High-dose tamoxifen added to concurrent biochemotherapy with decrescendo interleukin-2 in patients with metastatic melanoma.

BACKGROUND: In vitro cell culture data and preclinical models suggest that tamoxifen modulates tumor cell sensitivity to a wide range of therapeutic agents. In the current study, the authors examined whether high-dose tamoxifen (HDT) improved the overall and complete response in patients with metastatic melanoma who were treated with concurrent biochemotherapy. METHODS: Forty-nine patients were treated with a biochemotherapy regimen of dacarbazine, vinblastine, cisplatin, decrescendo interleukin-2, interferon-alpha-2b, and tamoxifen. The study had a 2-step design, beginning with a tamoxifen dose escalation from 40 mg to 320 mg (17 subjects) to evaluate safety and tolerability, followed by Phase II accrual of 32 patients to HDT (320 mg) to assess clinical efficacy. Efficacy was compared with a similar modified biochemotherapy regimen with low-dose tamoxifen (LDT). Pharmacokinetic studies were performed to determine in vivo tamoxifen levels. RESULTS: Tamoxifen dose escalation was completed without any reported dose-limiting toxicity. The overall response rate in the HDT group was 50% (95% confidence interval, 33.2%-66.8%), with a complete response rate of 6% and a median survival of 9.5 months. The overall response rate was not improved and the complete response and survival appeared inferior compared with that of patients recently treated with concurrent biochemotherapy and LDT. Serum tamoxifen levels were found to correlate with the dose administered, with a mean of 0.9 microM at the 40-mg dose to 4.6 microM at the 320-mg dose. Ultrafiltered protein-free sera demonstrated low (< 0.01 microM) concentrations of tamoxifen. CONCLUSIONS: The addition of HDT to a regimen of concurrent biochemotherapy did not appear to improve response rates or overall survival, despite reaching the targeted plasma concentration. Unknown drug interactions or high protein binding of tamoxifen may account for the lack of clinical effectiveness.

Vaccine therapy for patients with melanoma.

Investigation into the therapeutic use of vaccines in patients with metastatic melanoma is critically important because of the lack of effective conventional modalities. The most extensively studied melanoma vaccines in clinical trials are whole-cell preparations or cell lysates that contain multiple antigens capable of stimulating an immune response. Unfortunately, in the majority of studies, immune responses to these vaccines have not translated into a survival advantage. Advances in tumor cell immunology have led to the identification of candidate tumor cell antigens that can stimulate an immune response; this, in turn, has allowed for refinements in vaccine design. However, the exact tumor antigens that should be targeted with a specific vaccine are unknown. The univalent antigen vaccines, which have greater purity, ease of manufacturing, and reproducibility compared with polyvalent vaccines, may suffer from poorer efficacy due to immunoselection and appearance of antigen-negative clones within the tumor. Novel approaches to vaccine design using gene transfection with cytokines and dendritic cells are all promising. However, the induction of immune responses does not necessarily confer a therapeutic benefit. Therefore, these elegant newer strategies need to be studied in carefully designed clinical trials so that outcomes can be compared objectively with standard therapy. If survival is improved with these vaccine approaches, their ease of administration and lack of toxicity will firmly entrench active specific vaccine immunotherapy as a standard modality in the treatment of the melanoma patient.

Inhibition of endotoxin-induced TNF-alpha production in macrophages by 5Z-7-oxo-zeaenol and other fungal resorcylic acid lactones.

Resorcylic acid lactones are fungal metabolites that exhibit a wide range of biological properties which includes oestrogenic, antifungal, phytotoxic and anti-inflammatory activity. The capacity of 5Z-7-oxo-zeaenol, a resorcylic lactone of fungal origin and six naturally occurring analogues to inhibit lipopolysaccharide (LPS)-induced cytokine production in phorbol 12-myristate-13-acetate (PMA)-treated cultured myelomonocytic cells (U937) was compared. The activity of the natural analogues in the U937 assay varied over 10(4)-fold, with 5Z-7-oxo-zeaenol the most potent of those tested inhibiting tumour necrosis factor-alpha (TNF alpha) production in these cells with IC50 of 6 nM. The isomeric 7-oxo-zeaenol and structurally more distant monorden (radicicol) were the next most active compounds with IC50 approximately 500 nM, and zearalenone, the least active with IC50 > 400 microM. 5Z-7-oxo-zeaenol retained activity in LPS-stimulated peripheral blood mononuclear cells with an IC50 of 10-25 nM. This compound also inhibited LPS-induced TNF alpha production in whole blood experiments (IC50 100-1000 nM) and lowered serum levels of TNF alpha in mice when administered prior to LPS. 5Z-7-oxo-zeaenol was shown to inhibit the phosphorylation and activation of mitogen-activated protein kinase (MAPK) induced by LPS. These data are consistent with a mechanism of action at or upstream of MAPK with resultant downstream effects. This series of naturally occurring analogues represents an interesting group of compounds with diverse biological properties. Of this series, 5Z-7-oxo-zeanenol has exceptionally potent anti-inflammatory properties exhibited by its strong inhibition of cytokine production.

Advantages of concurrent biochemotherapy modified by decrescendo interleukin-2, granulocyte colony-stimulating factor, and tamoxifen for patients with metastatic melanoma.

PURPOSE: Concurrent biochemotherapy results in high response rates but also significant toxicity in patients with metastatic melanoma. We attempted to improve its efficacy and decrease its toxicity by using decrescendo dosing of interleukin-2 (IL-2), posttreatment granulocyte colony-stimulating factor (G-CSF), and low-dose tamoxifen. PATIENTS AND METHODS: Forty-five patients with poor prognosis metastatic melanoma were treated at a community hospital inpatient oncology unit affiliated with the John Wayne Cancer Institute (Santa Monica, CA) between July 1995 and September 1997. A 5-day modified concurrent biochemotherapy regimen of dacarbazine, vinblastine, cisplatin, decrescendo IL-2, interferon alfa-2b, and tamoxifen was repeated at 21-day intervals. G-CSF was administered beginning on day 6 for 7 to 10 days. RESULTS: The overall response rate was 57% (95% confidence interval, 42% to 72%), the complete response rate was 23%, and the partial response rate was 34%. Complete remissions were achieved in an additional 11% of patients by surgical resection of residual disease after biochemotherapy. The median time to progression was 6.3 months and the median duration of survival was 11.4 months. At a maximum follow-up of 36 months (range, 10 to 36 months), 32% of patients are alive and 14% remain free of disease. Decrescendo IL-2 dosing and administration of G-CSF seemed to reduce toxicity, length of hospital stay, and readmission rates. No patient required intensive care unit monitoring, and there were no treatment-related deaths. CONCLUSION: The data from this study indicate that the modified concurrent biochemotherapy regimen reduces the toxicity of concurrent biochemotherapy with no apparent decrease in response rate in patients with poor prognosis metastatic melanoma.

Overview of melanoma vaccines: active specific immunotherapy for melanoma patients.

Although a phase III trial has yet to show a statistically significant improvement in the disease-free or overall survival of melanoma patients receiving vaccine therapy, several phase II trials have shown enhanced disease-free and overall survival of patients who develop a humoral and/or cellular response to a melanoma vaccine. The challenge of active specific immunotherapy research is to determine which combination of humoral and cellular immune responses optimizes clinical outcome and how to monitor the immune response effectively. This review identifies key components of a successful melanoma vaccine, discusses new ways to modulate and stimulate the immune system, and summarizes some of the important clinical trials of active specific immunotherapy for patients with melanoma.

Is the survival of melanoma patients receiving polyvalent melanoma cell vaccine linked to the human leukocyte antigen phenotype of patients?

PURPOSE: An allogeneic polyvalent melanoma cell vaccine (PMCV) has been shown to be efficacious in improving overall survival of patients with malignant melanoma in a phase II clinical setting. The PMCV consists of three allogeneic melanoma cell lines. The objectives of the study were to determine (1) whether the survival of melanoma patients who received PMCV was related to the patient's human leukocyte antigen (HLA) class I phenotype matching the HLA class I phenotype of the PMCV, and (2) whether PMCV clinical efficacy was correlated to melanoma patients with a particular HLA phenotype(s). MATERIALS AND METHODS: PMCV was given to 69 melanoma patients with American Joint Committee on Cancer (AJCC) stage I to IV disease status. The PMCV and patients lymphocytes were typed for HLA-A and -B. A correlation was made between the HLA expression of PMCV lines and the HLA of patients to their survival status. A second correlation was made between the HLA of patients and survival independent of the PMCV HLA phenotype. RESULTS: Patients whose HLA phenotype (A3/11 and B7/44) matched the PMCV lines had a better overall survival (P < .029). Analysis of HLA expression of patients independent of PMCV HLA to survival showed that HLA-A25 phenotype patients had a significantly better overall survival (P = .006). HLA-B35 patients had a poorer survival outcome (P = .019). CONCLUSION: The studies indicate that overall survival following PMCV treatment in melanoma patients significantly correlates with their HLA phenotypes. These correlations may be related to the host immune response to the PMCV or due to differences in the clinical course of melanoma in patients with different HLA types.

Update on active specific immunotherapy with melanoma vaccines.

Although a randomized clinical trial has yet to show a statistically significant improvement in the survival of patients receiving vaccine therapy for malignant melanoma, several studies have shown enhanced survival of patients developing an immune response to a melanoma vaccine. The knowledge and techniques of modern molecular biology and immunology suggest multiple strategies to augment this response. The challenge of immunotherapy research is to determine which combination of approaches leads to a favorable clinical response and how to monitor that response effectively. This review identifies components of a successful vaccine, discusses new ways to modulate and stimulate the immune system, and summarizes some of the more interesting clinical trials of melanoma vaccine immunotherapy.

Differential effects on TNF alpha production by pharmacological agents with varying molecular sites of action.

This study describes the activation conditions for tumor necrosis factor-alpha (TNF alpha) production in myelomonocytic U937 cells and human primary peripheral blood monocytes in response to lipopolysaccharide (LPS) and/or phorbol 12-myristate 13-acetate (PMA). PMA itself induced only low levels of TNF alpha production with delayed kinetics (e.g. 0.758 +/- 0.128 ng/ml from U937 cells after 48 h) while LPS induced greater levels of TNF alpha production in less time (e.g. 2.083 +/- 0.96 ng/ml from monocytes in 24 h). Pharmacological agents with various molecular sites of action were used to validate the two systems, with the protein serine-threonine kinase inhibitors staurosporine and Ro-31-8220, the protein tyrosine kinase inhibitor herbimycin A (HBA) and dexamethasone exhibiting the greatest potency (IC50S 5-350 nM). In contrast to the effect on TNF alpha production, PMA induced strong phosphorylation/activation of p42/p44mapk in monocytes by 10 min determined in a mobility shift assay, while LPS was a weaker inducer. Additionally, staurosporine (to LPS and PMA) and HBA (to LPS only) inhibited the activation of these mitogen-activated protein kinase (MAPK) isoforms at doses 10-100 fold higher than those required to inhibit maximal TNF alpha production. These data indicate the involvement of the p42/p44mapk signalling pathway in LPS-induced pro-inflammatory cytokine production but suggest that other signalling pathways are also implicated in this phenomenon.

Detection of interleukin-1 signal transduction inhibitors: action of protein kinase inhibitors.

The dysregulated production of IL-1 has been shown to play an important role in many pathological processes. Despite the apparent value of compounds able to inhibit either the secretion of IL-1 or its signal transduction pathway in a specific manner, there are no such compounds suitable for clinical use. A major problem in identifying novel and specific inhibitors of signal transduction is the lack of knowledge of the intracellular events which mediate the cellular actions of IL-1. In this study a simple cellular assay has been established to screen natural product and synthetic compound libraries for low molecular weight inhibitors of the cytokine signalling pathways of potential therapeutic value. In addition, we have studied the action of several known modulators of signal transduction on the actions of IL-1.

A fungal metabolite which inhibits the interaction of CD4 with major histocompatibility complex-encoded class II molecules.

CD4, a cell-surface glycoprotein expressed on a subpopulation of T cells, is the receptor for class II molecules of the major histocompatibility complex (MHC II) and a receptor for the envelope glycoprotein (gp 120) of human immunodeficiency virus-1 (HIV-1). Screening of microbial metabolites for CD4-binding activity using an enzyme-linked immunosorbent assay based on the binding of the CD4-specific monoclonal antibody (mAb), anti-Leu3a, identified a family of compounds comprising several novel polyketides. The parent compound (411F, Vinaxanthone) is a C28 molecule probably arising from a dimerization of two C14 polyketide units. It strongly inhibited the interaction of anti-Leu 3a and that of several other D1/D2 epitope-specific mAb with CD4, but only weakly inhibited the binding of HIV-1 gp120. Binding of a representative MHC class II molecule, HLA-DRB*0401, was also inhibited by 411F with a comparable inhibitory concentration (IC50 = 1 microM). In functional assays 411F inhibited antigen-induced CD4-dependent T cell proliferative responses of peripheral blood mononuclear cells. At the clonal level 411F exhibited selectivity in that the compound inhibited peptide-induced CD4+ T cell proliferative responses but not alloantigen-induced CD8+ T cell proliferation. It is hypothesized that 411F, a polyanionic compound in aqueous solution at neutral pH, inhibits CD4-dependent functions by binding over a broad area of the positively charged amino-terminal D1 and D2 domains implicated in the interaction with MHC II molecules. 411F has the potential for development as an immunosuppressive agent with a novel mechanism of action.

Complications in the functional analysis of transfected MHC genes.

Analysis of the relationship between the structure of a protein molecule and its function often exploits the techniques of gene mutation and expression in transfected cell lines. This approach has been used extensively in the study of MHC molecules for testing predictions derived from structural models. Comparison of the functional properties of mutant molecules is difficult because MHC molecules interact with both peptides and T cell receptors. Functionality is commonly determined in biological assays which are dependent on T cell recognition of specific peptide/MHC complexes and measure secondary events triggered by T cell activation. In this study four L cell lines transfected with different combinations of alpha and beta chains from I-Ak and I-Au were used as antigen-presenting cells to activate two hen egg-white lysozyme-specific T cell clones. We compared several biological assays, namely, T cell proliferation, cytokine production, and cytotoxicity. Different assays indicated varying degrees of functionality for the same MHC molecule and thus demonstrated the difficulty in unambiguous interpretation of data from complex assays. Furthermore, we detected differences among the transfected lines which appeared unrelated to the expression of the introduced MHC genes.

Determinant spreading and the dynamics of the autoimmune T-cell repertoire.

In this article the authors propose a dynamic model of autoimmunity with T-cell recruitment and selection leading to changes in the specificity of the anti-self response during the course of disease. They argue that these changes are due to alterations in self-antigen presentation that lead to the display of previously cryptic self-determinants. Mechanisms that could underlie this differential self-presentation are proposed.

CD4-binding compounds: an assay to detect new classes of immunopharmacological agents.

The interaction of antibodies with protein antigens is accepted as a paradigm of protein-protein interactions. In searching for a new generation of immunomodulatory compounds based on the interaction of the T-cell surface glycoprotein CD4 with MHC class II antigens, a model assay has been developed in which MHC molecules have been substituted by a monoclonal antibody (anti-Leu3a) to the CD4 amino-terminal domain-specific epitope, Leu3a. This assay can detect diverse classes of molecules including proteins such as HIV envelope glycoprotein gp120 and low molecular weight compounds such as aurin tricarboxylic acid, dextran sulphate and Evans blue. The interaction of these molecules with CD4 in the assay appears to be identical to their interaction with native CD4 on intact cells. Other protein-antibody pairs could be substituted for CD4-anti-Leu3a enabling this assay format to be used for the detection of proteins or small organic compounds which interfere with a wide range of therapeutically-relevant macromolecular interactions.

Fluoxetine and methylphenidate in combination for treatment of attention deficit disorder and comorbid depressive disorder.

ABSTRACT Children and adolescents with attention deficit disorders (usually with comorbid conditions), who had shown inadequate therapeutic responses to methylphenidate, were treated by the addition of fluoxetine to methylphenidate. After 8 weeks in open trial, all 32 patients showed positive therapeutic responses in attention, behavior, and affect. Thirty of the 32 children showed clinically significant responses and the other two had statistically but not clinically significant responses. After 12 weeks of treatment, one patient showed a deterioration in clinical status. The children had improved report card grades in major academic subjects {p < 0.0001), and showed significant improvements (p < 0.0001) on the Children's Global Assessment Scale (C-GAS), Conners Parents Rating Scales (CPRS), and Children's Depression Inventory (CDI). Children who initially appeared more impaired on the C-GAS, CDI, CPRS, and GPA showed more improvement on the combined regimen. No significant side effects were observed, using a gradual elevation of fluoxetine dosage. About 40% of the patients showed substantial clinical effects with doses of fluoxetine below 20 mg daily. These preliminary results suggest that fluoxetine and methylphenidate in combination may be safe and effective for some children with attention-deficit hyperactivity disorder (and with comorbid anxiety or depressive symptoms) who do not show adequate responses to methylphenidate or fluoxetine alone.

The dominant self and the cryptic self: shaping the autoreactive T-cell repertoire.

Deletion of autoreactive T cells during the induction of self tolerance has been directly demonstrated. However, it is still relatively easy to detect self reactivity in normal healthy animals. In this article, Guy Gammon, Eli Sercarz and Gilles Benichou speculate on which T cells may escape tolerance induction and discuss how these cells could subsequently be involved in autoimmunity.

Induction of self-tolerance in T cells but not B cells of transgenic mice expressing little self antigen.

Self-tolerance to a transgene-encoded protein, hen egg lysozyme, was examined in the T and B cell repertoires of a series of lines of transgenic mice that expressed different serum concentrations of soluble lysozyme. T cells were tolerant in all lines in which lysozyme was expressed irrespective of the antigen concentration, whereas B cell tolerance did not occur when the serum lysozyme concentration was less than 1.5 nanograms per milliliter (0.1 nM). Induction of elevated transgene expression could restore B cell tolerance. These findings support the hypothesis that autoimmune disease may in some instances arise through a bypass of T cell tolerance.

T cell determinant structure: cores and determinant envelopes in three mouse major histocompatibility complex haplotypes.

T lymphocytes recognize discrete regions on an antigen. The specificity of the T cell responses in three mouse strains of differing major histocompatibility complex (MHC) haplotype to a protein antigen, lysozyme, was analyzed using a series of peptides that walk the antigen in single amino acid steps. These peptide series were synthesized using the pin synthesis system, which was modified to allow the peptides to be cleaved from the pins into a physiological buffer free of toxic compounds. This methodology overcomes many of the problems associated with the production of peptides for screening proteins for antigenic determinants. The T cell determinants for the three strains were markedly different. This result points out the limitations of algorithms predicting determinants without reference to the MHC, and the importance of the empirical methodology. This analysis of the T cell response to lysozyme constitutes the most complete study of reactivity to a foreign protein to date and illustrates many important features of antigen recognition by T cells, e.g., presence of major and minor determinant regions. The outer boundaries of each immunogenic region, the determinant envelope, are difficult to define from recently immunized lymph nodes because of the heterogeneity in T cell recognition. However, core sequences common to all the immunogenic peptides in a continuous sequence can be easily defined.

Does the presence of self-reactive T cells indicate the breakdown of tolerance?

There are many experimental systems in which autoreactive T cells can easily be demonstrated but where the host does not normally develop autoimmune disease. How do these animals avoid autoimmunity? Does the presence of these self-reactive cells indicate the failure of self-tolerance? To answer these questions it is necessary to consider how some T cells might escape tolerance induction and why they are not activated in the host. There are several different explanations which can be broadly placed into one of two categories. First, although autoreactive cells may be easily stimulated under experimental conditions, the requirements for activation and likewise deletion may not be met under physiological conditions. The self-antigen may be poorly presented by APC or sequestered in a particular body compartment; alternatively, these T cells may have low affinity receptors needing high levels of antigen. The second category is characterized by the need for immunoregulation. A random selection of T cells may escape clonal inactivation in the thymus but may be kept under constant suppression, which provides a fail-safe mechanism for deletional tolerance. In this review we will discuss these mechanisms and their possible importance in the prevention of autoimmunity.

T cell dominance and the vaccine problem: modifying effects on immunogenicity by residues at a distance from the site of T cell recognition.

The design of the optimal T cell-inducing component of a vaccine requires consideration of events both at, and distant from, the T cell recognition site. This area is approached here by analysis of T cell immunodominance. After describing the unpredictability of defining dominant T cell-inducing determinants and the preference of empirical methods, the question of designing a solely T determinant vaccine is explored. In this regard, evidence is presented that T helper cell-inducing and T proliferation-inducing determinants are not necessarily identical. With regard to the influence of other determinants on the molecule, three issues are discussed with relevance to vaccine design: (1) 'preferential partnerships' between T and B cells of certain specificities (2) the existence of suppressor T cells, reactive against non-overlapping determinants (3) competitive antigenic determinants, restricted to the same or to a different MHC molecule.