Adenoviral-mediated delivery of a viral chemokine binding protein blocks CC-chemokine activity in vitro and in vivo

Chemokines are important mediators of leukocyte recruitment and activation that play critical roles in the pathology of inflammatory diseases such as atherosclerosis, rheumatoid arthritis and asthma. The vaccinia virus (strain Lister) expresses a 35 kDa soluble protein ('35K') that binds and inactivates a wide range of CC chemokines. We generated a recombinant adenovirus encoding soluble 35K (Ad35K). Ad35K-infected cell culture medium, containing recombinant 35K, potently reduced migration of CCR5-transfected 293 cells by 95% in response to the CC-chemokine RANTES, but had no effect on cells transfected with the CX3CR1 fractalkine receptor. Delivery of Ad35K to mice in vivo via tail vein injection resulted in expression of recombinant 35K in plasma and increased serum RANTES and MIP-1alpha levels when quantified by ELISA. However, chemotaxis of both CCR5-transfected cells and primary macrophages was inhibited by more than 90% by plasma from Ad35K-infected animals compared with control plasma from animals injected with AdGFP. Furthermore, 35K delivered by intra-peritoneal injection more than halved biogel-induced inflammatory cell recruitment in peritoneal exudates compared to AdGFP medium. These studies identify broad-spectrum CC-chemokine blockade using in vivo adenoviral-mediated recombinant 35K expression as a promising strategy to reduce local and systemic inflammation.     

Contrasting in vivo and in vitro fates of glioblastoma cell subpopulations with amplified EGFR

Despite the high incidence of EGFR amplification in patient glioblastoma multiforme (GBM) tissues, only a single GBM cell line, of the many described in the literature, is known to contain and maintain amplified EGFR. Because EGFR mutations in GBM manifest primarily, if not exclusively, in amplified form, it follows that the availability of cell lines with mutation of endogenous EGFR would also be in short supply. In fact, there are no GBM cell lines harboring the common EGFR mutants described in patient GBMs. These observations suggest that in vivo environments select for EGFR amplification, whereas in vitro environments, specifically cell cultures, select against this gene alteration. To contrast directly the fates of EGFR amplification in vivo and in vitro, as well as to examine potential relationships between EGFR amplification and mutation, we have established and maintained GBM explants as xenografts by serial passaging in nude mice. Analysis of EGFR copy number and EGFR mutation status in 11 patient tumors and their corresponding xenografts, as well as the monitoring of EGFR copy number during the establishment of a GBM cell line from a xenograft with amplified EGFR, indicated that selection for EGFR amplification is an in vivo phenomenon. Furthermore, our data indicated that EGFR mutation occurs only in tumors with EGFR amplification and showed that the selection of amplified mutant EGFR over amplified wild-type EGFR as a xenograft occurred rapidly and completely during tumor propagation.     

Studies on cell motility in inflammation. II. The in vivo effect of anti-inflammatory and anti-rheumatic drugs on chemotaxis in vitro

Two systems were used to test the effect of anti-inflammatory and anti-rheumatic drugs on chemotactic activity of cell-free exudates and also on the chemotactic responsiveness of exudate leucocytes. (1) Inflammatory cell-free exudates from treated rats were tested for their chemotactic activity on exudate leucocytes from untreated rats. (2) Polymorph and mononuclear cells from treated rats were tested for their responsiveness to the chemotactic activity of cell-free exudates from untreated rats. Levamisole, coumarin and D-penicillamine were ineffective in (1) and (2). Colchicine reduced chemotaxis of polymorphs in both systems (1) and (2), whereas no effect was observed on mononuclears. Naproxen was more effective in reducing the chemotaxis of polymorphs compared with mononuclears in systems (1) and (2). In contrast, indomethacin and dexamethasone reduced the chemotaxis of both polymorph and mononuclear cells in systems (1) and (2). Of the drugs tested dexamethasone exhibited the highest potency. These results emphasize the necessity for studying both cellular and humoral factors in the evaluation of the action of anti-inflammatory drugs on chemotaxis.     

Contrasting effects of hydroxyurea on cell growth and reduction in Epstein-Barr virus genomes in EBV-infected epithelioid cell lines vs Burkitt's lymphoma cell lines

Eliminating Epstein-Barr virus (EBV) genomes from infected cells is an intriguing theoretical strategy in therapy for EBV-associated malignant diseases. Respective patterns were characterized for hydroxyurea (HU)-promoted loss of EBV genomes from EBV-infected epithelioid cell lines derived from the noncancerous portion of gastric carcinoma tissues and Burkitt's lymphoma (BL) cell lines. Epithelioid cell lines GT38 and PN were less sensitivity to HU than BL cell lines Akata, Raji, and Daudi in terms of cell growth inhibition and cell cycle arrest. On passage in medium with 50 microM HU, the fraction of EBV nuclear antigen (EBNA)-positive cells was reduced substantially in the BL cell lines, but only slightly in the epithelioid cell lines. EBV DNA was reduced in Akata, Raji, and Daudi cells upon passage in 50 microM HU by 95%, 70%, and 50%, respectively, but by only 10% in GT38 cells, in which EBV DNA reduction was enhanced at increased concentrations of HU. This indicates that EBV genome is more easily lost from BL cell lines than from epithelioid cell lines upon culturing in HU. These findings support the view that the elimination of EBV could be therapeutically effective in EBV-associated BL by HU.     

In vitro andin vivo effects of gold salts on chemotaxis and random migration of rat polymorphonuclear leucocytes

The effect produced by three gold salts (sodium aurothiomalate, allochrysine, auranofin) on chemotaxis and random migration of rat polymorphonuclear leucocytes (PMN) was investigated under various experimental conditions.The drug activity was examined after incubationin vitro or after administrationin vivo.PMNs were recruited after the induction of two acute inflammatory reactions (pleurisies induced by isologous serum or a suspension of calcium pyrophosphate (CaPP) crystals).The three gold salts administeredin vivo andin vitro inhibited the chemotactic responses of the two cell types. This action was dose-dependent. Auranofin was the most effective substance while sodium aurothiomalate was the least.The random migration was not always significantly depressed especially for CaPP-elicited cells.Reduction in neutrophil chemotaxis might be an important additional mechanism in the action of gold salts and their activity on inflammatory PMNs recruited at inflammatory foci might be beneficial in the treatment in rheumatic diseases in which PMN migration would be implicated.     

Contrasting in vitro effects of retinol and mononuclear cell factor on young and old human cartilage

Studies with young animal cartilage have shown that retinol and mononuclear cell-factor (MCF) cause in vitro breakdown of the cartilage, mediated by the living chondrocyte (indirect degradation). We studied the effects of retinol and MCF on healthy human articular cartilage of different ages, measuring the effects on proteoglycan (PG) content of the cartilage, and on PG synthesis during 8 days of culture. This study shows: Retinol and MCF induce indirect degradation of young, but not of old human cartilage of the humeral head; Both retinol and MCF suppress PG synthesis of young and stimulate PG synthesis of old cartilage; The effects of retinol and MCF on cartilage PG content and on PG synthesis are related to the metabolic state of the chondrocyte; Therefore mononuclear cell-factor may have a destructive or beneficial effect on cartilage depending on whether proteoglycan synthesizing activity is high or low, respectively.     

In vitro and in vivo studies of macrophage functions in amebiasis.

Experimental intrahepatic inoculation of the gerbil with Entamoeba histolytica trophozoites was used as a model of liver amebiasis to study the cellular immune response elicited by the parasite. It was shown that abscess-derived macrophages (5 to 20 days old) were deficient in their capacity to develop a respiratory burst, to secrete and express membrane-bound interleukin-1-like activity, and to kill E. histolytica trophozoites as well as to respond to lymphokines in vitro. However, macrophages isolated from the spleen and peritoneal cavities from the same infected animals were not significantly down regulated in these functions. Splenocytes from infected gerbils were shown to develop a strong responsiveness to amebic antigen, whereas their response to concanavalin A was suppressed. Crude E. histolytica extracts or conditioned medium down regulated murine BALB/c macrophage accessory and effector cell functions in vitro in a manner similar to abscess-derived macrophages, whereas crude extracts of the nonvirulent E. histolytica-like Laredo strain did not. Our results indicate that intrinsic or secreted products or both from E. histolytica are actively regulating macrophage functions at the abscess site and can possibly mediate other immunoregulatory mechanisms at distant targets.     

In vitro effects of interleukin-4 on interferon-gamma-induced macrophage activation.

Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) have been shown to be secreted by distinct T-helper cell subsets which have different roles in the determination of host resistance to infection. We studied the activity of these two cytokines on effector mechanisms of mouse macrophages. In vitro cultured bone marrow-derived macrophages from C57BL/6 mice were treated with IFN-gamma, IL-4, or a combination of both cytokines and the ability to secrete superoxide or nitrite or to restrict growth of Mycobacterium avium and Toxoplasma gondii was then evaluated. We found that IL-4 could inhibit the priming of macrophages for enhanced superoxide production induced by IFN-gamma although IL-4 when used alone did have some enhancing effect of its own. This effect of IL-4 on IFN-gamma-primed superoxide production was dose dependent and could be observed even if the treatment by IL-4 was done 24 hr after treatment by IFN-gamma. IL-4 did not, however, influence the enhanced production of nitrogen reactive intermediates, the induction of bacteriostatic activity against M. avium, or the restriction of T. gondii by IFN-gamma-treated macrophages, and did not have any effect of its own regarding these latter functions.     

Lymphocyte chemotaxis in inflammation. IX. Further characterization of lymphocyte chemotactic lymphokines produced by purified protein derivative-stimulation in vitro and in vivo

As recently reported, one lymphocyte chemotactic factor (beta-LCF, mol. wt. about 27,000) released from activated guinea-pig lymphocytes appeared to be identical to one of the LCFs (LCF-d) isolated from extract of purified protein derivative (PPD)-induced delayed-type hypersensitivity skin reaction sites in guinea-pigs with respect to antigenicity and chemotactic effect for T cells. However, the mol. wt. of LCF-d (about 300,000) was clearly distinct from beta-LCF. The experiments were undertaken to clarify the problem. beta-LCF appeared to be bound to some protein of normal guinea-pig serum (GPS) because the chemotactic activity was revealed in the fraction corresponding to that of LCF-d when the mixtures of beta-LCF with GPS were applied to a Sephadex G-200 column. Additionally, binding experiments using fluorescein isothiocyanate (FITC)-labelled beta-LCF were performed; fluorescence was only detected in the chemotactic fraction. It was thus assumed that the lymphokine (beta-LCF) would be released from activated lymphocytes around the inflammatory tissue, then bound with serum protein exuded in the site and function as LCF-d. The possibility was supported by the evidence that beta-LCF like-chemotactic substance (mol. wt. about 27,000) was dissociated from LCF-d under acid conditions. The factor dissociated from LCF-d was also bound with GPS protein under neutral conditions and converted to high molecular substance resembling LCF-d physiochemically. Furthermore, the chemotactic activity of LCF-d was almost completely absorbed by antibody against GPS. It is thus considered that the chemotactic activity of LCF-d may be attributed to beta-LCF released from activated lymphocytes and that some serum protein which binds beta-LCF may function as a carrier protein in the DTH sites.     

Inhibition of macrophage phagocytosis by Bacteroides fragilis in vivo and in vitro.

A number of investigators have provided experimental evidence for synergistic effects in mixed infections with Escherichia coli and Bacteroides fragilis. In vitro studies have suggested that competition for serum opsonins and diminished subsequent phagocytosis by polymorphonuclear leukocytes might explain these effects. In the present study we evaluated the effect of B. fragilis on macrophage phagocytosis. It was shown that peritoneal macrophages from mice injected intravenously 6 to 12 h earlier with 10(8) CFU of encapsulated B. fragilis were markedly suppressed in their phagocytic ability. Injections of laboratory-passaged, less-encapsulated B. fragilis, other bacteria, or latex particles were either not suppressive of macrophage phagocytosis or less effective. When peritoneal macrophages were treated in vitro for 24 h with the same challenge organisms prior to assessing their phagocytic capacity, encapsulated B. fragilis also proved significantly more suppressive than challenges with other organisms or latex particles. We conclude that suppression of macrophage phagocytosis by B. fragilis seems to be an important mechanism contributing to synergistic effects described for mixed aerobic and anaerobic infections.     

Solvent effects on beta protein toxicity in vivo.

Human beta (1-40) and rat beta (1-42) were dissolved in three different solvents and stereotaxically injected into rat hippocampus with the contralateral side injected with control reverse sequence peptide or vehicle alone. Results at 1 week showed gross toxicity of the 35% acetonitrile solvent which was markedly enhanced by 3 nmol of beta protein but not by reverse sequence peptide. Beta peptide in water also appeared more toxic than reverse sequence, but the results were less clear cut. In contrast, 3 nmol of beta peptide in a cyclodextrin/PBS solution produced no marked short-term toxic effects. Peripheral injection of substance P failed to prevent toxicity. We conclude that solvent effects play a major role in acute beta protein neurotoxicity.     

A hyperimmunoglobulin E syndrome with normal chemotaxis in vitro and defective leukotaxis in vivo.

A 26-yr-old male with a lifelong history of atopic dermatitis and recurrent severe staphylococcal abscesses was found to have hyperimmunoglobulinemia E. Evaluation of both the humoral and cellular aspects of chemotaxis in vitro showed both neutrophils and monocytes to be normal. However, quantitative neutrophil migration in vivo was significantly suppressed using the patient's own serum as the attractant. This defective migration in vivo was partially corrected by serum from normal donors as the attractant and also partially corrected following plasma infusion in this patient. Evaluation of quantitative leukocyte migration in vivo may be most useful in patients suspected of defects of leukocyte mobility.     

Inhibition by cholera toxin of rat polymorphonuclear leukocyte chemotaxis demonstrated in vitro and in vivo.

The effect of cholera toxin on the chemotaxis of rat polymorphonuclear leukocytes (PMN) was studied using a technique in which the movement of the cells towards a laser-lysed erythrocyte is followed under a phase-contrast microscrope. In vitro studies indicated that the intact toxin was capable of inhibiting PMN chemotaxis in a dose-dependent manner at doses ranging from 1 to 100 ng/ml. Subunits A and B of the toxin were without inhibitory activity when used alone, but after recombination their ability to inhibit chemotaxis was similar to that of the intact toxin, suggesting that the toxin is acting intracellularly. Cholera toxin has been reported to act in other systems via stimulation of adenyl cyclase with consequent elevation of intracellular cyclic adenosine 5'-monophosphate (cAMP) levels. It appears that this mechanism may also account for its ability to inhibit chemotaxis since there was a correlation, at all doses tested, between inhibition of chemotaxis and increased intracellular cAMP levels. Cholera toxin was also found to be active in vivo in that, after intrapleural injection of the toxin, the chemotaxis of cells subsequently recovered from the pleural cavity was markedly reduced. These results support previous findings which suggest that modification of leukocyte cAMP levels can influence the chemotactic responsiveness of these cells.     

Inhibitory effects of epi-sesamin on endothelial protein C receptor shedding in vitro and in vivo

Objective and design

Endothelial protein C receptor (EPCR) plays a pivotal role in augmenting Protein C activation by the thrombin–thrombomodulin complex. The activity of EPCR is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). The EPCR shedding is mediated by the tumor necrosis factor-α converting enzyme (TACE). Epi-sesamin (ESM), from the roots of Asarum siebodlii, is known to exhibit anti-allergic and anti-fungal activities. However, little is known about the effects of ESM on EPCR shedding.

Methods

We investigated this issue by monitoring the effects of ESM on phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and cecal ligation and puncture (CLP)-mediated EPCR shedding.

Results

Data showed that ESM induced potent inhibition of PMA, TNF-α, IL-1β, and CLP-induced EPCR shedding, likely through suppression of TACE expression. In addition, treatment with ESM resulted in a reduction of PMA-stimulated phosphorylation of p38, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK).

Conclusions

Given these results, ESM should be viewed as a candidate therapeutic agent for treatment of various severe vascular inflammatory diseases via inhibition of EPCR shedding.     

In vivo and in vitro effects of macrophage colony-stimulating factor (M-CSF) on bronchoalveolar macrophages for antihistoplasmal activity

The in vivo and in vitro effects of M-CSF on bronchoalveolar macrophages (BAM) activity against the intracellular fungal pathogen Histoplasma capsulatum (Hc) were studied. Three days after a single subcutaneous (s.c.) dose of M-CSF (2.5 mg/kg), enhanced ex vivo antifungal activity of BAM was measured. BAM from M-CSF-treated CD-1 mice significantly (P<0.01) inhibited the intracellular multiplication of He yeast cells in 20 h assays compared to BAM from control mice. This effect was not observed at days 1, 7, 11 or 21 post-treatment. A dose of 5 mg/kg s.c., but not 1 mg/kg, induced similar antifungal activity in BAM by day 3. Peritoneal macrophages (PM) from M-CSF-treated mice did not have enhanced antifungal activity at days and doses tested. BAM could also be activated for antihistoplasmal activity by M-CSF in vitro. M-CSF at 10,000 U/ml for 24 h or 5000 U/ml for 48 h induced significant (P<0.01) inhibition of intracellular multiplication of Hc. Interferon-gamma (IFN) plus lipopolysaccharide (LPS) activated BAM and PM in vitro to inhibit intracellular multiplication of He (P<0.001); the antihistoplasmal activity was completely inhibited by N^C-monomethyl l-arginine (N-MMA), indicating that an L-arginine-dependent nitric oxide-producing mechanism was operative. N-MMA could not inhibit the antihistoplasmal activity of BAM or PM activated by M-CSF in vitro. The mechanism by which M-CSF-activated macrophages inhibit intracellular multiplication of He remains to be determined.     

幽门螺杆菌cheA基因在细菌体外趋化和体内定植过程中的作用

目的 了解cheA基因在幽门螺杆菌体外趋化和体内定植中的作用.方法 从幽门螺杆菌NCTC11637株基因组DNA中扩增并克隆全长cheA和cheY基因.构建该两个基因原核表达系统,Ni-NTA亲和层析法提取目的 重组蛋白rCheA和rCheY.rCheA和rCheY免疫家兔制备抗血清,采用硫酸铵沉淀法及DEAE-52柱层析法制备rCheA-IgG和rCheY-IgG.构建cheA基因自杀质粒,根据同源重组交换原理利用该自杀质粒构建cheA基因敲除突变株(cheA-),采用PCR及测序对cheA-突变株进行鉴定.采用rcheA-IgG和rCheY-IgG锚定靶蛋白及蛋白磷酸化检测试剂盒,测定cheA-突变株与野生株CheA和CheY分子磷酸化水平.采用幽门螺杆菌趋化模型及BALB/c小鼠感染模型,比较cheA-突变株与野生株体外趋化及体内定植能力的差异.结果 PCR及测序结果证实cheA-突变株基因组中cheA基因被敲除.0.001～0.1 mol/L盐酸作用10 min,野生株CheA和CheY磷酸化水平分别从(59.6±11.5)和(55.5±10.2)μmol迅速下降至(10.8±2.6)和(5.5±1.2)μmol(P＜0.05),cheA-突变株CheY磷酸化水平均较低且无明显变化(P＞0.05).cheA-突变株对盐酸、硫酸和乙酸趋化聚集环直径[(10～20)±(2～3)mm]明显小于野生株[(16～24)±(2～3)mm],P＜0,05.野生株感染小鼠胃黏膜标本中幽门螺杆菌分离阳性率(90%)明显高于cheA-突变株(40%),P＜0.05;荧光定量PCR结果也显示野生株感染小鼠胃黏膜标本中幽门螺杆菌数量(6.3×103±2.1×103拷贝/mg)也明显高于突变株的(8.3×101±3.1 ×101拷贝/mg),P＜0.05.结论 cheA基因在幽门螺杆菌体外趋化和体内定植中有促进作用.     

Tea flavonoids: bioavailability in vivo and effects on cell signaling pathways in vitro

The elucidation of the potential health benefits of tea beverage continues to be a focus of research in many laboratories. Beneficial effects of tea have been particularly evident in animal tumorigenesis models, with green and black tea frequently demonstrating similar effectivity. Human data are now emerging to support a beneficial role for tea in cardiovascular disease, but the data with respect to cancer risk at various sites remain inconclusive. The constituent flavonoids of green and black tea beverage are known to be potent antioxidants, and although this may be a major factor in explaining their biological activity, it appears that the gallated flavonoids in particular (e.g., epigallocatechin gallate and the gallated theaflavins) impact on a wide range of molecular targets that influence cell growth and more specifically pathways such as those involving angiogenesis. Data on the pharmacokinetic properties of tea flavonoids, primarily on the catechins and therefore related most closely to green tea, have provided indications of the plasma levels and circulating molecular forms that may be expected in humans following tea consumption. The structural complexity of black tea flavonoids, in particular the thearubigins, has hindered efforts to describe their bioavailability and to perform mechanistic studies. Recent studies on the effects of catechins and theaflavins on growth factor-, nuclear factor-kappaB-, and stress-mediated signal transductions are described in this review, where possible in relation to their bioavailability in vivo. These studies indicate that effects that may be relevant to both cancer and atherosclerosis are often observed at tea flavonoid levels that could realistically be encountered in vivo. However, more studies need to be performed using those molecular forms of tea flavonoids (methylated, sulfated, and glucuronidated conjugates) that are the major circulating species encountered following tea consumption. Such studies, combined with further human epidemiological and interventional data, should ultimately elucidate the full beneficial potential of tea beverage on human health.