Treatment of advanced renal cell cancer with sequential intravenous recombinant interleukin-2 and subcutaneous alpha-interferon.

Starting from in vitro studies suggesting synergistic antitumour activity against renal cell cancer (RCC) of recombinant interleukin-2 (rIL-2) and alpha-interferon (IFN), a phase II trial was initiated to test the clinical activity of this combination. The two cytokines were administered sequentially, with the aim of reducing the risk of additive toxicity and enhancing the immunological reaction against the tumour. The original treatment schedule consisted of rIL-2 18 x 10(6) U/m2/day by continuous intravenous infusion for 120 h days 1-5, and alpha-IFN 2b, at a flat dose of 9 x 10(6) U by subcutaneous or intramuscular injection thrice in a week, from day 8 to 28. Treatment was planned to be continued for six or more 28-day cycles, depending on clinical response. 12 patients were treated according to this schedule; as some cardiovascular toxicity was experienced in this set of patients, 11 further patients were treated with half-dose rIL-2 (i.e. 9 x 10(6) U/m2/day). 17 out of 23 enrolled patients completed at least one cycle of treatment and were evaluated for response. We observed six major responses [one complete response (CR) + five partial responses (PR)] for an objective response rate of 35% [95% confidence interval (CI) 17-59%]. 5 additional patients achieved stabilisation of disease; one of them reached CR after surgical extirpation of a lung mass. Sites of response included lung, nodes and bone. Duration of response is 12+ months for CR; 17, 16, 12+, 9 and 9 months for PRs. Median survival is 16 months. Response was not significantly different between full-dose and half-dose rIL-2. Considering stable disease (SD) as responses, there seemed to be a higher chance of response for patients with smaller tumour burden (P = 0.032). The toxicity of rIL-2 treatment, mainly cardiovascular, was substantial; 9 patients experienced severe cardiotoxicity, consisting of major arrhythmias, myocardial ischaemia, reduction of ejection fraction measured with heart radionuclide scan, and were excluded from continuing treatment. Other rIL-2-related toxicities forcing exclusion from the study were severe thrombocytopenia (1 case), and generalised exfoliative dermatitis requiring steroids (1 case). Otherwise, treatment was well tolerated; rIL-2-related toxicities promptly recovered after rIL-2 discontinuation in the majority of cases, and no treatment-related deaths were reported. The half-dose rIL-2 regimen was significantly less toxic in terms of hypotension (P = 0.014), fever (P = 0.014), oliguria (P = 0.042), serum creatinine elevation (P = 0.009) and prothrombin time elongation (P = 0.038).(ABSTRACT TRUNCATED AT 400 WORDS)

Increased interleukin-10 serum levels in patients with solid tumours.

In 40 out of 99 patients (40.4%) with solid tumours of different tissue, but the same stage (IV), elevated serum levels of interleukin-10 were observed. The mean levels of the cytokine in patients with malignant melanoma (24.3 ng/ml), pancreatic (6.8 ng/ml) or gastric (6.3 ng/ml) adenocarcinoma were significantly higher than in healthy subjects (3.4 ng/ml) or in patients with uterine fibroma (1.7 ng/ml). Patients with colon (6.8 ng/ml) and renal (5.7 ng/ml) carcinoma had similar values of interleukin-10 but did not significantly differ from controls. Interleukin-10 is known to suppress the functions of both T lymphocytes and macrophages, working as a general dampener of the immune and inflammatory responses. The observation of increased circulating levels of interleukin-10 in cancer patients may have important implications for future investigations, immunological monitoring and therapeutic intervention on neoplastic patients, and suggests a mechanism for tumour cells escaping from immune surveillance.

Cell activation and death (apoptosis) induced by IL-2: ultrastructural evidence.

Peripheral blood cells of cancer patients with advanced renal carcinoma and treated with interleukin-2 by intravenous application were studied by electron microscopy at different intervals from the beginning of rIL-2 administration. Morphofunctional modifications of lymphocytes, circulating hystiocytes containing phagocitated bodies and polimorphonucleated cells undergoing phagocytosis were observed with a time dependent increase of altered cells. The analysis of blood cells cultured in the presence of recombinant IL-2 confirmed the in vivo results. Our data suggest that IL-2 induces apoptotic phenomena in the peripheral blood cells of treated patients.

Natural killer cell function in HIV-1 infected patients.

A cross-talk between dendritic cells (DC) and resting natural killer (NK) cells leads to the activation of both cell populations, a process requiring cell-cell contact. When the number of activated NK cells overwhelms surrounding DC, they became able to kill specifically immature DC, a feedback mechanism to shut off DC-mediated immune responses. DC, at the mucosal site, can capture HIV and transfer it to CD4+ T lymphocytes present in the regional lymph node thus giving rise to a productive infection; on the other hand, NK cells represent the first line of defence against viral infection. Our preliminary results suggest that during the early phases of an HIV infection, NK cell activity is not functionally compromised, but that infected cells might escape natural immune surveillance through several mechanisms, including a reduced lysis of autologous DC.

A prospective, randomized clinical trial on perioperative feeding with an arginine-, omega-3 fatty acid-, and RNA-enriched enteral diet: effect on host response and nutritional status.

BACKGROUND: The use of immune-enhancing enteral diets in the postoperative period has given contrasting results. The purpose of this prospective, randomized, double-blinded clinical study was to evaluate the effect of immunonutrition given perioperatively on cytokine release and nutritional parameters. METHODS: Patients with cancer of the stomach or colo-rectum were eligible. Subjects consumed 1 L/d of either a control enteral formula (n = 25; control group) or a formula supplemented with arginine, omega-3 fatty acids, and RNA (n = 25; verum group) for 1 week before surgery. Both formulas were given by mouth. Six hours after the operation, jejunal infusion with the same diets was started and maintained for 7 days. Blood was drawn at different time points to assess albumin, prealbumin (PA), transferrin, cholinesterase activity, retinol binding protein (RBP), interleukin-2 receptors alpha (IL-2Ralpha), IL-6, and IL-1 soluble receptors (IL-1RII). The composite score of delayed hypersensitivity response (DHR) to skin test also was determined (the higher the score, the lower the immune response). RESULTS: During the 7 days of presurgical feeding, none of the above parameters changed in either group. Eight days after operation, in the control group, the concentration of PA and RBP was lower than in the verum group (0.18 vs 0.26 g/L for PA and 30.5 vs 38.7 mg/L for RBP; p < .05). IL-2Ralpha concentration was 507 pg/mL in the verum group vs 238 pg/mL in the control group (p < .001), whereas IL-6 and IL-1RII were higher in the control group than in the verum group (104 vs 49 and 328 vs 183 pg/mL, respectively; p < .01). The DHR score was 0.68 in the control group vs 0.42 in the verum group (p < .05). CONCLUSIONS: Perioperative feeding with a supplemented enteral diet modulates cytokine production and enhances cell-mediated immunity and the synthesis of short half-life proteins.

Intrapleural administration of interleukin-2 and LAK cells in locally advanced non-small-cell lung cancer. A case report.

AIMS AND BACKGROUND: The systemic administration of recombinant interleukin-2 (rIL-2) and lymphokine-activated killer (LAK) cells is ineffective in non-small-cell lung cancer (NSCLC). However, there is some evidence that their intrapleural administration could be effective, since it increases the concentrations of the cytokine and the effector cells in the tumor area, thereby obtaining greater antitumor activity. STUDY DESIGN: We report the case of a patient affected by a locally advanced lung adenocarcinoma with pleural effusion (T4 N0 M0-stage IIIb) treated with repetitive courses consisting of a priming continuous i.v. infusion (48 h) of rIL-2 (18 MIU/m2/day) intraplural administration of LAK cells (3-9 x 10(9)/day), in a single daily bolus, for 3 consecutive days and concomitant administration of rIL-2 (1.8-7.2 x 10(6) IU/day), for 5 days. RESULTS: We observed early disappearance of neoplastic cells in the pleural effusion, progressive decrease until disappearance of the pleural effusion, cavitation of the primary lesion during the treatment, and its stabilization for 9 months until progression. Radiologic changes were accompanied by a marked eosinophilia (up to 50 x 10(9)/L), and the intrapleural route of administration of rIL-2 induced a relevant increase in eosinophil count in peripheral blood. Immunologic changes in lymphocyte subpopulation phenotypes were also observed. The performance status of the patient improved, and she was still alive and eupnoic 25 months from the diagnosis and 23 months from the start of treatment. CONCLUSIONS: This case suggests a therapeutic role for intrapleural rIL-2, and we believe that the relationship among intrapleural administration of rIL-2 and LAK cells, the development of peripheral eosinophilia, and clinical response should be further investigated.

Natural killer-cell cytotoxicity in HIV-positive and HIV-negative patients with and without severe course of hepatitis B virus infection.

Natural killer (NK) cells represent the first line of defence against viral infections but, in the case of hepatitis B virus (HBV), may also be involved in liver injury. We here compared NK-cell activity of 11 patients with acute HBV infection, either HIV-positive or HIV-negative, with that of 11 healthy subjects. One of the HIV-positive patients, characterized by a severe immunodeficiency, died 3 weeks after hospitalization for HBV-related fulminant hepatitis (FH). He displayed a remarkable NK-cell cytotoxicity against both cell lines and autologous dendritic cells, whereas the NK-cell activity of the remaining patients was significantly reduced as compared with healthy individuals. Our findings suggest that NK-cell-mediated cytotoxicity could contribute to the development of HBV-related acute liver failure in HIV-positive patients with severe immunodeficiency. An immunopathological model of FH in immunocompromised patients was proposed.

Salvage immunotherapy with subcutaneous recombinant interleukin 2 (rIL-2) and alpha-interferon (A-IFN) for stage D3 prostate carcinoma failing second-line hormonal treatment.

Immunotherapy with subcutaneous rIL-2 and alpha IFN was administered to stage D3 prostate cancer patients after failure of secondary treatment with oral estramustine phosphate. Of a total of 15 patients, 2 are in partial response, with estramustine maintained after 44+ and 36+ weeks, respectively. Response to estramustine was observed initially in 7 of 13 patients, with a median duration of 12 weeks (range 8-20). No response to estramustine was observed in the remaining 6 patients. After the failure of estramustine, 13 patients were treated with immunotherapy. After the first cycle, progression of disease no therapy was given to those patients. A reduction of PSA levels was observed during the first cycle in 2 patients (15.3%); levels subsequently increased during the second cycle of treatment. A partial response was observed in 4 patients (30.7%), with a reduction of PSA levels in 3. The duration of response was 28 and 32 weeks in 2 patients who survived after failure for 18 and 21 weeks, respectively. Two patients are still alive, with continued partial response at 62+ and 42+ weeks. Side effects were represented mainly by a flu-like syndrome, associated with fever and nausea in all patients. The serum concentration of IL-10 was measured in 8 patients under study and in 11 matched controls. Levels higher than mean + 2D of controls before, during, or after immunotherapy were correlated with treatment failure, whereas levels below 6 ng/ml were encountered among the patients who showed a clinical response and a reduction of PSA during treatment. Within the limitations of this pilot study, it appears difficult to distinguish between a spontaneously slowly progressing disease and a true response to therapy.

Reversible anergy in circulating lymphocytes of cancer patients during interleukin-2 therapy.

Interleukin-2 plays a crucial role in enhancing the antitumor immune response. Clinical trials, mainly in renal cell carcinoma and melanoma patients, have been carried out with encouraging results. Recent reports demonstrated that interleukin-2 therapy may depress the immune response either in vitro or in vivo. We decided to monitor, in nine renal cancer patients, the proliferative responses and the parallel variations in Ca2+ homeostasis of peripheral blood lymphocytes collected before, during and after the first cycle of a 3-day interleukin-2 systemic administration. The proliferative response to phytohemagglutinin or concanavalin A significantly dropped early during interleukin-2 infusion. Consistently, an impairment in mobilizing Ca2+, either from internal stores or via influx from outside, was observed. Results obtained with a mAb-alpha CD3 molecular complex strongly suggested that the TCR/CD3 signal transduction pathway was defective. In contrast, no major variations were observed in the general machinery controlling Ca2+ homeostasis nor in the total Ca(2+)-releasable pool. Patients' lymphocytes, cultured in vitro for 3 days in medium alone, showed an almost complete recovery in their ability to respond to mitogens. In conclusion, we show that interleukin-2 administration in cancer patients induces a reversible state of anergy in circulating lymphocytes, assessed both by the reduction in the proliferative response and the block of the mitogen-activated intracellular Ca2+ signalling.

Serum levels of soluble cell adhesion molecules (ICAM-1, VCAM-1, E-selectin) and of cytokine TNF-alpha increase during interleukin-2 therapy.

Circulating levels of soluble intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin were measured in 20 advanced cancer patients at different times during recombinant interleukin-2 immunotherapy. The concentration of all three molecules progressively increased as did the levels of circulating tumor necrosis factor-alpha (TNF-alpha), which is known to induce endothelial cell activation. A fair direct relationship (but not statistically significant) between the raised concentration of soluble cell adhesion molecules and TNF-alpha was observed. We suggest that elevated levels of soluble adhesion molecules and TNF-alpha in the blood of IL-2-treated patients may arise from a systemic inflammatory reaction producing endothelial cell activation.

Radical oncologic surgery affects the circulatory levels of interleukin 10.

BACKGROUND AND OBJECTIVE: Interleukin 10 (IL-10) has been shown to be elevated in the plasma of cancer-bearing patients. The source of systemic IL-10 may be the tumor microenvironment. We therefore tried to evaluate if ablative surgery for gastrointestinal cancer could affect the levels of circulating IL-10. METHODS: Plasma IL-10 concentration was measured in 45 patients with adenocarcinoma of the gastrointestinal tract. Forty healthy subjects, 15 women undergoing hysterectomy for uterine fibroma, and 15 patients undergoing palliative operation for pancreatic cancer were used as control groups. Plasma IL-10 was assessed 1 day before surgery (baseline) and 1, 4, and 8 days after operation. RESULTS: The baseline concentration of IL-10 was significantly higher in cancer patients than in healthy subjects and in women with fibroma (8.6 ng/mL, 2.1 and 1.8 respectively; P = 0.015). After radical surgery, the IL-10 levels significantly dropped in cancer patients (from 8.6 ng/mL to 3.8; P = 0.024), whereas in subjects undergoing palliative operation, the concentration remained elevated (8.5 ng/mL baseline versus 7.9 on day + 1). CONCLUSIONS: The origin of circulating IL-10 may be the tumor microenvironment.

Thymopentin modulates Th1 and Th2 cytokine response and host survival in experimental injury.

OBJECTIVE: To investigate the impact of thymopentin (Thy) on mortality and in vivo cytokine release in an animal model of gut-derived sepsis which includes different combinations of allogeneic blood transfusion (T) and burn injury plus bacterial gavage (BG). DESIGN: Randomly controlled experiments. MATERIAL: Two hundred sixteen Balb/c (H-2d) and 50 C3H/HeJ (H-2k) mice. INTERVENTIONS: In the first study 60 Balb/c mice were given Thy (1 mg/kg). The same day of therapy onset, 40 mice were transfused with allogeneic blood (from C3H/HeJ mice). The remaining 20 mice received aliquots of saline. Five days post-T, 20 of the 40 transfused mice were subjected to a 20% TBSA thermal injury and simultaneous gavage with 1 x 10(9) Escherichia coli and the other 20 mice underwent a sham burn. The 20 nontransfused mice also received a 20% burn plus bacterial gavage. In all animals Thy was administered for 15 days. Three control groups (n = 20 each) entered the same protocol design, but they did not receive Thy. In the second study 96 animals were randomized to six groups (n = 16 each) according to the above experimental design. Animals were sacrificed by exsanguination after burn or 5 days post-T in nonburned mice to measure TNF-alpha, IL-2, and IL-4 plasma levels. RESULTS: The highest mortality (70%) occurred when T was combined with BG. Thy significantly reduced mortality in both groups that underwent BG, regardless of the association with T. TNF-alpha was detectable in 30% of the tested samples, IL-2 in 50%, and IL-4 in 70%. Thy significantly reduced the levels of IL-4 and increased the production of IL-2. CONCLUSIONS: The protective effect of Thy in this experimental model may be mediated by modulation of cytokine release.

Study on the turnover of the receptor for the third component of complement on human lymphoid cells.

The in vitro turnover of the receptor for the third component of complement (C3) was studied in normal peripheral blood lymphocytes (PBL) and in lymphoblastoid cells from established cell cultures of both "normal" and "malignant" origin. The turnover was evaluated by studying i) the disappearance rate of the C3-receptor in cells in which the protein synthesis was blocked by cycloheximide and puromycin, ii) the reexpression rate of the C3-receptor after treatment of the cells with either rabbit antiserum against B lymphocytes or mouse C activated through the alternative pathway by inulin. The results show that the C3-receptor of all the lymphoid cells has roughly a half-life of about 3 to 4 hr. However, the cultured lymphoblastoid cells were less sensitive than normal PBL to inhibition by cycloheximide and showed a faster reexpression rate of the C3-receptor. A spontaneous release of the receptor was found to occur, since a receptor-like activity was detected in the spent culture medium of long-term cultured lymphoid cells.

LAK1 antigen defines two distinct subsets among human tumour infiltrating lymphocytes.

Both lymphokine activated killer (LAK) cells and specific cytotoxic T lymphocytes appear to play a role in tumour immunity. Tumour infiltrating lymphocytes (TIL) which display a CD56+ phenotype (both CD3+ and CD3-) are also likely to possess anti-tumour activity. We have previously described a 120 kDa surface antigen, termed LAK1, expressed on a subset of human peripheral blood lymphocytes (20-50%) with both NK and LAK activity. The aim of the present study was to determine whether LAK1 antigen is able to distinguish among TIL two populations of effector cells displaying either specific or non MHC-restricted (NK/LAK) activity. We showed that about 25% of freshly derived TIL were weakly stained with anti-LAK1 monoclonal antibody and most of them were also CD3+ CD56-. After culture in recombinant interleukin-2 the majority of TIL were CD3+ CD56- and the percentage of LAK1+ cells increased up to 50%. Among cloned TIL, only those lacking LAK1 antigen displayed a specific cytotoxicity against the autologous tumour, whereas the non-lytic clones were able to produce both tumour necrosis factor and gamma-interferon. Moreover, when TIL from a renal cell carcinoma were fractionated into LAK1- and LAK1+ populations, the specific lytic activity was mainly evident when LAK1- lymphocytes were used as effector cells. Conversely, LAK activity was confined to the LAK1+ subset.

Selection and characterization of T-cell variants lacking molecules involved in T-cell activation (T3 T-cell receptor, T44, and T11): analysis of the functional relationship among different pathways of activation.

A clone of the interleukin 2-producing Jurkat leukemia cell line termed JA3 (surface phenotype, T3+, Ti+, T44+, T11+, T40+) has been used to induce and select cell variants lacking surface molecules involved in T-cell activation. Following 200 rad of gamma-radiation (1 rad = 0.01 Gy), cells were treated with monoclonal antibodies (mAbs) directed to T3, Ti, T44, or T11 antigen and complement. After growth of the residual cells in culture, "negative" cells were cloned under limiting conditions. Depending on the specificity of the mAb used for the immunoselection, three groups of variants were obtained. (i) The use of mAbs directed to T3 or Ti resulted in cell variants that expressed the T3 Ti- T44+ Leu1+ T11+ T40+ 4F2+ HLA class I+ surface phenotype. (ii) Immunoselection with anti-T44 mAb resulted in 2 variants that shared the T3- Ti- T44- Leu1- T11+ T40+ 4F2+ HLA class I+ phenotype. (iii) Cell treatment with anti-T11 mAb resulted in 15 variants characterized by the lack of T11 antigen expression and of all the other T-cell-specific surface antigens. Therefore, it appears that the different sets of JA3 cell variants, like T cells at discrete stages of intrathymic differentiation, may follow a coordinated expression of surface differentiation antigens. Analysis of the functional responsiveness of the three distinct groups of JA3 cell variants to different stimuli showed that all produced interleukin 2 in response to A23187 calcium ionophore plus phorbol 12-myristate 13-acetate. The first group of variants (T3- Ti-) did not respond to stimulation with anti-T3, anti-Ti, or anti-T44 mAbs. Eight of 9 did not respond to phytohemagglutinin either; however, all responded to appropriate stimulatory combinations of anti-T11 mAbs (and to calcium ionophore). The second group of variants (T3-, Ti-, T44-, T11+), similar to the first group, did not respond to anti-T3, anti-Ti, anti-T44 mAbs, and phytohemagglutinin, but they were fully responsive to anti-T11 mAb. The last group of variants (lacking all the T-cell-specific surface antigens) only responded to calcium ionophore A23187.

Recombinant interleukin-2 and lymphokine-activated killer cells in renal cancer patients: II. Characterization of cells cultured ex vivo and their contribution to the in vivo immunomodulation.

Peripheral blood mononuclear cells, obtained from patients with renal cell cancer and cultured ex vivo, exhibit high natural killer (NK) and lymphokine-activated killer (LAK) activity (also against allogeneic fresh tumour cells), which is transcribed into the hosts' immune status after reinfusion. Phenotypic analysis shows a slight increase in the percentage of CD56+ and CD8+ lymphocytes, while CD4+ lymphocytes decrease slightly. As a sign of activation an increase of cells expressing DR and CD25 antigens is observed. At the peripheral blood level, mononuclear cells show an increase, compared to basal values, of NK and LAK activity, especially at the end of the first infusion cycle. Phenotypic analysis of the patients' PBMC shows a decrease of CD3+CD4+ T lymphocytes and an increase of NK cells (CD3-CD56+CD16+) and of cells expressing activation markers (DR and CD25), particularly evident by the end of the second infusion cycle. Finally, in addition to the changes induced by IL-2 alone, reinfusion of incubated cells results in an activation of CD56+ and LeuM3+ cells.