Anti-apoptotic seminal vesicle protein IV inhibits cell-mediated immunity.

The in vitro effect of seminal vesicle protein IV (SV-IV) on the cytotoxic activity of human natural or acquired cellular immunity has been investigated by standard immunological procedures, a (51)Cr-release cytotoxicity assay, and labeled-ligand binding experiments. The data obtained demonstrate that: (1) fluoresceinated or [(125)I]-labeled SV-IV binds specifically to the surface of human purified non-adherent mononuclear cells (NA-MNC); (2) SV-IV suppresses the cytotoxicity of natural killer (NK) cells against K562 target cells, that of IL-2-stimulated NK (LAK) cells against DAUDI target cells, and that of VEL antigen-sensitized cytotoxic T lymphocytes (CTLs) against VEL target cells; (3) treatment of K562 target cells alone with SV-IV decreases their susceptibility to NK-induced lysis. These findings indicate that the protein SV-IV has a marked in vitro inhibitory effect on NK, LAK and CTL cytotoxicity, providing a better understanding of its immune regulatory functions.

In vivo effect of an immunostimulating bacterial lysate on human B lymphocytes.

The aim of the present study is to investigate in humans the mechanism by which the oral vaccine Polyvalent Mechanical Bacterial Lysate (PMBL) can rapidly mobilize specific immune response and evaluate the efficacy of its immunostimulating activity in preventing recurrent infections of the upper respiratory tract (URTIs) in a group of patients with a medical history of URTI recurrence. Patients received, by sublingual route, PBML, an immunostimulating lysate obtained by mechanical lysis of the most common bacteria responsible for upper respiratory tract infections. The treatment was administered for 10 consecutive days/month for 3 consecutive months. After the end of the treatment period the patients were followed up for an additional 3 months. The frequency of IgM memory B cells and the expression of the activation marker CD25 in peripheral blood lymphocytes were measured using the flow cytometric method before the start and at days 30 and 90 of the treatment cycle. To correlate clinical results to immunological parameters, the patients were monitored at different time-points during the treatment and at the end of follow-up period. The results showed that PMBL exerts a therapeutic and preventing effect in acute and recurrent infections of the upper respiratory tract and that this effect correlated with the activation and enhancement of both IgM memory B lymphocytes (CD24+/CD27+ cells) and IL2 receptor-expressing lymphocytes (CD25+ cells) involved either in humoral or cellular immunity.

Effect of resveratrol on proliferation and telomerase activity of human colon cancer cells in vitro.

A number of studies performed in our laboratory and elsewhere, showed that resveratrol is able to prevent carcinogenesis and to impair tumor growth and progression. In order to provide additional information on the pleiotropic effects of resveratrol on malignant cells, the present study was performed to test the in vitro influence of the compound on the growth and TLMA of HT-29 and WiDr human colon cancer cell lines. The results confirmed that resveratrol has a direct, dose dependent, inhibitory effect on cell proliferation in both lines. In addition, for the first time, relatively high concentrations of this compound were found to be able to substantially down-regulate telomerase activity. These preliminary results further support the potential role of resveratrol in chemoprevention/chemotherapy of human colon tumor cells and provide the rational basis for novel strategies in cancer control.

Effect of morphine on cell-mediated immune responses of human lymphocytes against allogeneic malignant cells.

Opioid drugs, including morphine, are largely used as pain control in cancer patients at different stages of neoplastic growth and progression. Therefore, the possible influence of these drugs on host immunity appears to be of considerable interest. We have examined in vitro the effect of morphine on the generation of human cytotoxic T lymphocytes (CTL) against HTLV-I induced T-cell leukemia cells (MT-2 line). The results show that the drug, at graded concentrations (from 3 pg/ml to 32 microg/ml), that include those detectable in treated patients, enhances CTL activity whereas natural killer cell activity was unaffected. The enhancing effect is particularly evident when morphine was present at the onset of lymphocyte/MT-2 co-culture. On the contrary, the drug was ineffective when added on the last day of co-culture, thus indicating that morphine operates during the generation phase of CTL, but not on mature CTL. Flow cytometric analysis of intracellular cytokine expression showed that morphine increases the percentage of interferon gamma-producing CD8+ T cells in co-culture assay. Collectively, these results suggest that in our experimental model morphine enhances CTL responses by directly affecting the induction phase of T-dependent cell-mediated immunity.

In vitro effects of an immunostimulating bacterial lysate on human lymphocyte function.

MLBL is an oral immunostimulating vaccine consisting of bacterial standardized lysates obtained by mechanical lysis of different strains of Gram-positive and Gram-negative bacteria that can cause acute and chronic infections of the respiratory tract. Previous studies suggested a stimulating effect of MLBL both on humoral and cellular immune responses. In the present study, the in vitro effects of MLBL on human lymphocyte effector functions and its mechanisms of action were evaluated. The results show that the most remarkable effects of MLBL on the immune system are: i) activation of the IL-2 receptor (IL-2Ralpha) on different lymphocyte subsets (B, CD4+ T and CD8+ T cells) involved both in humoral and cellular immune responses; ii) induction of cytokine synthesis (IL-2, IL-10, IL-12, IFNgamma) in the immune competent cells that induce and regulate immune responses; iii) generation of CD4+ and CD8+ effector T cells. Overall, these results suggest that the therapeutic effect of MLBL on acute and recurrent infections of the respiratory tract is related to its ability to activate the responses of different subsets of immune competent cells both for humoral and cellular immunity. Moreover, these effects can be induced either by direct immune cell activation or through the generation and activation of immune effector cells.

DNA repair enzymes and cytotoxic effects of temozolomide: comparative studies between tumor cells and normal cells of the immune system.

O6-alkylguanine-DNA alkyltransferase (OGAT) and the mismatch repair system (MRS) play a crucial role in the susceptibility of tumor cells to the cytotoxic effects of agents that generate O6-methylguanine in DNA, including the triazene compound temozolomide (TMZ). Studies performed with peripheral blood mononuclear cells (MNC) showed that TMZ was scarcely active on lymphocyte functions not dependent on cell proliferation (e.g. NK activity and cytokine-mediated induction of CD1b molecule in adherent MNC). In contrast, TMZ depressed proliferation and lymphokine activated killer (LAK) cell generation in response to IL-2. In this case, a reasonably good inverse relationship was found between OGAT levels of MNC and their susceptibility to TMZ. This study also analyzed the ratio of the toxic effect of TMZ on MNC and on tumor cells (i.e. "Tumor-Immune Function Toxicity Index", TIFTI). A particularly favorable TIFTI can be obtained when OGAT levels are extremely high in MNC and markedly low in tumor cells. This holds true for MRS-proficient neoplastic cells, but not for MRS-deficient tumors. In conclusion, strategies aimed at modulating OGAT and MRS may improve the clinical response to TMZ. However, the use of OGAT inhibitors to potentiate the antitumor activity of TMZ might result in a concomitant increase of the immunosuppressive effects of the drug, thus reducing the relative TIFTI.

Effects of resveratrol on human immune cell function.

Resveratrol (3,5,4'-trihydroxystilbene), a polyphenol found in grapes and grape products such as red wine, has been reported to exhibit a wide range of biological and pharmacological activities both in vitro and in vivo. Because many of the biological activities of resveratrol, like the inhibition of cyclooxygenase, induction of CD95 signaling-dependent apoptosis, effects on cell division cycle and modulation of NF-kB activation, suggest a possible effect on the immune system, we evaluated the in vitro effects of resveratrol in three immune response models: i) development of cytokine-producing CD4+ and CD8+ T cells induced by stimulation of peripheral blood mononuclear cells (PBMC) with anti-CD3/anti-CD28; ii) specific antigen-induced generation of cytotoxic T lymphocytes; iii) natural killer (NK) activity of PBMC. The results showed that in vitro exposure to resveratrol produces a biphasic effect on the anti-CD3/anti-CD28-induced development of both IFN-gamma- IL2- and IL4-producing CD8+ and CD4+ T cells, with stimulation at low resveratrol concentrations and suppression at high concentrations. Similarly, the compound was found to induce a significant enhancement at low concentrations and suppression at high concentrations of both CTL and NK cell cytotoxic activity. On the whole, the results of the study indicate that resveratrol modulates several human immune cell functions and suggest that this activity may be related to its effects on cytokine production by both CD4+ and CD8+ T cells.

In vitro effect of hyperthermia on natural cell-mediated cytotoxicity.

It is well known that hyperthermia (HY), which is used for the treatment of cancer, depresses natural cell-mediated immunity in vitro. Experiments were performed to confirm the inhibitory effect of HY (42 degrees C for 1 hour) on natural killer (NK) activity and to evaluate the influence of HY on the generation and cytotoxic activity of interleukin-2 (IL-2)-activated NK cells. Additional experiments were also carried out to evaluate the effect of a simultaneous exposure of effector and target cells to HY. The results showed that HY profoundly reduced the lytic activity of NK cells and demonstrated that this inhibition was transient and not due to an apoptosis-induced reduction of the number of effector cells. Moreover, the exposure of mononuclear cells to HY before IL-2 stimulation did not affect the generation of IL-2-activated NK cells, whereas, the hyperthermic treatment of IL-2-activated NK cells produced a marked reduction of their cytotoxic activity. The results also showed that the simultaneous exposure of effector and target cells to HY, during the cytotoxicity assay, produced a marked reduction of lytic activity of NK and IL-2-activated NK cells, and that this impairment was specific for effector cells. In this context, heat-exposure of target cells alone, did not substantially modify their susceptibility to lysis induced by either NK or IL-2-activated NK cells. These results add further evidence of HY-induced inhibition of natural cell-mediated immunity, and suggest that, in the course of therapeutic HY, immune response could be significantly altered.

In vitro infection of CD4+ T lymphocytes with HTLV-I generates immortalized cell lines coexpressing lymphoid and myeloid cell markers.

The human T cell leukemia/lymphoma virus (HTLV-I) is the etiologic agent of adult T cell leukemia (ATL). CD4+ lymphocytes are the preferential targets of infection, even though other cell types can be infected in vitro by the virus. Although ATL cells show CD3 and CD4 surface markers, some ATL-derived cell lines were reported to express also myeloid antigens. In order to analyze possible phenotypic changes induced by HTLV-I after infection of human lymphocytes, CD4+ cells were isolated from peripheral blood of three healthy donors, by separation through immunomagnetic beads. CD4+ lymphocytes were then infected by coculture with irradiated HTLV-I producing MT-2 cells. The phenotypic profile of infected cells was studied by flow cytometric analysis using monoclonal antibodies against lymphoid (CD3, CD4, TCR alpha/beta) and myelomonocitic markers (CD13, CD14, CD15, CD33, CD34). The results show that HTLV-I immortalized cell lines coexpressed CD13, CD33 and lymphoid markers. No expression of CD14, CD15 and CD34 was observed. These data suggest that the presence of both myeloid and lymphoid phenotype in HTLV-I infected T cells is the results of an induction rather than a selection mechanism.

Effect of 4-hydroxynonenal, a product of lipid peroxidation, on natural cell mediated cytotoxicity.

Lipid peroxidation of cell membrane yields a variety of final products whose a quantitatively important component is 4-hydroxynonenal (HNE). Previous studies performed in our laboratory suggest that HNE may play a physiological role in the control of cellular proliferation and/or differentiation. This appears to be further supported by our recent findings showing that pre-treatment of K562 cells with a physiological concentration of HNE leads to a marked reduction of susceptibility of NK cells. The observed regulatory effects of HNE on tumor cell growth and susceptibility to natural immune resistance, led us to try to better understand the immunotoxicological properties of this aldehyde. The present study analyses the effects of HNE on NK-mediated cytotoxicity. Treatment of MNC as effector cells with concentrations of HNE ranging from 0.001 to 1 microM for 1 h, did not produce noticeable effects on NK activity. Therefore, this aldehyde at physiological concentrations is able to differentiate tumor cells and to down-regulate target susceptibility to NK effectors from one side. On the other side, it is not able to modify the efficiency of the NK function. Moreover, HNE concentrations higher than 1 microM showed significant and concentration-dependent inhibition of NK activity. However, this effect is reversible and can be antagonized, at least in part, by treatment of effector cells with HNE in combination with beta-interferon.

2'-2'-difluorodeoxycytidine: in vitro effects on cell-mediated immune response.

Little or no data are available on the immunotoxicity of the new deoxycytidine analogue, gemcitabine (2'-2'-Difluorodeoxycytidine, dFdC). The drug was tested on natural killer (NK), interleukin 2-activated killer (LAK) and antigen-dependent cytotoxic effector cells (CTL) activity. NK cells were treated for 16 hours and then tested against K562 cell line. LAK cells were pretreated for 16 hours before or after IL2 stimulation, and tested against DAUDI cells on day 4. CTL were pretreated for 16 hours on day-1 or on day 4 of coculture, and then tested against MT-2 on day 5. Cytotoxic activity was evaluated by a 4 hours 51Cr-release assay. The results indicate that dFdC inhibits markedly LAK or CTL generation, but does so less efficiently on "mature" LAK or CTL lymphocyte function and only slightly on NK cell activity. Therefore dFdC can be considered immunotoxic for either natural or antigen-dependent cell-mediated immunity.

Interferons antagonize gamma-ray-induced depression of natural immunity.

PURPOSE: The aim of this study was to determine the inhibitory effects of in vitro radiation on the number and function of natural killer (NK) cells and to investigate the capability of interferons (IFNs) to restore the activity of NK, depressed by gamma-rays. METHODS AND MATERIALS: Mononuclear cells (MNC) were obtained from intact or in vitro irradiated (20 Gy) peripheral blood collected from healthy donors. Alternatively, MNC were irradiated (20 Gy) after separation from intact whole blood. The in vitro treatment of MNC with IFNs (alpha, beta, or gamma, 200 UI/ml) was performed at different times after or before radiation. The NK activity (4 h-51Cr release test), the percentage of CD16+/CD56+ cells and apoptosis (cytometric analysis), and binding (microscopic observation) were evaluated on Days 0, 1, 2, and 5 from gamma-ray exposure and IFNs treatment. RESULTS: The in vitro treatment of irradiated MNC with betaIFN after radiation completely reverses the inhibitory effects of gamma-rays on human NK activity. BetaIFN do not reduce the apoptosis induction by radiation and don't modify the number of CD16- or CD56-positive cells. The binding between irradiated effectors and tumor cells (K562) appears partially increased in betaIFN-treated MNC. CONCLUSIONS: The results of the present investigation suggest a possible role of betaIFN in reversing the detrimental effect of radiation on human natural immunity and provide a rational basis for in vivo use of betaIFN in cancer radiotherapy.

SV-IV, a major protein secreted from rat seminal vesicle epithelium, promotes lymphocyte cytotoxic activity against the lymphoblastoid Raji cell line in human peripheral blood mononuclear cells.

The treatment of human peripheral blood mononuclear cells (PBMC) with micromolar concentrations of SV-IV, a major protein secreted from the rat seminal vesicle epithelium, promotes in this cell population a marked cytotoxic activity against the Raji lymphoblastoid cell line. This activity is apparently due to cell-to-cell contact interactions. The expression of HLA DR on Raji cells has a modulatory effect on the SV-IV-induced cytotoxic activity. The experimental evidence strongly suggests that the cytotoxic effector cells are functionally activated NK cells.

In vitro tumour cell growth inhibition: a comparative study between allosensitized cytotoxic T lymphocytes and lymphokine activated killer cells.

There is general agreement that several distinct subpopulation of lymphocytes, including major histocompatibility complex (MHC)-restricted T lymphocytes and non-restricted natural killer, or lymphokine-activated killer (LAK), cells are active in lysing neoplastic cells. In this study experiments were designed to compare the inhibitory effects of LAK cells and allosensitized cytotoxic T lymphocytes (CTL) on in vitro growth of an Epstein-Barr virus-transformed B-cell line (BSM) and of a HTLV-I producer T-cell line (MT-2). It was found that allosensitized CTL are more efficient at inducing BSM, or MT-2, cell growth inhibition than LAK cells. These results are consistent with the hypothesis that MHC-restricted T effector cells could mediate higher tumour suppressive effects than non-MHC restricted LAK cells.

In vitro combined effects of human interferons and interleukin-2 on natural cell-mediated cytotoxicity.

In vitro modulation of natural cell-mediated cytotoxicity (NCMC), following sequential treatment of human mononuclear cells (MNC) with cytokines was investigated. Recombinant Interleukin-2 (IL2) used in combination with interferons (IFNs) induced variable effects on the cytolytic function of different MNC preparations obtained from 16 healthy donors. When MNC were treated with IFNs on day 4, after IL2 induction of LAK cells, increase or no change in cytotoxic activity was found. On the other hand, either no change or decrease in LAK activity occurred when MNC were treated with IFNs on day 0 before exposure to IL2. In this case the effect of IFNs on NCMC did not correlate with their activity on cell proliferation or on TAC antigen expression. In conclusion the present study points out that the NCMC of MNC of healthy donors, subjected to IL2 treatment in vitro, can be significantly increased by IFNs. However this effect is largely schedule-dependent (i.e. detectable with IL2-IFNs but not with IFNs-IL2 sequence), and can be obtained in a relatively limited number of cases. Moreover it is suggested that these in vitro studies could provide preclinical bases for a rational approach to in vivo treatment with cytokine cascade in a clinical setting.

Immuno-chemotherapy of advanced colorectal cancer with alpha-2a interferon and 5-fluorouracil. Immunopharmacological studies.

Twelve patients with metastatic colorectal cancer received alternating cycles of low immunomodulating doses of alpha-IFN + 5-Fluorouracil (5-FU) or 5-FU alone. Hematological, biochemical and physical evaluation showed that both treatment cycles were well tolerated. However, transient fever and moderate flu-like symptoms were observed following alpha-IFN administration. Treatment with 5-FU alone produced long-lasting inhibition of CD8+ T lymphocytes, but did not depress NK activity (NKA). Combined treatment with alpha-IFN produced a short-term increase of NKA and antagonized the effect of 5-FU on CD8+ cells on day 5 of the cycle. Parallel studies on in vitro models showed antiproliferative effects of 5-FU on PHA-stimulated MNC and confirmed the preferential inhibition of CD8+ cells. Pretreatment with alpha-IFN did not reverse the effect of 5-FU on CD8+ lymphocytes, but partially protected MNC from the toxic effects of the drug. This was presumably due to the cytostatic effects induced by alpha-IFN on MNC before exposure to the cycle-specific antineoplastic agent. This investigation suggests that alpha-IFN could play a positive role in immuno-chemotherapy of colorectal cancer through multiple mechanisms not entirely related to direct antitumor effects of the agent.

Drug-mediated increase of susceptibility of human lung cancer to NK or LAK effector cells.

Previous studies in murine models have shown that in vivo or in vitro treatment of tumor cells with mutagenic triazene compounds (TZC) lead to the appearance of novel drug-mediated tumor antigens (DMTA) capable of eliciting graft resistance in syngeneic hosts. This phenomenon, defined as 'chemical xenogenization' (CX), could be of potential value for immunochemotherapy of human neoplasias. It was also shown that TZC modulate NK sensitivity of murine tumor cells. Therefore, experiments were conducted to evaluate whether susceptibility of a human lung adenocarcinoma cell line (H125) to natural cytotoxic effectors could be affected by treatment with an in vitro active TZC. The results showed that drug treatment of H125 line leads to heritable increase of susceptibility to NK and LAK cells. Moreover, increased binding between effector and drug-treated target cells was observed. Additional studies on HLA antigens showed that changes in HLA-ABC molecule expression were probably not involved in TZC-induced increase of NK/LAK susceptibility. These results suggest that TZC treatment of a human tumor could result in increased expression of membrane structures recognized by natural cytotoxic effector cells. Further studies are required to explore whether these changes are generated by mutational events correlated with TZC-induced CX of human cancer cells.

N-methylformamide affects spontaneous metastases of 3LL lines and increases natural killer activity of tumor-bearing mice.

The antitumor activity of the polar solvent N-methylformamide (NMF) was evaluated on three lines derived from the Lewis lung carcinoma (3LL), endowed with different metastatic potential. Two administration schedules were tested, these being repeated regimens of NMF (200 mg/kg per dose) for 12 consecutive days, starting 24 h or 6-10 days after tumor implantation (early or late treatment, respectively). The results of the present work can be summarized as follows: (1) NMF regimens did not greatly affect tumor growth behavior of 3LL lines; conversely, they markedly influenced their spontaneous colonizing ability in the lungs, either by delaying early metastatic spread or by reducing the number and size of pulmonary metastases already implanted. (2) A significant increase of NK cell activity during and after early treatment with NMF was observed in the more-metastasizing lines, thus suggesting the possibility of an immunomodulating effect of NMF.

Role of biological response modifiers in immunochemotherapy of solid tumors and retroviral-induced leukemia.

Modulation of the immune response by the use of biological response modifiers (BRM) is aimed at amplifying the host resistance against cancer. Studies on inhibition of tumor growth on an in vitro model, in which human breast carcinoma (HBL-100) and human lung carcinoma (H125) cells were used as target tumor cells, confirmed that interferons (IFNs) alpha and beta can amplify the antineoplastic effects of immunochemotherapy by enhancing the cytotoxic activity of effector cells and by antagonizing the immunodepressive effects of radiation or anticancer drugs. Moreover, data obtained from a pilot clinical trial, designed to test the effect of low concentrations of beta-IFN on natural cell-mediated cytotoxicity, pointed out a good correlation between the in vitro and in vivo responsiveness to beta-IFN in cancer patients. The immunomodulating and antiproliferative effects of BRM were also evaluated in a model of viral leukemogenesis in vitro, after infection of cord blood derived mononuclear cells (CB-MNC) with the human leukemic retrovirus HTLV-I. Alpha-and beta-IFN were previously shown to regulate differentially the antiviral competence of recipient CB-MNC, by interfering with viral replication and delaying the emergence of the transformed clone(s). One of the mechanisms of IFN action that contributes to control HTLV-I infection in vitro can be ascribed to their property of partially counteracting the depression of cell-mediated cytotoxicity that follows exposure to HTLV-I. In the light of data previously and herein described, it seems that alpha- and beta-IFN can be considered potential candidates to define combined therapy with antiviral drugs, to control the early stages of retrovirus-associated disease in human pathology.