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Superantigens are potent activators of the immune system, causing a variety of diseases, ranging from food poisoning to septic shock. Here, we examined the effects of different toxic shock syndrome toxin 1 (TSST-1) concentrations on the activation, proliferation and synthesis of interferon-γ (IFN-γ) and interleukin-4 (IL-4) in purified naïve human CD4+ T cells in a serum-free in vitro system. TSST-1 given in low doses (1–10 pg/ml) generates a pronounced T helper 2 (Th2)-like cytokine profile, characterized by elevated IL-4-expressing T-cell populations and reduced IFN-γ-producing populations, whereas higher doses (100 pg/ml) induce a Th1-like profile, with increased expression of IFN-γ and reduced expression of IL-4. These patterns were even more pronounced by adding exogenous cytokines like IL-12 and IL-4 and by the type of antigen-presenting cells (APCs). Thus, B cells induced Th2 shifts, whereas monocytes favoured Th1 induction. Moreover, IL-12 in conditions with B cells counteracted their Th2 bias. Interestingly, in purified naïve T-cell cultures, containing a small population of HLA-DR+ T cells, Th1/Th2 differentiation can be induced by TSST-1 too. There, Th-cell polarization is strongly dependent on TSST-1 concentration, indicating that this is a key parameter in regulating the differentiation of T cells. In conclusion, our data show that Th1/Th2 differentiation of TSST-1-stimulated naïve T cells is controlled by the type of APCs, and in APC-depleted cultures, it depends on the presence of HLA-DR+ cells and TSST-1 concentration. [ABSTRACT FROM AUTHOR]

Monocyte-T-cell interactions were studied in pokeweed mitogen (PWM)-activated cell cultures. We addressed the question of monocyte changes in PWM-stimulated cultures of T cells and monocytes and found, by flow cytometric analysis, that PWM activation led to a loss of cells with monocyte or macrophage phenotype (CD14, HLA-DR, HLA-DQ) within 48 hr of culture in the presence of T cells (CD4+ T cells), but not in cultures of pure monocytes. Chemiluminescence measurements revealed that phagocytic stimulation of monocytic superoxide release was impaired in PWM-stimulated cultures of monocytes plus T cells, but not in PWM-stimulated cultures of pure monocytes. Furthermore, PWM induced the secretion of interferon-gamma (IFN-γ) in primary cultures oft cells supplemented with 20% of monocytes, whereas in subsequent secondary cultures of these cells PWM induced IFN-γ only when monocytes were added. We conclude from these flow cytometric and functional analyses that monocytes are efficiently eliminated from PWM-activated T-cell/monocyte cultures by CD4+ T lymphocytes. [ABSTRACT FROM AUTHOR]

Soluble egg antigens of the parasitic helminth Schistosoma mansoni (S. mansoni egg antigen [SEA]) induce strong Th2 responses both in vitro and in vivo. However, the specific molecules that prime the development of Th2 responses have not been identified. We report that omega-1, a glycoprotein which is secreted from S. mansoni eggs and present in SEA, is capable of conditioning human monocyte-derived dendritic cells in vitro to drive T helper 2 (Th2) polarization with similar characteristics as whole SEA. Furthermore, using IL-4 dual reporter mice, we show that both natural and recombinant omega-1 alone are sufficient to generate Th2 responses in vivo, even in the absence of IL-4R signaling. Finally, omega-1-depleted SEA displays an impaired capacity for Th2 priming in vitro, but not in vivo, suggesting the existence of additional factors within SEA that can compensate for the omega-1-mediated effects. Collectively, we identify omega-1, a single component of SEA, as a potent inducer of Th2 responses.

Recently we described a new signal transduction-based tumor therapeutic strategy involving first sensitization of tumor cells by trichostatin A (TSA), an inhibitor of histone deacetylation, and thereafter efficient apoptotic triggering by ribotoxic agents, which activate stress-activated protein kinases. In the present work we investigate the molecular basis of the sensitization step in this therapeutic model system and describe TSA-induced changes in mRNA and protein expression of several candidate genes identified previously by complex hybridization. Furthermore, activities of 15 different protein kinases were followed after TSA application, using a new filter-based technique (PhosphoSpots-Assay). The obtained data suggest that TSA induces pro-apoptotic genes like ID1, ID2, ID3, and down-regulates anti-apoptotic genes like Hsp27 and Bcl-xL, thereby shifting the cellular equilibrium from life to death. Furthermore, activities of calcium/calmodulin-dependent kinase II and protein kinase C, which have been assigned pro-apoptotic function in other systems, are induced.

Lung adenocarcinoma cells treated for 16 h with trichostatin A (TSA), an inhibitor of histone deacetylases, and untreated cells were analyzed with respect to differential gene expression. Complex hybridization of cDNA arrays revealed repression of Bcl-xL, CRAB2 and TFIID/TAFII31 as well as induction of p21waf1/cip1, GATA-2, hsp86, ID1, ID2 and ID3 mRNA expression, which could be verified by Northern blotting. ID2 induction was further confirmed by Taqman realtime quantitative RT-PCR. The described alterations of gene expression due to TSA renders the lung adenocarcinoma cells susceptible to induction of apoptosis.

We describe a procedure that sensitizes chemotherapy-and tumor necrosis factor-resistant human tumor cell populations in vitro and in nude mouse transplants to the immediate triggering of high rates of cell death by anisomycin, an agent causing activation of stress-activated protein kinases [SAPKs, as defined by P. Cohen (Trends Cell Biol., 7: 353-361, 1997)] including p38/RK and c-jun NH2-terminal kinase homologues, following its binding to ribosomal 28S RNA (M. S. Iordanov et al, Mol. Cell. Biol., 17: 3373-3381, 1997). Sensitization is effected by successive application of an inhibitor of histone deacetylation (trichostatin A, butyrate) and of flavopiridol, known as an inhibitor of cyclin dependent kinases and evaluated presently in clinical trials. Effective concentrations of anisomycin, flavopiridol, and trichostatin A are in the submicromolar range. Tumor cell death can be prevented by epidermal growth factor (EGF), if added before flavopiridol or after anisomycin but not if applied between the additions of these agents, suggesting that flavopiridol interrupts an EGF-activated survival pathway and that anisomycin, besides triggering cell death, guards this pathway against the interference by flavopiridol. In contrast to EGF, dibutyryl-cAMP exerts protection that is flavopiridol-insensitive. For triggering cell death, anisomycin cannot be replaced by DNA- or mitotic spindle-targeted drugs in this system. The present findings, that a combination of transcriptional and signal transduction-targeted modulators sensitizes tumor cells synergetically to stress-mediated triggering of cell death and that ribotoxic stress is more efficient in this respect than genotoxic or spindle-targeted stress, bear important implications for the therapeutic exploitation of cellular stress responses. The stepwise sensitization and triggering of cell death in the present system allow separate analysis and manipulation of processes contributing to cellular death susceptibility and of the mechanism responsible for triggering cell death, thus providing the operational basis for further development of this therapeutic approach.

A new T(H1)/T(H2) in vitro model for mechanistic studies and drug screening in human T cells was established working with ficoll-separated PBMCs or elutriated lymphocytes cultured in serum-free medium. Human T cells could be kept viable and reactive in this medium for several months. In this model, superantigens (SAs) were used to activate exclusively those T cell clones which were known to express specifically SA-binding Vbeta-chains of the T cell receptor. It was possible to identify the activated SA-specific T cells among the whole T cell population by using monoclonal antibodies against these Vbeta-chains and to follow responses involving receptor regulation and cytokine expression. The flow cytometric analysis revealed, that SA exposure caused an upregulation of the IL-2 receptor selectively in the SA-specific subpopulation. Only the T cells of this subpopulation could be shifted towards T(H1) or T(H2) differentiation, which was determined by the distribution of IFN-gamma and IL-4 positive cells. Regulation of IFN-gamma could be detected by flow cytometry after 18 h and that of IL-4 on the third day of cell culture. The differentiation status could be influenced by various measures: T(H1) shifts were achieved in the presence of IL-12 and anti-IL-4, whereas, T(H2) shifts were induced more slowly with monocyte-reduced elutriated lymphocytes in the presence of IL-4, IL-6, anti-IL-12, 1alpha,25-dihydroxy-vitamin-D3 or combinations thereof. It was found that sialidase stimulated whereas TGF-beta and pentoxifylline suppressed both kinds of T cell response. The T(H1)/T(H2) differentiation persisted for several weeks after primary activation if cells were expanded in IL-2 containing serum-free culture medium. Therefore, this human T(H1)/T(H2) in vitro model should be ideal for studying early and late events of infection, allergy, and autoimmunity as well as for investigating the cellular interactions involved. In addition, the early detection of the response pattern makes this model potentially useful for drug screening.

In the present work, cytokine-mediated induction of cell death was investigated by flow cytometry in cell cycle-synchronous human tumor cell populations gained by centrifugal elutriation or by cell cycle blockade with mimosine and aphidicolin. Attention was payed to the question of whether the effector phase of cell death takes place in the same phase of the cell cycle in which the death signal is received. Another point of interest was the question whether synchronization of cell populations with respect to the cell cycle leads to increased synchronicity of the death phase. The results demonstrate that supernatants from monocyte/tumor cell interaction cultures containing tumor necrosis factor-alpha, interferons, and interleukins-1 and -6 or appropriate combinations of pure cytokines cause cell cycle arrest predominantly in G1 and to a lesser extent in G2. Cell death is initiated from both arrest points. Cytokine-treated G1 cells do not enter S phase. They die within the same G1 phase in which they receive the death signal. In contrast, a high proportion of cytokine-treated G2 cells pass through mitosis and are arrested and die in the subsequent G1 phase, whereas only a smaller proportion of cells are arrested and die in G2. The synchronicity of the death phase cannot be increased by the diverse methods of cell cycle synchronization applied. Interestingly, aurintricarboxylic acid, an agent known for inhibitory effects on nucleolytic activities and other protein/nucleic acid interactions, not only prevents cell death, but also cell cycle arrest.

It is shown in a serum-free system with human cells that during monocyte/tumor cell interactions signals are generated which restore the principles of density-dependent inhibition of growth in tumor cell populations. Tumor cells that fail to comply with these principles are induced to die. This cell death is an active process in target cells involving protein biosynthesis and tumor cell-derived signals.

In a serum-free culture system, the release of TNF, lI-1, lI-6, IFN-alpha, and IFN-beta during interaction of elutriated human monocytes (MO) with human tumor cells (TC) was studied by ELISA-technique. Contributions of these cytokines to inhibition of TC-growth and to induction of TC-death by supernatants (SU) gained from such MO/TC-interaction cultures were investigated using affinity chromatography for removal of individual cytokines. Although the TC used are relatively insensitive to recombinant human TNF, withdrawal of TNF causes 50% to 75% reduction of SU-induced TC-death rates, suggesting that susceptibility to TNF is raised during MO/TC-interaction by the other cytokines. Individual removal of other cytokines does not cause reduction of SU-mediated TC-death. However, combined withdrawal of lI-1 and IFN-alpha/beta causes in 2 of 4 TC-lines significant reduction of TC-death. Combined removal of TNF, IFN-alpha/beta, lI-1, and lI-6 leads to complete prevention of SU-mediated growth inhibitory and lytic effects, suggesting that besides these cytokines other signals are not involved significantly. SU-effects can be mimicked by appropriate combinations of authentic cytokines. The response of TC to SU- or cytokine-exposure is strikingly dependent on TC-density, leading at subconfluent TC-density exclusively to inhibition of growth and at postconfluent TC-density to induction of cell death. The principal effect of SU or cytokine combinations in this context seems to be the activation of growth inhibitory signal transduction pathways leading to TC-death in postconfluent TC-populations exclusively if growth stimulatory pathways are activated at the same time. Mouse L cells do not follow this reaction pattern: Their death is exclusively dependent on the presence of TNF in SU and they die upon SU-exposure at postconfluent as well as at subconfluent cell density.

The mechanisms controlling "spontaneous" cellular death rates in normal and tumorigenic tissues are largely unknown. An important parameter in this respect is the susceptibility of the target cell to induction of the lytic pathway by appropriate signals. In the present article it is demonstrated in a serum-free in vitro system that the susceptibility of human tumor cells (TC) to induction of lysis by cytokine signals generated during interaction of TC with elutriated human monocytes (MO) is a highly dynamic parameter subject to modulation by hormones, growth factors, and tumor cell density. It was found that growth stimulatory signals such as insulin, and especially epidermal growth factor (EGF), increase lytic susceptibility, whereas hydrocortisone, which does not exert significant growth modulatory effects in these examples, protects TC against the induction of lysis. Increasing TC density above confluence dramatically enhances lytic susceptibility, suggesting interactions between TC to be involved in the induction of their death. In conjunction with previous data demonstrating the insusceptibility of TC, which are forced out of the cell cycle into the quiescent state (G0), the hypothesis is put forward that growth stimulatory factors increase a TC's lytic susceptibility by preventing its transit from G1 to G0 in response to growth inhibitory signals generated during MO/TC interaction. The data support the concept that TC susceptibility to the induction of cell death is a consequence of simultaneously activated growth stimulatory and growth inhibitory signalling pathways.

In a recently established serum-free in vitro system it has been demonstrated that the susceptibility of various human tumor cells to the induction of cell death by elutriated human monocytes is critically dependent on tumor cell density and growth state. In the present work it is shown by flow cytofluorometric analysis of bromodeoxyuridine incorporation rates and of expression of the proliferation-associated nuclear antigen Ki-67, that tumor cells forced out of the cell cycle into the quiescent state (G0), which can be accomplished by treatment with supernatant from monocyte/tumor cell interaction cultures, are no longer susceptible to the induction of cell death by monocytes. This suggests that processes essential for the lytic pathway cannot take place in quiescent cells. It is furthermore demonstrated that tumor cells are driven into G0 during interaction with monocytes and that the rate of transit from G1 to G0 increases with increasing monocyte dosage. This explains our earlier finding that maximum rates of tumor cell death are induced at rather low monocyte:tumor cell ratios of around 1:2 and that lysis is suppressed at higher monocyte dosages (van der Bosch et al.:Exp Cell Res 187:185-192, 1990). The potential significance of these findings for the supposed function of mononuclear phagocytes in tumor defense lies in the notion that tumor cells driven into G0 might escape this control and that signals involved in monocyte/tumor cell-interaction contribute to the accumulation of tumor cells in G0.

In the present article a hypothesis is put forward and supporting data are referred that explain the elimination of tumorigenic cells as a consequence of their failure to comply with the rules of ubiquitous negative growth control mechanisms involving among others also mononuclear phagocytes as effector cells. This hypothesis does not invoke the recognition of altered cell surface structures on tumorigenic cells as the basis for discriminating them from normal cells and thus avoids one of the most irritating problems of hypothetical tumor defense mechanisms involving the recognition of spontaneously arising tumor cells as 'non-self' by immunologic effector cells.

The population development of five human tumor cell lines is examined under the influence of elutriator-prepared human monocytes in a serum-free hormone- and growth factor-supplemented medium. Analysis was performed by electronic counting and sizing of tumor cell nuclei and flow cytometric detection of cell cycle phases. Tumor cell death is triggered at rather low monocyte:tumor cell ratios (1:2 to 1:4) whereas it is strongly reduced at high monocyte densities. Furthermore, it is shown that confluence of the target cell population is a necessary prerequisite for lysis. The data suggest that in monocyte/tumor cell cocultures the decision on target cell lysis is not made by the effector cell, but rather by the target cell and that the criterion for this decision is the target cell's ability or inability to respond to a monocyte challenge by arresting the cell cycle in G1. Interactions between target cells play an important role in determining the result of this decision process. A common basis is suggested for this kind of density-dependent monocyte-triggered lysis and density-dependent cell death in 3T3 cell cultures as described previously.

Improved procedures are described for the seeding of primary cultures from human colon adenocarcinoma and for the use of these cultures in the evaluation of drug effects. Two of the specimens studied were xenografts maintained in athymic (nude) mice, while the other six were biopsies obtained directly from patients. Tumor cells obtained directly from the patients proliferated in defined hormone-supplemented medium to the exclusion of other cells. In drug-response studies with cultures from a colon tumor biopsy all four drugs studied (4'-deoxydoxorubicin, 4'-O-methyldoxorubicin, 5-fluorouracil, and 1,3-bis-[chloroethyl]-1-nitrosourea) inhibited growth of the cells within 3-6 days after drug treatment. On an equitoxic dose basis (LD10 in mice), 4'-deoxydoxorubicin appeared to be the most active drug. This drug also showed dose-dependent activity against one of the xenografted tumors in vitro. In dose-response studies with cultures from another patient's colon tumor, doxorubicin and 5-fluorouracil showed significant activity against the tumor 10 days after the drug treatment with concentrations at 1X and 10X average peak plasma levels.

In vitro responses of 4 xenografted human colon tumors (T183, T219, T245, and T348) to various doses of 4'-deoxydoxorubicin have been investigated. The individual tumors showed marked differences in drug responsiveness, ranging from high sensitivity at low doses (T219; 125 ng/ml) to very low sensitivity at high doses (T245; 4000 ng/ml). The sensitivity ranking deduced from these in vitro experiments correlates well with the ranking deduced earlie (Guiliani et al., Int. J. Cancer, 27: 5-13, 1981) from in vivo drug treatments of transplants of these tumors in the nude mouse. The effect of in vitro drug treatment (4'-deoxydoxorubicin; 250 ng/ml; 1-hr incubation) on the in vivo growth of one of the tumors, T219, in nude mice was investigated. Growth of the tumor in nude mice was markedly delayed by pretreatments in vitro with 4'-deoxydoxorubicin. Furthermore, in vitro responsiveness of the T219 tumor was investigated following in vivo and in vitro treatment of the tumor with 4'-deoxydoxorubicin. Both of the pretreatments produced very similar decreases in drug responsiveness to all of the doxorubicin derivatives tested (4'-deoxydoxorubicin, 4'-O-methyldoxorubicin, 4'-epidoxorubicin, 4-demethoxydoxorubicin, and N-trifluoroacetyldoxorubicin-14-valeriate).

The adherence to human uroepithelial cells of 23 Escherichia coli strains belonging to three groups with different levels of virulence was investigated, and the mechanism of adherence was studied. It was found that strains belonging to the most virulent group adhered better to human uroepithelial cells than did avirulent strains. Adherence of loss virulent but supposedly nephropathogenic strains was more variable. These results suggest that adherence is an important virulence factor, especially in the group of strains with the highest but a more general virulence. Piliated strains adhered better than did nonpiliated strains. We found strong evidence for the existence of at least two different mechanisms of adherence: (i) mannose-sensitive adherence by piliated strains, very likely mediated by type I pili because this mannose-sensitive adherence was associated with mannose-sensitive hemagglutination of guinea pig erythrocytes by broth cultures of the strains; (ii) mannose-resistant adherence by piliated strains, very likely mediated by non-type I pili because this mannose-resistant adherence was invariably associated with mannose-resistant hemagglutination of human group A erythrocytes by the strains, whether grown in broth or on plates. Additionally, one strain without pili and without hemagglutinating activity adhered well. Thus in most cases adherence seemed to be mediated by bacterial pili, although different types might be involved.

3T3 cells are shown to reduce SV40-3T3 cell population growth in a denisty-dependent manner in co-cultures of 3T3 and SV40-3T3 cells. The development of this inhibitory activity roughly parallels the development of density-dependent inhibition of growth in homogeneous 3T3 control cultures. The extent of reduction of SV40-3T3 growth can be manipulated by pretreatment of 3T3 cells with a high serum concentration. SV40-3T3 growth rates are reduced by factors between 10 and 20 under optimum inhibitory conditions as compared to SV40-3T3 growth in control cultures.

A simple three-enzyme treatment of collagenase, dispase and hyaluronidase on finely minced chick oviduct yields clumps of 50-150 cells. These cells attach to collagen-treated dishes and survive in culture for at least 2 weeks without subculturing. Oviduct cell cultures can also be induced to grow. Estradiol or epidermal growth factor (EGF) induce a 40% increase in cells in 4 days when cultures are grown in serum levels that do not support growth. Serum from estrogen-stimulated chicks promotes rapid cellular proliferation (doubling times of 1-2 days). Sera from estrogen withdrawn chicks, laying hen or horse do not support as rapid proliferation. The oviduct growth-promoting factors in serum from estrogen-stimulated chicks are not steroids or fibroblast growth factors (FGF). Removal of steroids from these sera by charcoal treatment or delipidization does not decrease the rate of growth. The addition of 1-100 nM estradiol does not increase a serum's ability to promote growth. Purified FGF or platelet-derived growth factor (PDGF) do not induce oviduct proliferation. These results were reproduced in oviduct cell cultures started from estrogen-stimulated and withdrawn chicks as well as laying hens. Thus the factors in serum from estrogen-stimulated chicks that promote rapid oviduct growth are induced by estrogen treatments in vivo, but do not seem to be only steroids.

Procedures are described for the preparation of reproducible primary cultures from human colorectal tumors transplanted in the athymic nude mouse and for the quantitative evaluation of growth by means of counting suspensions of nuclei from these cultures with a Coulter counter. Growth curves of primary cultures from 11 colorectal tumors in serum-free medium are shown and discussed with respect to the in vitro conditions to be met for the propagation of in vivo stem cell populations.

Lowered extracellular [Ca2+] cause low growth rates and low stationary cell densities in 3T3 cell cultures as compared to physiological [Ca2%]. Under otherwise constant conditions the extracellular [Ca2+] determines a stable stationary cell density, which can be reached by increase of net cell number or decrease of net cell number, depending on cell density at the time of [Ca2+] adjustment. SV40-3T3 cells do not show this [Ca2+] dependency. At 39 degrees C 3T3 and SV40-3T3 cell populations show an increased growth rate at low cell densities as compared to cell populations at 35 degrees C. Approaching the stationary density the growth rate of both cell sorts is reduced faster at 39 degrees C than at 35 degrees C, leading to lower stationary cell densities at 39 degrees C than at 35 degrees C. A temperature change from 39 degrees C to 35 degrees C or in the opposite direction can affect the stationary cell density of 3T3 cell populations only if applied before reduction of growth rate by density-dependent growth-inhibiting principles has taken place.

Improved procedures for the seeding of primary cultures from human colon tumors are described. Responsiveness of primary cultures from human colon carcinoma T348 to a crude preparation of human leukocyte alpha-interferon and a DNA-intercalating experimental anticancer drug, 4'-deoxydoxorubicin, has been investigated. The effect of the interferon preparation on such cultures is shown to be growth state specific; i.e., stationary populations are killed, whereas fast-growing cultures are reversibly growth inhibited by the same doses of interferon. In contrast, 4'-deoxydoxorubicin kills growing populations, whereas stationary cultures are barely affected by the same concentration of this drug. Interferon antagonizes the cytotoxic effect of 4'-deoxydoxorubicin on a growing colon tumor cell population when applied immediately after 4'-deoxydoxorubicin treatment. Implications of this finding for the combined application of several drugs in cancer therapy are discussed.