Lymphotoxin production by human lymphocytes. III. The action of adenovirus antigen]

In lymphocyte cultures (LC) from 5 immune donors blast transformation occurred as a result of the stimulting action of adenovirus antigen; the supernatants obtained from these cultures produced a cytotoxic effect on L cells, thus suggesting the presence of lymphotoxin (LT). No blast transformation and no LT production were detected in nonstimulated LC used for control, as well as in LC from 9 nonimmune donors. LT production was shown to depend on the time elapsed after recovery from acute adenovirus infection. LT production was found to be most intensive during the first 2 months after recovery.     

Regulation and function of the protein inhibitor of nitric oxide synthase (PIN)/dynein light chain 8 (LC8) in a human mast cell line

The protein inhibitor of nitric oxide synthase (PIN) was independently identified as an inhibitor of nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS), and as a member of the cellular dynein light chain family, dynein light chain 8 (LC8), responsible for intracellular protein trafficking. Mast cells (MC) are involved in several homeostatic and pathological processes and can be regulated by NO. This study describes the expression of PIN/LC8 in the human MC line HMC-1. We also studied if PIN/LC8 binds nNOS, and what role this might have in leukotriene (LT) production. We found that PIN/LC8 mRNA and protein was expressed in HMC-1. Using a GST-PIN construct, we showed PIN binds to nNOS, but not endothelial (e)NOS in HMC-1; in our studies HMC-1 did not express inducible (i)NOS. Intracellular delivery of anti-PIN/LC8 antibody enhanced ionophore (A23187)-induced LT production through an unknown mechanism. Thus we established for the first time expression of PIN/LC8 in human MC, its ability to bind nNOS, and the effect that blocking it has on LT production in a human MC lines.     

Studies on the mechanism of action of guinea pig lymphotoxin. I. Membrane active substances prevent target cell lysis by lymphotoxin.

The effect of various pharamacologic agents on lysis of L cells by guinea pig lymphotoxin (LT) was studied. Among the many drugs tested, only those that affect plasma membrane functions were found to interfere with the cytolytic action of LT. Dimethyl sulfoxide or lidocaine potentiated L cell resistance against lysis. Stronger protection was provided by ouabain. Addition of ouabain to cells previously injured by LT was also effective in reducing cell death. Attempts to detect metabolic disturbances in cells before LT cytolysis were unsuccessful. The biosynthetic rate of DNA, RNA, or protein, and the total cellular content of ATP were not significantly changed in LT-treated cells. The results suggest that LT disturbs some plasma membrane functions of the target cell, perhaps ion transport systems, and consequently induces ionic imbalances between the intra-and extracellular milieu which eventually cause cell death.     

Induction of autophagy by anthrax lethal toxin

Autophagy is an evolutionary conserved intracellular process whereby cells break down long-lived proteins and organelles. Accumulating evidences suggest increasing physiological significance of autophagy in pathogenesis of infectious diseases. Anthrax lethal toxin (LT) exerts its influence on numerous cells and herein, we report a novel effect of LT-induced autophagy on mammalian cells. Several autophagy biochemical markers including LC3-II conversion, increased punctuate distribution of GFP-LC3 and development of acidic vesicular organelles (AVO) were detected in cells treated with LT. Analysis of individual LT component revealed a moderate increase in LC3-II conversion for protective antigen-treated cells, whereas the LC3-II level in lethal factor-treated cells remained unchanged. In addition, our preliminary findings suggest a protective role of autophagy in LT intoxication as autophagy inhibition resulted in accelerated cell death. This study presents a hitherto undescribed effect of LT-induced autophagy on cells and provides the groundwork for future studies on the implication of autophagy in anthrax pathogenesis.     

Differential production of interferon and lymphotoxin by human tonsil lymphocytes.

Lymphotoxin (LT) production, interferon (IF) production, and DNA synthesis were investigated after mitogen stimulation and in the mixed lymphocyte culture (MLC) reaction using human tonsil lymphocytes. Both LT and IF were assayed in parallel and from the same lymphocyte supernatants. An analysis of the PHA, PWM, one-way and two-way MLC reactions showed that the amounts of LT and IF produced could not be correlated. Polyriboinosinic: polyribocytidylic acid (poly(I: C)) failed to induce either LT production or [³H]TdR incorporation but did induce IF production. Removal of glass-adherent cells (GAC) had no effect on mitogen induced LT production but their removal reduced LT production in MLC reactions. GAC were necessary for IF production and optimal [³H]TdR incorporation in both mitogen stimulated cultures and in MLC reactions. IF and LT activities were shown to be the result of different molecules by using a Sephadex G-75 column. These results indicate that mitogen stimulation differs from MLC reactions in the cell type or control mechanisms involved for LT production, and that in mitogen stimulated cultures all three of these in vitro phenomena are probably the results of either different cell types or of different cell to cell interactions.     

Regulatory substances produced by lymphocytes. III. Evidence that lymphotoxin and proliferation inhibitory factor are identical and different from the inhibitor of DNA synthesis.

L cell mutant lines which were considerably more resistant to rat lymphotoxin (LT) than the original cell line were obtained by periodic additions of LT, partially purified from sensitized lymph node cell culture supernatants by DEAE-cellulose chromatography and Sephadex gel filtration. Addition of actinomycin D to cultures of these mutant cells abrogated resistance to LT, suqgesting that resistance was not due to a loss of LT receptors but probably to increased activity of a repair mechanisms. These mutant lines were also more resistant to the proliferation inhibitory effect of LT in low concentration and to that of diluted culture supernatants of lymph node cells stimulated with antigen (ovalbumin) than the original cell line, but they remained as sensitive to inhibitor of DNA synthesis (IDS) as the original line. The mutant lines also remained fully sensitive to both complement-dependent lysis by antibody and antibody-dependent cellular cytotoxicity, but showed increased resistance to the killing effect of rat lymph node cells sensitized with orginal L cells in vivo. These findings suggest that the lymphotoxic substance partially purified and characterized in this and in the previous paper may be an important mediator of T cell-mediated cytotoxicity.     

Mouse lymphotoxin.

The addition of PHA to C3H mouse spleen cells in tissue culture led to the production of lymphotoxin (LT). Cytotoxicity was assayed by addition of the culture fluids to syngeneic target cells labeled with tritiated thymidine; after an incubation period of 72 hr the amount of radioactivity released into the supernatant was measured. The LT activity in unfractionated culture fluids survived lyophilization, remained unchanged for many weeks at 4 degrees C, and progressively decreased on heating at 56 degrees C for periods from 1 to 9 hr. Based on the G-200 Sephadex distribution coefficients for several preparations, the m.w. of mouse lymphotoxin was about 41,000 daltons. Lymphotoxin from three different spleen cell production runs was recovered from isoelectric focusing columns in sharply focused peaks, the pH of which ranged from 4.4 to 4.8.     

Production of interleukin-1 alpha and interleukin-2 by mononuclear cells from children with growth delay in relation to the degree of growth hormone deficiency: effects of substitutive treatment

Interleukin-1 alpha (IL-1 alpha) and interleukin-2 (IL-2) levels were measured by radioimmunoassay in samples of conditioned medium from mononuclear cells taken from 20 normal subjects (14 adults ranging in age from 20 to 45 years and 6 children ranging in age from 3 to 11 years) and from 49 children with growth delay. Cultures were performed with 10(6) cells/ml in medium containing 1% normal human serum and 4.8 g/l phytohemagglutinin M. The incubation was performed for 48 h in an atmosphere containing 5% CO2. In normal subjects, the production of IL-1 alpha was 38.5 +/- 9.8 fmol/ml of conditioned medium (mean +/- SEM) in 14 adults and 41.6 +/- 3.0 fmol/ml in 6 children. The production of IL-2 was 46.9 +/- 6.5 and 57.3 +/- 10.5 fmol/ml, respectively. In the 16 patients with growth hormone (GH) deficiency studied before treatment, the production of ILs was significantly decreased in relation to the degree of deficiency. We observed a positive correlation between the production of IL-1 alpha and the values of insulin-like growth factor I but not with serum GH values. IL-1 alpha production was normalized after 15 days of substitutive GH therapy and IL-2 was normalized after 3 months of therapy. In 10 other patients with GH deficiency (4 with total and 6 with partial isolated GH deficiency) studied after long-term GH treatment (5 months or more), the mean of IL production was not significantly different from that of GH-deficient children treated for 3 months.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro depression of cellular immunity by Friend virus leukemic spleen cells.

Four distinct sublines of mouse L 929 cells (termed alpha, beta, gamma, and delta) were derived and shown to differ markedly in their in vitro sensitivity to human lymphotoxin (LT). The alpha L cell is most sensitive and is rapidly destroyed by very low dilutions of LT. This cell is 100 times more sensitive to LT than the most resistant (delta) L cell. The highly lymphotoxin-sensitive alpha cell makes it possible to reproducibly detect LT activity in as little as 0.0005 ml of supernatant medium. Additional studies revealed a direct correlation between the sensitivities of the four L cell sublines to LT and to direct cytolysis mediated by mitogen-stimulated human lymphocytes. The alpha, beta, gamma, and delta L cells were shown to be equally sensitive to antibody-mediated complement-dependent lysis, indicating that the sequence of sensitivities of these L cell sublines to the direct lymphocyte and to LT does not merely reflect a general susceptibility to cell destruction. These results lend further support to the view that lymphotoxin is an important mediator of in vitro target cell destruction by human effector lymphocytes.     

Cytotoxic lymphokines produced by cloned human cytotoxic T lymphocytes. II. A novel CTL-produced cytotoxin that is antigenically distinct from tumor necrosis factor and alpha-lymphotoxin

We have cloned lines of IL 2-dependent human T cells derived from alloantigen, soluble antigen (tetanus toxoid), mitogen, or IL 2-stimulated peripheral blood lymphocytes and characterized their surface marker expression and cytolytic activity. The surface phenotype and cytolytic function was compared with the ability of these T cell clones to release cytotoxic lymphokines in response to mitogenic lectins. The cytotoxins released by these CTL clones were detected on the murine L929 target cells in a 16-hr assay. All of the T cell clones, whether stimulated by HLA alloantigens, tetanus toxoid, or mitogens, exhibited killer cell activity and the capacity to secrete a soluble cytotoxin(s). Specific polyclonal antisera to recombinant human tumor necrosis factor (rTNF) and human alpha-lymphotoxin (alpha LT) were unable to neutralize the cytotoxic activity released by most of these CTL clones. These results indicate that human CTL produce a novel antigenic form(s) of cytotoxin that we have termed CTL-toxin. Supernatants from several CTL clones yielded a cytotoxic activity that was partially neutralized (10 to 40%) by saturating levels of anti-TNF (but not anti-alpha LT) indicating that human CTL may be capable of producing a TNF-like molecule. Only two out of 60 CTL clones studied thus far produced a cytotoxic activity that was partially neutralized by anti-alpha LT (20 to 40%). Collectively, these results suggest that although both the CD4 and the CD8 subpopulations of human cytotoxic T cells may be capable of releasing several types of cytotoxins in response to mitogenic signals, the predominant cytotoxin is distinct from alpha LT and TNF.     

In vitro development of Trichostrongylus axei, from infective larvae to young adults.

Advanced stages of Trichostrongylus axei were grown from exsheathed infective larvae in roller culture systems consisting of a complex cell-free medium, API-6, when used alone or supplemented for 8- or 21-day intervals with pepsin or reducing agents, or both. Young adult males and females were the most advanced stages attained. This stage was attained by days 18--21, when medium API-6 contained pepsin for 8 or 21 days, with or without reducing agents for the first 8 days; and by days 24--26, when medium API-6 was nonsupplemented. Trichostrongylus axei did not advance beyond early fourth stage or fourth molt when API-6 contained reducing agents for 8 or 21 days or reducing agents and pepsin for 21 days. The optimal culture system for development to young adults required medium API-6 supplemented with pepsin for 21 days and reducing agents for the first 8 days. This system produced maximal yields of less than 1% young adults in populations of about 100,000 T. axei by days 21--28.     

Production and release of heat-labile toxin by wild-type human-derived enterotoxigenic Escherichia coli

Production and release of heat-labile toxin (LT) by wild-type enterotoxigenic Escherichia coli (ETEC) strains, isolated from diarrheic and asymptomatic Brazilian children, was studied under in vitro and in vivo conditions. Based on a set of 26 genetically diverse LT(+) enterotoxigenic E. coli strains, cell-bound LT concentrations varied from 49.8 to 2415 ng mL(-1). The amounts of toxin released in culture supernatants ranged from 0% to 50% of the total synthesized toxin. The amount of LT associated with secreted membrane vesicles represented <5% of the total toxin detected in culture supernatants. ETEC strains secreting higher amounts of LT, but not those producing high intracellular levels of cell-bound toxin, elicited enhanced fluid accumulation in tied rabbit ileal loops, suggesting that the strain-specific differences in production and secretion of LT correlates with symptoms induced in vivo. However, no clear correlation was established between the ability to produce and secrete LT and the clinical symptoms of the infected individuals. The present results indicate that production and release of LT by wild-type human-derived ETEC strains are heterogeneous traits under both in vitro and in vivo growth conditions and may impact the clinical outcomes of infected individuals.     

Genetic diversity of heat-labile toxin expressed by enterotoxigenic Escherichia coli strains isolated from humans

The natural diversity of the elt operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either LT(+) (25 strains) only or LT(+)/ST(+) (26 strains), isolated from asymptomatic (24 strains) or diarrheic (27 strains) subjects, were subjected to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types of the H10407 strain were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes. Additionally, the single-nucleotide polymorphic analysis revealed 50 base changes in the elt operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified, including LT1, expressed by the H10407 strain and 23 other strains belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not other toxin types, showed reduced toxic activities measured either in vitro with cultured cells (Y-1 cells) or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LTs produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogeneses of the strains and the epidemiology of the disease.     

High-level production of erythritol by mutants of osmophilic Moniliella sp.

An erythritol-producing osmophilic yeast-like fungus, Moniliella sp. 440, was isolated from honey and then successively mutated with iterative rounds of N-methyl-N′-nitro-N-nitrosoguanidine (NTG) treatment and selection. Six generations of mutants, named N12115-6, N21105-6, N31074-3, N42208-2, N53199-9, and N61188-12, were selected for and produced erythritol at 151.0, 157.2, 177.8, 191.4, 196.6, and 237.8 g/L, respectively, while the wild type strain produced 113.0 g/L erythritol in media containing 40% glucose and 1% yeast extract. The mutant cells were found to have a short rod-like shape, while the wild type cells have a long rod-like shape. The most efficient erythritol producer, N61188-12, assimilated myo-inositol and weakly assimilated erythritol. However, the wild type strain did not assimilate myo-inositol and assimilated erythritol well. In 250-L and 2000-L pilot-scale fermentors, the erythritol production by N61188-12 was 151.4 g/L and 152.4 g/L, respectively. A simple fed-batch culture of strain N61188-12 in a 2000-L fermentor increased erythritol production to 189.4 g/L after 10 days fermentation.     

Filaria-induced immune evasion: suppression by the infective stage of Brugia malayi at the earliest host-parasite interface

To assess the physiologic interactions between the infective stage of Brugia malayi--one of the extracellular parasites responsible for lymphatic filariasis in humans--and the APC with which they come in contact during their development and routes of travel, we have investigated the interaction between the infective stage (L3) of B. malayi and human Langerhans cells (LC) in the skin. Our data indicate that live L3 result in increased migration of LC from the epidermis without affecting the viability of these cells and up-regulation of the IL-18 cytokine involved in LC migration. Live L3 also result in down-regulation of MHC class I and II on the LC cell surface. Additionally, microarray data indicate that live L3 significantly down-regulated expression of IL-8 as well as of multiple genes involved in Ag presentation, reducing the capacity of LC to induce CD4(+) T cells in allogeneic MLR, and thus resulting in a decreased ability of LC to promote CD4(+) T cell proliferation and production of IFN-gamma and IL-10. These data suggest that L3 exert a down-regulatory response in epidermal LC that leads to a diminished capacity of these cells to activate CD4(+) T cells.     

Regulation of lymphotoxin production by the p21ras-raf-MEK-ERK cascade in PHA/PMA-stimulated Jurkat cells

Although the production of lymphotoxin (LT) from activated Th1 lymphocytes has been reported extensively, the intracellular signaling mechanisms that regulate this T cell function remain totally undefined. We have examined whether the p21ras-raf-1-mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) kinase (MEK)-ERK cascade plays a role in regulating the production of LT, because the activity of these signaling molecules is up-regulated in activated T lymphocytes. Transfection of Jurkat leukemic T cells with a dominant negative mutant of p21ras (ras17N or ras15A), raf-1 (raf 1-130), or ERK1 (Erk1-K71R) resulted in the suppression of the mitogen/phorbol ester-stimulated production/secretion of LT. This suppression was accompanied by a parallel inhibition of mitogen-stimulated ERK activation. The selective antagonist of MEK1 activation, PD98059, also attenuated the mitogen-stimulated or anti-CD3 Ab and phorbol ester-stimulated production of LT from Jurkat cells or peripheral blood T lymphocytes. This study provides, for the first time, direct evidence that the p21ras-raf-MEK-ERK cascade plays a vital role in regulating the production of LT.     

Cytolytic activity of Ia-restricted T cell clones and hybridomas: evidence for a cytolytic mechanism independent of interferon-gamma, lymphotoxin, and tumor necrosis factor-alpha

Ten different helper T cell (Th) hybridomas that are specific to Ia or antigen plus Ia were found to express nonspecific cytolytic activity toward the cytotoxin (CT)-resistant P815 cells upon activation with either Con A or a monoclonal anti-T3 antibody (T3-mAb). In contrast to cytolytic Th1 clones which secrete high levels of interferon-gamma (IFN-gamma) and cytotoxin (CT) (lymphotoxin (LT, also known as TNF-beta) or tumor necrosis factor-alpha (TNF-alpha], these Th hybridomas produce low or undetectable levels of IFN-gamma and CT. No inhibitory activity of IFN-gamma and CT was observed in culture supernatants of activated Th hybridomas. Double-chamber experiments demonstrated that CT-sensitive L929 cells when physically separated from activated Th1 clones were killed by membrane-permeable CT. Under identical experimental conditions, lysis of P815 cells did not occur. Moreover, activation of Th hybridomas directly in wells containing the CT-sensitive L929 cells failed to induce target cell lysis. This confirms that these Th hybridomas produce little CT and argues against high local concentrations of CT being responsible for Th hybridoma-mediated killing of P815 cells. Finally, a polyclonal rabbit antiserum to rTNF-alpha, which strongly and specifically inhibited CT-mediated and Th1 clone-mediated killing of L929 cells, failed to inhibit P815 lysis by activated Th1 clones and Th hybridomas. These observations establish that a cytolytic mechanism independent of IFN-gamma, LT, and TNF-alpha is responsible for lysis of CT-resistant target cells.     

培养山羊痘病毒常用细胞在X-gal环境中的显色差异分析*

目的：为筛选出表达LacZ基因重组山羊痘病毒的适宜培养细胞系。方法：将常用于培养山羊痘病毒(GPV)的乳仓鼠肾细胞(BHK21)、羊肾细胞(SK)和羔羊睾丸细胞(LT)培养至单层,之后单层细胞用含X-gal的培养基继续培养,观察比较各细胞在X-gal环境中呈现的蓝色。提取单层细胞的RNA,进行LacZ基因的RT-PCR,回收PCR产物、连接载体、转化E.coli,挑阳性克隆测序分析。将单层细胞继续培养72 h,检测各细胞产生的β-半乳糖苷酶。结果：随着培养时间的延长,固体培养的BHK21、LT细胞孔中胶颜色变蓝且逐渐加深,显微镜观察可见细胞蓝斑,而SK细胞、空白、无X-gal孔中的胶未变色,镜检无细胞蓝斑;液体培养的单层细胞逐渐脱壁,培养液未见蓝色。RT-PCR产物电泳条带与预期分子量大小相符,测序结果显示目标DNA与LacZ基因序列相似性值达100%,表明3种细胞均有LacZ基因的转录本。β-半乳糖苷酶测定结果显示3种细胞均表达此酶,细胞产酶能力比较为BHK21>LT>SK,尤以SK细胞产β-半乳糖苷酶量极低。结论：显色差异法筛选出的羊肾细胞适宜培养表达LacZ基因的重组山羊痘病毒,结果可为山羊痘病毒新型疫苗研发提供一些参考。