+/-6%) with HDAC (P = 0.09). The overall survival rates at four years were 38% (+7%) and 48% (+7%), respectively (P = 0.10). In multivariate analysis HDAC significantly reduced the hazard of relapse by 39% compared to SDAC (hazard ratio = 0.61, 95% CI: 0.37-0.99; P = 0.049). CONCLUSIONS: We conclude that early consolidation of adult AML in CR with a single course of HDAC is superior in terms of outcome to one cycle of SDAC. The results of our intensive, single course HDAC group compare favourably with less intensive, repetitive HDAC cycles, suggesting that Ara-C dose intensity may be more important than total dosage. In addition, our treatment strategy is much less toxic and less expensive.

Intensification of chemotherapy for the treatment of solid tumours: feasibility of a 3-fold increase in dose intensity with peripheral blood progenitor cells and granulocyte colony-stimulating factor.

Dose intensity may be an important determinant of the outcome in cancer chemotherapy, but is often limited by cumulative haematological toxicity. The availability of haematopoietic growth factors such as granulocyte colony-stimulating factor (G-CSF) and of peripheral blood progenitor cell (PBPC) transplantation has allowed the development of a new treatment strategy in which several courses of high-dose combination chemotherapy are administered for the treatment of solid tumours. PBPCs were mobilised before chemotherapy using 12 or 30 micrograms kg-1 day-1 G-CSF (Filgrastim) for 10 days, and were collected by 2-5 leucaphereses. The yields of mononuclear cells, colony-forming units and CD34-positive cells were similar at the two dose levels of Filgrastim, and the numbers of PBPCs were sufficient for rescue following multiple cycles of chemotherapy. High-dose chemotherapy (cyclophosphamide 2.5 g m-2 for 2 days, etoposide 300 mg m-2 for 3 days and cisplatin 50 mg m-2 for 3 days) was administered sequentially for a median of three cycles (range 1-4) to ten patients. Following the 30 evaluable cycles, the median duration of leucopenia < or = 0.5 x 10(9) l-1 and < or = 1.0 x 10(9) l-1 was 7 and 8 days respectively. The median time of thrombopenia < or = 20 x 10(9) l-1 was 6 days. There was no cumulative haematological toxicity. The duration of leucopenia, but not of thrombopenia, was inversely related to the number of reinfused CFU-GM (granulocyte-macrophage colony-forming units). In the majority of patients, neurotoxicity and ototoxicity became dose limiting after three cycles of therapy. However, the average dose intensity delivered was about three times higher than in a standard regimen. The complete response rate in patients with small-cell lung cancers was 66% (95% CI 30-92%) and the median progression-free survival and overall survival were 13 months and 17 months respectively. These results are encouraging and should be compared, in a randomised fashion, with standard dose chemotherapy.

High levels of the shed form of L-selectin are present in patients with acute leukemia and inhibit blast cell adhesion to activated endothelium.

L-selectin is expressed by most leukocytes and mediates the initial step of adhesion to vascular endothelium. A feature of this adhesion receptor is to be shed from the cell surface. We report here the presence of high levels of the shed form of L-selectin (sL-selectin) in plasma from patients with acute leukemia. We also show that sL-selectin purified from acute leukemia plasma exhibits functional activity. The mean (+/- 1 SD) plasma level of sL-selectin among 100 healthy individuals was 2.1 +/- 0.7 micrograms/mL. This value was increased (> 2 SD above the mean) in 63% of 58 patients with acute lymphoblastic leukemia (ALL) and 59% of 93 patients with acute myelogenous leukemia ([AML] P < .001). Repeated measurements in 24 patients showed normal-range levels in 16 of 16 patients in complete remission and high levels in eight of eight patients with therapy-resistant acute leukemia or leukemia relapse. Furthermore, elevated sL-selectin levels were detected in cerebrospinal fluid of three patients with ALL suffering from a relapse limited to the central nervous system. Epitope mapping with monoclonal antibodies demonstrated that L-selectin shedding from leukemic blasts was accompanied by conformational changes of its epidermal growth factor-like domain. A functional role for sL-selectin purified from leukemic plasma was supported by its ability to completely inhibit L-selectin-dependent adhesion of blast cells to tumor necrosis factor-alpha (TNF-alpha)-activated endothelium in vitro. These results suggest that sL-selectin may have an important role in the regulation of leukemic cell adhesion to endothelium. In addition, monitoring of the sL-selectin level may be useful for evaluating leukemia activity, in particular for the detection of leukemia relapse.

Rapid induction of cytolytic T cells via CD28 stimulation for cellular immunotherapy.

One approach to adoptive cancer immunotherapy is based on the use of bispecific monoclonal antibodies (mAb) capable to redirect ex vivo generated cytolytic T lymphocytes (CTL) onto tumour cells. The efficiency of the CD28 T-cell activation pathway to induce CD3-dependent cytolytic activity was investigated while avoiding modulation of the TCR/CD3 complex needed for targeting by bispecific mAb. When used e.g. in conjunction with anti-CD2 antibodies or diacylglycerol derivatives, the in vitro stimulation of T cells with anti-CD28 mAb resulted within 36 h in high levels of CD3-dependent cytolysis (tested on a FcR+ target in the presence of anti-CD3 mAb) and sustained lymphokine production, such as TNF alpha, IFN gamma and IL-2, which may affect tumour growth when delivered locally by the transferred T cells. Rapid activation may reduce costly in vitro procedures, preserve homing capacities of retransfused T cells, and thus facilitate implementation of clinical trials based on the use of bispecific antibodies.

CD11b/CD18 expression, adherence, and chemotaxis of granulocyte in adult respiratory distress syndrome.

The accumulation of granulocytes in the pulmonary microvasculature is generally thought a cardinal event in the pathology of adult respiratory distress syndrome (ARDS). However, the mechanism by which granulocytes are sequestered in the pulmonary vascular bed remains largely unknown. Because the CD11b/CD18 membrane receptors mediate various adhesion-dependent functions, their expression was investigated in granulocytes from patients during the course of ARDS development in relation to adherence and chemotaxis. CD11b expression of ARDS resting granulocytes was increased within 24 h of ARDS onset by a factor of two in comparison with control patients (p < 0.05) and remained significantly increased 72 to 120 h later. In contrast, the stimulated expression was significantly decreased only within 24 h of ARDS onset. Adherence was not modified within 8 h of the onset of ARDS, but was increased at Days 1, 3, and 5. The time course of granulocyte chemotaxis shows a decreased chemotaxis capacity during the first 3 d of ARDS, followed by normalization at Day 5. The dynamic changes observed in the various functions studied indicate a possible relationship between the modulation of the CD11b expression and a hyperadhesive state of granulocytes in ARDS. These sticky granulocytes may potentially contribute to the microvascular injury.

Intensive induction/consolidation therapy without maintenance in adult acute lymphoblastic leukaemia: a pilot assessment. Working Party on Leukaemia of the Swiss Group for Epidemiologic and Clinical Cancer Research (SAKK).

Maintenance chemotherapy for up to 3 years is traditionally given to patients with acute lymphoblastic leukaemia (ALL) achieving complete remission. We questioned the value of such maintenance therapy in adult patients treated with intensive induction/consolidation. In a phase II study (SAKK 33/86) 63 patients between 17 and 72 years of age (median 27 years) with newly diagnosed ALL were treated with three intensive cycles of marrow-ablative chemotherapy. All subtypes were included. No maintenance phase was added. 53 patients (84%) entered a complete remission (CR) and 21 (33%) continue to be in unmaintained remission for 11-69 months (median 21 months). The disease-free survival of patients achieving CR and completing all three cycles is 40% at 3 years, with a 95% confidence interval of +/- 19%. These findings are comparable to the results of conventional studies. We conclude that maintenance therapy might not be needed in all adult ALL patients. Its value should be tested in a randomized trial. For patients failing, novel approaches are needed to improve outcome in adult ALL.

Cytolysis of leukemic B-cells by T-cells activated via two bispecific antibodies.

Bispecific monoclonal antibodies can be used in the activation of effector cells to lyse autologous tumor cells. We analyzed the activation of human T-cells in vitro with bispecific monoclonal antibodies, which were generated by hybridoma-hybridoma fusion. Preactivated allogeneic and autologous T-cells could be triggered to lyse tumoral B-cells in the presence of CD3 x CD19 bispecific antibodies. In addition, the combined use of two CD3 x CD19 plus CD28 x CD22 bispecific antibodies induced optimal interleukin 2 secretion by Jurkat T-cell acute lymphocytic leukemia cells in the presence of target B-cells. The same antibody combination was able to generate cytolytic effector cells without prior activation, when resting T-cells were cocultured with freshly isolated autologous leukemic B-cells in the presence of the bispecific antibodies. The results suggest that signals required to activate cytolytic T-cell precursors can be provided by the two bispecific antibodies. Although activation of resting T-cells can be achieved by CD3 x CD19 bispecific antibodies in association with monospecific bivalent CD28 antibodies, the second bispecific antibody, CD28 x CD22, further increases the specificity of the target cell dependent activation of T-cells. When used for immunotherapy of B-cell malignancies, the CD3 x CD19 and CD28 x CD22 bispecific antibody combination may avoid the need for ex vivo activated effector cells, because the antibodies may induce T-cell activation directly at the tumor site.

Effect of prestorage leukocyte reduction on proteins of platelets obtained by apheresis.

The effect of prestorage leukocyte reduction was evaluated on platelet concentrates (PCs) obtained by apheresis using the 'surge' technique. Two hours after collection, the PCs were divided into 2 equal units. One unit was filtered through a Sepacell PL-10a, producing a filtered PC (FPC). The second unit constituted a non-filtered PC (NFPC). FPCs and NFPCs were stored at room temperature in 1,400-ml CLX bags on a horizontal agitator up to 7 days. We analyzed platelet samples obtained during storage from NFPCs and FPCs at days 1, 3 and 7. The expression of membrane glycoproteins (GP)Ib and GPIIb/IIIa (assessed by flow cytometry), platelet response to thrombin and ristocetin (aggregometry) and global platelet protein pattern (studied by high-resolution two-dimensional gel electrophoresis) remained stable over the 7 days of storage in NFPCs as well as in FPCs. However, in both preparations, the expression of GMP-140 (flow cytometry) progressively increased during storage. Our in vitro study indicates that early leukocyte reduction by filtration of apheresis PCs does not induce modifications in platelet GPs and protein patterns.

Production of tumor necrosis factor-alpha by naive or memory T lymphocytes activated via CD28.

While it is well established that activated T cells can produce tumor necrosis factor alpha (TNF-alpha), it is less clear whether this function is confined to a given subset, e.g., memory cells. To approach this question, we investigated the production of TNF-alpha by human peripheral blood T lymphocytes activated with anti-CD28 mAb since this activation pathway is known to potentiate cytokine production. Under the culture conditions used, the amount of TNF-alpha produced was markedly enhanced compared to that obtained after activation with immobilized anti-CD3 mAb. The enhancement of TNF-alpha production was already apparent after incubation of T cells for 6 hr. Up to 5 ng/ml of TNF-alpha was measured on Day 2 in supernatants of cultures of 10(4) T lymphocytes. To determine the source of the cells producing high amounts of TNF-alpha, T lymphocytes were separated into two subpopulations, namely naive cells (expressing the CD45RA isoform) and memory cells (expressing the CD45RO isoform). While both subpopulations proliferated equally well after stimulation with anti-CD28 mAb, up to 90% of the TNF-alpha produced under these conditions originated from memory T cells. These results thus document that T cell activation via CD28 results in a marked increase in TNF-alpha production without affecting the functional disparity that exists between naive and memory T cells.

[Persistent B-cell polyclonal lymphocytosis: a benign lymphoproliferative syndrome]. / La lymphocytose B polyclonale persistante: un syndrome lymphoprolifératif bénin.

A further case of persistent polyclonal B-cell lymphocytosis is reported. This recently identified distinct clinicopathologic entity is frequently associated with the presence of the HLA-DR7 antigen. It follows a benign course and has so far been reported only in women smokers. The disorder is characterized by mild chronic peripheral lymphocytosis, the presence of characteristic binucleate lymphocytes on peripheral blood smears, and a polyclonal increase in serum IgM. In some cases, lymphadenopathies and/or splenomegaly are observed. Surface marker studies of peripheral lymphocytes demonstrate the polyclonal B-cell nature of this entity.

Cytotoxic potential despite impaired activation pathways in T lymphocytes infiltrating nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is an epithelial tumor consistently associated with EBV. The histological picture is characterized by a strikingly abundant lymphocytic infiltrate. Furthermore, the epithelial tumor cells present several immunological characteristics which suggest an important role for tumor-infiltrating lymphocytes (TIL) in the biology of this tumor. The present study reports the phenotypic and functional characterization of TIL from NPC obtained after enzymatic digestion of 15 NPC biopsies. Flow cytometric analysis of TIL suspensions indicated that most TIL were mature CD3+ T lymphocytes (mean = 60%) with a variable CD4/CD8 ratio. Most TIL were TCR alpha/beta-positive (mean = 55%) and only a few TCR gamma-delta-positive cells could be identified. A small percentage (mean = 9%) displayed an activated phenotype (CD25+, HLA class II+). Using limiting dilution analysis, we found that the average frequency of proliferative T-lymphocyte precursors (PTL-P) is lower among TIL (1/40) than in autologous (1/7) or normal PBL (1/1.4). Moreover, sorting experiments have shown that this defect is significantly more pronounced in the CD8+ than in the CD4+ TIL subset. Accordingly, the TCR and the CD2-mediated antigen-independent pathways of activation were impaired. Different types of cytotoxic precursor could be detected. These included lectin-dependent cell cytotoxicity (LDCC) and NK-like or lymphokine-activated killer (LAK) activity. Interestingly, some TIL from NPC were able to lyse an NPC tumor (C15) maintained in nude mice. Thus, despite impaired activation pathways, the cytolytic potential of proliferating TIL in NPC is preserved.

[The treatment of acute lymphatic leukemia in adults: pilot project with intensive early therapy without a maintenance phase]. / Behandlung der akuten lymphatischen Leukämie beim Erwachsenen: Pilotversuch mit intensiver Frühtherapie und ohne Erhaltungsphase.

Acute lymphoblastic leukemia (ALL) is conventionally treated in three phases: remission induction, consolidation and maintenance. In adults initial remission rates of nearly 80% can be achieved. The 3 to 5 year leukemia free survival is 20 to 40%. Most relapses occur during maintenance, despite continuous therapy for 2 to 3 years. The value of maintenance therapy following intensive induction in adults is not documented. In this pilot study we treated 34 patients with ALL in five Swiss centers between 1986 and 1989 with intensive induction/consolidation therapy alone. Three induction/consolidation courses were applied: course I, 43 days, consisting of daunomycin, vincristine, prednisone, methotrexate, L-asparaginase and intrathecal CNS prophylaxis; course II, 6 days, consisting of high dose cytosine arabinoside and VP-16; course III, three non randomized arms, either allogeneic or autologous bone marrow transplantation (BMT) or high dose cyclophosphamide and repeated intrathecal therapy alone. 32 patients (94%) reached complete remission (CR): 24 after course I (71%), 7 after course II (cumulative 91%) and one after course III only (cumulative 94%). 3 patients had early, relapses before course III, 3 died of infection, and 2 of graft-versus-host disease. One patient had refractory leukemia in all three courses. 26 patients (76%) were in CR after completion of therapy. 16 relapsed within 1 to 17 months (median 5). 10 patients are free of disease after 10 to 44 months (median 24): 5 had had allogeneic BMT, 3 autologous BMT and 2 cyclophosphamide in course III.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of bone metastases from breast cancer and myeloma with pamidronate.

28 patients with progressing painful bone metastases (18 breast cancer, 9 myeloma and 1 low grade lymphoma) received pamidronate 60 mg by 24 h continuous infusion for at least 2 courses (range 2-12). In patients urinary calcium and hydroxyproline excretion significantly decreased in relation to diminution of bone resorption. 9 of 18 breast cancer patients and 8 of 9 evaluable patients with myeloma had symptomatic improvement. Sclerotic areas of previously lytic lesions appeared in 8 breast cancer patients and in 1 myeloma patient. Transient fever developed in 1 patient and local phlebitis in 2. Among the 28 patients, 15 did not receive any anticancer treatment or have any change of the anticancer therapy during pamidronate administration. Of 7 with breast cancer, 4 had an improvement of symptoms and 4 sclerosis on radiographs. Impressive control of symptoms was the major feature of 8 myeloma patients, but only 1 had radiographic sclerosis.

Effect of transforming growth factor beta on the EL4 thymoma variant EL4/6.1: dissociation of inhibition of proliferation from expression of IL-1 and IL-2 receptors.

In order to further characterize the action of transforming growth factor beta (TGF-beta) on lymphoid cells, we investigated the effects of porcine TGF-beta 1 and -2 on the IL-1 sensitive EL4/6.1 thymoma cell line. The proliferation of EL4/6.1 thymoma cells was inhibited by TGF-beta 1 and TGF-beta 2 (1 ng/ml) to a similar degree, the population doubling time was increased by 50-60%, total inhibition was not achieved. This decrease of proliferation was associated with an increase of the number of cells in the G0/G1 compartment of the cell cycle. TGF-beta-mediated inhibition could not be overcome by adding exogenous rIL-1 nor was the binding capacity for IL-1 reduced. In addition, TGF-beta did not interfere with the induction of IL-2 receptors by a combination of Ionomycin+PMA+IL-1. The data suggest that TGF-beta mediated inhibition of thymocyte/lymphocyte proliferation is not associated with an inhibition of the expression or the induction of expression of IL-2 or IL-1 receptors.

Stimulation of FACS-analysed CD4+ and CD8+ human tumour-infiltrating lymphocytes with ionomycin + phorbol-12,13-dibutyrate does not overcome their proliferative deficit.

Human tumour-infiltrating lymphocytes (TIL) were prepared by enzyme digestion from a series of different tumours and were purified on a fluorescence-activated cell sorter (FACS II) according to their CD4+ and CD8+ phenotype. CD4+ and CD8+ TIL were stimulated separately in a low density microculture system with phytohaemagglutinin (PHA) or with ionomycin plus phorbol-12, 13-dibutyrate (PDBu). The PHA-induced proliferation of TIL was highly decreased when compared with control peripheral blood lymphocytes. A decreased proliferation of TIL was also observed when cells were stimulated with ionomycin plus PDBu, a combination which is thought to circumvent early events associated with lymphocyte activation. Some TIL were also plated in limiting dilution where they showed decreased frequencies of proliferating T cell precursors. The data suggest that one component of the inhibition of TIL must be acting 'downstream' of the early events of lymphocyte activation.