Cytokine-induced selective increase of high-molecular-weight bFGF isoforms and their subcellular kinetics in cultured rat hippocampal astrocytes

Cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and epidermal growth factor (EGF) are probable factors responsible for up-regulation of basic fibroblast growth factor (bFGF) expression in reactive astrocytes following brain damage, however the effect of these cytokines on the expression of each bFGF-isoform has not been elucidated. Western biot analysis revealed the expression of 18, 22 and 24-kD bFGF isoforms in cultured rat hippocampal astrocytes, and the expression of high molecular weight (HMW)-isoforms (22 and 24-kD isoforms) but not of 18-kD isoform was selectively increased by cytokines. Immunofluorescent analysis demonstrated that bFGF content in the cytoplasm of astrocytes is initially increased by cytokines followed by nuclear targeting and localization in agreement with the previous evidence that HMW-isoforms possess a nuclear targeting signal. The present results suggest the important role of HMW-bFGF isoforms in the response of nervous tissue to injury.     

Doxorubicin plus tumor necrosis factor α combination treatments in EL4-lymphoma-bearing C57BL/6 mice

 The therapeutic efficacy of a total of 42 single-agent or combination protocols involving doxorubicin (Adriamycin, ADM) and tumor necrosis factor α (TNFα) were evaluated in the syngeneic murine lymphoma model, C57BL/6-EL4. Combination treatments were the most effective and the therapeutic effects were schedule-dependent; e.g. it was generally advantageous for ADM to precede TNFα administration. Two protocols selected for further study were 4 mg/kg ADM i.v. on days 1 and 8 plus TNFα, i.v., at either 16 000 U (7 μg)/injection, on days 1 and 8 or 4000 U (1.7 μg)/injection, on days 11–15. Survival of mice bearing one of four EL4 sublines having different in vitro drug sensitivities was assessed. These sublines were E10 (ADM-sensitive/TNFα-resistant), E16 (sensitive/sensitive), ER2 (ADM-resistant/TNFα-sensitive) and ER13 (resistant/resistant). Between 80% and 100% long-term survivors (i.e. tumor free on day 60) were obtained with the two treatments in mice bearing ADM-sensitive sublines, even though one of these sublines, E10, was resistant to TNFα in vitro. Induction of long-term survival appeared, therefore, to correlate with in vitro defined sensitivity/resistance to ADM, but not to TNFα. Treatment-induced modulations of tumoricidal immune effector functions were also examined. Taken together, the results indicated that induction of long-term survival involved complex interactions of: (1) ADM-induced tumor modifications, including, but not limited to, tumor debulking, (2) combination-treatment-induced modifications of splenic cytolytic T cell and macrophage activities, and (3) the restoration of thymus cellularity. Finally, when long-term survivors resulting from treatment of E10- or E16-bearing mice were implanted with ER2 on day 120, the majority survived, indicating that long-term immune memory, capable of recognizing drug resistant variants, had been established. Received: 14 August 1997 / Accepted: 2 October 1997     

Transforming growth factor β , (TGFβ) secreted by immunogenic ex vivo human carcinoma cells, counteracts the activation and inhibits the function of autologous cytotoxic lymphocytes. Pretreatment with interferon γ and tumor necrosis factor α reduces the production of active TGFβ

 We present the results obtained with an in vitro model system that resembles the in vivo tumour microenvironment, where malignant cells are in close contact with the infiltrating lymphocytes. Unmanipulated blood lymphocytes were cytotoxic against the autologous ex vivo tumour cells in 3/19 patients and this function was generated in 6-day mixed cultures in five additional cases. Production of transforming growth factor β (TGFβ) by the freshly separated tumour cells was determined in parallel. Cytotoxicity was generated by a small number of tumour cells (2–5/100 lymphocytes), while a large number (10–20/100 lymphocytes) inhibited not only the generation but also the existing lytic activity. The presence of a neutralising TGFβ-specific mAb (2G7) potentiated the activation of lymphocytes and suspended the suppression inflicted by the tumour cells. In those tumours, which expressed relatively high levels of MHC class I and ICAM-1 molecules, the quantity of secreted TGFβ interfered with the ability of tumour cells to generate cytotoxic lymphocytes. In the tumours with low expression of class I, such a correlation was not detected, indicating the primordial role of MHC class I expression in the regulation of autologous tumour recognition. Our results demonstrate the involvement of TGFβ in the impaired lymphocyte-mediated reactivity against immunogenic tumours and support a mechanism that contrasts the tolerance or anergy. Since presence of TGFβ in the microenvironment of tumours counteracts the function of cytotoxic T lymphocytes, production of this cytokine can contribute to the failure of immunotherapy. Received: 19 June 1997 / Accepted: 14 August 1997     

Peripheral natural killer cell activity and intraperitoneal soluble p55 tumor necrosis factor receptor in patients with malignant ascites: two possible indicators for response to intraperitoneal combined tumor necrosis factor α and interferon γ treatment

 Tumor necrosis factor α (TNFα) and interferon γ (IFNγ) are important immunomodulators. They are capable of acting in a synergistic manner on tumor cells in vitro and in vivo. In a clinical phase I study 13 patients with malignant ascites due to abdominal spread of different primary tumors received intraperitoneally (i. p.) TNFα and IFNγ once weekly over 3 – 8 weeks in order to evaluate the effect of locoregionally administered TNFα/IFNγ on ascites formation. Therefore some peripheral and local immunological functional parameters of peripheral blood and malignant ascites were investigated. Mononuclear lymphocytes and natural killer (NK) cell activity of peripheral blood and ascites, TNF-inhibitory activity, soluble p55 and p75 TNF receptors, and prostaglandin E₂ values in ascites were measured immediately before and 24 h after each TNFα/IFNγ infusion. Peripheral mononuclear lymphocytes and NK activity decreased significantly 24 h after i. p. TNFα/IFNγ application. However, over the entire treatment schedule, peripheral NK activity in all responders showed a continuous increase, when compared to pre TNFα/IFNγ treatment levels. In contrast, NK activity in non-responders constantly decreased. In contrast to non-responders, TNF-inhibitory activity and soluble p55 TNF receptor levels, determined in ascites, decreased in responders. Taken together, our findings suggest, that successful locoregional i. p. TNFα/IFNγ therapy induces systemic immunological reactions possibly after saturation of soluble p55 TNF receptors in ascites, which leads to an increase of peripheral NK activity. Received: 28 September 1995 / Accepted: 16 November 1995     

Endotoxaemia, production of tumour necrosis factor alpha and polymorphonuclear neutrophil activation following strenuous exercise in humans

To examine whether endotoxaemia accompanying long-term, strenuous physical exercise is involved in exercise-induced increase in plasma tumour necrosis factor alpha (TNF-alpha) concentration and polymorphonuclear neutrophil (PMN) activation, 14 male recreational athletes [mean age 28 (SEM 1) years] were studied. Exercise consisted of a 1.5-km river swim, a 40-km bicycle race, and a 10-km road race. Mean time to complete the race was 149.8 (SEM 4.8) min. The plasma concentrations of granulocyte myeloperoxidase (MPO) and TNF-alpha were significantly higher than baseline values immediately and 1 h after exercise (P<0.001). Both variables returned to pre-race levels the day after exercise. Marked, transient decreases in plasma concentrations of anti-lipopolysaccharide (LPS) immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies directed against a panel of selected smooth gram-negative LPS were observed after the race, reaching in most cases minimal values in the blood sample drawn immediately following the completion of the triathlon. There was no significant correlation between the magnitude of PMN activation, as assessed by the increase in plasma concentrations of MPO, and the humoral markers of endotoxaemia and TNF-alpha. An inverse, highly significant relationship between the increase in plasma TNF-alpha concentrations and the changes in circulating anti-LPS IgM antibodies concentrations was observed (r = -0.7; P<0.01). These findings suggest that exercise-induced endotoxaemia was involved in the release of TNF-alpha, that the magnitude of the TNF-alpha response to exercise was down-regulated by anti-LPS antibodies of the IgM class, and that the production of TNF-alpha and endotoxaemia did not seem to play a role in the activation of circulating PMN in the exercising subjects.     

Effect of tumor necrosis factor-α on intracellular Staphylococcus aureus in vascular endothelial cells

Although tumor necrosis factor-α (TNF-α) is an important host factor against intracellular bacteria, little is known about the effect of TNF-α on the persistence of intracellular Staphylococcus aureus in vascular endothelial cells. It was investigated whether recombinant human TNF-α influences the survival of intracellular S. aureus (ATCC 29213) in human umbilical vein endothelial cells (HUVEC) under a condition with an antistaphylococcal agent, and its mechanism. The HUVECs were incubated with TNF-α, oxacillin, or both in 24-well plates for up to 48 h following internalization of S. aureus (10⁶ CFU well⁻¹) into HUVECs for 1 h. TNF-α (1 ng mL⁻¹) significantly reduced the number of intracellular S. aureus in HUVECs, and TNF-α plus oxacillin eliminated more intracellular S. aureus in HUVEC than oxacillin alone. The LDH viability assay and quantification of apoptosis using photometric enzyme-immunoassay showed that TNF-α preferentially induced cell death and apoptosis of HUVECs infected with S. aureus compared with noninfected HUVECs. These results indicate that TNF-α helps antistaphylococcal antibiotics to eliminate intracellular S. aureus in vascular endothelial cells, partly because TNF-α preferentially induces apoptosis of endothelial cells infected by S. aureus .     

The TACE zymogen

The tumor necrosis factor-α-converting enzyme (TACE) is a member of the disintegrin family of metallopro-teinases (ADAMs) that plays a central role in the regulated shedding of a host of cell surface proteins. TACE is biosynthesized as a precursor protein with latent proteolytic activity (zymogen). TACE’s zymogen inhibition is mediated by its Pro domain, a 197-amino acid region that serves this function as well as aiding in the secretion of this enzyme through the secretory pathway. We have discovered that a conserved “cysteine switch” consensus motif within TACE’s Pro domain is, contrary to expectations, not required for maintenance of the inactive precursor state or for the secretion of this metalloproteinase in its functional form. The only role for this motif seems to be in decreasing TACE’s susceptibility to proteolytic degradation during its biogenesis and maturation within the secretory pathway. Interestingly, the Pro domain of TACE seems to carry both its inhibitory and secretory functions through the same mechanism: it seems to prevent the Catalytic domain from accessing its native, functional state, resembling the function of true molecular chaperones. Recent evidence suggests that TACE may also be switched out of the active conformation even by small, drug-like molecules such as the synthetic compound SB-3CT. These findings point at the possibility of developing, in the near future, a new generation of anti-inflammatory, noncompetitive TACE inhibitors that would exert negative allosteric modulation over the activity of this key enzyme, mediating several inflammatory diseases and certain cancers.     

Dehydroepiandrosterone and melatonin prevent Bacillus anthracis lethal toxin-induced TNF production in macrophages

The lethal toxin of Bacillus anthracis, which is composed of two separate proteinaceous exotoxins, namely protective antigen and lethal factor, is central to the pathogenesis of anthrax. Low levels of this toxin are known to induce release of cytokines such as tumor necrosis factor α (TNF-α). In the present study we investigated the effect of dehydroepiandrosterone (DHEA), melatonin (MLT), or DHEA + MLT on production of lethal toxin-induced TNF-α in mouse peritoneal macrophages. We found that treatment with DHEA significantly inhibited the TNF-α production caused by anthrax lethal toxin. Exposure of MLT to anthrax lethal toxin-treated macrophages also decreased the release of TNF-α to the extracellular medium as compared to the control. However, combined use of DHEA and MLT also inhibited TNF-α release, but not more than single therapies. These results suggest that DHEA and MLT may have a therapeutic role in reducing the increased cytokine production induced by anthrax lethal toxin. This revised version was published online in July 2006 with corrections to the Cover Date.     

The mycotoxin fumonisin B1 transiently activates nuclear factor-κB, tumor necrosis factor α and caspase 3 via protein kinase Cα-dependent pathway in porcine renal epithelial cells

Fumonisin B1 (FB1) is a toxic mycotoxin produced by Fusarium verticillioides, predominantly present in corn. The principal biochemical responses of FB1 involve disruption of sphingolipid metabolism from the inhibition of ceramide synthesis leading to accumulation of free sphingoid bases, particularly sphinganine. The ability of FB1 to modulate signal transduction pathways plays a role in its toxicity. We recently reported that FB1 selectively and transiently activates protein kinase Calpha (PKCalpha) in porcine renal epithelial cells (LLC-PK1). The aim of current study was to investigate the effect of PKCalpha activation by FB1 on NF-kappaB activation and subsequently on TNFalpha gene expression and caspase 3 induction in LLC-PK1 cells. FB1 (1 micromol/L for 5 min) transiently activated PKCalpha and increased nuclear translocation of NF-kappaB, followed by their down-regulation at later time points. Preincubating the cells with the PKC inhibitor, calphostin C, prevented the activation of NF-kappaB by FB1. TNFalpha mRNA expression was increased after 15 min exposure to FB1 or the PKC activator, phorbol 12-myristate 13-acetate. In addition, an increase in caspase 3 activity was observed after addition of FB1 for 1 h. Calphostin C prevented both the FB1-induced increase in TNFalpha expression and caspase 3 activation. In summary, we hereby demonstrate that the FB1 activation of NF-kappaB and sequential induction of TNFalpha expression resulting in the subsequent increase in caspase 3 activity are all dependent on PKCalpha stimulation in LLC-PK1 cells.     

肿瘤坏死因子致肺微血管内皮细胞单层通透性损伤的实验研究

目的和方法：用针头式滤器检测肿瘤坏死因子（TNF）作用前后及三种药物干预时大鼠肺微血管内皮细胞（RPMVEC）单层通透性的变化,并用免疫组化的方法检测TNF作用前后细胞F－肌动蛋白的改变。结果：TNF作用30min、60min、90min通透系数Kf值较致伤前显著增高;分别加福莫特罗（FOR）、山莨菪碱或霍乱毒素（CTX）干预时Kf值均显著低于TNF组。而TNF作用90min,RPMVEC F-肌动蛋白发生明显解聚：分别加POR、山莨菪硷或CTX干预时F－肌动蛋白无明显变化。结论：TNF诱导RPMVEC单层通透性增高的机制与细胞F－肌动蛋白解聚有关,FOR、山莨菪碱和CTX可能通过抑制F－肌动蛋白解聚而抑制NF诱导的RPMVEC单层通透性增高。     

Differential activation of phospholipases during necrosis or apoptosis: A comparative study using tumor necrosis factor and anti-Fas antibodies

Phospholipases generate important secondary messengers in several cellular processes, including cell death. Tumor necrosis factor (TNF) can induce two distinct modes of cell death, viz. necrosis and apoptosis. Here we demonstrate that phospholipase D (PLD) and cytosolic phospholipase A₂ (cPLA₂) are differentially activated during TNF-induced necrosis or apoptosis. Moreover, a comparative study using TNF and anti-Fas antibodies as cell death stimuli showed that PLD and cPLA₂ are specifically activated by TNF. These results indicate that both the mode of cell death and the type of death stimulus determine the potential role of phospholipases as generators of secondary messengers. J. Cell. Biochem. 71:392–399, 1998. © 1998 Wiley-Liss, Inc.     

Effects of weak laser radiation (632.8 nm) on isolated mouse immune cells

The dose dependence of in vitro effects of low-intensity radiation of a He-Ne laser (632.8 nm, 0.2 mW/cm²) on the functional activity of peritoneal macrophages and lymphocytes of mouse spleen was studied. The exposure of isolated cells was varied from 5 to 180 s. If the exposure did not exceed 60 s, stimulation of secretory activity was observed: increased production of interleukin 2, interferon γ, and interleukin 6 in lymphocytes; increased production of tumor necrosis factor α, nitric oxide, and interleukin 6 in macrophages; and enhanced activity of natural killer cells. A longer exposure (up to 180 s) either had no effect on the synthesis of certain cytokines (interleukin 2 in lymphocytes and interleukin 6 in macrophages) or inhibited it, which was expressed in decreased production of interleukin 6 and interferon γ in lymphocytes and nitric oxide in macrophages, as well as in suppression of the activity of natural killer cells. Conversely, the production of interleukin 3 decreased after a short-term exposure but increased after 180-s irradiation. The high sensitivity of cells to extremely weak laser light also manifested itself as a considerable increase in expression of the inducible heat shock protein 70; this effect was observed at all doses studied, including the 5-s exposure. In contrast, expression of the heat shock protein 90 slightly decreased after irradiation of cells with laser light.     

Cytotoxic Triterpenoids from Roots of Actinidia chinensis

Phytochmical investigation of roots of Actinidia chinensisPlanch . led to the isolation triterpenoids 1 – 16 , including a new compound 2α,3α,23,24‐tetrahydroxyursa‐12,20(30)‐dien‐28‐oic acid ( 1 ). Their structures were identified on the basis of spectroscopic analysis, including 1D‐ and 2D‐NMR, HR‐ESI mass spectrometry, and by comparison with the literatures. The cytotoxicities of triterpenoids 1 – 16 against a panel of cultured human cancer cell lines (HepG2, A549, MCF‐7, SK‐OV‐3, and HeLa) were evaluated. The new compound 1 exhibited moderate antitumor activities with IC₅₀ values of 19.62 ± 0.81, 18.86 ± 1.56, 45.94 ± 3.62, 62.41 ± 2.29, and 28.74 ± 1.07 μm , respectively. The experiment data might be available to explain the use of roots of A. chinensis to treat various cancers in traditional Chinese medicine.     

Population density and area: the role of between- and within-patch processes

The relationship between population density and the size of host plant patches was investigated for the red milkweed beetle Tetraopestetraophthalmus inhabiting unmanipulated patches of Asclepias syriaca. The resource concentration hypothesis proposes that density-area patterns, specifically that of increasing herbivore density with patch size, are primarily a function of movement between host plant patches. This research investigated the degree to which movement accounted for density-area patterns. Poisson regression analysis of beetle abundance versus milkweed patch size revealed that beetle density tended to increase with patch size. The pattern of density and patch size resulted from local reproduction and residence time. The density of emerging beetles tended to increase with patch size while emigration rates were unrelated to patch size. Immigration rates were constant with patch size for male beetles, and decreased with patch size for female beetles. Net flux of beetles (immigration – emigration) did not vary with patch size for male beetles and decreased with patch size for female beetles. Comparisons are made between this system and previously studied systems where movement plays a significant role in forming density area patterns. Additionally, several hypotheses are presented which may account for greater in situ recruitment and residence time in large patches. Received: 23 February 1996 / Accepted: 8 January 1997     

Oral administration of milk fermented with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 to DBA/1 mice inhibits secretion of proinflammatory cytokines

We have previously shown that oral administration of skimmed milk(SM) fermented with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (OLL1073R-1/SM) to DBA/1 mice inhibited the development of collagen-induced arthritis (CIA). In this study, our aim was to examine possible mechanisms of inhibiting the development of CIA. We studied the effect of OLL1073R-1/SM on cytokine secretion from cells of popliteal lymph nodes (lymph node cells; LNC) of mice. The results showed that feeding OLL1073R-1/SM inhibited secretion of proinflammatory cytokines such as interferon γ (IFN-γ), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and the chemokine, monocyte chemoattractant protein 1 (MCP-1). The most prominent effect was inhibition of TNF-α. Secretion of IL-2 and IL-4 were not influenced. Feeding OLL1073R-1/SM inhibited secretion of proinflammatory cytokines produced by accessory cells, but not T cells. We conclude that CIA may be prevented via down regulation of secretion of proinflammatory cytokines such as IL-6, TNF-α and IFN-γ, and of the chemokine of MCP-1. This revised version was published online in August 2006 with corrections to the Cover Date.