Neurosyphilis Increases Human Immunodeficiency Virus (HIV)-associated Central Nervous System Inflammation but Does Not Explain Cognitive Impairment in HIV-infected Individuals With Syphilis.

BACKGROUND: Individuals infected with human immunodeficiency virus (HIV) who have previously had syphilis may have cognitive impairment. We tested the hypothesis that neurosyphilis causes cognitive impairment in HIV by amplifying HIV-related central nervous system (CNS) inflammation. METHODS: HIV-infected participants enrolled in a study of cerebrospinal fluid (CSF) abnormalities in syphilis underwent the mental alternation test (MAT), venipuncture, and lumbar puncture. CSF concentrations of chemokine (C-X-C motif) ligand 10 (CXCL10), chemokine (C-C motif) ligand 2 (CCL2), and neurofilament light (NFL) were determined by commercial assays. The proportion of peripheral blood mononuclear cells (PBMCs) and of CSF white blood cells (WBCs) that were activated monocytes (CD14+CD16+) was determined by flow cytometry. Neurosyphilis was defined as detection of Treponema pallidum 16S RNA in CSF or CSF white blood cells (WBCs) >20/uL or a reactive CSF-Venereal Disease Research Laboratory (VDRL) test; uncomplicated syphilis was defined as undetectable CSF T. pallidum, CSF WBCs ≤5/uL and nonreactive CSF-VDRL. MAT <18 was considered low. RESULTS: Median proportion of PBMCs that were activated monocytes (16.6 vs. 5.3), and median CSF CXCL10 (10658 vs. 2530 units), CCL2 (519 vs. 337 units) and HIV RNA (727 vs. 50 c/mL) were higher in neurosyphilis than in uncomplicated syphilis (P ≤ .001 for all comparisons). Neurosyphilis was not related to low MAT scores. Participants with low MAT scores had higher median CSF CXCL10 (10299 vs. 3650 units, P = .008) and CCL2 (519 vs. 365 units, P = .04) concentrations than those with high MAT scores. CONCLUSIONS: Neurosyphilis may augment HIV-associated CNS inflammation, but it does not explain cognitive impairment in HIV-infected individuals with syphilis.

Enhancement of CD3AK cell proliferation and killing ability by α-Thujone.

Thujone is a monoterpene ketone natural substance found mainly in wormwood and sage. Previous studies have shown that Thujone has various pharmacological effects, such as anti-tumor, analgesic, and insecticide. The effect of α-Thujone to human immune cells is still unknown. Our study focuses on investigating the effects and mechanism of α-Thujone to CD3AK (anti- CD3 antibody induced activated killer) cells proliferation and cytotoxicity to colon cancer cell lines. With cell proliferation and FCM assay, it is found that α-Thujone could significantly enhance CD3AK cell proliferation and expression of CD107a in a dose-dependent manner. The cytotoxicity to colon cancer cells detected by CCK-8 assay is also improved. The expressions of TNF-α and FasL detected with ELISA assay were not significantly changed. Mechanically, the study shows that α-Thujone could enhance the expression of p-ERK1/2 and p-Akt. In addition, α-Thujone has no cytotoxicity to HCT116 and SW620 cells proliferation. In a word, α-Thujone enhances CD3AK cell proliferation and cytotoxicity via the improvement of expression of CD107a, p-Akt and p-ERK1/2.

Human monocytes in the presence of interferons alpha2a and gamma are potent killers of serous ovarian cancer cell lines in combination with paclitaxel and carboplatin.

Interferons (IFNs) play an important role in immune surveillance of tumors; however, their efficacy in the treatment of malignancies has been limited. Monocytes are mononuclear phagocytes that are critical to the generation of an innate immune response to tumors. The authors and others have shown that treatment of tumor cell lines in vitro and in vivo with human monocytes primed with type I and type II IFNs results in killing. We now expand on this work, in an extended panel of ovarian cancer cell lines. In this study, we hypothesized that there would be variable sensitivity amongst cell lines to the killing properties of monocytes and IFNs. To this end, we explored the interactions of IFN primed monocytes in conjunction with the standard of therapy for ovarian cancer, taxane, and platinum-based chemotherapeutics. Using 6 ovarian cancer cell lines, we demonstrated that there is variation from cell line to cell line in the ability of IFN-α2a and IFN-γ primed monocytes to synergistically kill target tumor cells, and further, there is an additive killing effect when target cells are treated with both IFN primed monocytes and chemotherapy.

Combinatorial cytotoxicity of gemcitabine and cytokine-activated killer cells in hepatocellular carcinoma via the NKG2D-MICA/B system.

AIM: Natural-killer group 2, member D (NKG2D) is an activating receptor on natural killer cells and activated T-cells, designated cytokine-activated killer (CAK) cells here. The MHC class I chain-related A and B (MICA and MICB, respectively) are ligands of NKG2D and are expressed on various human tumor cells, including hepatocellular carcinoma (HCC) cells. Here, we investigate whether gemcitabine, a chemotherapeutic agent, affects MICA/B expression in HCC. MATERIALS AND METHODS: We used ELISA, RT-PCR and adherent target detachment assays to determine expression of MICA/B in HepG2 HCC cells and the level of cellular cytotoxicity generated by treatment with gemcitabine and/or CAK cells. RESULTS: Surface expression of MICA/B was evident after gemcitabine treatment, and MICB-specific mRNA was up-regulated. Pre-treatment with gemcitabine and subsequent exposure to CAK cells induced greater cytotoxicity than either treatment alone. Inclusion of soluble MICB significantly reduced cytotoxicity. CONCLUSION: Gemcitabine induced MICA/B expression in HepG2 cells, resulting in synergistic enhancement of the cytotoxic effects of NKG2D-high CAK cells. The combination of gemcitabine and CAK cells may have clinical therapeutic significance for HCC.

Adenosine inhibits the release of arachidonic acid in activated human peripheral mononuclear cells. A proposed model for physiologic and pathologic regulation in systemic lupus erythematosus.

In the current work, the pathways are presented and reviewed showing how adenosine acts on the production and release of arachidonic acid (AA) in activated human monocytes by the involvement of various phospholipase A2 (PLA2) and protein kinase C (PKC) enzymes in physiological (normal) conditions and in a pathologic state in systemic lupus erythematosus (SLE). Two molecules of activated monocytes mainly determine the actual amounts of AA released: (1) interleukin-1 beta (IL-1 beta) increasing and (2) adenosine (Ado) suppressing this process. The AA production of monocytes mainly depends on two (IV and VI) types of PLA2 enzymes. PKC alpha phosphorylates the cytosolic, Ca2+-dependent and steroid-sensitive PLA2 (type IV), whereas PKC delta phosphorylates the Ca2+-independent PLA2 (type VI). By the suppression of IL-1 beta production in the activated human monocytes, adenosine can decrease the release of AA causing a diminished phosphorylation of both PKC isoenzymes. In SLE monocytes, the disease-specific decreased release of AA that we found earlier could be related to the decreased expression of PKC delta. These pathways are summarized in a proposed model.

Near eradication of clinically relevant concentrations of human tumor cells by interferon-activated monocytes in vitro.

We have previously reported that low concentrations of interferon (IFN)-activated monocytes exert near-eradicative cytocidal activity against low concentrations of several human tumor cells in vitro. In the present study, we examined 7 human tumor cell lines and 3 diploid lines in the presence or absence of 10 ng/mL IFNα2a and monocytes. The results confirmed strong cytocidal activity against 4 of 7 tumor lines but none against 3 diploid lines. To model larger in vivo tumors, we increased the target cell concentration and determined the concentration of IFNα2a and monocytes, required for cell death. We found that increasing the tumor cell concentration from 10- to 100-fold (10(5) cells/well) required an increase in the concentration of IFNs by over 100-fold and monocytes by 10-fold. High concentrations of monocytes could sometimes kill tumor or diploid cells in the absence of IFN. We may conclude that killing of high concentrations of tumor or diploid cells required high concentrations of monocytes that could sometimes kill in the absence of IFN. Thus, high concentrations of tumor cells required high concentrations of IFN and monocytes to cause near eradication of tumor cells. These findings may have clinical implications.

Innate immune activation by the viral PAMP poly I:C potentiates pulmonary graft-versus-host disease after allogeneic hematopoietic cell transplant.

Respiratory viral infections cause significant morbidity and increase the risk for chronic pulmonary graft-versus-host disease (GVHD) after hematopoietic cell transplantation (HCT). Our overall hypothesis is that local innate immune activation potentiates adaptive alloimmunity. In this study, we hypothesized that a viral pathogen-associated molecular pattern (PAMP) alone can potentiate pulmonary GVHD after allogeneic HCT. We, therefore, examined the effect of pulmonary exposure to polyinosinic:polycytidylic acid (poly I:C), a viral mimetic that activates innate immunity, in an established murine HCT model. Poly I:C-induced a marked pulmonary T cell response in allogeneic HCT mice as compared to syngeneic HCT, with increased CD4+ cells in the lung fluid and tissue. This lymphocytic inflammation persisted at 2 weeks post poly I:C exposure in allogeneic mice and was associated with CD3+ cell infiltration into the bronchiolar epithelium and features of epithelial injury. In vitro, poly I:C enhanced allospecific proliferation in a mixed lymphocyte reaction. In vivo, poly I:C exposure was associated with an early increase in pulmonary monocyte recruitment and activation as well as a decrease in CD4+FOXP3+ regulatory T cells in allogeneic mice as compared to syngeneic. In contrast, intrapulmonary poly I:C did not alter the extent of systemic GVHD in either syngeneic or allogeneic mice. Collectively, our results suggest that local activation of pulmonary innate immunity by a viral molecular pattern represents a novel pathway that contributes to pulmonary GVHD after allogeneic HCT, through a mechanism that includes increased recruitment and maturation of intrapulmonary monocytes.

CVT-E002 stimulates the immune system and extends the life span of mice bearing a tumor of viral origin.

The present study evaluated the dose-related effects of CVT-E002, a proprietary extract of Panax quinquefolius (CV Technologies Inc., Edmonton, AB), in the treatment of a tumor of viral origin, that is, erythroleukemia, in mice. Three treatments including ingestion of 2, 40, and 120 mg/d were compared. The study revealed that the dose of 40 mg/d was particularly effective in stimulating cells mediating nonspecific immunity and extending the life span of tumor-bearing mice. This study represents the first in vivo demonstration of the anticancer efficacy of CVT-E002 in an animal model. CVT-E002 treatment significantly elevated the absolute numbers of natural killer cells and monocytes and reduced the number of tumor cells in the bone marrow and spleen. This study has shown that (1) approximately 30 to 50% of tumor-bearing mice administered CVT-E002 at a dose of 40 mg/d achieved a significantly extended life span, and (2) dosage is critical in producing these ameliorative effects.

Lysis of endogenously infected CD4+ T cell blasts by rIL-2 activated autologous natural killer cells from HIV-infected viremic individuals.

Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK) cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However, it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous, endogenously HIV-1-infected CD4+ T cells. Here, we stimulate primary CD4+ T cells, purified ex vivo from HIV-1-infected viremic patients, with PHA and rIL2 (with or without rIL-7). This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that, subsequent to the selective down-modulation of MHC class-I (MHC-I) molecules, HIV-1-infected p24(pos) blasts become partially susceptible to lysis by rIL-2-activated NK cells, while uninfected p24(neg) blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However, the degree of NK cell cytolytic activity against autologous, endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and -B alleles and against heterologous MHC-I(neg) cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs) and with the high frequency of the anergic CD56(neg)/CD16(pos) subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively, our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24(pos) blasts derived from primary T cells.

Prospective phase II study of post-surgical adjuvant chemo-immunotherapy using autologous dendritic cells and activated killer cells from tissue culture of tumor-draining lymph nodes in primary lung cancer patients.

BACKGROUND: The efficacy and toxicity of adjuvant chemo-immunotherapy using dendritic cells and activated killer cells are not clear in post-surgical primary lung cancer patients. PATIENTS AND METHODS: Pathologically diagnosed N2 lung cancer patients were selected for postsurgical adjuvant chemo-immunotherapy. The activated killer cells and dendritic cells (AKT-DC) obtained from tissue cultures of tumor-draining lymph nodes (TDLN) or from TDLN co-cultured with peripheral blood lymphocytes (TDLN-Pb) were used for the adoptive transfer of immunotherapy. The patients received 4 courses of chemotherapy along with immunotherapy every 2 months for 2 years. RESULTS: There were 31 N2 patients eligible for the study. Three cases were excluded because of refusal by the patients after 1-2 courses of immunotherapy. For the 28 cases treated, a total of 313 courses of immunotherapy were administered. The main toxicities were fever (78.0%), chill (83.4%), fatigue (23.0%) and nausea (17.0%) on the day of cell transfer. The 2- and 5-year survival rates were 88.9 % (95.9-81.9; 95% confidence interval, C.I.) and 52.9% (76.4-29.4; C.I.). CONCLUSION: Adoptive transfer of activated killer cells and dendritic cells from the tumor-draining lymph nodes of primary lung cancer patients is feasible and safe, and a large-scale multi-institutional study is necessary for evaluation of the efficacy of this treatment.

Ca2+/calmodulin-dependent kinase kinase alpha is expressed by monocytic cells and regulates the activation profile.

Macrophages are capable of assuming numerous phenotypes in order to adapt to endogenous and exogenous challenges but many of the factors that regulate this process are still unknown. We report that Ca(2+)/calmodulin-dependent kinase kinase alpha (CaMKKalpha) is expressed in human monocytic cells and demonstrate that its inhibition blocks type-II monocytic cell activation and promotes classical activation. Affinity chromatography with paramagnetic beads isolated an approximately 50 kDa protein from nuclear lysates of U937 human monocytic cells activated with phorbol-12-myristate-13-acetate (PMA). This protein was identified as CaMKKalpha by mass spectrometry and Western analysis. The function of CaMKKalpha in monocyte activation was examined using the CaMKKalpha inhibitors (STO-609 and forskolin) and siRNA knockdown. Inhibition of CaMKKalpha, enhanced PMA-dependent CD86 expression and reduced CD11b expression. In addition, inhibition was associated with decreased translocation of CaMKKalpha to the nucleus. Finally, to further examine monocyte activation profiles, TNFalpha and IL-10 secretion were studied. CaMKKalpha inhibition attenuated PMA-dependent IL-10 production and enhanced TNFalpha production indicating a shift from type-II to classical monocyte activation. Taken together, these findings indicate an important new role for CaMKKalpha in the differentiation of monocytic cells.

Leukotriene B4 activates T cells that inhibit B-cell proliferation in EBV-infected cord blood-derived mononuclear cell cultures.

Epstein-Barr virus (EBV)-specific cellular memory is not transferred from mother to child. Therefore, EBV-induced B-cell proliferation in in vitro-infected cord blood mononuclear cell cultures is not inhibited. However, by addition of immunomodulators, polysaccharide K (PSK) or truncated thioredoxin (Trx80) that activate monocytes, EBV-specific T-cell response could be generated in such cultures. Presently, we demonstrate that leukotriene B(4) (LTB(4)) is involved in the effect of the immunomodulators. LTB(4) was detected in the medium, and T-cell activation was compromised by addition of leukotriene biosynthesis inhibitors. Moreover, we found that LTB(4) added to infected cultures, which did not receive the immunomodulators, induced functional activation of the T cells. LTB(4) activated the monocytes and acted directly on the T cells. In consequence, addition of LTB(4) inhibited the EBV-induced proliferation of B lymphocytes. Specific cytotoxicity could be generated by restimulation of the T cells. The experiments showed successive stages of T-cell activation in acquisition of their immunologic effector function. This is orchestrated by complex cellular interactions, and autocrine loops mediated by soluble factors-here interferon (IFN)-gamma, interleukin (IL)-15, IL-12, and LTB(4). Importantly, the results indicate that endogenous LTB(4) can induce T-cell activation that inhibits the EBV-induced proliferation of B lymphocytes.

HIF-1 alpha expression is associated with an atheromatous inflammatory plaque phenotype and upregulated in activated macrophages.

OBJECTIVE: Angiogenesis and inflammation are important features in atherosclerotic plaque destabilization. The transcription factor hypoxia-inducible factor-1 alpha (HIF-1 alpha) is a key regulator of angiogenesis and is also involved in inflammatory reactions. We studied HIF-1 alpha expression in different atherosclerotic plaque phenotypes. METHODS AND RESULTS: HIF-1 alpha expression was observed in 18/37 (49%) carotid and in 9/15 (60%) femoral endarterectomy specimens. Expression of HIF-1 alpha was associated with the presence of a large extracellular lipid core (P=0.03) and macrophages (P=0.02). HIF-1 alpha co-localized with vascular endothelial growth factor (VEGF), an important downstream target of HIF-1 alpha. In addition, a strong association was observed between expression levels of HIF-1 alpha and VEGF (P=0.001). The average number of plaque microvessels was higher in plaques with no or minor HIF-1 alpha staining than in plaques with moderate or heavy HIF-1 alpha staining (P=0.03). In human macrophages, lipopolysaccharide activation induced HIF-1 alpha expression. In embryonic fibroblasts derived from wild-type mice, lipopolysaccharide activation induced an increase in HIF-1 alpha mRNA, whereas in Toll-like receptor 4 defective embryonic fibroblasts no effect was observed after lipopolysaccharide stimulation. CONCLUSIONS: In atherosclerotic plaque, the transcription factor HIF-1 alpha is associated with an atheromatous inflammatory plaque phenotype and with VEGF expression. HIF-1 alpha expression is upregulated in activated macrophages under normoxic conditions.

Clinical model: interferons activate human monocytes to an eradicative tumor cell level in vitro.

Eradicative levels of antitumor activity by cytokines and leukocytes have not yet been reached experimentally and are needed clinically. Only a limited number of human cancers respond to therapy with interferon (IFN), other cytokines, or mononuclear leukocytes despite significant antitumor activity in vitro. We studied the IFN and monocytic cell conditions that would lead to an eradicative effect using human cells in vitro. Targets of the IFN-activated monocytic cells were either four human tumor cell lines (human osteosarcoma [HOS], LOX melanoma, A549 lung tumor, and SNB-19 glioblastoma) or two diploid cell lines (WI38 and MRC5). An average of 30-90 colony-forming tumor target cells were cultured overnight in 96-well tissue culture plates prior to treatment with serially diluted IFN with or without activated elutriation-purified monocytes or lymphocytes. The target cell colonies were treated for 3 days. The colonies were then stained with crystal violet to determine the levels of antitumor activity. IFN-activated human monocytes reached an eradicative level (95%-100%) against three of four tumor cell lines. The eradicative level (1) was induced best in human monocytes activated by combined type I and II IFNs, (2) was effective against tumor cells that were growing for 24 h, (3) was specific for human tumors, as diploid human cells were not inhibited, and (4) required contact between the macrophage and the tumor cells. Also, for the first time, the minimal effective concentration (MEC) of IFNs to activate monocytes can approach those needed for antiviral activity. To our knowledge, this is the first report of near total eradication of many tumor cells, but not diploid cells, by IFN-activated monocytes. Because of its potency and specificity, the IFN-activated monocyte arm of the innate immune system may be a candidate for therapy of established tumors.

IL-21-induced Bepsilon cell apoptosis mediated by natural killer T cells suppresses IgE responses.

Epidemiological studies have suggested that the recent increase in the incidence and severity of immunoglobulin (Ig)E-mediated allergic disorders is inversely correlated with Mycobacterium bovis bacillus Calmette Guerin (BCG) vaccination; however, the underlying mechanisms remain uncertain. Here, we demonstrate that natural killer T (NKT) cells in mice and humans play a crucial role in the BCG-induced suppression of IgE responses. BCG-activated murine Valpha14 NKT cells, but not conventional CD4 T cells, selectively express high levels of interleukin (IL)-21, which preferentially induces apoptosis in Bepsilon cells. Signaling from the IL-21 receptor increases the formation of a complex between Bcl-2 and the proapoptotic molecule Bcl-2-modifying factor, resulting in Bepsilon cell apoptosis. Similarly, BCG vaccination induces IL-21 expression by human peripheral blood mononuclear cells (PBMCs) in a partially NKT cell-dependent fashion. BCG-activated PBMCs significantly reduce IgE production by human B cells. These findings provide new insight into the therapeutic effect of BCG in allergic diseases.

TNF-alpha activates human monocytes for Paracoccidioides brasiliensis killing by an H2O2-dependent mechanism.

Human monocytes activated by recombinant tumor necrosis factor alpha (TNF-alpha) exhibited significant fungicidal activity on the yeast cells of a highly virulent strain of Paracoccidioides brasiliensis. This process was significantly inhibited in the presence of catalase (CAT - a scavenger of H2O2), but not in the presence of superoxide-dismutase (SOD - a scavenger of superoxide anion) or NG-monomethyl-L-arginine (NG-MMLA - a nitric oxide inhibitor). Furthermore, there was a direct association between the intracellular killing of the fungus and the production of H2O2 by activated cells. These results strongly suggest a role for H2O2 in the killing of highly virulent strains of P. brasiliensis by TNF-alpha-activated human monocytes.

Mechanisms of bacillus Calmette-Guerin mediated natural killer cell activation.

PURPOSE: Natural killer (NK) cells are of crucial importance for bacillus Calmette-Guerin (BCG) mediated antitumor effects. We defined the mechanisms of BCG mediated NK cell activation in vitro. MATERIALS AND METHODS: A standard Cr release assay was used to measure the cytotoxicity of BCG activated NK cells. Using the MACS system (Miltenyi Biotec, Bergisch-Gladbach, Germany) we depleted various immune cell subpopulations from BCG stimulated peripheral blood mononuclear cells to phenotype activated NK cells. During the stimulation process anticytokine antibodies and recombinant cytokines were added to define their role in NK cell activation. For costimulation studies peripheral blood mononuclear cells were separated into lymphocytes and monocytes by counterflow-centrifugation (elutriation). Inhibitory NK cell receptor expression on activated NK cells was measured by flow cytometry by antiCD3, antiCD56 and anti-inhibitory NK cell receptor triple staining. RESULTS: The accessory function of monocytes was indispensable for BCG mediated NK cell activation. However, the stimulatory potential of monocytes did not require direct cell-cell contact to NK cells or major histocompatibility complex dependent antigen presentation to T cells. Monocyte derived interleukin (IL)-12 and to a lesser extent interferon (IFN)-alpha were key mediators for stimulating BCG induced NK cell cytotoxicity and IFN-gamma production. In contrast, IL-10 inhibited NK cell cytotoxicity and IL-18 did not show any effect. Exogenous recombinant IFN-alpha and IL-12 enhanced BCG mediated secretion of IFN-gamma and yet BCG induced NK cell cytotoxicity remained unchanged. While the CD158a and CD158b subsets did not have a significant role, NKG2A cells represented the predominant cytolytic subset in BCG activated NK cells. CONCLUSIONS: Following BCG stimulation the monocyte derived TH1 cytokines IL-12 and IFN-alpha activate tumor cytotoxic CD3/CD56/NKG2A NK cells. Our results elucidate NK activating mechanisms that are operative during BCG immunotherapy for bladder cancer and are relevant for an early, innate antimycobacterial immune response.

Host-oriented peptide evaluation using whole blood assay for generating antigen-specific cytotoxic T lymphocytes.

A whole blood assay using antigenic peptide was established to predict host cytotoxic T lymphocyte (CTL) precursor status. Blood samples from HLA-A24 donors and colorectal cancer patients were directly diluted with RPMI-1640 medium to a 20% blood concentration, then distributed to tubes and a peptide of an HLA-A24-restricted CEA peptide panel (20 microM) was added to the tubes. Incubation was performed for 4-5 days and supernatants were subjected to ELISA specific for IFN-gamma protein. It was observed that certain CEA peptides could stimulate the diluted blood samples to produce IFN-gamma. Only the peripheral blood mononuclear cells (PBMCs) that were purified from the IFN-gamma-positive samples of the whole blood assay showed positive spots, detected with IFN-gamma ELISPOT assay, and could proliferate with the stimulation of immobilized anti-CD3 antibody plus interleukin-2 (CD3/IL-2 system). The proliferating PBMCs expressed cytotoxic activity against HLA-A24+ CEA-expressing tumor cells and the TISI target cells pulsed with the CEA peptide that had been used to stimulate the PBMCs to produce IFN-gamma, but they did not kill the target cells pulsed with peptides that had failed to stimulate IFN-gamma production, nor did they kill the target cells alone. Theses findings suggest that the IFN-gamma production of the blood samples detected by the whole blood assay identifies the peptide that can induce the CEA antigen-specific CTL response. Detection of IFN-gamma gene expression using real-time-PCR analysis could identify the peptide within 6 hours, which is earlier than the protein analysis by ELISA. The whole blood assay using the CEA peptide panel for healthy donors and colorectal cancer patients revealed that IFN-gamma-inducible peptides were different among the individual samples tested, indicating that the CEA peptides that should be used for generating CTLs are different in individual patients. The whole blood assay using a CEA antigen peptide panel is simple and beneficial for identifying candidate peptides. The host-oriented peptide evaluation (HOPE) approach may provide hope for the augmentation of clinical efficacies for peptide-based cancer immunotherapy.

Cytotoxicity of normal and activated rat monocytes analyzed by flow cytometry.

BACKGROUND: The cytotoxic potential of activated monocytes might play an important role during severe systemic immune reactions and thus needs further elucidation. As established cytotoxicity tests are not suitable for this purpose, we developed a flow cytometry-based method. METHODS: During acute renal allograft rejection in the rat, monocytes were harvested by vascular perfusion and then purified by Percoll density gradient centrifugation and subsequent immunomagnetic negative selection. For comparison, natural killer (NK) cells were similarly isolated from spleen homogenates. Cytotoxicity was determined by flow cytometry using the fluorescein isothiocyanate-labeled NK-sensitive lymphoma Yac-1 as target. Necrotic cells were identified by propidium iodide, and apoptotic cells were identified by MC 540. Cytotoxicity was determined by the calculation of a cytotoxicity coefficient, zeta. The zeta coefficient describes the interrelation between the reciprocal proportion of target cells in a sample and the specific cytotoxicity, simultaneously allowing estimation of the contribution of contaminating NK cells. RESULTS: The method showed a substantial cytotoxicity of activated monocytes and indicated different or additional cytotoxic mechanisms compared with NK cells. Our assay permitted a detailed study of effector and target cells and took cytotoxicity of contaminating cells into account. CONCLUSIONS: The method is nonradioactive, easy to perform, and thus helpful in investigating the role of monocytes in several diseases.

Effect of cytokines on the in vitro fungicidal activity of monocytes from paracoccidioidomycosis patients.

Peripheral blood monocytes obtained from paracoccidioidomycosis patients and healthy individuals were preactivated with recombinant gamma interferon (IFN-gamma) in different concentrations (250, 500 and 1000 U/ml) and evaluated for fungicidal activity against Paracoccidiodes brasiliensis strain 18 (Pb 18, high-virulence strain) and strain 265 (Pb 265, low-virulence strain) by plating of cocultures and counting of colony-forming units, after 10 d. Monocytes from healthy individuals failed to present fungicidal activity against P. brasiliensis even after IFN-gamma activation at the three concentrations. However, patient monocytes activated with IFN-gamma (1000 U/ml) showed a significant fungicidal activity when compared to that obtained with non-activated or activated cells with other IFN-gamma concentrations (250 and 500 U/ml). Moreover, patient monocytes presented higher fungicidal activity than the control, even before the activation process. These results may be explained by the activation state of patients' cells as a function of the in vivo contact with the fungus, which was confirmed by their higher capacity to release H(2)O(2) in vitro. Unlike the results obtained with Pb 18, patient and control cells presented a significant fungicidal activity against Pb 265, after priming with IFN- gamma. These results are explained by the higher levels of TNF-alpha in supernatants of cultures challenged with Pb 265. Moreover, higher levels of the cytokine were obtained in patient cell supernatants. Taken together, our results suggest that for effective killing of P. brasiliensis by monocytes, an initial activation signal induced by IFN-gamma is necessary to stimulate the cells to produce TNF-alpha. This cytokine may be involved, through an autocrine pathway, in the final phase activation process. The effectiveness of this process seems to depend on the virulence of the fungal strain and the activation state of the challenged cells.