The roles of homologous recombination and the immune system in the genomic evolution of cancer.

A variety of factors, whether extracellular (mutagens/carcinogens and viruses in the environment, chronic inflammation and radiation associated with the environment and/or electronic devices/machines) and/or intracellular (oxidative metabolites of food, oxidative stress due to inflammation, acid production, replication stress, DNA replication/repair errors, and certain hormones, cytokines, growth factors), pose a constant threat to the genomic integrity of a living cell. However, in the normal cellular environment multiple biological pathways including DNA repair, cell cycle, apoptosis and the immune system work in a precise, regulated (tightly controlled), timely and concerted manner to ensure genomic integrity, stability and proper functioning of a cell. If damage to DNA takes place, it is efficiently and accurately repaired by the DNA repair systems. Homologous recombination (HR) which utilizes either a homologous chromosome (in G1 phase) or a sister chromatid (in G2) as a template to repair the damage, is known to be the most precise repair system. HR in G2 which utilizes a sister chromatid as a template is also called an error free repair system. If DNA damage in a cell is so extensive that it overwhelms the repair system/s, the cell is eliminated by apoptosis. Thus, multiple pathways ensure that genome of a cell is intact and stable. However, constant exposure to DNA damage and/or dysregulation of DNA repair mechanism/s poses a risk of mutation and cancer. Oncogenesis, which seems to be a multistep process, is associated with acquisition of a number of genomic changes that enable a normal cell to progress from benign to malignant transformation. Transformed/cancer cells are recognized and killed by the immune system. However, the ongoing acquisition of new genomic changes enables cancer cells to survive/escape immune attack, evolve into a more aggressive phenotype, and eventually develop resistance to therapy. Although DNA repair (especially the HR) and the immune system play unique roles in preserving genomic integrity of a cell, they can also contribute to DNA damage, genomic instability and oncogenesis. The purpose of this article is to highlight the roles of DNA repair (especially HR) and the immune system in genomic evolution, with special focus on gastrointestinal cancer.

MiR-29b antagonizes the pro-inflammatory tumor-promoting activity of multiple myeloma-educated dendritic cells.

Dendritic cells (DCs) have a key role in regulating tumor immunity, tumor cell growth and drug resistance. We hypothesized that multiple myeloma (MM) cells might recruit and reprogram DCs to a tumor-permissive phenotype by changes within their microRNA (miRNA) network. By analyzing six different miRNA-profiling data sets, miR-29b was identified as the only miRNA upregulated in normal mature DCs and significantly downregulated in tumor-associated DCs. This finding was validated in primary DCs co-cultured in vitro with MM cell lines and in primary bone marrow DCs from MM patients. In DCs co-cultured with MM cells, enforced expression of miR-29b counteracted pro-inflammatory pathways, including signal transducer and activator of transcription 3 and nuclear factor-κB, and cytokine/chemokine signaling networks, which correlated with patients' adverse prognosis and development of bone disease. Moreover, miR-29b downregulated interleukin-23 in vitro and in the SCID-synth-hu in vivo model, and antagonized a Th17 inflammatory response. All together, these effects translated into strong anti-proliferative activity and reduction of genomic instability of MM cells. Our study demonstrates that MM reprograms the DCs functional phenotype by downregulating miR-29b whose reconstitution impairs DCs ability to sustain MM cell growth and survival. These results underscore miR-29b as an innovative and attractive candidate for miRNA-based immune therapy of MM.

miR-23b/SP1/c-myc forms a feed-forward loop supporting multiple myeloma cell growth.

Deregulated microRNA (miR)/transcription factor (TF)-based networks represent a hallmark of cancer. We report here a novel c-Myc/miR-23b/Sp1 feed-forward loop with a critical role in multiple myeloma (MM) and Waldenstrom's macroglobulinemia (WM) cell growth and survival. We have found miR-23b to be downregulated in MM and WM cells especially in the presence of components of the tumor bone marrow milieu. Promoter methylation is one mechanism of miR-23b suppression in myeloma. In gain-of-function studies using miR-23b mimics-transfected or in miR-23b-stably expressing MM and WM cell lines, we observed a significant decrease in cell proliferation and survival, along with induction of caspase-3/7 activity over time, thus supporting a tumor suppressor role for miR-23b. At the molecular level, miR-23b targeted Sp1 3'UTR and significantly reduced Sp1-driven nuclear factor-κB activity. Finally, c-Myc, an important oncogenic transcription factor known to stimulate MM cell proliferation, transcriptionally repressed miR-23b. Thus MYC-dependent miR-23b repression in myeloma cells may promote activation of oncogenic Sp1-mediated signaling, representing the first feed-forward loop with critical growth and survival role in myeloma.

Targeting IL-17A in multiple myeloma: a potential novel therapeutic approach in myeloma.

We have previously demonstrated that interleukin-17A (IL-17) producing T helper 17 cells are significantly elevated in blood and bone marrow (BM) in multiple myeloma (MM) and IL-17A promotes MM cell growth via the expression of IL-17 receptor. In this study, we evaluated anti-human IL-17A human monoclonal antibody (mAb), AIN457 in MM. We observe significant inhibition of MM cell growth by AIN457 both in the presence and the absence of BM stromal cells (BMSCs). Although IL-17A induces IL-6 production, AIN457 significantly downregulated IL-6 production and MM cell adhesion in MM-BMSC co-culture. AIN457 also significantly inhibited osteoclast cell differentiation. More importantly, in the SCIDhu model of human myeloma administration of AIN457 weekly for 4 weeks after the first detection of tumor in mice led to a significant inhibition of tumor growth and reduced bone damage compared with isotype control mice. To understand the mechanism of action of anti-IL-17A mAb, we report, here, that MM cells express IL-17A. We also observed that IL-17A knockdown inhibited MM cell growth and their ability to induce IL-6 production in co-cultures with BMSC. These pre-clinical observations suggest efficacy of AIN457 in myeloma and provide the rationale for its clinical evaluation for anti-myeloma effects and for improvement of bone disease.

A multiepitope of XBP1, CD138 and CS1 peptides induces myeloma-specific cytotoxic T lymphocytes in T cells of smoldering myeloma patients.

We evaluated a cocktail of HLA-A2-specific peptides including heteroclitic XBP1 US184-192 (YISPWILAV), heteroclitic XBP1 SP367-375 (YLFPQLISV), native CD138260-268 (GLVGLIFAV) and native CS1239-247 (SLFVLGLFL), for their ability to elicit multipeptide-specific cytotoxic T lymphocytes (MP-CTLs) using T cells from smoldering multiple myeloma (SMM) patients. Our results demonstrate that MP-CTLs generated from SMM patients' T cells show effective anti-MM responses including CD137 (4-1BB) upregulation, CTL proliferation, interferon-γ production and degranulation (CD107a) in an HLA-A2-restricted and peptide-specific manner. Phenotypically, we observed increased total CD3(+)CD8(+) T cells (>80%) and cellular activation (CD69(+)) within the memory SMM MP-CTL (CD45RO(+)/CD3(+)CD8(+)) subset after repeated multipeptide stimulation. Importantly, SMM patients could be categorized into distinct groups by their level of MP-CTL expansion and antitumor activity. In high responders, the effector memory (CCR7(-)CD45RO(+)/CD3(+)CD8(+)) T-cell subset was enriched, whereas the remaining responders' CTL contained a higher frequency of the terminal effector (CCR7(-)CD45RO(-)/CD3(+)CD8(+)) subset. These results suggest that this multipeptide cocktail has the potential to induce effective and durable memory MP-CTL in SMM patients. Therefore, our findings provide the rationale for clinical evaluation of a therapeutic vaccine to prevent or delay progression of SMM to active disease.

Identification of novel myeloma-specific XBP1 peptides able to generate cytotoxic T lymphocytes: a potential therapeutic application in multiple myeloma.

The purpose of these studies was to identify human leukocyte antigen (HLA)-A2(+) immunogenic peptides derived from XBP1 antigens to induce a multiple myeloma (MM)-specific immune response. Six native peptides from non-spliced XBP1 antigen and three native peptides from spliced XBP1 antigen were selected and evaluated for their HLA-A2 specificity. Among them, XBP1(184-192), XBP1 SP(196-204) and XBP1 SP(367-375) peptides showed the highest level of binding affinity, but not stability to HLA-A2 molecules. Novel heteroclitic XBP1 peptides, YISPWILAV or YLFPQLISV, demonstrated a significant improvement in HLA-A2 stability from their native XBP1(184-192) or XBP1 SP(367-375) peptide, respectively. Cytotoxic T lymphocytes generated by repeated stimulation of CD3(+) T cells with each HLA-A2-specific heteroclitic peptide showed an increased percentage of CD8(+) (cytotoxic) and CD69(+)/CD45RO(+) (activated memory) T cells and a lower percentage of CD4(+) (helper) and CD45RA(+)/CCR7(+) (naïve) T cells, which were distinct from the control T cells. Functionally, the cytotoxic T lymphocytes (CTL) demonstrated MM-specific and HLA-A2-restricted proliferation, interferon-γ secretion and cytotoxic activity in response to MM cell lines and importantly, cytotoxicity against primary MM cells. These data demonstrate the distinct immunogenic characteristics of unique heteroclitic XBP1 peptides, which induce MM-specific CTLs and highlights their potential application for immunotherapy to treat the patients with MM or its pre-malignant condition.

Biological pathways and in vivo antitumor activity induced by Atiprimod in myeloma.

Atiprimod (Atip) is a novel oral agent with anti-inflammatory properties. Although its in vitro activity and effects on signaling in multiple myeloma (MM) have been previously reported, here we investigated its molecular and in vivo effects in MM. Gene expression analysis of MM cells identified downregulation of genes involved in adhesion, cell-signaling, cell cycle and bone morphogenetic protein (BMP) pathways and upregulation of genes implicated in apoptosis and bone development, following Atip treatment. The pathway analysis identified integrin, TGF-beta and FGF signaling as well as Wnt/beta-catenin, IGF1 and cell-cycle regulation networks as being most modulated by Atip treatment. We further evaluated its in vivo activity in three mouse models. The subcutaneous model confirmed its in vivo activity and established its dose; the SCID-hu model using INA-6 cells, confirmed its ability to overcome the protective effects of BM milieu; and the SCID-hu model using primary MM cells reconfirmed its activity in a model closest to human disease. Finally, we observed reduced number of osteoclasts and modulation of genes related to BMP pathways. Taken together, these data demonstrate the in vitro and in vivo antitumor activity of Atip, delineate potential molecular targets triggered by this agent, and provide a preclinical rational for its clinical evaluation in MM.

A method for the dispersal and characterization of leukocytes from the human female reproductive tract.

PROBLEM: The isolation of human female reproductive tract (RT) cells that maintain viability and are representative of the entire population is essential for a thorough evaluation of mucosal immunity in the reproductive tract mucosa. Here, we describe the isolation of RT cells in high yields and with high viability from the Fallopian tube, uterine endometrium, endocervix, ectocervix and vagina. METHOD OF STUDY: This cell dispersion method uses an enzyme cocktail composed of pancreatin, hyaluronidase, and collagenase (PHC), and employs a 250-microm mesh screen to facilitate cell dispersion. RESULTS: The yields of cells isolated per gram of tissue in the presence of this PHC cocktail were compared and found to be strikingly higher relative to the yields obtained with other enzyme cocktails or in the absence of enzymes. Flow cytometry was used to characterize leukocyte subsets isolated from uterine endometrium in the presence of the various enzyme cocktails. The common leukocyte antigen marker CD45, pan T-cell marker CD3, monocyte/macrophage marker CD14 and B-cell marker CD19 were retained after exposure to the PHC cocktail of enzymes. The expression of CD8 and CD4 was lost after exposure to added enzymes but regained after culture overnight. CONCLUSION: These studies demonstrate the feasibility of using enzymatic digestion for the isolation of whole populations of Fallopian tube, endometrial, cervical and vaginal cells, including leukocyte subsets in high yields, and provide a foundation for investigating mucosal immune cell function in the human female RT.

Antigen-presenting cells in the human female reproductive tract: analysis of antigen presentation in pre- and post-menopausal women.

PROBLEM: To determine whether cells in the female reproductive tract (FRT) are functionally capable of presenting antigen to T cells. METHOD OF STUDY: Analysis was done by determining the proliferation of purified autologous T cells to antigen, following co-incubation with non-proliferating cell suspensions isolated from the uterus and prepared by enzymatic digestion of reproductive tract tissues from hysterectomy patients with benign disease. RESULTS: All uterine preparations analyzed were functionally capable of presenting antigen; the ability to present antigen was independent of pre- and post-menopausal status. In contrast, some, but not all, tissues from the ovary, Fallopian tube, cervix, and vagina were capable of presenting antigen. CONCLUSION: These results suggest that the human FRT is an inductive site for immune responses. Regulation of antigen presentation in the reproductive tract may be important for protection against sexually transmitted diseases.

Regulation by human uterine cells of PBMC proliferation: influence of the phase of the menstrual cycle and menopause.

To determine the influence of human uterine cells recovered at different stages of the menstrual cycle and following menopause on the proliferation of peripheral blood mononuclear cells (PBMC), whole cell suspensions of uterine tissues were co-cultured with autologous and heterologous PBMC. PBMC proliferation in response to tetanus toxoid (TT) or Con A was inhibited by uterine endometrial cells and was dependent on the phase of the menstrual cycle. Inhibition by cells from the proliferative phase was significantly greater than by cells from the secretory phase. Uterine cells isolated from post-menopausal women also inhibited proliferation of PBMC. Cell fractionation studies indicated that epithelial cells are the primary source of uterine inhibitory activity. When epithelial cells and PBMC were cultured in separate compartments, epithelial cells released a soluble factor(s) that inhibited the PBMC proliferation. These results suggest that uterine epithelial cells produce cytokines that down-regulate the proliferation of PBMC in response to antigens and mitogens. This may be important for the control of uterine immune responses, as well as the growth of the reproductive tract in preparation for implantation during the secretory phase of the menstrual cycle.

The mucosal immune system in the human female reproductive tract: potential insights into the heterosexual transmission of HIV.

Using isolated cell suspensions and in situ techniques, we have partially characterized the organization, functional capacity, and sex hormone regulation of the mucosal immune system in the human female reproductive tract. Isolated cells suspensions have been used to demonstrate that the uterus contains antigen-presenting cells that are functionally able to present antigen to autologous tetanus toxoid-specific T cells. Immunophenotypic analyses of the female reproductive tract by three-color immunofluorescent staining has been used to show that lymphoid aggregates, which are absent in postmenopausal women, develop in the uterine endometrium during the menstrual cycle in premenopausal women. Lymphoid aggregates are composed of a B lymphocyte core surrounded by numerous CD8+CD4- T lymphocytes and an outer halo of macrophages. Macrophages, CD4+ and CD8+ T cells, and CD56+ NK cells are distributed throughout the uterine endometrium. In contrast, the Fallopian tube, cervix, and vagina, which lack lymphoid aggregates, contain CD8+ and CD4+ T cells as well as macrophages. The female reproductive tract has also been analyzed for the presence of antigen-independent CD3+ T lymphocyte cytolytic function by an anti-CD3 MAb-mediated redirected lysis assay. High levels of CD3+ T lymphocyte cytolytic activity were demonstrated in cervix and vagina and independent of stage of the menstrual cycle. In the uterus, cytolytic activity changed with endocrine state. In postmenopausal women the uterine endometrium had CD3+ T lymphocytes with high cytolytic activity, whereas premenopausal women had CD3+ T lymphocytes with moderate cytolytic potential during the proliferative phase to low/no cytolytic activity during the secretory phase of the menstrual cycle. In studies to determine whether the upper reproductive tract could be infected with HIV-1, we found on the basis of nef expression and p24 release that epithelial cells from the Fallopian tube, and from the uterus and cervix, are infectable. These studies demonstrate that the human female reproductive tract is an inductive site for immune responses and the cell-mediated immunity is present throughout the female reproductive tract. These studies further indicate that the Fallopian tube and uterus are potential entry sites for HIV-1 infection and that uterine immune cell architecture as well as cytolytic activity are under hormonal control.

Sex hormone and IL-6 regulation of antigen presentation in the female reproductive tract mucosal tissues.

The purpose of this study was to determine whether Ag presentation takes place in the female reproductive tract and if the stage of the reproductive cycle, sex hormones, and IL-6 regulate uterine Ag presentation. Purified epithelial and mixed stromal cells were incubated with sensitized lymph node T lymphocytes in the presence of OVA. Ag presentation was highest at proestrus, the stage of the reproductive cycle that immediately precedes ovulation when estradiol levels peak, and lowest at estrus, when sperm enter the uterus in preparation for fertilization. Further, these studies demonstrate that estradiol given to ovariectomized rats increases uterine cell Ag presentation relative to that in controls. In the presence of mouse anti-rat Ia Ab, Ag presentation was blocked, indicating that Ag presentation by uterine cells is MHC class II restricted. In studies to analyze Ag presentation at sites distal to the female reproductive tract, we found that Ag presentation by adherent cells from the spleen is elevated at proestrus relative to that at other stages of the estrous cycle. When IL-6 was placed in the uterine lumen of ovariectomized rats, Ag presentation by epithelial and mixed stromal cells as well as mitogenesis by spleen cells were increased significantly relative to those in controls. These studies demonstrate that the female reproductive tract is an inductive site for immune responses and that mucosal immune protection may be either enhanced or suppressed depending on the endocrine balance when the female reproductive tract is exposed to pathogens.

Influence of estrous cycle and estradiol on mitogenic responses of splenic T- and B-lymphocytes.

Previous studies from our laboratory have shown that the stage of the estrous cycle and hormone treatment regulates the presence of immune cells in the female reproductive tract. The purpose of the present study was to determine whether sex hormones influence spleen cell responses to mitogens. The present study demonstrates that spleen cell responses to mitogens vary with the stage of the estrous cycle in intact rats. Further, our findings indicate that estradiol administered to ovariectomized animals increases the mitogenic response of spleen cells to both B and T lymphocyte mitogens.

Influence of beta-carotene on immune functions.

Recent reports have demonstrated that beta-carotene, a nontoxic carotenoid, is able to stimulate immune functions in humans. The purpose of this study is to understand the mechanisms of immunoenhancement by carotenoids in order to explain their anticancer effects. We have evaluated the clinical efficacy of beta-carotene, given 30 mg/day orally, for treatment of oral leukoplakia patients. Patients who responded to beta-carotene treatment showed increased plasma levels of TNF-alpha. Epithelial cells from these patients were characterized in vitro. These results may lead to a better understanding of the therapeutic use of beta-carotene in humans.

A preliminary trial of beta-carotene in subjects infected with the human immunodeficiency virus.

beta-Carotene is a nontoxic carotenoid with immunomodulating properties in animals and humans. Based on our observations in normal immunocompetent subjects, we studied the effects of this compound in 11 patients infected with the human immunodeficiency virus (HIV). Each subject received 60 mg of beta-carotene daily for 4 mo. Clinical and laboratory studies were obtained at baseline, every month while on treatment and for 2 mo after treatment. Increases in the percent of cells expressing Leu 11 (natural killer cells), Ia antigen and transferrin receptor (activated lymphocytes) were observed after 3 mo of treatment with beta-carotene and diminished thereafter. Major changes were not seen in total lymphocyte count or in the percent of cells expressing CD11, CD8 or CD4 antigens. No clinical toxicity was observed. These data suggest that beta-carotene can modulate certain immune markers in HIV-infected subjects. Further study of this compound in HIV infection may be warranted.

Cytokine regulation of the mucosal immune system: in vivo stimulation by interferon-gamma of secretory component and immunoglobulin A in uterine secretions and proliferation of lymphocytes from spleen.

The secretory immune system in the female reproductive tract is known to be regulated by sex hormones and antigen. The purpose of the present study was to examine the control by interferon-gamma (IFN gamma) of the secretory component (SC), the polymeric immunoglobulin A (IgA) receptor, and IgA in uterine secretions and to determine whether IFN gamma influences the proliferation of spleen cells in response to mitogens. When measured by RIA, SC levels in uterine secretions were elevated when increasing doses of IFN gamma (1000-5000 U/uterus) were placed in the uterine lumen of ovariectomized rats. In contrast, IFN alpha-beta (5000 U/uterus) placed in the uterine lumen had no effect on uterine SC levels. To determine whether IgA movement increases in response to IFN gamma, animals were treated with estradiol to increase uterine tissue IgA levels without stimulating the accumulation of IgA or SC in uterine secretions. Under these conditions, intrauterine IFN gamma increased SC and IgA levels in uterine secretions, suggesting that in vivo IFN gamma stimulation of uterine SC increases the transport of IgA from tissue to lumen. Analysis of uterine tissues indicated that intrauterine IFN gamma had no apparent effect on epithelial cell morphology. In contrast, intraepithelial lymphocytes and polymorphonuclear leucocytes, which were sparse in control tissues, increased markedly with IFN gamma treatment. This increase was antagonized when estradiol was administered along with IFN gamma. In other studies, IFN gamma placed in the uterine lumen significantly increased spleen cell proliferation in response to Concanavalin-A, phytohaemagglutinin, and lipopolysaccharide mitogens relative to those in spleen cells from control animals. These studies demonstrate that in vivo treatment with IFN gamma stimulates the mucosal immune system in the female reproductive tract by increasing SC and IgA levels in the uterine lumen and promoting the infiltration of intraepithelial lymphocytes and polymorphonuclear leucocytes into uterine tissue. Further, it suggests that antigen, in stimulating a local uterine response, may act through cytokines, particularly IFN gamma, to regulate the transport of IgA into uterine secretions as well as modulate lymphocyte proliferation at sites distal to the uterus.

The effects of 13-cis-retinoic acid and beta-carotene on cellular immunity in humans.

Deficiency of vitamin A and/or its precursors has been associated with increased cancer risk in animals and humans. Therapeutic trials of vitamin A and related compounds have demonstrated activity in several cancerous and precancerous conditions. The authors measured the effects of a retinoid, 13-cis-retinoic acid, and a carotenoid, beta-carotene, on the human immune system in vivo in conjunction with their use in ongoing clinical trials. Immune cell subpopulations were analyzed by quantifying the expression of markers using flow cytometric study. Both compounds produced significant effects on immune cell populations. 13-cis-Retinoic acid resulted in an increase in the percentage of peripheral blood lymphoid cells expressing surface markers for T-helper cells with only minimal effect on natural killer cell marker expression. In contrast, beta-carotene produced an increase in the percentage of cells expressing natural killer cell markers with smaller effect on T-helper markers. Modest increases in the percentage of cells expressing Ia antigen, transferrin, and interleukin-2 receptors were produced by both drugs. These results suggest that retinoids and carotenoids can produce major changes in immune cellular marker expression in vivo in humans at doses relevant to their potential clinical use.

In vivo response of secretory component in the rat uterus to antigen, IFN-gamma, and estradiol.

Intrauterine immunization of ovariectomized rats with SRBC is known to elicit pronounced IgA and IgG antibody responses in uterine secretions of immunized uteri. To determine whether secretory component (SC), the receptor for transporting polymeric IgA from tissues to mucosal surfaces, was also influenced by Ag, ovariectomized rats were immunized and boosted by placing SRBC into the lumena of individual uterine horns. In response to Ag, the levels of polymeric IgA, as well as free SC and SC bound to polymeric IgA, increased in uterine secretions. When ovariectomized animals were treated with estradiol, a fivefold increase in SC levels was observed in the immunized horns, indicating that a hormone response is superimposed on the Ag-induced stimulation of uterine SC. To determine whether IFN-gamma influences the presence of SC in uterine secretions, IFN-gamma was placed in the uterine lumena of ovariectomized nonimmunized rats. When uterine secretions were analyzed, significantly higher levels of SC were found in IFN-gamma-exposed uteri than were present in saline treated control animals. In contrast, intrauterine instillation of IFN-gamma had no effect on the levels of IgA in uterine secretions. This response was specific for IFN-gamma in that IFN-alpha/beta had no effect on uterine SC or IgA levels. These results indicate that intrauterine instillation of Ag, in addition to evoking pronounced antibody responses, stimulates the production of SC, which may be responsible for the transport of polymeric IgA from tissue to uterine secretions. Furthermore, they indicate that IFN-gamma placed in the uterine lumen stimulates SC production and suggest that the uterine SC response to Ag may be mediated by the action of IFN-gamma on uterine epithelial cells.

Effect of beta-carotene on lymphocyte subpopulations in elderly humans: evidence for a dose-response relationship.

The effects of various doses (0, 15, 30, 45, and 60 mg/d) of supplementary beta-carotene were evaluated. The percentage of lymphoid cells with surface markers for T-helper and natural killer (NK) cells and cells with interleukin 2 (IL-2) and transferrin receptors were significantly and substantially increased in peripheral blood mononuclear cells collected from older human adult volunteers after supplementation with greater than or equal to 30 mg beta-carotene/d for 2 mo. The increase in the percentage of cells with markers of NK cells and in expression of IL-2 receptors was dose dependent. The plasma concentrations of beta-carotene were also elevated significantly; however, there was no increase in the amount of retinol present in plasma. This indicated that immunomodulation induced by beta-carotene may be due to the carotenoid rather than to an increased amount, and hence actions, of vitamin A. These results support the role of immunostimulation as a potential mechanism of action of beta-carotene with cancer-prevention potential.