Adsorption of coliphages T1 and T7 to clay minerals.

Coliphages T1 and T7 of Escherichia coli were absorbed by kaolinite (K) and montmorillonite (M). Maximum adsorption of T7 (96%) to M was greater than that of T1 (84%), but the adsorption of both coliphages to K was the same (99%). Positively charged sites (i.e., anion exchange sites) on the clays appeared to be primarily responsible for the adsorption of T1 to K but only partially responsible for the adsorption of T1 to M; equilibrium adsorption isotherms of T1 to K and M did not show a correlation between adsorption and the cation exchange capacity of the clays, and the reduction in adsorption caused by sodium metaphosphate (a polyanion that interacts with positively charged sites on clay) was more pronounced with K than with M. The equilibrium adsorption isotherms of T7 to K and M suggested a correlation between adsorption and the cation exchange capacity of the clays. However, studies with sodium metaphosphate indicated that T7 also adsorbed to positively charged sites on the clays, especially on K. Adsorption of the coliphages to positively charged sites was greater with K than with M, probably because the ratio of positively charged sites to negatively charged sites was greater on K than on M.     

EGFR T790M Mutation as a Possible Target for Immunotherapy; Identification of HLA-A*0201-Restricted T Cell Epitopes Derived from the EGFR T790M Mutation

Treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, has achieved high clinical response rates in patients with non–small cell lung cancers (NSCLCs). However, over time, most tumors develop acquired resistance to EGFR-TKIs, which is associated with the secondary EGFR T790M resistance mutation in about half the cases. Currently there are no effective treatment options for patients with this resistance mutation. Here we identified two novel HLA-A*0201 (A2)-restricted T cell epitopes containing the mutated methionine residue of the EGFR T790M mutation, T790M-5 (MQLMPFGCLL) and T790M-7 (LIMQLMPFGCL), as potential targets for EGFR-TKI-resistant patients. When peripheral blood cells were repeatedly stimulated in vitro with these two peptides and assessed by antigen-specific IFN-γ secretion, T cell lines responsive to T790M-5 and T790M-7 were established in 5 of 6 (83%) and 3 of 6 (50%) healthy donors, respectively. Additionally, the T790M-5- and T790M-7-specific T cell lines displayed an MHC class I-restricted reactivity against NSCLC cell lines expressing both HLA-A2 and the T790M mutation. Interestingly, the NSCLC patients with antigen-specific T cell responses to these epitopes showed a significantly less frequency of EGFR-T790M mutation than those without them [1 of 7 (14%) vs 9 of 15 (60%); chi-squared test, p  =  0.0449], indicating the negative correlation between the immune responses to the EGFR-T790M-derived epitopes and the presence of EGFR-T790M mutation in NSCLC patients. This finding could possibly be explained by the hypothesis that immune responses to the mutated neo-antigens derived from T790M might prevent the emergence of tumor cell variants with the T790M resistance mutation in NSCLC patients during EGFR-TKI treatment. Together, our results suggest that the identified T cell epitopes might provide a novel immunotherapeutic approach for prevention and/or treatment of EGFR-TKI resistance with the secondary EGFR T790M resistance mutation in NSCLC patients.     

Factors influencing the replication of somatic coliphages in the water environment

The potential replication of somatic coliphages in the environment has been considered a drawback for their use as viral indicators, although the extent to which this affects their numbers in environmental samples has not been assessed. In this study, the replication of somatic coliphages in various conditions was assayed using suspensions containing naturally occurring somatic coliphages and Escherichia coli WG5, which is a host strain recommended for detecting somatic coliphages. The effects on phage replication of exposing strain WG5 and phages to a range of physiological conditions and the effects of the presence of suspended particles or other bacteria were also assayed. Phage replication was further tested using a strain of Klebsiella terrigena and naturally occurring E. coli cells as hosts. Our results indicate that threshold densities of both host bacterium and phages should occur simultaneously to ensure appreciable phage replication. Host cells originating from a culture in the exponential growth phase and incubation at 37 degrees C were the best conditions for phage replication in E. coli WG5. In these conditions the threshold densities required to ensure phage replication were about 10(4) host cells/ml and 10(3) phages/ml, or 10(3) host cells/ml and 10(4) phages/ml, or intermediate values of both. The threshold densities needed for phage replication were higher when the cells proceeded from a culture in the stationary growth phase or when suspended particles or other bacteria were present. Furthermore E. coli WG5 was more efficient in supporting phage replication than either K. terrigenae or E. coli cells naturally occurring in sewage. Our results indicate that the phage and bacterium densities and the bacterial physiological conditions needed for phage replication are rarely expected to be found in the natural water environments.     

Inactivation of the T7 coliphage by monofunctional alkylating agents. Action of phage adsorption and injection of its DNA.

Alkylation by ethyl or methyl methanesulfonate to an extent that inactivates more than 99.5% of T7 coliphages has no effect on phage adsorption on Escherichia coli B cells, but decreases the amount of phage DNA injected into the host cells. Depurination interferes with the injection of the phage DNA. Failure to inject the whole phage genome thus appears to be a cause of the immediate as well as of the delayed inactivation of the T7 coliphage treated by monofunctional alkylating agents; the hypothesis that it is the only cause of inactivation, although not very likely, cannot be excluded at the present time.     

A Comparative Study of CN-Resistant Respiration in Different Cultures of Tobacco Callus

The callus of Nicotiana rustica cv Gansu yellow flower and N. tabacum cv willow leaf were cultured on ordinary subculture medium (M-1) and on regeneration medium (M-2), respectively. No differentiation was observed in Gansu yellow flower tobacco callus cultures grown on both M-1 and M-2 medium. The respiration of both cultures was partially resistant to cyanide and markedly inhibited by m-chlorobenzhydroxamic acid. The relative contributions of alternative and cytochrome pathway were 31% and 47% of the total respiration, respectively, in M-1 callus cultures. The relative O₂ uptake of the two pathways was not changed significantly in M-2 callus cultures. In subcultured M-1 callus cultures of Willow leaf tobacco, the respiration mediated via alternative pathway was about 29 to 38% of the total respiration, and the cytochrome pathway still was the major respiratory pathway. In M-2 callus cultures in which differentiation occurred, the relative contribution of alternative pathway increased to 41 to 47% of the total respiration, and the cytochrome pathway decreased considerably. These results suggested that the change of respiratory electron transport pathway was probably related to the differentiation of tobacco callus cultures.     

Charcoal as a habitat for microbes and its effect on the microbial community of the underlying humus

Wildfires produce a charcoal layer, which has an adsorbing capacity resembling activated carbon. After the fire a new litter layer starts to accumulate on top of the charcoal layer, which liberates water‐soluble compounds that percolate through the charcoal and the unburned humus layer. We first hypothesized that since charcoal has the capacity to adsorb organic compounds it may form a new habitat for microbes, which decompose the adsorbed compounds. Secondly, we hypothesized that the charcoal may cause depletion of decomposable organic carbon in the underlying humus and thus reduce the microbial biomass. To test our hypotheses we prepared microcosms, where we placed non‐heated humus and on top one of the adsorbents: non‐adsorptive pumice (Pum), charcoal from Empetrum nigrum (EmpCh), charcoal from humus (HuCh) or activated carbon (ActC). We watered them with birch leaf litter extract. The adsorbing capacity increased in the order Pumorg in the litter extract, respectively. After one month, all adsorbents harboured microbes, but their amount and basal respiration was largest in EmpCh and HuCh, and smallest in Pum. In addition, different kinds of microbial communities with respect to their phospholipid fatty acid and substrate utilization patterns were formed in the adsorbents. The amount of microbial biomass and number of bacteria did not differ between humus under different adsorbents, although different microbial communities developed in humus under EmpCh compared with Pum, which is obviously related to the increased pH of the humus under EmpCh, and also ActC. We suggest that charcoal from burning can support microbial communities, which are small in size but have a higher specific growth rate than those of the humus. Although the charcoal layer induces changes in the microbial community of the humus, it does not reduce the amount of humus microbes.     

Specific binding of T lymphocytes to macrophages. I. Kinetics of binding.

Peritoneal exudate lymphocytes obtained from immune guinea pigs and cultured for 1 week on antigen-pulsed autologous macrophages were tested for their ability to bind to fresh antigen-pulsed autologous macrophages or to macrophages pulsed with an irrelevant antigen. Up to 30% of the lymphocytes bound to macrophages bearing the relevant antigen whereas only 2 to 5% remained nonspecifically bound to macrophages after vigorous washing. Specific binding was observed in cultures as early as 1 hr. Analysis of the kinetics of binding suggests that the observed nonspecific binding is not a step in specific binding. The possibility that weaker antigen-independent association between lymphocytes and macrophages precedes specific binding cannot be excluded. No evidence was obtained that serum antibody adsorbed to the macrophage or T cell plays a role in this cell interaction or that the T cell can bind antigen directly. We suggest that the observed specific binding represents the initial event in stimulation of T lymphocytes by antigen.     

Enhanced anthraquinones production from adsorbent-treated Morinda elliptica cell suspension cultures in production medium strategy

Continuous removal of anthraquinones (AQ) by Amberlite polymeric adsorbents (XAD-4, XAD-7 and XAD-16) through in situ adsorption in Morinda elliptica cell suspension cultures is studied for product recovery and improvement of the overall titre. Ethanol was the best eluting solvent for effective recovery of AQ from all adsorbents. Pre-treatment of XAD-4 with sodium acetate not only enhanced intracellular AQ, but also AQ release and subsequent recovery from the adsorbent. The addition of sodium acetate pretreated XAD-4 on day 18 for 6-day contact period, achieved comparable cell growth to control (41 g/L), but with 1.3-fold higher intracellular AQ (124 mg/g DW) and two-fold increase in extracellular AQ (14.3 mg/L). High amount of adsorbent and longer contact period for the cultures entering stationary growth phase, stimulated AQ release and recovery but at the expense of cell growth. With 5–8.3 g XAD-4 adsorbent per litre M. elliptica culture in production (P) medium, between 60 and 90% AQ was recovered from extracellular AQ after 24–26 days of culture period.     

Lymphocytes cytotoxic to uveal and skin melanoma cells from peripheral blood of ocular melanoma patients

Summary To study antitumor immunity in patients with choroidal melanoma, T cells were generated from the peripheral blood of choroidal melanoma patients by mixed lymphocyte/tumor cell culture (MLTC). Because autologous tumors are generally unavailable, an allogeneic choroidal melanoma cell line, OCM-1, was used as the specific stimulus. Lymphocyte cultures from 27 patients were characterized by cell-surface phenotypes, patterns of reactivity towards cells of the melanocytic origin and T-cell-receptor gene usage. Antimelanoma reactivity was found in cell-sorter-purified CD4⁺ and CD8⁺ T cell subsets. To analyze this reactivity, sorter-purified CD4⁺ and CD8⁺ cells from a MLTC were cloned by limiting dilution in the presence of exogenous interleukin-2 and interleukin-4 as well as irradiated OCM-1. Under these conditions, CD4⁺ T cells did not proliferate, perhaps because of the absence of antigen-presenting cells. However, CD8⁺ grew vigorously and 29 cytolytic CD8⁺ T cell clones were isolated. On the basis of their pattern of lysis of OCM-1, a skin melanoma cell line M-7 and its autologous lymphoblastoid cell line LCL-7, the clones were categorized into three groups. Group 1, representing 52% of the clones, lysed all three target cells, and are alloreactive. However, since OCM-1 and M-7 did not share class I antigens, these clones recognized cross-reactive epitope(s) of the histocompatibility locus antigen (HLA) molecule. Group 2, constituting 28% of the clones, lysed both the ocular and skin melanoma cell lines but not LCL-7, and were apparently melanoma-specific. Unlike classical HLA-restricted cytolytic T lymphocytes, these T cells might mediate the lysis of melanoma cells via other ligands or a more degenerate type of HLA restriction. For the latter, the HLA-A2 and -A28 alleles would have to act interchangeably as the restriction element for shared melanoma-associated antigen(s). Group 3, representing only 10% of the T cell clones, was cytotoxic only to OCM-1, but not to M-7 or LCL-7. These clones may recognize antigens unique to ocular melanoma cells. Our data suggest that choroidal melanoma patients can recognize melanoma-associated antigens common to both ocular and cutaneous melanoma cells, and presumbly their autologous tumor. Thus, choroidal melanoma, like its skin counterpart, may be responsive to immunotherapeutic regimens such as active specific or adoptive cellular immunotherapy.This work is supported by National Institutes of Health research grants CA 36 233 and EY 9031, the Lucy Adams Memorial Fund and support from the Concern Foundation     

Influence of fungal elicitors on production of ajmalicine by cell cultures of Catharanthus roseus

Suspension cultures of Catharanthus roseus (C. roseus) were elicited with fungal cell wall fragments of Aspergillus niger (A. niger), Fusarium moniliforme (F. moniliforme), and Trichoderma viride (T. viride). The effects of elicitor dosage, exposures time, and age of subculture on ajmalicine accumulation were studied. A higher concentration of elicitor extract responded positively to C. roseus suspension cultures. Ajmalicine accumulation increased by about 3-fold when cells were treated with A. niger, F.moniliforme, and T. viride. The maximum ajmalicine production (75 microg g(-1) dry weight (DW)) was observed in cells treated with T. viride. Cell cultures were elicited with 5% preparation of A. niger, F. moniliforme, and T. viride and exposed for 24, 48, 72, and 96 h. for elicitation. Suspension cultures elicited with T. viride for 48 h showed a 3-fold increase (87 microg g(-1) DW) in ajmalicine contents, whereas A. niger and F. moniliforme synthesized a 2-fold increase in alkaloid and yielded 52 and 56 microg g(-1) DW ajmalicine, respectively. C. roseus cells of different age (5,10, 15, 20, and 25 days old) were treated with a 5% elicitor of A. niger, F. moniliforme, and T. viride and investigated elicitors activity at different age of cell cultures. Maximum yield 166 microg g(-1) DW of ajmalicine was synthesized in 20 day old suspension cultures treated with T. viride. A longer period of incubation of cell cultures with elicitors adversely affected the ajmalicine synthesis.     

Characterization of Effector and Memory T Cell Subsets in the Immune Response to Bovine Tuberculosis in Cattle

Cultured IFN-γ ELISPOT assays are primarily a measure of central memory T cell (Tcm) responses with humans; however, this important subset of lymphocytes is poorly characterized in cattle. Vaccine-elicited cultured IFN-γ ELISPOT responses correlate with protection against bovine tuberculosis in cattle. However, whether this assay measures cattle Tcm responses or not is uncertain. The objective of the present study was to characterize the relative contribution of Tcm (CCR7⁺, CD62L^hi, CD45RO⁺), T effector memory (Tem, defined as: CCR7^-, CD62L^low/int, CD45RO⁺), and T effector cells (CCR7^-, CD62L^-/low, CD45RO^-), in the immune response to Mycobacterium bovis. Peripheral blood mononuclear cells (PBMC) from infected cattle were stimulated with a cocktail of M. bovis purified protein derivative, rTb10.4 and rAg85A for 13 days with periodic addition of fresh media and rIL-2. On day 13, cultured PBMC were re-stimulated with medium alone, rESAT-6:CFP10 or PPDb with fresh autologous adherent cells for antigen presentation. Cultured cells (13 days) or fresh PBMCs (ex vivo response) from the same calves were analyzed for IFN-γ production, proliferation, and CD4, CD45RO, CD62L, CD44, and CCR7 expression via flow cytometry after overnight stimulation. In response to mycobacterial antigens, ~75% of CD4⁺ IFN-γ⁺ cells in long-term cultures expressed a Tcm phenotype while less than 10% of the ex vivo response consisted of Tcm cells. Upon re-exposure to antigen, long-term cultured cells were highly proliferative, a distinctive characteristic of Tcm, and the predominant phenotype within the long-term cultures switched from Tcm to Tem. These findings suggest that proliferative responses of Tcm cells to some extent occurs simultaneously with reversion to effector phenotypes (mostly Tem). The present study characterizes Tcm cells of cattle and their participation in the response to M. bovis infection.     

Enhanced in vitro production of Ruta graveolens L. coumarins and rutin by mannitol and ventilation

Ruta graveolens shoot cultures were maintained on static medium supplemented with 0, 1, 2 and 3?% mannitol. The cultures were grown in vessels that ensured a ventilation rate of 7.44, 10.82 or 62.83 air exchanges per day (V1, V2 or V3, respectively). The growth index and fresh weight were significantly increased at 1?% mannitol and decreased with increasing mannitol concentrations, whereas the dry weight (DW) and DW?% increased at higher concentrations of mannitol. Improving the culture ventilation significantly increased all of these parameters. A higher concentration of mannitol resulted in a higher proline content and percentage of coumarins and rutin, but the final accumulation of these bioactive molecules decreased. The coumarins, calculated as xanthotoxin, were increased from 8.15 to 13.60?mg?g^?1 DW using (V1 and mannitol-free medium) and (V2 with medium enriched with 2?% mannitol), respectively. Rutin was linearly increased by raising the mannitol concentrations, achieving the highest content of 54.87?mg?g^?1 DW using V2 and medium supplemented with 3?% mannitol. The lowest accumulation of coumarins and rutin (32, 144?mg vessel^?1, respectively) were found on mannitol-free medium using V1, whereas the highest rutin contents were found on medium with 1?% mannitol using V3. A GC analysis revealed the presence of five main compounds in all of the cultures, coumarin, 7-hydroxucoumarin, scopoletin, xanthotoxin and bergapten, whereas pasoralen was not detected when the cultures were maintained on mannitol-free medium, regardless of the type of vessel. Moreover, the concentrations of these compounds varied according to the mannitol concentration and ventilation.     

Adsorption of reovirus by minerals and soils.

Adsorption of [35S]methionine-labeled reovirus by 30 dry soils, minerals, and finely ground rocks suspended in synthetic freshwater at pH 7 was investigated to determine the conditions necessary for optimum virus removal during land application of wastewaters. All of the minerals and soils studied were excellent adsorbents of reovirus, with greater than 99% of the virus adsorbed after 1 h at 4 degrees C. Thereafter, virus remaining in suspension was significantly inactivated, and within 24 h a three to five log10 reduction in titer occurred. The presence of divalent cations, i.e., Ca2+ and Mg2+, in synthetic freshwater enhanced removal, whereas soluble organic matter decreased the amount of virus adsorbed in secondary effluent. The amount of virus adsorbed by these substrates was inversely correlated with the amount of organic matter, capacity to adsorb cationic polyelectrolyte, and electrophoretic mobility. Adsorption increased with increasing available surface area, as suspended infectivity was reduced further by the more finely divided substrates. However, the organic content of the soils reduced the level of infectious virus adsorbed below that expected from surface area measurements alone. The inverse correlation between virus adsorption and substrate capacity for cationic polyelectrolyte indicates that the adsorption of infectious reovirus particles is predominately a charged colloidal particle-charged surface interaction. Thus, adsorption of polyelectrolyte may be useful in predicting the fate of viruses during land application of sewage effluents and sludges.     

Overexpression of a Catharanthus tryptophan decarboxylase (tdc) gene leads to enhanced terpenoid indole alkaloid (TIA) production in transgenic hairy root lines of Rauwolfia serpentina

To enhance the production of terpenoid indole alkaloids in Rauwolfia serpentina, Catharanthus tryptophan decarboxylase (Crtdc) gene was over-expressed in transgenic hairy root cultures using Agrobacterium rhizogenes-mediated transformation. Among six transgenic hairy root lines, line RT4 accumulated the highest alkaloid content, with 0.1202 % dry weight (DW) reserpine and 0.0064 % DW ajmalicine, after 10 weeks of culture. Whereas, wild-type roots accumulated 0.0596 ± 0.003 % DW reserpine and 0.0011 ± 0.001 % DW ajmalicine. Transgenic hairy root line RT7 produced the lowest alkaloid content (reserpine: 0.0896 ± 0.002 % DW; ajmalicine: 0.002 ± 0.0 % DW). On the basis of alkaloid content the six hairy root lines were grouped as RT4/RT2 > RT3/RT5 > RT7/RT8. Analysis of gene expression profile indicated that Crtdc was expressed at a higher level in transgenic lines, which could be correlated with enhanced metabolite accumulation in roots. This study confirms that over-expression of Crtdc is a superlative method to improve the biosynthetic potential of Rauwolfia hairy root cultures. Enhanced reserpine and ajmalicine production can serve as an alternative choice to provide resources for relative pharmaceutical industries.     

Accumulation of hydroxybenzoic acids and other biologically active phenolic acids in shoot and callus cultures of Aronia melanocarpa (Michx.) Elliott (black chokeberry)

Phenolic acids are plant metabolites important in phytotherapy and also in cosmetology. In this study, proliferating shoot and callus cultures of Aronia melanocarpa were established and maintained on Linsmaier and Skoog (L-S) medium containing different levels of α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA), ranging from 0.1 to 3.0 mg l^?1. Methanolic extracts from the biomass of these cultures and from the fruits of soil-grown plants were used to determine the amounts of free phenolic acids and cinnamic acid using the high-performance liquid chromatography (HPLC) method. Out of a total of twelve analyzed compounds, all of the extracts contained four of them: caffeic acid, p-hydroxybenzoic acid, syringic acid, and vanillic acid. Moreover, shoot extracts also contained salicylic acid (o-hydroxybenzoic acid), while callus extracts contained p-coumaric acid. On the other hand, fruit extracts also contained both salicylic acid and p-coumaric acid. The total amount of the analyzed compounds in extracts from both shoot and callus cultures depended on the L-S medium used, and varied between 103.05 and 150.95 mg 100 g^?1 dry weight (DW), and between 50.23 and 81.56 mg 100 g^?1 DW, respectively. Both types of culture contained higher levels of phenolic acids than the fruit extracts (32.43 mg 100 g^?1 DW). In shoot cultures, p-hydroxybenzoic acid and salicylic acid were the predominant metabolites (reaching 55.14 and 78.25 mg 100 g^?1 DW, respectively), while in callus cultures, p-hydroxybenzoic acid (25.60 mg 100 g^?1 DW) and syringic acid (41.20 mg 100 g^?1 DW) were the main compounds. In fruit extracts, salicylic acid (15.60 mg 100 g^?1 DW) and p-hydroxybenzoic acid (5.29 mg 100 g^?1 DW) were predominant.     

Toxicity of zinc to fungi, bacteria, and coliphages: influence of chloride ions.

A 10 mM concentration of Zn2+ decreased the survival of Escherichia coli; enhanced the survival of Bacillus cereus; did not significantly affect the survival of Pseudomonas aeruginosa, Norcardia corallina, and T1, T7, P1, and phi80 coliphages; completely inhibited mycelial growth of Rhizoctonia solani; and reduced mycelial growth of Fusarium solani, Cunninghamella echinulata, Aspergillus niger, and Trichoderma viride. The toxicity of zinc to the fungi, bacteria, and coliphages was unaffected, lessened, or increased by the addition of high concentrations of NaCl. The increased toxicity of zinc in the presence of high concentrations of NaCl was not a result of a synergistic interaction between Zn2+ and elevated osmotic pressures but of the formation of complex anionic ZnCl species that exerted greater toxicities than did cationic Zn2+. Conversely, the decrease in zinc toxicity with increasing concentrations of NaCl probably reflected the decrease in the levels of Zn2+ due to the formation of Zn-Cl species, which was less inhibitory to these microbes than was Zn2+. A. niger tolerated higher concentrations of zinc in the presence of NaCl at 37 than at 25 degrees C.     

Activation of immunoglobulin allotype-specific T cells by B cells

To investigate the role of Ia and immunoglobulin (Ig) molecules of B cells in alloantigen-specific and nominal antigen-specific T-cell activations, the ability of B cells to stimulate Ig allotype-specific T cells was examined. T15-primed B10.BR T cells responded to MOPC 315 (IgA myeloma protein derived from BALB/c) as well as T15 but not to MOPC31c (IgG, myeloma protein). These T cells were stimulated by papain-digested Fc fragment of T15. Thus, T15-primed B10.BR T cells were shown to be specific for Ig allotype of T15, that is, Igh-2a. T15-specific B10.BR T cells were selected by 10-day cultures with T15 in vitro. They responded to BALB.K spleen cells without addition of soluble T15 antigen to the assay culture. Stimulator cells in this mixed lymphocyte reaction (MLR)-like response between T15-specific B10.BR T cells and BALB.K spleen cells were Thy-1⁻, Ia⁺ cells and these responses were blocked by anti-Ia^κ antibodies. Furthermore, Sephadex G-10-passed BALB.K B cells stimulated the proliferation of T15-specific B10.BR T cells, while they failed to stimulate allogeneic BALB/c spleen cells. The stimulating ability of B cells in this MLR-like response of T15-specific B10.BR T cells was shown to be genetically restricted, namely, both H-2 and non-H-2 genes are involved in the manifestation of the stimulating ability. This system will provide a useful model for studying the role of B-cell surface Ig and Ia molecules in the activation of antigen-specific T cells and alloreactive T cells.     

T cell responses to allogeneic human mesenchymal stem cells: immunogenicity,tolerance, and suppression

Human mesenchymal stem cells (MSCs) were evaluated for their ability to activate allogeneic T cells in cell mixing experiments. Phenotypic characterization of MSCs by flow cytometry showed expression of MHC Class I alloantigens, but minimal expression of Class II alloantigens and costimulatory molecules, including CD80 (B7-1), CD86 (B7-2), and CD40. T cells purified from peripheral blood mononuclear cells (PBMCs) did not proliferate to allogeneic MSCs. Lack of response was not due to a deficiency of costimulation, since retroviral transduction of MSCs with either B7-1 or B7-2 costimulatory molecules did not result in lymphoproliferation. Although these results suggested that MSCs were immunologically inert or potentially tolerogenic, T cells cultured with MSCs produced IFN- and displayed secondary kinetics to restimulation with PBMCs, indicating alloantigen priming rather than tolerance induction by the MSCs. To determine whether MSCs suppressed alloreactive T cells, MSCs were added to primary mixed lymphocyte reaction (MLR) cultures. MSCs suppressed cell proliferation when added at the initiation of culture or when added to an ongoing MLR culture. Suppression was dose-dependent, genetically unrestricted, and occurred whether or not MSCs were pretreated with IFN-. MSCs in transwell chambers suppressed primary MLR cultures, indicating that suppression was mediated by soluble molecules. Analysis of cytokines in suppressed MLR cultures demonstrated up-regulation of IFN- and IL-10, and down-regulation of TNF- production relative to control cultures. We conclude that MSCs can initiate activation of alloreactive T cells, but do not elicit T cell proliferative responses due to active suppressive mechanisms.     

Production of butanol from glucose and xylose with immobilized cells of Clostridium acetobutylicum

Pretreated cotton towels were used as carriers to immobilize Clostridium acetobutylicum CGMCC 5234 cells for butanol or ABE production from glucose and xylose. Results showed that cell immobilization was a promising method to increase butanol concentration, yield and productivity regardless of the sugar sources compared with cell suspension. In this study, a high butanol concentration of 10.02 g/L with a yield of 0.20 g/g was obtained from 60 g/L xylose with 9.9 g/L residual xylose using immobilized cells compared with 8.48 g/L butanol and a yield of 0.141 g/g with 20.2 g/L residual xylose from 60 g/L xylose using suspended cells. In mixed-sugar fermentation (30 g/L glucose plus 30 g/L xylose), the immobilized cultures produced 11.1 g/L butanol with a yield of 0.190 g/g, which were 28.3% higher than with suspended cells (8.65 g/L) during which 30 g/L glucose was utilized completely using both immobilized and suspended cells while 3.46 and 13.1 g/L xylose maintained untilized for immobilized and suspended cells, respectively. Based on the results, we speculated that immobilized cells showed enhanced tolerance to butanol toxicity and the cultures preferred glucose to xylose during ABE fermentation. Moreover, the cultures showed obvious difference when grown between high initial concentrations of glucose and those of xylose. Repeated-batch fermentations from glucose with immobilized cells showed better long-term stability than from xylose. At last, the morphologies of free and immobilized cells adsorbed on pretreated cotton towels during the growth cycle were examined by SEM.     

Contrasting effects of T cell growth factors on T cell responses to melanoma in vitro

 Determinants of T cell responses to tumor cells remain largely unknown. In the present study we have used long-term cultures of human melanoma cells and autologous peripheral blood lymphocytes to examine the influence of cytokines with T cell growth activity on the phenotype and cytotoxic and proliferative response of T cells to melanoma. It was found that addition of interleukin-4 (IL-4) inhibited the response of CD8⁺ T cells and promoted the response of the CD4 subset. IL-2 or IL-7 was effective in increasing melanoma-specific cytotoxic T lymphocyte (CTL) activity in cultures where CD8 T cells were predominant, whereas IL-4 followed by IL-2 was most effective in cultures where CD4 T cells predominated. IL-10 or IL-12 inhibited proliferation and CTL activity against melanoma in long-term cultures. The effects of IL-12 were reproduced in long-term cultures of T cells stimulated with mAb against CD3 and were shown to depend on prior exposure of T cells to IL-12 before IL-2. As yet unidentified factors, such as co-factor expression on melanoma, appear to be as important as exogenous cytokines in determining the nature of T cell responses to melanoma. These results suggest that analysis of responses in long-term culture may assist in defining the role of key cytokines and other determinants of immune responses to melanoma. Received: 4 June 1996 / Accepted: 12 November 1996