Energy and protein utilisation by broiler chickens fed diets containing cottonseed meal and supplemented with a composite enzyme product.

1. The present study examined the potential of new-generation microbial enzymes to improve the utilisation of energy and protein of cottonseed meal (CSM)-containing diets, with the aim of increasing its inclusion level in broiler chickens diets. 2. Four hundred and eighty, one-day-old Ross 308 male broilers were used to assess the utilisation of energy and protein by broiler chickens fed diets containing four graded levels of CSM - none, low (4, 8, 12%), medium (5, 10, 15%) or high (6, 12, 18%) in the starter, grower, and finisher phases, respectively, supplemented with 100 mg/kg of a composite enzyme product (xylanase and ß-glucanase). 3. Inclusion of CSM improved (P < 0.01) apparent metabolisable energy (AME), with further improvement (P < 0.001) seen in the enzyme-supplemented diets. Inclusion of CSM reduced (P = 0.002) the metabolisable energy intake (MEI), but this was increased (P < 0.05) with enzyme supplementation. 4. Enzyme addition increased (P < 0.001) the net energy of production (NEp), while heat production (HP) decreased (P < 0.001) with CSM inclusion. More energy was retained as fat (P < 0.05) and protein in birds fed diets with the enzyme, but this was reduced (P < 0.029) by CSM. 5. There was an increase (P < 0.05) in efficiencies of ME use for energy, lipid and protein retention, with higher CSM levels. The enzyme improved (P < 0.013) efficiency of ME use for lipid retention. 6. Feeding diets containing CSM to the broilers enhanced (P < 0.05) protein intake (PI) and protein efficiency ratio (PER). Positive effects (P < 0.05) of enzyme were observed on protein gain (PG) and net protein utilisation (NPU). 7. Results obtained from this study suggested that nutrient utilisation of diets containing CSM by broiler chickens can be improved by enzyme supplementation.

Dietary hydrolysed yeast cell wall extract is comparable to antibiotics in the control of subclinical necrotic enteritis in broiler chickens.

1. The aim of this study was to examine the effect of yeast cell wall (YCW) on performance and physiological responses of broiler chickens under subclinical necrotic enteritis challenge.2. Six treatments in a 2 × 3 factorial arrangement (non-challenged or challenged plus no supplement, YCW or antibiotics (AB)) was used. Each treatment was replicated eight times with 12 birds per replicate. The treatments included: (1) Positive control (PC; no additive, not challenged); (2) Negative control (NC; no additive, with challenge); (3) YCWN = yeast cell wall (2.0 g/kg diet, not challenged; (4) YCWC = yeast cell wall (2.0 g/kg diet, challenged); (5) ABN = zinc bacitracin 50 ppm + Salinomycin 60 ppm, not challenged); (6) ABC = zinc bacitracin 50 ppm + Salinomycin 60 ppm, challenged).3. Eimeria challenge at 9 d of age did not affect feed intake (FI), body weight gain (BWG), FCR or liveability at 10 d. The BWG and FCR at 10 d were greater (P < 0.05) in birds fed YCW or AB (AB) diets relative to the PC or NC groups. On 24 and 35 d, FI, BWG, FCR and flock uniformity (28 d) were greater (P < 0.05) in the challenged groups fed YCW or AB diets compared to NC group.4. Supplementation with YCW ameliorated the negative effects of NE on liver, spleen and bursa weight of birds.5. Necrotic enteritis challenge decreased (P < 0.05) caecal Lactobacillus and Bifidobacterium spp. counts, and increased ileum lesion score and caecal Clostridium perfirngens counts. This was reversed by the addition of either YCW or AB.6. Supplementation with YCW and AB resulted to a greater (P < 0.05) dressing percentage and meat yield (35 d).7. The results indicated that YCW plays a vital role in improving the physiological response and performance of broiler chickens under subclinical necrotic enteritis challenge.

Influence of autolyzed whole yeast and yeast components on broiler chickens challenged with salmonella lipopolysaccharide.

The objective of this study was to assess the effect of dietary yeast products on broiler chickens challenged with salmonella lipopolysaccharide (LPS). The chicks were divided into 8 treatments with 6 replicates and 9 birds per replicate. The treatments consisted of a positive control (PC) [without supplementation and not challenged]; negative control (NC) [without supplementation but challenged]; whole yeast and challenged; yeast cell wall and challenged; yeast glucan and challenged; yeast mannan and challenged; zinc bacitracin and challenged; and Salinomycin and challenged. Whole yeast or Yeast cell wall was included at 2.0 g/kg diet. Yeast glucan or mannan was added at 0.20 g/kg diet. Zinc bacitracin (ZNB) and Salinomycin (SAL) was included at 50 and 60 ppm, respectively. Dietary treatments had no effect (P > 0.05) on feed intake (FI) at day 10. Supplementation with yeast and its derivatives improved (P < 0.05) body weight gain (BWG) and feed conversion ratio (FCR) on day 10. On days 24 and 35, LPS challenge declined FI, BWG, FCR, and flock uniformity (day 28) in the NC group compared to the PC group. Yeast products and antibiotics improved (P < 0.05) FI, BWG, FCR, and flock uniformity in LPS-challenged birds. On day 24, spleen weight increased while bursa weight decreased in the NC group relative to the PC group; this effect was reversed (P < 0.05) by feeding all yeasts and antibiotics. On day 24, application of all the dietary treatments ameliorated the changes observed in white blood cell, lymphocyte and monocyte counts as well as albumin and immunoglobulin G of NC birds. On day 35, all yeasts additives, ZNB and SAL improved (P < 0.05) the meat yield of broilers challenged with LPS. In conclusion, supplementation of diets with yeast and its derivatives can ameliorate the negative effects of salmonella LPS challenge on broiler chicks, thus improving the performance, flock uniformity, and meat yield.

Interleukin-12 and interleukin-18 synergistically induce murine tumor regression which involves inhibition of angiogenesis.

The antitumor effect and mechanisms activated by murine IL-12 and IL-18, cytokines that induce IFN-gamma production, were studied using engineered SCK murine mammary carcinoma cells. In syngeneic A/J mice, SCK cells expressing mIL-12 or mIL-18 were less tumorigenic and formed tumors more slowly than control cells. Neither SCK.12 nor SCK.18 cells protected significantly against tumorigenesis by distant SCK cells. However, inoculation of the two cell types together synergistically protected 70% of mice from concurrently injected distant SCK cells and 30% of mice from SCK cells established 3 d earlier. Antibody neutralization studies revealed that the antitumor effects of secreted mIL-12 and mIL-18 required IFN-gamma. Interestingly, half the survivors of SCK.12 and/or SCK.18 cells developed protective immunity suggesting that anti-SCK immunity is unlikely to be responsible for protection. Instead, angiogenesis inhibition, assayed by Matrigel implants, appeared to be a property of both SCK.12 and SCK.18 cells and the two cell types together produced significantly greater systemic inhibition of angiogenesis. This suggests that inhibition of tumor angiogenesis is an important part of the systemic antitumor effect produced by mIL-12 and mIL-18.

Prospective randomized trial of high-dose interleukin-2 alone or in conjunction with lymphokine-activated killer cells for the treatment of patients with advanced cancer.

BACKGROUND: Treatment using interleukin-2 (IL-2) alone or in conjunction with lymphokine-activated killer (LAK) cells has been shown to mediate disease regression in selected patients with advanced cancer. PURPOSE: This prospective randomized trial was designed to determine whether the administration of LAK cells in conjunction with high-dose IL-2 alters response and survival rates, compared with those for IL-2 alone, in patients with advanced cancer. METHODS: The 181 patients who had metastatic cancer that had failed to respond to standard therapy or who had disease for which no effective therapy existed received treatment with high-dose IL-2 alone or with LAK cells plus IL-2. Both treatment groups were to receive the same dose of IL-2 administered according to the same schedule. IL-2 doses were omitted depending on the tolerance of the patient. Of the 181 patients, 97 had renal cell cancer and 54 had melanoma. RESULTS: Median potential follow-up was 63.2 months. There were 10 complete responses among the 85 assessable patients who received IL-2 plus LAK cells, compared with four among the 79 who received IL-2 alone. There were 14 and 12 partial responses, respectively. Complete response continues in seven patients at 50-66 months. The 36-month actuarial survival with IL-2 plus LAK cells was 31%, compared with 17% with IL-2 alone (two-sided P value [P2] = .089). A trend toward improved survival was seen for patients with melanoma who received IL-2 plus LAK cells, compared with those who received IL-2 alone (24-month survival: 32% versus 15%; 48-month survival: 18% versus 4%; P2 = .064 [corrected]). None of 26 patients with melanoma who received IL-2 alone are alive; five of 28 who received IL-2 plus LAK cells are alive, and three continue in complete response. No difference in survival was seen in patients with renal cell cancer in the two treatment groups. There were six treatment-related deaths (3.3%); three were due to myocardial infarction. Other toxic effects resolved by discontinuation of IL-2. Many toxic effects were related to increased vascular permeability induced by IL-2. CONCLUSIONS: Some patients with metastatic cancer have prolonged remission when they are treated with high-dose IL-2 alone or in conjunction with LAK cells. Our results suggest a trend toward increased survival when IL-2 is given with LAK cells in patients with melanoma, but no trend was observed for patients with renal cell cancer. IMPLICATIONS: As these studies continue, efforts are underway to develop improved immunotherapies using tumor-infiltrating lymphocytes (TIL) and gene-modified TIL.

Economic implications of hepatic arterial infusion chemotherapy in treatment of nonresectable colorectal liver metastases. Meta-Analysis Group in Cancer.

BACKGROUND: Approximately 20% of patients with colorectal cancer die of metastases confined to the liver. A meta-analysis recently performed by our group confirmed that in these patients hepatic arterial infusion of 5-fluoro-2'-deoxyuridine, compared with intravenous chemotherapy with fluoropyrimidines or supportive care (including symptom palliation when necessary), improved tumor response. PURPOSE: Because of the high cost of hepatic arterial infusion, we undertook a cost-effectiveness analysis that related the cost of such therapy to its medical efficacy. METHODS: The patient population was drawn from the seven randomized clinical trials included in the meta-analysis and included individual data on 654 patients. Of these seven trials, five compared hepatic arterial infusion and intravenous chemotherapy and two compared hepatic arterial infusion and a control group in which some patients could be left untreated. Patients assigned to receive hepatic arterial infusion made up the hepatic arterial infusion group; the other patients constituted the control group. The measures of efficacy were survival and tumor response. Health-care costs (in 1995 U.S. dollars) were computed over the duration of patient follow-up and were derived from actual costs in two centers, one at Henri Mondor Hospital (Paris, France) and the other at Stanford University Medical Center (Palo Alto, CA). The total cost of treatment included the initial procedure, chemotherapy cycles, and main complications. RESULTS: The mean gain in life expectancy in the hepatic arterial infusion group compared with the control group was 3.2 months (standard error = 1.0 month). For patients treated by hepatic arterial infusion in Paris, the hepatic arterial infusion pump, initial hospitalization, and the entire process (including follow-up and complications) cost, on average, $8400, $15172, and $29562, respectively; in Palo Alto, these costs were $4700, $13784, and $25 208, respectively. For patients in the control groups in Paris and Palo Alto, the total treatment costs were, on average, $9926 and $5928. The additional costs of hepatic arterial infusion over control treatment were $19636 in Paris and $19280 in Palo Alto. The cost-effectiveness (i.e., the additional cost divided by the additional benefit) with respect to survival of the patients in the hepatic arterial infusion group compared with the patients in the control group was $73635 per life-year in Paris and $72300 per life-year in Palo Alto. CONCLUSIONS AND IMPLICATIONS: The cost-effectiveness of localized chemotherapy for colorectal liver metastases is within the range of accepted treatments for serious medical conditions, although it might be considered borderline by policy-makers in some countries. Prospective clinical trials should be conducted to more definitively answer this question.

Phenotype analyses and cellular mechanisms of the pre-effector T-lymphocyte response to a progressive syngeneic murine sarcoma.

Lymph nodes draining the progressively growing, weakly immunogenic, MCA 105 sarcoma contained tumor-sensitized but not fully functional pre-effector T-cells. These cells could further differentiate to acquire full antitumor effector function for adoptive therapy in an established in vitro sensitization (IVS) procedure. In this study, we utilized selective depletion with antibodies of lymphocyte subsets bearing the L3T4 (CD4) or Lyt-2 (CD8) antigen and of cells bearing the asialo-GM1 (ASGM-1) glycosphingolipid to identify the phenotype of pre-effector cells elicited during progressive tumor growth. Cells from lymph nodes draining a progressive MCA 105 tumor in the footpad were treated with antibodies plus complement prior to IVS. The antitumor efficacy of resulting IVS cells was assessed in adoptive therapy of 3-day established pulmonary MCA 105 metastases. Depletion of Lyt-2+ cells eliminated in vivo antitumor reactivity with concurrent elimination of in vitro cytotoxic activity against the MCA 105 tumor, whereas depletion of L3T4+ cells did not have an impact on either in vivo or in vitro antitumor reactivities. Treatment with ASGM-1 antiserum plus complement was also found to abrogate therapeutic efficacy. However, the in vitro cytotoxic activity was not affected. These results indicate that the pre-effector cells were Lyt-2+, L3T4-, and ASGM-1+. We next examined whether the sensitization of pre-effector cells in vivo required the participation of L3T4+ helper cells. To approach this, mice were depleted of L3T4+, Lyt-2+, or ASGM-1+ cells by antibody injections before tumor inoculation. Treatment with Lyt-2 monoclonal antibody abrogated the pre-effector cell response in the draining lymph nodes, as evidenced by failure to generate therapeutically effective cells following IVS. On the other hand, neither L3T4 nor ASGM-1 antibody treatment affected the generation of pre-effector cells. Thus, sensitization of Lyt-2+ pre-effector cells in response to progressive tumor occurred in the absence of L3T4+ helper cells.

Cellular interactions in effector cell generation and tumor regression mediated by anti-CD3/interleukin 2-activated tumor-draining lymph node cells.

Previous studies have demonstrated that progressive growth of the weakly immunogenic MCA 106 murine sarcoma stimulated, in the draining lymph nodes, the production of tumor-sensitized but not fully functional preeffector lymphocytes. These lymphocytes could develop into specific immune effector cells after sequential in vitro activation with anti-CD3 monoclonal antibody and interleukin 2 (IL-2). In this study, we analyzed cellular requirements for in vivo sensitization of preeffector cells, for generation of immune effector cells by the method of anti-CD3/IL-2 activation, and for adoptive immunotherapy mediated by activated cells. By selective depletion of T-cell subsets in vivo, we found that tumor regression after systemic adoptive immunotherapy required the collaboration of activated CD4+ and CD8+ cells. It was further demonstrated that CD8+ immune cells alone could mediate antitumor effects if exogenous IL-2 was provided in vivo. These results suggest that CD8+ cells served as immediate effector cells, whereas CD4+ immune cells provided a helper function via the secretion of IL-2. During in vitro anti-CD3/IL-2 activation, generation of effector cells depended on the collaborative interaction between previously sensitized CD4+ and CD8+ preeffector cells. At the stage of in vitro activation, the addition of IL-2 could not substitute the function of CD4+ cells. We next examined whether the sensitization of preeffector cells in the draining lymph nodes required cellular interactions between CD4+ and CD8+ T-cells. By in vivo depletion of T-cell subsets during tumor growth, we found that CD4+ cells were sensitized independently of CD8+ cells. More interestingly, in vivo sensitization of CD8+ preeffector cells also occurred independently in the absence of a CD4+ helper cell response. The lack of T-cell-T-cell interactions in vivo may explain the failure of effector cell generation during progressive tumor growth. Taken together, these results demonstrate that the anti-CD3/IL-2 activation defines an immune response distinct from many previously described mechanisms of antitumor immune responses.

Reduced efficacy of allogeneic versus syngeneic fibroblasts modified to secrete cytokines as a tumor vaccine adjuvant.

We examined the relative efficacy of allogeneic versus syngeneic fibroblasts admixed with tumor cells as a vaccine to induce antitumor T-cell reactivity. Allogeneic (3T3) or syngeneic (BLK) fibroblasts transfected to secrete equivalent amounts of GM-CSF were admixed with either D5 melanoma or MCA 207 sarcoma and inoculated s.c. into the flanks of C57BL/6 mice. Vaccine-primed lymph node (LN) cells were examined for in vivo antitumor reactivity in an adoptive transfer model. At fibroblast: tumor cell ratios of < or=1, allogeneic and syngeneic granulocyte macrophage colony-stimulating factor-secreting fibroblasts enhanced T-cell reactivity to tumor cells. However, at ratios of 2.4, the adjuvant effect induced by granulocyte macrophage colony-stimulating factor was not evident. Instead, we observed increased alloreactivity of primed LN cells against 3T3 targets as assessed by cytotoxicity and cytokine release assays, which was not observed with syngeneic fibroblasts. Moreover, with increasing numbers of allogeneic fibroblasts, there was a skewing of the T-cell Vbeta repertoire. These latter cells responded to tumor stimulation with the release of greater amounts of interleukin 10, which may account for the diminished antitumor reactivity observed in vivo. Allogeneic fibroblasts transduced to secrete interleukin 2 or IFN-gamma also induced diminished tumor reactivity of primed LN cells. Syngeneic fibroblasts are superior to allogeneic fibroblasts as vehicles to deliver cytokines in tumor vaccines.

Cross-reactivity of anti-CD3/IL-2 activated effector cells derived from lymph nodes draining heterologous clones of a murine tumor.

Cells from lymph nodes (LN) draining progressively growing tumors can differentiate into immune effector cells upon in vitro stimulation with anti-CD3 monoclonal antibodies followed by interleukin-2. The adoptive transfer of these activated LN cells to tumor-bearing mice mediates potent tumor-specific therapeutic effects. In this study, we sought to further characterize the antitumor efficacy and specificity mediated by the anti-CD3/IL-2 activated tumor-draining LN cells against heterologous clones derived from the murine MCA 106 sarcoma. Ten clones of divergent characteristics with regard to morphology, in vivo growth rate, ability to establish pulmonary metastases, MHC class I (H-2) antigen expression, susceptibility to lysis by allogeneic cytotoxic T-lymphocytes, as well as sensitivity to doxorubicin were selected and analyzed. In adoptive immunotherapy experiments, pulmonary metastases derived from each clone were found to be sensitive to the therapeutic effects of activated cells derived from LN draining the parental MCA 106 tumor. The antigenic cross-reactivity was evident from the observation that activated cells from LN draining each of the individual tumor clones were capable of mediating the regression of parental tumor metastases. The specificity of the antitumor reactivities mediated by LN cells draining MCA 106 clones was demonstrated by a lack of in vivo efficacy against metastases derived from the antigenically distinct MCA 205 sarcoma. Additionally, selected clones were tested for their ability to stimulate draining LN against other cloned tumors or used as targets for therapy with activated LN cells draining different clones. In all 29 adoptive immunotherapy experiments, there was complete cross-reactivity between different MCA 106 tumor clones. These findings suggest that the MCA 106 tumor-specific antigen(s) that stimulates draining LN in vivo and recognized by the anti-CD3/IL-2 activated cells is present on most if not all tumor cells. However, in the absence of a demonstrably resistant tumor clone, a very highly polymorphic antigen with many cross-reactive, but distinct epitopes might be operative and attributable to these observations.

Differences in the effects of host suppression on the adoptive immunotherapy of subcutaneous and visceral tumors.

A syngeneic transplantable sarcoma induced in C57BL/6 mice, MCA 105, was used in studies to examine host suppression on the adoptive immunotherapy of established intradermal and experimentally induced pulmonary and hepatic metastases. Fresh immune splenocytes were generated from mice immunized to the MCA 105 tumor by a mixture of viable tumor cells and Corynebacterium parvum. The adoptive immunotherapy of intradermal MCA 105 tumor with immune cells required prior immunosuppression of the recipient by sublethal irradiation with 500 R or T-cell depletion. The effect of whole-body sublethal irradiation appeared to eliminate a systemic host suppression mechanism, since partialbody irradiation involving the tumor-bearing area did not permit successful immunotherapy. Host irradiation was not required to achieve successful immunotherapy of experimentally induced pulmonary or hepatic metastases. In nonirradiated recipients bearing both intradermal and pulmonary tumors, host suppression did not affect the function of transferred immune cells to induce regression of pulmonary metastases. Thus, suppression of adoptive immunotherapy appears to be relevant to tumors confined to the skin and subcutaneous tissue but not to tumor in visceral sites, such as the lung and liver.

Recombinant interleukin 12 enhances cellular immune responses to vaccination only after a period of suppression.

Adjuvant use of recombinant murine IL-12 (rmIL-12) was examined in mice vaccinated with irradiated syngeneic tumor or allogeneic cells. rmIL-12 given to A/J mice vaccinated with irradiated SCK tumor cells engineered to secrete granulocyte/macrophage-colony-stimulating factor resulted in significantly better protection from tumor challenges 28 days after vaccination but, unexpectedly, severely compromised host protection 14 days after vaccination. Immune suppression was rmIL-12 dose dependent and manifested as reduced splenic CTL activity, stimulated cytokine release and ability to reject SCK cells. Transient immune suppression was also seen with rmIL-12 given during vaccination of C3H/HeN mice with irradiated K1735 melanoma cells and of C57BL/6 mice with irradiated allogeneic HKB cells. The period of suppression coincided with transiently reduced splenic T-cell mitogenic responses to concanavalin A and IL-2, suggesting that they may be causally related. Suppression appears to be due to impaired immune effector mechanisms rather than impaired host immunization, which is actually enhanced as evidenced by the enhanced reaction to immunogens when hosts are challenged later after rmIL-12 administration. Demonstration that rmIL-12, as it is frequently used, induces a transient period of impaired immune response that can compromise host protection suggests that the unquestioned effectiveness of rmIL-12 against murine tumors is primarily due to activation of mechanisms other than antigen-specific tumor immunity (e.g., antiangiogenic effects) and that use of human IL-12 should be monitored for similar effects.

Characteristics and in vivo homing of long-term T-cell lines and clones derived from tumor-draining lymph nodes.

Lymph nodes draining a progressively growing tumor contain T-cells which can be activated sequentially by anti-CD3 and IL-2 to differentiate into tumor-specific effector cells. In this study, long-term cultured T-cell lines were established from activated MCA 106 tumor-draining lymph node cells by periodic stimulation with irradiated tumor cells in the presence of low concentrations of IL-2 (< or = 60 International units/ml). Such long-term cultured cell lines maintained therapeutic effects when transferred to tumor-bearing mice. Although the initial anti-CD3/IL-2-activated T-cells displayed a broad distribution of T-cell antigen receptor beta chain variable region (V beta) usages, long-term cultured cells were dominated by T-cells expressing a few V beta elements. Of six cell lines, only three V beta phenotypes (V beta 5, 11, 13) were identified, and individual cell lines frequently expressed a single V beta gene product. Despite restricted V beta expression, each cell line mediated tumor-specific reactivity in adoptive immunotherapy. Many T-cell clones were isolated from long-term cell lines. Three V beta 13 T-cell clones demonstrated specific in vivo antitumor effects, whereas two V beta 11 and two V beta 5 clones revealed a significant degree of cross-reactivity against the antigenically distinct MCA 205 tumor. Although the initial anti-CD3/IL-2-activated cells lacked demonstrable cytotoxic reactivity, T-cell clones derived from them exhibited cytotoxic effects to the MCA 106 tumor cells. The specificity of the cytotoxicity mediated by each clone reflected its in vivo antitumor effects. Furthermore, studies of in vivo localization of cloned T-cells demonstrated tumor-specific infiltration of the 5A2 (V beta 13) clone to the MCA 106 tumor metastases, whereas clone 9H6 (V beta 5) revealed some accumulation in the MCA 205 tumor. Again, the in vivo antitumor effects of the 9H6 clone correlated with its in vivo infiltration into the specific MCA 106 and the nonspecific MCA 205 metastases. Taken together, the long-term culture of anti-CD3/IL-2-activated tumor-draining lymph node cells resulted in selective expansion of a few T-cells as evidenced by the limited T-cell receptor V beta expression. Our results also demonstrated that systemically administered antitumor T-cell clones gained access and accumulated at metastatic tumor sites, and the degree of infiltration correlated with the specificity of the in vivo antitumor effect as well as the in vitro cytotoxic activity.

Clinical observations on adoptive immunotherapy with vaccine-primed T-lymphocytes secondarily sensitized to tumor in vitro.

The adoptive immunotherapy of human malignancy requires reliable methods to sensitize and expand patients' T-cells reactive to autologous tumors. In animal studies, we have generated therapeutic effector cells against a poorly immunogenic tumor by a two-step procedure: vaccination of the host followed by the secondary stimulation of vaccine-primed lymph node (LN) cells by in vitro sensitization (IVS) with tumor in the presence of interleukin 2 (IL-2). Based on these observations, we performed a clinical trial in patients with advanced cancer to evaluate the antitumor efficacy of vaccine-primed LN cells which were similarly activated in vitro. Patients were vaccinated with irradiated autologous tumor admixed with Bacillus Calmette-Guérin and had draining LN excised 10 days later for IVS culture. During IVS culture, LN cells expanded up to 14-fold (average of 8.4-fold). A mean of 6.7 x 10(9) cells was infused in ten patients (seven melanoma, three renal cell cancer) along with the concomitant i.v. administration of IL-2 (180,000 IU/kg every 8 h for 5 days). Phenotype analysis of IVS-LN cells revealed 78 +/- 4% CD3+ T-cells which were predominantly CD4+ (67 +/- 5%) with expression of HLA-DR and IL-2 receptor. IVS-LN cells displayed relative specificity of autologous tumor lysis in four of ten cases compared to zero of seven IVS-peripheral blood lymphocytes derived from the same patients as measured by the 51Cr release assay. One mo after therapy, seven of nine patients treated with IVS-LN cells and IL-2 developed delayed-type hypersensitivity reactivity to autologous tumor compared to zero of nine patients treated with tumor vaccination and IL-2 only (P < 0.002). These observations suggest that antitumor reactivity was passively transferred with the IVS-LN cells. Major toxic side effects including fever, hepatic dysfunction, and weight gain associated with the capillary leak syndrome were associated with exogenous IL-2 administration. Tumor vaccination and cell transfer were well tolerated without significant complications. Of the ten patients treated with IVS-LN cells and IL-2, there were one partial and one minor response, and one patient has had stable disease for 27+ mo. There was no evidence of tumor response in ten patients treated with tumor vaccination and IL-2 only. Further clinical studies evaluating the antitumor reactivity of vaccine-primed LN cells are warranted.

Vaccination of pediatric solid tumor patients with tumor lysate-pulsed dendritic cells can expand specific T cells and mediate tumor regression.

Dendritic cells (DCs) have been shown to be a promising adjuvant for inducing immunity to cancer. We evaluated tumor lysate-pulsed DC in a Phase I trial of pediatric patients with solid tumors. Children with relapsed solid malignancies who had failed standard therapies were eligible. The vaccine used immature DC (CD14-, CD80+, CD86+, CD83-, and HLA-DR+) generated from peripheral blood monocytes in the presence of granulocyte/monocyte colony-stimulating factor and interleukin-4. These DC were then pulsed separately with tumor cell lysates and the immunogenic protein keyhole limpet hemocyanin (KLH) for 24 h and then combined. A total of 1 x 10(6) to 1 x 10(7) DC are administered intradermally every 2 weeks for a total of three vaccinations. Fifteen patients (ages 3-17 years) were enrolled with 10 patients completing all vaccinations. Leukapheresis yields averaged 2.8 x 10(8) peripheral blood mononuclear cells (PBMC)/kg, and DC yields averaged 10.9% of starting PBMC. Patients with neuroblastoma, sarcoma, and renal malignancies were treated without obvious toxicity. Delayed-type hypersensitivity (DTH) response was detected in 7 of 10 patients for KLH and 3 of 6 patients for tumor lysates. Priming of T cells to KLH was seen in 6 of 10 patients and to tumor in 3 of 7 patients as demonstrated by specific IFN-gamma-secreting T cells in unstimulated PBMCs. Significant regression of multiple metastatic sites was seen in 1 patient. Five patients showed stable disease, including 3 who had minimal disease at time of vaccine therapy and remain free of tumor with 16-30 months follow-up. Our results demonstrate that it is feasible to generate large numbers of functional DC from pediatric patients even in those highly pretreated and with a large tumor burden. The DC can be administered in an outpatient setting without any observable toxicity. Most importantly, we have demonstrated the ability of the tumor lysate/KLH-pulsed DC to generate specific T-cell responses and to elicit regression of metastatic disease.

Injection of complementary DNA encoding interleukin-12 inhibits tumor establishment at a distant site in a murine renal carcinoma model.

Interleukin (IL-12) protein has been shown to elicit diverse immunological responses and potent antitumor activity. We demonstrate here that intradermal injection of IL-12 cDNA induces systemic biological effects characteristic of the cytokine in vivo. Intradermal injection of IL-12 cDNA resulted in local expression of IL-12 mRNA, which correlated with a 10-fold increase in natural killer activity and a 3-4-fold increase in anti-CD3-induced IFN-gamma production in cultured splenocytes. Furthermore, when challenged with Renca tumor cells at a distant site, the day of tumor emergence was significantly delayed, and tumor growth was reduced in mice that received IL-12 cDNA, compared to mice given injections of plasmid vector alone. A number of the mice receiving IL-12 cDNA injections remained tumor free months after tumor challenge. In contrast to mice receiving recombinant IL-12 protein, no splenomegaly was detected when natural killer activity was significantly induced in mice receiving injections of IL-12 cDNA. Because purified plasmid DNA is more economical to prepare and has a longer shelf-life than recombinant proteins, and intradermal administration of cDNA encoding IL-12 did not cause splenomegaly, our findings suggest that the in vivo injection of cDNA encoding IL-12 may be a less toxic and more cost-effective alternative to IL-12 protein therapy in some clinical or experimental therapeutic applications.

Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids.

In order to evaluate the efficacy of dexamethasone (dex) in reducing the toxicity of therapy with lymphokine-activated killer (LAK) cells and interleukin-2 (IL-2), we treated six patients receiving this form of immunotherapy with intravenous (IV) dex, 4 mg every six hours. Compared with a control group of 27 patients not receiving dex with their immunotherapy, these corticosteroid-treated patients were able to tolerate the administration of more IL-2, yet experienced significantly less toxicity. Dyspnea, confusion, fever, mean peak serum creatinine, and bilirubin levels during treatment were significantly reduced in corticosteroid-treated patients, with a corresponding decrease in pruritus in this group as well. Overall weight gain was not different between groups, although a curtailment of weight gain temporally related to dex treatment was seen in some patients. Hematologic side effects, including anemia, eosinophilia, and thrombocytopenia, were not reduced by dex. These results suggest that dex can inhibit at least some of the toxic side effects of LAK cell and IL-2 therapy. Because of the concern that the therapeutic effect may also be abrogated, future studies combining corticosteroids with this form of immunotherapy should be undertaken with caution.

A phase I study of intraarterial iododeoxyuridine in patients with colorectal liver metastases.

Regional delivery of iododeoxyuridine (IdUrd) to patients with colorectal liver metastases was examined in a phase I study. The maximum-tolerated intraarterial (IA) dose (MTD) was 1,333 mg/m2/d administered continuously for 14 days. The dose-limiting toxicity was thrombocytopenia. Thrombocytopenia and leukopenia were correlated with the amount of IdUrd incorporated into DNA of peripheral granulocytes. In contrast to our experience with 5-fluorodeoxyuridine, there was no evidence of hepatobiliary toxicity. In 11 patients who received IA IdUrd alone, seven had a greater than or equal to 50% decrease in carcinoembryonic antigen (CEA) levels, with five having tumor volume reductions of 65%, 48%, 46%, 44%, and 27%. Thus, IA IdUrd alone has antitumor efficacy. Patients subsequently received IdUrd in combination with external beam radiation to a total dose of 2,400 cGy without acute local toxicity. In addition to these favorable clinical findings, we have previously shown that IdUrd is selectively incorporated into tumor DNA compared with normal liver in these patients. Further phase II evaluations of this approach are warranted.

Adoptive immunotherapy with vaccine-primed lymph node cells secondarily activated with anti-CD3 and interleukin-2.

PURPOSE: In preclinical studies, we have reported the ability to induce immune T cells in lymph nodes (LN) primed by in vivo vaccination with tumor cells admixed with a bacterial adjuvant. These LN cells can be activated and expanded ex vivo for the successful immunotherapy of established tumors. We have applied these methods to generate vaccine-primed LN in patients with advanced melanoma and renal cell cancer (RCC) for therapy. MATERIALS AND METHODS: Irradiated autologous tumor cells admixed with bacille Calmette-Guérin (BCG) were used to vaccinate patients. Seven days later, draining LN were removed for activation with anti-CD3 monoclonal antibody (mAb) followed by expansion in interleukin-2 (IL-2). Activated LN cells were administered intravenously (IV) with the concomitant administration of IL-2. RESULTS: A total of 23 patients were evaluated (11 melanoma and 12 RCC). Vaccine-primed LN were expanded ex vivo with a mean of 8.4 x 10(10) cells administered per patient. Among 20 patients assessed, 15 demonstrated minimal cytotoxicity of autologous tumor cells by the activated LN cells, with the remaining mediating nonspecific cytotoxicity. By contrast, a majority of the activated LN cells showed highly specific release of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon gamma (IFN-gamma) to autologous but not allogeneic tumor stimulation. This tumor-specific cytokine release was found to be major histocompatibility complex (MHC) class I-restricted, which indicates the involvement of CD8+ cells. Among 11 melanoma patients, one had a partial tumor response. Among 12 RCC patients, two had complete and two partial responses. A trend (P = .066) between the enhancement of delayed-type hypersensitivity (DTH) reactivity to autologous tumor after therapy and tumor regression was observed. CONCLUSION: Tumor vaccines can be used to induce immunologically specific T-cell responses against melanoma and RCC in draining LN. Anti-CD3/IL-2 activation of primed LN cells can be reliably performed for clinical therapy and appears to have activity in patients with metastatic RCC.

Immunotherapy of patients with advanced cancer using tumor-infiltrating lymphocytes and recombinant interleukin-2: a pilot study.

Clinical investigations using the adoptive transfer of lymphokine-activated killer (LAK) cells and recombinant interleukin-2 (rIL-2) to treat patients with advanced cancer have yielded encouraging results. We have thus sought ways to enhance the effectiveness of adoptive immunotherapy while minimizing its toxic side effects. Murine experiments have identified tumor-infiltrating lymphocytes (TIL) as killer cells more effective than LAK cells and less dependent on adjunctive systemically administered IL-2 to mediate antitumor effects. Accordingly, we performed a pilot protocol to investigate the feasibility and practicality of administering IL-2-expanded TIL to humans with metastatic cancers. Twelve patients, including six with melanoma, four with renal cell carcinoma, one with breast carcinoma, and one with colon carcinoma, were treated with varying doses and combinations of TIL (8.0 X 10(9) to 2.3 X 10(11) cells per patient), IL-2 (10,000 to 100,000 U/kg three times daily to dose-limiting toxicity), and cyclophosphamide (CPM) (up to 50 mg/kg). Two partial responses (PR) to therapy were observed: pulmonary and mediastinal masses regressed in a patient with melanoma, and a lymph node mass regressed in a patient with renal cell carcinoma. One additional patient with breast cancer experienced a partial regression of disease in lymph nodal and cutaneous sites with complete elimination of malignant cells from a pleural effusion, although cutaneous disease recurred at 4 weeks. The toxicities of therapy were similar to those ascribed to IL-2; no toxic effects were directly attributable to TIL infusions. In five of six melanoma patients, TIL demonstrated lytic activity specific for the autologous tumor target in short-term chromium-release assays, distinct from the nonspecific lytic activity characteristic of LAK cells. This study represents an initial attempt to identify and use lymphocyte subsets with enhanced tumoricidal capacity in the adoptive immunotherapy of human malignancies.