Experimental allergic encephalitis. Dissociation of cellular immunity to brain protein and disease production.

The encephalitogenic determinant of brain protein, a nonapeptide having the amino acid sequence Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys, has been characterized and synthesized. In a previous study, analogues of this encephalitogenic peptide were synthesized and some were shown to be encephalitogenic while others were not. Guinea pigs were immunized with encephalitogenic peptides having amino acid sequences different from that in the native protein. These guinea pigs did not show cellular immunity in vivo (skin reactivity) or in vitro (lymphocyte stimulation or macrophage migration inhibition) to the encephalitogenic brain protein (EP) although they did show cellular immunity to the immunizing antigenic peptide. Guinea pigs immunized with an encephalitogenic peptide having the same amino acid sequence as the brain protein, or with a nonencephalitogenic peptide having the same amino acid sequence as the native protein but lacking the terminal lysine, did develop cellular immunity to the EP. Animals immunized with EP showed cellular immunity to this protein, but not to the encephalitogenic peptides. Animals immunized with nonencephalitogenic protein (NEP), prepared by altering the tryptophan residue of EP, did not develop disease but did show cellular immunity in vitro and in vivo to the EP. Animals protected from disease by immunization with NEP similarly showed cellular immunity to EP. Thus, the results suggest a dissociation between cellular immunity to EP and the production of experimental allergic encephalitis (EAE). Animals immunized with the encephalitogenic peptides develop EAE, but do not show cellular immunity to EP, and animals immunized with NEP show cellular immunity to EP but do not develop EAE. A fresh approach to the examination of the pathogenesis of EAE is now possible through the use of these well-characterized antigens.

Osteogenic sarcoma. Immunologic parameters before and during immunotherapy with tumor-specific transfer factor.

18 patients with osteogenic sarcoma were followed by serial measurements in vitro of tumor-specific cell-mediated cytotoxicity and of "active" and total rosette-forming T-cells. 13 of these patients have had or are currently receiving injections of osteogenic sarcoma-specific dialyzable transfer factor derived from healthy donors. In three patients with very small lesions, cytotoxicity was high before amputation and decreased within 2 mo after removal of tumor. Cytotoxicity was low at time of diagnosis in all patients with large tumor masses. The cytotoxicity of the patients' lymphocytes increased after administration of tumor-specific transfer factor in all patients so treated. Patients receiving nonspecific transfer factor showed evidence of declining cell-mediated cytotoxicity. Tumor-specific transfer factor may produce an increase in cell-mediated cytotoxicity to the tumor in patients with osteogenic sarcoma. This possibility is suggested by the pain and edema that occurred in the area of the tumor in patients who had metastatic disease when therapy was started and by lymphocytic infiltrates in the tumor, as well as by the increase in cell-mediated cytotoxicity and the increase in percentage of active rosette-forming cells from subnormal to normal. Serial measurements of cell-mediated cytotoxicity are helpful in monitoring the efficacy of transfer factor and other modes of therapy in these patients, and these measurements are the best available criteria for selection of donors of tumor-specific transfer factor.

The Wiskott-Aldrich syndrome. Results of transfer factor therapy.

12 patients with Wiskott-Aldrich syndrome were treated with therapeutic doses of transfer factor in an attempt to induce cellular immunity. Clinical improvement was noted after transfer factor therapy in 7 of the 12 patients treated. Because this disease has a variable course and temporary spontaneous improvement can occur, the observed improvement cannot necessarily be attributed to the transfer factor. However, in two patients repeated remissions consistently followed transfer factor administration on repeated occasions. This included freedom from infections, regression of splenomegaly, and clearing of eczema. An unexpected finding was a decrease in bleeding in 3 of the 10 patients who had bleeding. Conversion of skin reactivity was obtained in all seven patients who clinically seemed to respond to transfer factor. In vitro studies performed after the administration of transfer factor demonstrated that the lymphocytes of the patients now produced migration inhibitory factor in response to appropriate test antigens, but did not undergo increased radioactive thymidine incorporation in response to the same antigens. A defect in the monocyte IgG receptors has been found in certain patients with the disease, and the current study shows that all patients with defective monocyte IgG receptors responded to transfer factor, whereas only one patient with normal receptors showed any response. This test may thus prove to be useful in predicting the results of transfer factor therapy in patients with Wiskott-Aldrich syndrome, although evaluation of a larger series of patients will be necessary to confirm this point. We conclude that cellular immunity can be induced, that there appears to be clinical benefit in certain patients with Wiskott-Aldrich syndrome by the use of transfer factor, and that this mode of therapy warrents trial in these patients and others with defects of cellular immunity.

Effects of levamisole on in vivo and in vitro murine host response to syngeneic transplantable tumor.

The effects of levamisole were studied in vivo and in vitro on two murine tumors, B16 melanoma and adenocarcinoma 15091, syngeneic to the mouse strains used. Administration of levamisole before tumor transplantation enhanced the early appearance of neoplasms but did not affect the overall incidence or course of tumor growth as compared with that observed in controls given saline injections or animals given levamisole with lethally X-irradiated tumor cells. Administration of the drug 1 day before iv injection of tumor cells significantly reduced the incidence of pulmonary nodules, but if the drug was given 3 or 5 days before tumor challenge, the incidence of nodules was increased. Lymphocytes or macrophages from normal mice given levamisole had no effect on tumor cells in vitro, whereas lymphocytes incubated with levamisole in vitro enhanced tumor cell growth. When lymphocytes and tumor cells were mixed in vitro, lymphocytes from animals treated with the drug formed larger multicell clumps with tumor cells than did those from normal controls. We concluded that levamisole did not protect the mice against the tested tumors.

Toxicity and immunogenicity of monoclonal antimelanoma antibody-ricin A chain immunotoxin in rats.

This study was performed to assess the subacute toxicity and immunogenicity in rats of XOMAZYME-MEL, an antimelanoma monoclonal antibody-ricin A chain immunotoxin. Female Sprague-Dawley rats received 14 consecutive daily i.v. injections of XOMAZYME-MEL at doses of 5 mg/kg/day, 1 mg/kg/day, or normal saline. Animals from each dose group were sacrificed on days 8, 15, and 22. The low dose of immunotoxin was well tolerated and produced only minimal signs of toxicity. Side effects in animals receiving the high dose of immunotoxin consisted of transient weight loss, peripheral edema, leukocytosis, hypoalbuminemia, and mildly elevated liver function tests. Histological findings in these animals included cytoplasmic vacuolization of hepatocytes, focal myocardial and skeletal muscle degeneration, and renal deposits of proteinaceous casts. The administration of immunotoxin resulted in the appearance of anti-mouse and antiricin A chain immunoglobulin binding activity in the sera of treated animals. This study documents the systemic effect of the multiple-dose administration of a ricin A chain immunotoxin in rats.

Therapy of patients with malignant melanoma using a monoclonal antimelanoma antibody-ricin A chain immunotoxin.

We conducted a trial of a murine monoclonal antimelanoma antibody-ricin A chain immunotoxin (XOMAZYME-MEL) in 22 patients with metastatic malignant melanoma. The dose of immunotoxin administered ranged from 0.01 mg/kg daily for 5 days to 1 mg/kg daily for 4 days (total dose: 3.2 to 300 mg). Side effects observed in most patients were a transient fall in serum albumin with an associated fall in serum protein, weight gain, and fluid shifts resulting in edema. In addition, patients experienced mild to moderate malaise, fatigue, myalgia, decrease in appetite, and fevers. There was a transient decrease in voltage on electrocardiograms without clinical symptoms, change in serial echocardiograms or elevation of creatine phosphokinase MB isozyme levels. Symptoms consistent with mild allergic reactions were observed in three patients. The side effects were related to the dose of immunotoxin administered and were generally transient and reversible. Encouraging clinical results were observed, even after a single course of a low dose of immunotoxin. In addition, localization of antibody and A chain to sites of metastatic disease was demonstrated by immunoperoxidase staining of biopsy specimens. Additional studies are being conducted to continue the evaluation of safety and efficacy of immunotoxin therapy for malignancy.

A randomized trial of levamisole versus placebo as adjuvant therapy in malignant melanoma.

We conducted a long-term follow-up (median, 10.5 years) of patients included in a randomized trial of levamisole versus placebo as surgical adjuvant therapy in 203 patients with malignant melanoma. Of the patients randomized, 104 received levamisole, and 99 received placebo. The results show that there is no difference between the treatment and control groups with regard to any of the three end points analyzed. These included disease-free interval, time to appearance of visceral metastasis, and survival. Moreover, there was no significant difference between the treatment and control groups after adjusting for age, sex, or stage of disease.

Adjuvant therapy of stage III and IV malignant melanoma using granulocyte-macrophage colony-stimulating factor.

PURPOSE: To evaluate granulocyte-macrophage colony-stimulating factor (GM-CSF) as surgical adjuvant therapy in patients with malignant melanoma who are at high risk of recurrence. PATIENTS AND METHODS: Forty-eight assessable patients with stage III or IV melanoma were treated in a phase II trial with long-term, chronic, intermittent GM-CSF after surgical resection of disease. Patients with stage III disease were required to have more than four positive nodes or a more than 3-cm mass. All patients were rendered clinically disease-free by surgery before enrollment. The GM-CSF was administered subcutaneously in 28-day cycles, such that a dose of 125 microg/m(2) was delivered daily for 14 days followed by 14 days of rest. Treatment cycles continued for 1 year or until disease recurrence. Patients were evaluated for toxicity and disease-free and overall survival. RESULTS: Overall and disease-free survival were significantly prolonged in patients who received GM-CSF compared with matched historical controls. The median survival duration was 37.5 months in the study patients versus 12.2 months in the matched controls (P <.001). GM-CSF was well tolerated; only one subject discontinued drug due to an adverse event (grade 2 injection site reaction). CONCLUSION: GM-CSF may provide an antitumor effect that prolongs survival and disease-free survival in patients with stage III and IV melanoma who are clinically disease-free. These results support institution of a prospective, randomized clinical trial to definitively determine the value of surgical adjuvant therapy with GM-CSF in such patients.

Transfer factor therapy for histoplasmosis in a patient with Hodgkin's disease.

A patient with recurrent chronic histoplasmosis was diagnosed also as having Hodgkin's disease. Studies of cell-mediated immunity (CMI) demonstrated no reaction to histoplasmin by skin test, lymphocyte transformation (LT), or leukocyte inhibition factor (LIF) assay. Clinical and immunologic studies were performed during treatment with 19 doses of dialyzable transfer factor (TF) prepared from a normal donor with strong CMI against histoplasmin. Transfer of CMI to the patient was demonstrated by all three tests. All tests reverted to nonreactive during the period of observation. Repeated doses of dialyzable TF were followed by reconversion of skin tests. The LIF assay was most reactive. Reactivation of histoplasmosis occurred during antimetabolic therapy for Hodgkin's disease; however, the lesions cleared rapidly when TF was added to amphotericin B. Amphotericin B was administered at a dosage of 25 mg three times each week during the entire study.

Coexistent lymphoid interstitial pneumonia, pernicious anemia, and agammaglobulinemia.

Immunologic factors have been incriminated in the pathogenesis of lymphoid interstitial pneumonia. The discovery of a patient with coexistent lymphoid interestitial pneumonia, pernicious anemia, and common variable hypogammaglobulinemia focused attention on the possible autoimmune nature of this pulmonary disease. Extensive immunologic studies demonstrated a noticeably impaired bonemarrow-dependent (B cell) system and intact thymus-dependent (T cell) system. No evidence of humoral or cellular hypersensitivity to homologous lung determinants was found.

Malignant melanoma.

Recent advances in our understanding of the pathology and prognosis of malignant melanoma make possible rationally designed immunotherapeutic studies. A number of immunologic studies suggest that there may be a specific immune response to melanoma-associated antigens in patients with melanoma; however, other studies have shown lack of specificity, so this issue remains to be definitively resolved. New immunotherapeutic agents, including BCG, TF, and levamisole, among others, offer the potential for improving therapy for patients.

Immunotherapy in infectious disease, autoimmunity and cancer.

The clinical and immunologic effects of transfer factor and of levamisole were evaluated in over 200 patients with a variety of diseases. With transfer factor, the most encouraging results were observed in patients with the Wiscott-Aldrich syndrome, chronic mucocutaneous candidiasis, coccidioidomycosis, Behcet's disease and malignant melanoma. With levamisole, the most promising results were observed in patients with recurrent aphthous stomatitis, rheumatoid arthritis and herpes simplex infections, especially ocular herpes. Immunologically, transfer factor usually caused conversion of skin test reactivity and conversion of in vitro tests of cellular immunity as well, whereas levamisole caused increases in skin test reactivity without a parallel change in in vitro para meters, suggesting that the two agents may have different mechanisms of action. In a limited number of patients with multiple sclerosis, reactivity to three viral antigens was found to be lower than that in normal subjects, as measured by lymphocyte stimulation. Following levamisole therapy, this reactivity increased to normal levels, but the patients did not show clinical benefit.

Short-term autologous tumor cell lines for the active specific immunotherapy of patients with metastatic melanoma.

We established short-term cell lines for 108/170 (64%) patients with metastatic melanoma. Tumor cell numbers were expanded to 10(8), then cells were irradiated, aliquoted, and cryopreserved for clinical use. Vaccines have been used to treat 69 patients with clinical follow up for 33 who had measurable metastatic disease at the time vaccine therapy was initiated (METS), and 33 who had no evidence of disease (NED) at the time of vaccine therapy following surgical resection of metastases. The protocol called for a baseline test of delayed tumor hypersensitivity (DTH), three weekly injections, a repeat of the DTH test, then monthly injections for an additional 5 months. Objective tumor responses were noted in 3/26 (12%) patients who received a minimum of three vaccinations, one complete, and two partial, with survivals of 36, 46+, and 78+ months. Only 6/64 (9.4%) had a positive DTH (>10 mm) at baseline, including three METS, all of whom progressed within 4 months and died within a year, and three who are still NED after more than 5 years. Conversion of DTH from negative to positive was documented in 18/44 (41%) patients who were tested at week 0 and 4. At a median follow up of greater than 5 years, the median overall survival (OS) was 40 months for "NED" with a 5-year survival rate of 39%, and 8.6 months with a 5-year survival rate of 10% for "METS" The 18 patients who had conversion of their DTH had a median event-free survival (EFS) of 15.8 months and 5-year EFS of 32% compared to 4.2 months and 9% for the 26 non-converters (P=0.012, two-tailed, log-rank test). Among patients who were NED when treatment started, the 12 patients whose DTH converted had a median overall survival of 61.4 months with 5-year survival of 63% compared to 9.7 months and 0% for the 13 non-converters (P=0.0026). This treatment approach is feasible, produces minimal toxicity, and is associated with long-term survival in a significant subset of patients.

Immunologic approaches to the treatment of prostate cancer.

The presence of several organ-specific molecules that could serve as immunogens or targets of an immune attack, the nonessential nature of the prostate gland, the substantial failure rate after treatment of the primary tumor, and the lack of effective chemotherapy for metastatic disease make prostate cancer an ideal candidate for immunotherapy. This report reviews the current status of two novel approaches to the treatment of prostate cancer. The first is an effort to induce antitumor immunity by enriching the cytokine environment within the primary cancer by intraprostatic injection of Leukocyte Interleukin (Cel-Sci Corp, Vienna, VA), a mixture of natural cytokines that includes interleukin-1 beta (IL-1beta), IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha). The second approach uses OncoVax-P (Jenner Biotherapies, Inc, San Ramon, CA), a vaccine consisting of liposome-encapsulated recombinant prostate-specific antigen (PSA) and lipid A. When administered as an emulsion or in association with bacillus Calmette-Guérin (BCG)/cyclophosphamide or GM-CSF with or without IL-2/cyclophosphamide, immunologic tolerance is broken as evidenced by the generation of humoral and cellular immunity. Both of these approaches have been shown to be feasible and safe, and are now being tested in patients with less advanced disease to determine if manipulation of the immune system can favorably influence clinical outcome.

Transfer factor therapy in the Wiskott-Aldrich syndrome. Results of long-term follow-up in 32 patients.

Thirty-two patients with the Wiskott-Aldrich syndrome have been treated with transfer factor provided by this laboratory. Apparent clinical benefit was observed in 44 per cent of them. The mean age of the patients who showed clinical benefit was significantly greater than that of the patients who showed no benefit. Conversion of immunologic reactivity correlated with clinical benefit. Thirteen of the patients who received transfer factor are alive, and 17 have died (43 per cent survival). Clinical benefit was correlated with survival. The median survival was greater than five years in the patients who showed clinical benefit, whereas it was 18 months in those who did not show clinical benefit. We conclude that transfer factor caused conversion of immunologic parameters, apparent clinical benefit and prolonged survival in some, but not all, patients with the Wiskott-Aldrich syndrome.

Nephropathy in the Wiskott-Aldrich syndrome.

Nephropathy was detected in five of 32 patients with the Wiskott-Aldrich syndrome who were participating in a study of transfer factor (TF) therapy. In two patients, nephropathy was present before TF and did not appear changed by TF therapy. One of these patients subsequently developed progressive renal failure requiring dialysis beginning 5 1/2 years after TF therapy. In two patients, decreased renal function appeared very soon after the administration of TF. One patient showed gradually decreasing renal function beginning after two years of TF therapy. An additional patient was identified who died with renal failure without having received TF. The results suggest that renal failure occurs in the Wiskott-Aldrich syndrome more frequently than generally recognized and that administration of TF may precipitate or accelerate the renal disease in patients with this syndrome.

Improved immune-specificity in monoclonal radioimmunoimaging using dual radionuclide color functional maps.

Diagnostic radioimmunoimaging is potentially limited by tissue localization of radiolabeled antibody products through mechanisms other than antigen binding. Comparing the distributions of reactive and nonreactive products can distinguish tracer in targeted and nontargeted tissues. To achieve this in a single imaging procedure, dual photopeak scintigraphy was performed using 111In and 67Ga products. Melanoma-bearing athymic mice were coadministered intravenously subtype-matched 111In melanoma-reactive and 67Ga melanoma-nonreactive murine monoclonal antibodies. Paired images from 245 and 93 keV windows were processed with a unique dual parameter color display program. The display algorithm expresses pixel counts from paired photo-peak images in polar coordinates and color-encodes angle as hue and magnitude as intensity. The color functional maps permitted ready distinction of immune from nonimmune uptake. Compared with single tracer imaging methods, this technique better depicts antigen distribution.

Transfer factor in immunodeficiency diseases.

Results of therapeutic trials of transfer factor in a number of laboratories suggest clinical benefit and enhancement of immunological reactivity in patients with primary or secondary immunodeficiency diseases. Long term follow-up of 32 patients with the Wiskott-Aldrich syndrome suggested that transfer factor caused conversion of immunologic reactivity, apparent clinical benefit, and prolonged survival in some, but not in all patients. In 18 patients with disseminated (Stage III) malignant melanoma treated with surgery and transfer factor, survival was better than would ordinarily be expected for disseminated disease (78% with mean follow-up of 2 years). A randomized trial has been initiated which will answer the question of the efficacy of transfer factor as surgical adjuvant therapy in malignant melanoma. Studies in human subjects suggested that transfer factor does not cause enhancement of reactivity in normal subjects, when evaluated in a controlled, double-blind fashion. Similar controlled studies in immunodeficient patients are necessary to ascertain whether transfer factor does cause enhancement of immune responses in these patients. Based on these observations, a guinea pig model was developed in which transfer factor caused abrogation of tolerance to ABA-Tyrosine.

Combined immunotherapy of malignant melanoma. Unusual survival following cerebral metastasis.

An 18-year-old woman was found to have solitary cerebral, choroidal, and pulmonary metastases of malignant melanoma three years after excision of a primary malignant melanoma. The cerebral metastasis was excised, and the patient's condition was treated with CNS irradiation followed by combined immunotherapy with transfer factor and Bacille bilié de Calmette-Guérin. The transfer factor donor was her father, who showed cellular immunity to melanoma extracts on in vitro testing. Histologic examination of the pulmonary nodule, which was excised after the initiation of immunotherapy, revealed a dense lymphocytic infiltrate associated with the metastatic melanoma. The patient is currently free of detectable melanoma more than three years after the cerebral metastasis. Studies in a second patient also demonstrated the appearance of inflammatory infiltrate in metastatic melanoma following transfer factor therapy.

Oil-in-water liposomal emulsions: characterization and potential use in vaccine delivery.

Emulsification of mineral oil by phospholipids donated by liposomes composed of dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, cholesterol, and lipid A by extrusion resulted in the formation of oil-in-water liposomal emulsions containing a substantial number of intact liposomes. Increasing the proportion of liposomes from 25 mM to 150 mM phospholipid and increasing the oil content from 2.5% (v/v) to 42.5% (v/v) changed the flow characteristics of the emulsions from fluid liquid-like to viscous. Likewise, the degree of stability of the emulsions was liposomal phospholipid concentration-dependent, ranging from partial emulsification in the range 25-100 mM to complete stabilization in the range 125-150 mM. Despite some loss of liposome integrity, as evidenced by the release of liposomal trapped glucose, emulsification of liposomes containing encapsulated prostate-specific antigen (PSA) exhibited antigen-specific immunostimulation in mice. These results suggest that liposomes containing encapsulated antigen can serve as constituents for the formulation of oil-in-water vaccines.