A male genital tract-specific carbohydrate epitope on human CD52: implications for immunocontraception.

The identification of unique sperm surface epitopes that are not expressed or exposed in the female reproductive tract is a key element in the development of antibody-based contraceptives. Western blotting and immunohistochemistry were performed to define the tissue distribution of the S19 epitope, which has been proposed as a target for immunocontraception. S19 is an IgG1 murine monoclonal antibody (mAb) directed to an N-linked carbohydrate epitope on a 15-25 kDa glycoprotein, sperm agglutination antigen-1 (SAGA-1), containing a peptide core identical to that of the lymphocytic surface protein CD52. In this study, the S19 epitope was shown to be absent from human lymphocytes, demonstrating a distinction between this epitope and the CAMPATH epitope that is recognized by an antibody against the terminal tripeptide and GPI-anchor of CD52. Further tissue specificity analysis identified the S19 epitope in the epithelium of the human epididymis and vas deferens, as well as on both epididymal and ejaculated spermatozoa. In contrast, the S19 epitope was absent in the five human female reproductive tract and 18 other somatic tissues tested. These results support the use of the S19 epitope as a contraceptive immunogen and the suitability of the S19 mAb as an intravaginal contraceptive. To test the agglutinating activity of the S19 mAb in a formulation designed for vaginal use, S19 mAb were bound to the surface of Novasomes, a multilamellar liposome delivery vehicle. S19-Novasome formulations agglutinated human spermatozoa and were as effective as unbound S19 mAb, demonstrating the feasibility of spermistatic contraceptives targeted to the male reproductive tract specific carbohydrate epitope.

Manipulation of avidity to improve effectiveness of adoptively transferred CD8(+) T cells for melanoma immunotherapy in human MHC class I-transgenic mice.

The adoptive transfer of tumor-reactive CD8(+) T cells into tumor-bearing hosts provides an attractive alternative to vaccination-based active immunotherapy of melanoma. The development of techniques that result in the preferential expansion of tumor-reactive T cells is therefore of great importance. In this study, we report the generation of HLA-A*0201-restricted CD8(+) T cell populations that recognize either tyrosinase(369-376) or gp100(209-217) from tolerant human class I MHC-transgenic mice by using single amino acid-substituted variant peptides. Low peptide concentration or restimulation with the parent peptide was used to enhance the functional avidity, defined by stimulation of IFN-gamma accumulation, and cross-reactivity of the resulting T cell populations. We found a direct correlation between the ability of a T cell population to respond in vitro to low concentrations of the precise peptide expressed on the tumor and its ability to delay the outgrowth of B16 melanoma after adoptive transfer. Surprisingly, we found that some T cells that exhibited high functional avidity and were effective in controlling tumor outgrowth exhibited low structural avidity, as judged by MHC-tetramer staining. Our results establish strategies for the development and selection of CD8(+) T cell populations that persist despite peripheral tolerance, and that can control melanoma outgrowth. Furthermore, they support the use of human MHC class I-transgenic mice as a preclinical model for developing effective immunotherapies that can be rapidly extended into therapeutic settings.

Immune responses to the HLA-A*0201-restricted epitopes of tyrosinase and glycoprotein 100 enable control of melanoma outgrowth in HLA-A*0201-transgenic mice.

Many of the Ags recognized by human melanoma-reactive CTL are derived from proteins that are also expressed in melanocytes. The possibility of self-tolerance to these epitopes has led to questions about their utility for antitumor immunotherapy. To investigate the issue, we established a preclinical model based on transgenic mice expressing a recombinant HLA-A*0201 molecule and B16 melanoma transfected to express this molecule. HLA-A*0201-restricted epitopes from the melanocyte differentiation proteins (MDP) tyrosinase and gp100 are expressed in both tumor cells and melanocytes, and the former is associated with self-tolerance. However, adoptive transfer of tyrosinase or gp100-reactive CTL developed from tolerant mice delayed tumor outgrowth, as did immunization with MDP peptide-pulsed dendritic cells. Protection was enhanced by the use of peptide ligands containing conservative substitutions that were cross-reactive with the original Ags. These data establish that CTL populations reactive against MDP-derived self-Ags can be activated to mount effective antitumor immunity and strongly support their continued development for tumor immunotherapy in humans.

Tumor cell-derived TGF-beta and IL-10 dysregulate paclitaxel-induced macrophage activation.

Paclitaxel (TAXOL) activates in vitro macrophage (Mø) expression of proinflammatory and cytotoxic mediators, including IL-12, tumor necrosis factor alpha (TNF-alpha), and nitric oxide (NO). However, tumors dysregulate Mø through soluble suppressor molecules, and it is possible that tumors evade paclitaxel-mediated immune effector function through the production of immunomodulatory molecules and inhibition of Mø function in situ. Because Mø activation in the tumor microenviroment is a desirable goal of anti-tumor immunotherapy, we evaluated whether tumor-derived immunomodulatory factors dysregulate paclitaxel-mediated Mø activation. Tumor cell-derived supernatant suppressed paclitaxel's capacity to induce IL-12, TNF-alpha, and NO production by RAW264.7 Mø. Tumor factors also dysregulated paclitaxel-induced expression of a HIV-LTR, promoter-driven luciferase construct in RAW264.7 Mø, suggesting that tumors may inhibit a broad range of Mø functionality. Depletion studies revealed that IL-10 and transforming growth factor-beta1 (TGF-beta1), but not prostaglandin E2 (PGE2), impaired paclitaxel-mediated activation, suggesting that abrogation of these factors in situ might restore paclitaxel's activating capacity and enhance anti-tumor efficacy.

Direct identification of human tumor-associated peptide antigens and a preclinical model to evaluate their use.

Although the arsenal of a healthy immune system includes both circulating antibodies and cellular components such as T cells, the latter seem to be particularly important in tumor immunology. Under normal conditions, the immune system does not react to the body's cells, which may be described as expressing "self" antigens on the cell surface. When a cell becomes cancerous, however, novel antigens are expressed on the cell surface. These novel "tumor" antigens are recognized as foreign by the body's immune system, and the cells that express them are destroyed or incapacitated. Whereas antibodies may react directly with protein antigens, T cells instead recognize peptide antigens presented by class I and class II molecules of the major histocompatibility complex (MHC). All cells normally break down proteins that they have made. The class I antigen-processing pathway has evolved to display peptides produced by this breakdown process as a way to provide information to cytotoxic T cells about what the cell is making. The display of new peptides as a result of infection or transformation can stimulate cytotoxic T cells to kill the cell. In addition, antigen-processing cells such as dendritic cells engulf dead or dying cells and degradeproteins into peptide fragments. These peptides are then displayed by the MHC class II molecules and presented to T helper cells, which augment the activity of the cytotoxic T cells. Cytotoxic T lymphocytes have recently been isolated from human tumors (especially melanoma) and are critical to the development of promising immunotherapeutic agents. As we shall discuss, these cells can recognize antigens that are common to tumors from different patients. We shall also explore how advances in instrumentation and the use of transgenic mice have increased our understanding of tumor-associated peptides to the point where we can begin to strive for a peptide-based therapeutic vaccine. The caveats for such therapy will also be addressed.

Self-tolerance to the murine homologue of a tyrosinase-derived melanoma antigen: implications for tumor immunotherapy.

The human tyrosinase-derived peptide YMDGTMSQV is presented on the surface of human histocompatibility leukocyte antigen (HLA)-A*0201(+) melanomas and has been suggested to be a tumor antigen despite the fact that tyrosinase is also expressed in melanocytes. To gain information about immunoreactivity and self-tolerance to this antigen, we established a model using the murine tyrosinase-derived homologue of this peptide FMDGTMSQV, together with transgenic mice expressing the HLA-A*0201 recombinant molecule AAD. The murine peptide was processed and presented by AAD similarly to its human counterpart. After immunization with recombinant vaccinia virus encoding murine tyrosinase, we detected a robust AAD-restricted cytotoxic T lymphocyte (CTL) response to FMDGTMSQV in AAD transgenic mice in which the entire tyrosinase gene had been deleted by a radiation-induced mutation. A residual response was observed in the AAD(+)tyrosinase(+) mice after activation under certain conditions. At least some of these residual CTLs in AAD(+)tyrosinase(+) mice were of high avidity and induced vitiligo upon adoptive transfer into AAD(+)tyrosinase(+) hosts. Collectively, these data suggest that FMDGTMSQV is naturally processed and presented in vivo, and that this presentation leads to substantial but incomplete self-tolerance. The relevance of this model to an understanding of the human immune response to tyrosinase is discussed.

Paclitaxel enhances macrophage IL-12 production in tumor-bearing hosts through nitric oxide.

Tumor-induced macrophages (Mphis) mediate immunosuppression, in part, through increased production of factors that suppress T cell responsiveness and underproduction of positive regulatory cytokines. Pretreatment of tumor-bearing host (TBH) Mphis with the anticancer agent paclitaxel (Taxol) partially reverses tumor-induced Mphi suppressor activity, suggesting that paclitaxel may restore TBH Mphi production of proimmune factors. Because paclitaxel demonstrates LPS-mimetic capabilities and increased production of the LPS-induced immunostimulatory cytokine IL-12 could account for enhanced T cell responsiveness, we investigated whether paclitaxel induces Mphi IL-12 production. Tumor growth significantly down-regulated Mphi IL-12 p70 production through selective dysregulation of IL-12 p40 expression. LPS stimulation failed to overcome tumor-induced dysregulation of p40 expression. In contrast, paclitaxel significantly enhanced both normal host and TBH Mphi IL-12 p70 production in vitro, although TBH Mphi IL-12 production was lower than that of similarly treated normal host Mphis. Paclitaxel enhanced p40 expression in a dose-dependent manner. Through reconstituted Mphi IL-12 expression, paclitaxel pretreatment relieved tumor-induced Mphi suppression of T cell alloreactivity. Blocking Mphi NO suppressed paclitaxel's ability to induce IL-12 production. This suggests that paclitaxel-induced activities may involve a NO-mediated autocrine induction pathway. Collectively, these data demonstrate that paclitaxel restores IL-12 production in the TBH and ascribe a novel immunotherapeutic component to the pleiotropic activities of NO. Through its capacity to induce IL-12 production, paclitaxel may contribute to the correction of tumor-induced immune dysfunction.

Tumor growth modulates macrophage nitric oxide production following paclitaxel administration.

The antineoplastic agent paclitaxel (Taxol) mimics bacterial lipopolysaccharide (LPS) in normal host macrophages (Mphis), enhancing antitumor cytotoxicity in vitro. Because paclitaxel is used as an antitumor chemotherapeutic agent and tumor growth alters Mphi phenotype and function, we assessed effector molecule production and cytotoxic activity by normal host and tumor-bearing host (TBH) Mphis following paclitaxel administration. Paclitaxel treatment, duplicating human chemotherapeutic regimens, primed normal host splenic Mphis for enhanced production of the cytotoxic mediator nitric oxide (NO); in contrast, paclitaxel's NO-inducing activity was significantly suppressed in TBHs. In contrast to NO regulation, Mphi capacity for tumor necrosis factor-alpha (TNF-alpha) production in both normal hosts and TBHs was enhanced by paclitaxel administration. Although tumor growth modulated paclitaxel-induced Mphi NO production, paclitaxel administration enhanced both normal host and TBH Mphi cytotoxic antitumor activity. Blocking NO with a competitive inhibitor abrogated Mphi cytotoxicity, suggesting paclitaxel-induced TBH Mphi NO production, although suboptimal, remains sufficient to mediate antitumor activity. These data demonstrate that paclitaxel's in vivo immune activities are differentially regulated during tumor burden and suggest that paclitaxel's immunotherapeutic functions may contribute to its success as an anticancer agent.

Interleukin-12 overcomes paclitaxel-mediated suppression of T-cell proliferation.

The antineoplastic agent paclitaxel (TAXOL) is a potent inhibitor of tumor cell division and a useful chemotherapeutic for the treatment of refractory ovarian and breast carcinoma. Multiple immune system actions have been ascribed to paclitaxel, including the capacity to induce macrophage antitumor cytotoxic molecule production. However, T-cells are susceptible to paclitaxel's cytostatic functions, and no studies have investigated the effects of direct paclitaxel administration on lymphocyte function in the tumor-bearing host (TBH). Because paclitaxel is currently used as an antitumor chemotherapeutic agent and tumor growth alters leukocyte functions, we assessed T-cell function following chemotherapeutic-type paclitaxel treatment. Paclitaxel administration significantly compromised the proliferative capacity of both normal host and TBH lymphocytes in vitro. Although tumor growth impaired T-cell interferon-gamma (IFN-gamma) production, paclitaxel treatment did not alter IFN-gamma. We speculate that the immunostimulatory cytokine interleukin-12 (IL-12), which promoted T-cell activation and proliferation, was capable of reversing paclitaxel-mediated immunosuppression. Exogenous IL-12 fully reconstituted proliferative reactivity and enhanced IFN-gamma production by both normal host and TBH lymphocytes in vitro. Collectively, these data suggest that chemotherapeutic paclitaxel regimens impart significant but reversible inhibition of lymphocyte populations, and IL-12 may be a useful ancillary immunotherapeutic to overcome paclitaxel-induced modulation of lymphocyte activities.

Tumor-induced immune dysfunction: the macrophage connection.

Although macrophages (Mphis) mediate tumor cytotoxicity, display tumor-associated antigens, and stimulate antitumor lymphocytes, cancer cells routinely circumvent these host-mediated immune activities, rendering the host incapable of mounting a successful antitumor immune response. Evidence supporting a direct causal relationship between cancer and immune dysfunction suggests that the presence of neoplastic tissue leads to immunologic degeneration. Furthermore, substantial data demonstrate that tumor growth adversely alters Mphi function and phenotype. Thus, although Mphis can serve as both positive and negative mediators of the immune system, the importance of Mphis in tumor-induced immune suppression remains controversial. This review focuses on the evidence that tumor-derived molecules redirect Mphi activities to promote tumor development. Tumors produce cytokines, growth factors, chemotactic molecules, and proteases that influence Mphi functions. Many tumor-derived molecules, such as IL-4, IL-6, IL-10, MDF, TGF-beta1, PGE2, and M-CSF, deactivate or suppress the cytotoxic activity of activated Mphis. Evidence that tumor-derived molecules modulate Mphi cytotoxicity and induce Mphi suppressor activity is presented. This information further suggests that Mphis in different in vivo compartments may be differentially regulated by tumor-derived molecules, which may deactivate tumor-proximal (in situ) Mphi populations while concurrently activating tumor-distal Mphis, imparting a twofold insult to the host's antitumor immune response.

Taxol-mediated changes in fibrosarcoma-induced immune cell function: modulation of antitumor activities.

The anticancer drug taxol (paclitaxel) inhibits tumors through multiple cytotoxic and cytostatic mechanisms. Independently of these mechanisms, taxol induces distinct immunological efficacy when it acts as a second signal for activation of tumoricidal activity by interferon gamma (IFN gamma)-primed murine normal host macrophages. We reported that tumor-distal macrophages, which mediate immunosuppression through dysregulated nitric oxide (NO) and tumor necrosis factor alpha (TNF alpha) production, are differentially regulated by taxol. Because taxol influences tumor cell growth dynamics and activates immune cell populations, we assessed the ex vivo immunosuppressive and antitumor activities of taxol-treated normal host and tumor-bearing host (TBH) macrophages. Pretreatment of such cells with taxol partly reconstituted T cell alloantigen reactivity, suggesting that taxol mediates a limited reversal of TBH macrophage immunosuppressive activity. Taxol-treated TBH macrophages significantly suppressed the growth of fibrosarcoma cells (Meth-KDE) through soluble effector molecules and promoted direct cell-mediated cytotoxicity, indicating that taxol enhanced tumor-induced macrophage antitumor activities. Tumor-induced helper T cells, however, showed a higher sensitivity to direct taxol-induced suppression. These data demonstrate that taxol exerts pleiotropic effects on antitumor immune responses with the capacity to abate the immunosuppressive activities of macrophages and promote macrophage-mediated antitumor activities simultaneously, but also directly modulating T cell reactivity. Collectively, these studies suggest that the antineoplastic drug taxol may impart antitumor activity through an immunotherapeutic capacity.

Taxol, a microtubule-stabilizing antineoplastic agent, differentially regulates normal and tumor-bearing host macrophage nitric oxide production.

Taxol, a potent antitumor chemotherapeutic, promotes in vitro cytotoxic antitumor activities by normal host macrophage (M phi s). Because tumor growth induces functional changes among M phi populations, we determined whether fibrosarcoma growth (Meth-KDE) modified M phi responsiveness to the activating agent taxol. Tumors induce tumor-distal M phi populations to become immune suppressor cells, partially through overproduction of the cytotoxic and proinflammatory molecules nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha). Beneficial to the tumor-bearing host (TBH) when released by tumor-proximal M phi s, NO and TNF-alpha suppress lymphoproliferation and fail to impart antitumor activity when expressed in tumor-distal compartments. We report that taxol differentially regulated normal host and TBH M phi production of the immunosuppressive molecule NO by tumor-distal M phi populations. In response to IFN-gamma-priming and taxol triggering, TBH M phi s increase their production of NO as compared to resting M phi s; however, unlike normal host M phi s, taxol-induced TBH M phi NO production was significantly suboptimal. Modulation of TBH M phi NO production in tumor-distal compartments may alleviate M phi-mediated suppression of T-cell proliferative responses, yet promote sufficient NO production by tumor-associated M phi s to affect cytotoxicity. Collectively, these data leave implications for immunotherapeutic activities by the anticancer drug taxol.

The science literacy crisis, philosophical issues, and the origin sciences.

Since 1983, when the National Commission on Excellence in Education released its much-awaited report, A Nation at Risk, the United States has perceived itself to be in the midst of a serious education crisis. While nearly everyone is in agreement that most areas of the educational enterprise are in need of some revision and revitalization, problems related to science education have clearly received the lion's share of reform-minded attention. The present essay reviews the various symptoms of the so-called science literacy (education) crisis; speculates on the unique role that the philosophical underpinnings of the "cosmic" (Weisskopf, 1994) or origin sciences may play in these problems; and offers some suggestions as to how academic research scientists interested in these areas might more readily and effectively involve themselves in efforts to improve science education at all levels of the educational system.

Effect of hyperthermia and doxorubicin on nucleoid sedimentation and poly (ADP-ribose) polymerase activity in L1210 cells.

We report on the individual and combined effects of doxorubicin (DOX) and hyperthermia (HYP) on nucleoid sedimentation and poly (ADP-ribose) polymerase (PARP) activity of L1210 cells. The effects of HYP and DOX on nucleoid sedimentation (increased sedimentation) were similar and correlated with cell viability. No correlation of PARP activity with cell toxicity was evident; the activity of PARP was inhibited by HYP (42 degrees C; 1-3 h) and stimulated by DOX (1-10 microM; 30 min). The HYP-induced inhibition of PARP was actually ameliorated by simultaneous exposure to DOX. Although separate studies have previously suggested that chromatin alterations or the inhibition of PARP might play a role in the effect of HYP, the correlation of nucleoid changes (rather than PARP activity) with cell viability emphasizes the contribution of the former. Furthermore, the results suggest that the nucleoid technique may prove useful in screening potential treatment modalities.

Restoration of hyperthermia-associated increased protein to DNA ratio of nucleoids.

The sedimentation of L1210 nucleoids has been used to demonstrate a hyperthermia-associated increased protein to DNA ratio and an apparent inhibition of processes involved in the restoration of the protein to DNA ratio. The distance of nucleoid sedimentation increased as a function of exposure temperature and exposure time, and was proportional to an increased protein to DNA ratio in the nucleoids. Studies in which control and hyperthermia-treated cells were mixed prior to nucleoid preparation indicated that the association of protein with the nucleoid occurred during hyperthermia treatment and not during nucleoid preparation. However, double-labelling studies suggested an interaction between control and hyperthermia-treated cells since the presence of control cells during lysis resulted in near normalization of nucleoid sedimentation. Treatment with proteinase K also restored the nucleoid sedimentation. Incubation at 37 degrees C following hyperthermia revealed a rapid restoration of nucleoid sedimentation and a slower restoration of the protein to DNA ratio. Ethidium bromide-induced changes in nucleoid sedimentation were altered by the hyperthermia-associated increased protein content of nucleoids and the alterations were overcome by enzymatic digestion of the protein prior to the ethidium bromide exposure. Thus, hyperthermia caused, and inhibited the repair of, an increased protein content of nucleoids. The restoration of the increased protein possibly occurs by a heat-sensitive proteolytic enzyme. The temporal use of an appropriate chemotherapy agent to inhibit the restoration of hyperthermia-associated changes may be a useful treatment option.

Aliphatic amino acid side chains associate with the "face" of the adenine ring.

We have synthesized the free amino acid adenylate anhydrides of phenylalanine, leucine, isoleucine and valine. These activated compounds are very labile at high pH, but at low pH they become more stable. Proton NMR spectra of these adenylates show that in every case, the hydrophobic side chains, even in these small molecules at low pH and low concentration, are associated with the "face" of the adenine ring. Although aromatic rings are known to associate with adenine in this fashion, to our knowledge this is the first report of an intercalative-type interaction of aliphatic side chains with nucleic acid bases. Since adenine is the most hydrophobic base, these interactions are of a hydrophobic character, and occur in spite of the fact that the adenine ring is protonated. These results may have implications regarding recognition processes in DNA-protein and RNA-protein interactions.

Comparative rates of esterification of 5'-AMP with hydrophobic amino acids: relevance to the genetic-code assignments.

We have continued our program aimed at understanding the origin and evolution of the genetic code and the process of protein synthesis by comparing the rates of esterification of 5'-AMP by a series of hydrophobic N-acetylamino acids. The reaction clearly shows differences in reaction rate (AcPhe greater than AcLeu greater than AcVal greater than AcIle) among the amino acids having A as middle letter of their anticodons. However, there were no significant differences in reaction rate between AcLeu, AcNorleu, and Ac-alpha-aminobutyric acid, and AcGly reacted faster than all of these and AcPhe. Consequently, this simple reaction with AMP can distinguish only among those amino acids that actually have A as the middle anticodonic nucleotide. The relevance of these studies to the origins of the process of protein synthesis and of the genetic code is discussed in conjunction with results from other studies of a similar nature.

Rationalization of some genetic anticodonic assignments.

The genetic code appears to be a logic matrix in which, generally speaking, there is a correlation between the hydrophobicities of amino acids and their anticodonic nucleotides. There are several exceptions to this generality, however, and using previous data on hydrophobicity and binding constants, coupled with new data on reaction rates, we rationalize several of the anticodonic assignments.

Chirally selective, intramolecular interaction observed in an aminoacyl adenylate anhydride.

All earthly creatures use only L-amino acids in template directed protein synthesis. The reason for this exclusive use of the L-isomer is not yet apparent, although recent experiments by Usher and his colleagues have shown some stereoselectivity in the aminoacylation of di- and polynucleotides. We have separately reported on intramolecular interactions between hydrophobic amino acid side chains and the adenine ring in aminoacyl adenylates. There was a preferential association of Phe greater than Leu = Ile greater than Val with the adenine in these studies, but we made no attempts to address the question of D, L selectivity. Recently, in 1H NMR studies of N-acetylphenylalanyl adenylate anhydride, we noticed evidence that both D- and L-isomers of the amino acid were present and, furthermore, that one isomer seemed to be associating with the adenine ring more strongly than the other. Using HPLC, we have separated the two diastereoisomers and have enzymatically determined that the isomer which associates more strongly is the biologically important one, the L-isomer. We present those studies here and discuss the evolutionary significance of this finding.