Efficacy of Bioindexing for Graft-Transmissible Citrus Pathogens in Mixed Infections.

Biological indexing for graft-transmissible pathogens of citrus in the presence of additional pathogens was investigated. The probability for symptom expression, the efficacy of the bio-indexing tests, and the number of citrus indicators required for pathogen detection were statistically evaluated. Multiple infections did not preclude symptom expression or reduce the diagnostic efficacy of the primary indexing hosts for Citrus tristeza virus (CTV), Citrus psorosis virus (CPsV), and Citrus tatter leaf virus (Apple stem grooving virus). Symptoms of Citrus vein enation virus (CVEV) and the diagnostic efficacy of Mexican lime were suppressed by the T30 group CTV isolates, but not by other CTV isolates tested. CPsV suppressed symptom expression and diagnostic efficiency of Dweet tangor and sweet orange for concave gum. The application of alternate bioassay hosts for indexing was also investigated. Dweet tangor, sweet orange, and Citrus excelsa are not typically used for bioindexing of CVEV, however, Dweet tangor and C. excelsa detected CVEV in single infections, whereas in sweet orange, CVEV was detected only when CPsV, concave gum, or citrus viroids were present. CTV was readily detected using the alternative indicator C. excelsa, whereas only shock reacting CPsV isolates were effectively indexed by Mexican lime.

Finger Imprint of Poncirus trifoliata: A Specific Interaction of a Viroid, a Host, and Irrigation.

The unusual symptom, "finger imprint", described exclusively on Poncirus trifoliata, has been reported in only a single field trial investigating the effects of citrus viroids on crop performance. With this, the question has persisted whether the observed growth abnormality was a disease symptom induced by Citrus viroid IIIb (CVd-IIIb) or a consequence of mechanical damage caused by the handling of young trees during propagation or cultural practices in the field. The recurrence of finger imprint symptoms on trees after 5 years in the field in which no abnormal growth features were previously noted now supports the proposition of a viroid-induced disease. The symptom expression results from an unusual etiology of a complex relationship of the specific viroid CVd-IIIb on the specific rootstock P. trifoliata only when supplemental water is applied by sprinkler irrigation.

Recombinant vaccinia interleukin-2-infected tumor cell vaccines in immunotherapy of murine colon adenocarcinoma.

A recombinant vaccinia virus (vCF13) containing and expressing the gene for human interleukin (IL)-2(vCF13) was compared to a recombinant vaccinia transfection control strain containing the LacZ gene at the same insertion site (vTFCLZ-1) for their ability to augment the immunogenicity of murine colon adenocarcinoma cell lines CT26 and CA51 in Balb/c mice. Both recombinant vaccinia strains abolished tumorigenicity of 10(5) CT26 or CA51 tumor cells. vCF13-infected tumor cells that secreted human IL-2 as measured by both CTLL-2 and enzyme-linked immunosorbent assays induced delay in tumorigenesis when administered in two weekly subcutaneous injections 1 week prior to challenge with 10(5) uninfected tumor cells. Although three of five vCF13-CT26 immunized mice developed tumors by day 14 after challenge, intralesional injection of these tumors with vCF13 induced rapid regression, whereas all five tumors that developed in vTFCLZ-1 immunized mice showed no response to intralesional vTFCLZ-1. These preliminary results provide support for the potential utility of recombinant vaccinia/IL-2 in tumor immunotherapy.

Active specific immunotherapy with vaccinia colon oncolysate enhances the immunomodulatory and antitumor effects of interleukin-2 and interferon alpha in a murine hepatic metastasis model.

The role of cytokines as primary or adjuvant antineoplastic agents has been well established. Interleukin-2 (IL-2) and the interferons have, particularly, proven to be effective antitumor agents when given alone, and seem to act synergistically on the eradication of metastases from immunogenic tumors. Active specific immunotherapy, in the form of viral oncolysates, has also shown effectiveness in cancer therapy. Bearing this in mind, we decided to combine these agents in an adjuvant triple regimen and compare their effectiveness to other treatments in terms of tumor burden and survival in a murine colon cancer hepatic metastases model. BALB/c mice were injected with CC-36, a weakly immunogenic murine colon adenocarcinoma, intrasplenically, to produce artificial liver metastases. The animals were divided into one control group and seven treatment groups receiving either vaccinia colon oncolysate (VCO), IL-2, interferon-alpha (IFN alpha) alone, or combinations of these agents. Half the animals were followed for survival and the other half were sacrificed at the end of the experiment for quantification of tumor burden. The blood of the sacrificed animals was utilized in a series of immunological tests in order to demonstrate the cytolytic potential of the peripheral blood lymphocytes (PBL) in each treatment group, as well as to characterize phenotypically the cells acting as effectors. The triple-adjuvant regimen group was by far the most effective treatment group, demonstrating 100% survival and a significant reduction in tumor burden when compared to other groups. Furthermore, the PBL from the animals in this group showed 69.4% lysis of the CC-36 target cells in vitro. These effector lymphocytes were characterized as ASMG1-/Lyt2.2+ cytolytic lymphocytes. We conclude that these lymphocytes were stimulated by the administration of VCO and further augmented by the immunomodulation of the cytokines given in the triple regimen, and that such a regimen might prove beneficial in the treatment of established hepatic metastases from weakly immunogenic tumors.

Vaccinia virus-infected C-C36 colon tumor cell lysates stimulate cellular responses in vitro and protect syngeneic Balb/c mice from tumor cell challenge.

Vaccinia virus (VV) was used to infect and lyse the Balb/c colon tumor line C-C36 to prepare oncolysate (VCO) with augmented immunogenicity. Mice treated with VCO and challenged with C-C36 were significantly protected against tumor growth as compared to untreated controls (P less than 0.001) and mice treated with CO (P less than 0.01). Moreover, protection induced by VCO was specific when growth inhibition of C-C36 was compared to that of meth-A (P = 0.027). Splenocytes from mice stimulated with VCO in vitro showed greater proliferation than splenocytes stimulated with CO alone or VV alone, suggesting induction of a unique VCO component. Additional evidence for a specific response was suggested by the observation that splenocytes stimulated with VCO in vitro demonstrated augmented cytolysis of C-C36 but did not show cytolytic activity against unrelated target cells. However, augmented cytolysis of the natural killer (NK)-sensitive YAC-1 by VCO-stimulated splenocytes was also observed. These results suggest that in vivo resistance to tumor challenge induced by VCO treatment may result from stimulation of both specific and nonspecific effector cells.

Melanoma patient antibody responses to melanoma tumor-associated antigens defined by murine monoclonal antibodies.

Sera from melanoma patients undergoing immunization with three distinct immunogens were evaluated for development of IgG antibody specific for the melanoma tumor-associated antigens (TAAs) GD3 and 250-kd glycoprotein (high molecular weight; HMW). Of 99 patients receiving either irradiated allogeneic melanoma cells admixed with bacillus Calmette-Guérin, vaccinia viral oncolysate melanoma cell membranes, or cholesterol hemisuccinate-modified melanoma cells, 12 were determined to have responded against GD3 and 17 against the HMW TAA. This reactivity was measured using both direct binding to purified TAA and specific inhibition of binding of murine monoclonal antibodies R24 and Me-1-14 for GD3 and HMW TAA, respectively. Preimmune sera from these patients did not react with these TAAs, nor did preimmune sera or follow-up sera from melanoma patients electing not to receive immunotherapy. These results suggest that melanoma patients can be immunized against these TAAs as presented on the melanoma cell membrane in an immunotherapy setting and that immunization using purified TAAs might likewise be feasible.

Treatment of murine hepatic metastases with vaccinia colon oncolysates and IL-2.

To evaluate the feasibility and utility of vaccinia colon oncolysates (VCO) and low-dose interleukin-2 (IL-2) immunotherapy for advanced colon cancer, we have developed a murine model and tested the efficacy of combined treatment regimens. We employed intrasplenic injection of cultured colon adenocarcinomas (C-C36) in syngeneic Balb/c mice to produce experimental hepatic metastases. In the first set of experiments, animals were challenged with 5 X 10(5) tumor cells and sacrificed 14 days following tumor challenge. In the second set of experiments, animals were challenged with 2 X 10(5) tumor cells and followed for survival over the ensuing 90 days. In the first set of experiments, animals were treated prophylactically with VCO (40 micrograms, sc, 14 and 7 days prior to challenge) and/or therapeutically with IL-2 (25,000 u, Hoffmann-LaRoche rIL-2, ip BID, on Days 1-3 following challenge). In the second set of experiments, animals were treated with either the identical regimens or therapeutically with VCO (same dose, sc, 2 and 10 days following challenge) and/or IL-2 (same dose, ip BID, on Days 9-11 following challenge). Tumor burden data from sacrificed animals was in agreement with survival data and showed significant tumor burden reduction in the combined treatment group as assessed by liver weight and tumor nodule enumeration. Survival data demonstrated highly significant survival advantage for animals treated with the two biological response modifiers: VCO (P less than 0.0021), and IL-2 (P less than 0.0017). The data presented suggest a synergistic effect for these two agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Vaccinia colon oncolysate immunotherapy for murine hepatic metastases can be modulated with low-dose interleukin-2. Third place winner: Conrad Jobst Award.

A murine colon cancer hepatic metastases model was developed via intrasplenic injection of C-C36 tumor cells in syngeneic Balb/c mice to determine the potential efficacy of vaccinia colon oncolysate (VCO) immunoprophylaxis and therapy with and without low-dose interleukin-2 (IL-2) immunomodulation. Mice were injected with 40 micrograms VCO subcutaneously, either prophylactically or therapeutically. IL-2 (Hoffman-La Roche, Nutley, NJ) was administered at a dose of 25,000 units intraperitoneally twice daily for three consecutive days, prophylactically, therapeutically immediately after tumor challenge (early), or 9 days after tumor challenge (late). Mice were followed for 50 days after tumor challenge, and mortalities were recorded. Mice receiving VCO alone did not demonstrate better survival than controls. However, mice receiving VCO with IL-2 immunomodulation demonstrated consistently better survival than mice treated with IL-2 alone or controls. The group receiving VCO therapy with late IL-2 modulation (75% survival demonstrated improved survival over controls (0% survival, P less than 0.00001), VCO-treated mice (0% survival, P less than 0.005), and IL-2-treated mice (29% survival, P = 0.07). In vitro assays revealed enhanced NK activity and suggested cytotoxic T-lymphocyte (CTL) induction as possible mechanisms responsible for these biologic effects. Combined VCO and IL-2 immunotherapy may be of potential benefit to patients with metastatic colon cancer, but further research is required to optimize treatment regimens.

Positive relationship of clinical and serologic responses to vaccinia melanoma oncolysate.

In this phase Ia/Ib trial, vaccinia melanoma oncolysate (VMO) is a virus-augmented melanoma cell membrane vaccine that has been shown to be safe and to stimulate the production of antimelanoma antibodies in high-risk melanoma patients treated in a surgical adjuvant setting. One patient with stage I and 38 patients with stage II melanoma were entered in the study between December 1984 and October 1985, with a mean follow-up of approximately 17 months. Each patient received a smallpox booster injection followed one week later by the first of 13 weekly intradermal injections of 2.0 mg of VMO. At the end of 13 weeks, injections were given every other week for 12 months or until recurrence. Clinical results show that 25 of the 39 patients had no evidence of disease as of December 1986. Moreover and more importantly, statistical comparison of patients in this study with 39 matched controls shows a significant increase in disease-free survival for the patients treated with VMO. Serum obtained prior to treatment and at three-month intervals during treatment was tested in a Staphylococcus protein A rosette assay for reactivity with melanoma cell lines. All pretreatment samples (39/39) were negative, and 64% became positive by 12 months after appropriate dosage escalations. Moreover, enzyme-linked immunosorbent assay showed a positive correlation between anti-melanoma IgG antibody titer and disease-free survival.

Serological evaluation of melanoma patients in a phase I/II trial of vaccinia melanoma oncolysate (VMO) immunotherapy.

Vaccinia melanoma oncolysates (VMO) were tested in a Southeastern Cancer Study Group (SECSG)-sponsored phase I/II multiinstitutional trial. Forty-eight patients with stage I or II disease were placed on study at six different dose levels of VMO and two different dose schedules, immediate or delayed. Patients' sera, obtained before treatment and every 3 months following initiation of treatment, were tested for antimelanoma antibodies using a Staphylococcus protein A (SpA) assay. Pretreatment sera were negative in 46 of 47 patients, and only two of 19 patients on delayed treatment developed reactivity by 6 months. However, 13 of 23 on immediate treatment developed reactivity, including eight of eight at the higher doses (1.5 and 2.0 mg). Neither anti-HLA antibody tested by a standard microcytotoxicity assay nor circulating immune complexes measured by both Clq and conglutinin binding assays were produced as a result of the immunization. The demonstration of immunogenicity of VMO at the 2 mg dose and immediate schedule supported the rationale for the use of this dose and schedule for the ongoing second phase Ia/Ib trial and for the future phase III randomized prospective study.

A Southeastern Cancer Study Group phase I/II trial with vaccinia melanoma oncolysates.

Vaccinia melanoma oncolysates (VMO) were tested in a Southeastern Cancer Study Group (SECSG) Phase I/II trial. Forty-eight patients with high-risk Stage I or pathologic Stage II disease were placed on study at six different dose levels and two different treatment regimens. Patients were monitored for toxicity to the VMO after each injection. Patients' sera were tested for anti-human melanoma reactivity with the Staphylococcus Protein A (SpA) assay. Toxicity was minimal at all doses tested. In only 2 of 19 patients on delayed treatment did reactivity develop in the SpA assay by 6 months. However, 13 of 23 patients on immediate treatment showed reactivity, including 8 of 8 at the two highest doses. Since the VMO appears to be safe at all of the doses tested, and because of the immunogenicity of the VMO at the higher doses as demonstrated by the SpA assay, the 2-mg dose level, for immediate treatment, was chosen for use in future trials.

The effect of treatment with Corynebacterium parvum on the development and growth of experimental hematogenic metastases of schwannoma in the rat.

Single i.v. administration of Corynebacterium parvum 5 days before i.v. injection of 10(6) tissue cultured syngeneic schwannoma cells in Lewis rats resulted in extension of survival time (P less than 0.05). There was a significant decrease in metastatic tumor incidence for lung, heart, and kidney and decreased lung tumor growth with approximately 50% of the lung tumor burden of untreated controls (P less than 0.05). Rats treated similarly with C. parvum 10 days after tumor cell injection showed no enhanced survival; to the contrary, their survival was shortened. Moreover, tumor incidence in the post-treated group was not significantly different from the control but significantly increased in comparison to the pretreated group. Enhanced lung tumor growth resulted in a final tumor burden about twice that of untreated controls (P less than 0.05).

Cellular immunosenescence in F344 rats: decreased natural killer (NK) cell activity involves changes in regulatory interactions between NK cells, interferon, prostaglandin and macrophages.

Natural killer (NK) activity of F344 rat spleen cells remained constant between 1 and 18 months of age under specific pathogen-free (SPF) conditions. Between 18 and 24 months of age, however, there was a dramatic decline in activity which remained at a low baseline throughout the normal lifespan. Removal of adherent cells on G-10 Sephadex columns revealed age-related changes in adherent cell regulation of NK activity. Young (4-6 week) NK activity was consistently decreased by adherent cell removal while old (24-30 month) NK activity was slightly but reproducibly increased. Moreover, splenic macrophages from old rats purified by adherence to microexudate-coated surfaces were highly suppressive to young nonadherent NK activity. A role for endogenous prostaglandin (PG) in suppressed old rat NK activity was suggested by the effectiveness of anti-PGE2 in vivo to boost old NK activity. Although old rat NK activity was boosted to a relatively greater extent by interferon (IFN) in vitro than was young NK activity, IFN-boosted NK activity of old rats was much more sensitive to PGE2 inhibition than was IFN-boosted young rat NK activity. IFN treatment in vitro or poly(I:C) treatment in vivo induced protection against PGE2 inhibition of NK activity in young rats, while no resistance to PGE2 inhibition was induced in old rat NK cells by similar treatments. In vivo, the same protocol of IFN administration which boosted young rat NK activity further suppressed old rat activity. These results support the hypothesis that immunosuppression related to aging, which supersedes the boosting effect of IFN, involves the combined effects of suppressor macrophages (via PGE2) and intrinsic changes in effector (NK) cells which render them more sensitive to PGE2 inhibition.

Cellular immunosenescence in F344 rats: decline in responsiveness to phytohemagglutinin involves changes in both T cells and macrophages.

Spleen cells from F344 male rats showed decreased DNA synthetic responsiveness [( 3H]thymidine incorporation) with age to the T cell mitogen phytohemagglutinin (PHA). Decreased proliferative responses were associated with decreased production of interleukin-2 (IL-2) and could be partially restored by providing exogenous IL-2. Responses of spleen cells from aged rats could also be enhanced by removal of Sephadex G-10 adherent cells. Furthermore, co-culture of adherent cell-containing spleen cells from aged rats with nonadherent spleen cells from young rats resulted in suppression of responses. Purified peritoneal exudate macrophages (PEM) from aged rats showed potent regulatory effects on PHA responses of young and old nonadherent spleen cells resulting in suppression above 2.5% macrophage/nonadherent spleen cell ratios. PEM from young rats enhanced the response of young T cells but failed to affect the response of aged T cells. T cell proliferation in the presence of prostaglandin (PGE1) showed age-dependent differences in regulation such that at 10(-6) to 10(-7) M young responses were enhanced and old responses were suppressed. These results suggest that decreased responsiveness of T cells to PHA with age is a complex phenomenon involving changes in both production of regulatory mediators by T cells (IL-2) and macrophages (PGE) as well as changes in T cell responsiveness to these signals.

Immunomodulatory activities of human leukocyte interferon in advanced cancer patients.

Natural killing and antibody-dependent cellular cytotoxicity of peripheral blood lymphocytes from 33 advanced cancer patients were monitored during the course of treatment with human leukocyte interferon in a phase I trial. Ten patients receiving 10 to 60 X 10(6) international units (IU)/m2 in a single injection showed augmentation of cytolytic activity above pretreatment values. The most typical response consisted of a decrease in activity at day 1 followed by a significant increase on day 3 with a return to baseline at day 7. No clearcut minimal immunomodulatory dose was achieved, although four of six patients treated at 60 X 10(6) IU/m6 showed increased activity at day 3. Of these, three subsequently received repeated 60 X 10(6) IU/m2 doses on a weekly basis, and two of these showed repetitive augmentation after 3-4 weeks.