Modeling inflammation–drug interactions in vitro: A rat Kupffer cell-hepatocyte coculture system

Xenobiotic–inflammation interactions lead to hepatotoxicity in vivo. Selected xenobiotic agents (acetaminophen, APAP; chlorpromazine, CPZ; allyl alcohol, AlOH; monocrotaline, MCT) for which this occurs were evaluated for ability to elicit the release of Kupffer cell (KC)-derived inflammatory mediators and to modulate lipopolysaccharide (LPS)-stimulated release of these mediators. Using KCs and hepatocytes (HPCs) isolated from rat, KC/HPC cocultures were treated with either LPS, xenobiotic, vehicle or a combination. Six hours later, the release of inflammatory mediators was assessed. LPS alone caused a concentration-dependent increase in TNF- release but had no significant effect on the release of PGE₂. APAP by itself did not alter release of TNF-, PGE₂, IL-10, Gro/KC or IFN-γ; however, in the presence of LPS, APAP enhanced LPS-induced TNF- and Gro/KC release. APAP also attenuated LPS-induced increases in IL-10 and MCP-1. CPZ alone caused a concentration-dependent increase in TNF- release, which was approximately additive in the presence of LPS. AlOH alone did not affect TNF- release, but decreased TNF- production in the presence of LPS. AlOH increased PGE₂ production, and this effect was potentiated in the presence of LPS. MCT by itself did not affect release of TNF- but increased the response to LPS. Neither MCT, LPS, nor the combination affected production of PGE₂. These results demonstrate that KC/HPC cocultures can be used to evaluate interactions of xenobiotics with LPS. Furthermore, data from these studies qualitatively mirror reported data from whole animal studies, suggesting that this model could be useful for predicting aspects of xenobiotic–inflammation interactions in vivo.     

失血性休克后小鼠肺组织TLR4受体表达及其意义

目的探讨单纯性失血性休克(无复苏)后肺组织TLR4表达变化及意义。方法C57BL/6小鼠随机分为失血性休克组、脂多糖(LPS)刺激组、假手术组。复制各组动物模型,在不同时间点取出肺组织,通过免疫组化方法,观察TLR4在肺组织的表达变化。结果在失血性休克和LPS刺激后肺部出现明显的中性粒细胞浸润、红细胞渗出。在失血性休克1、2及4 h后,肺巨噬细胞、肺泡上皮细胞及肺间质TLR4表达逐渐增加,6 h开始下降;而LPS刺激后1、2、4及6 h肺TLR4表达逐渐增加。假手术组未见TLR4表达。结论失血性休克后肺TLR4变化可能与急性肺损伤(ALI)的发生有关。失血性休克及LPS刺激后肺组织TLR4变化增强了机体的非特异性免疫能力,同时增加了宿主对随后各种刺激的易感性,过度表达的TLR4可能造成组织、器官结构和功能的损害。     

烫伤、内毒素对豚鼠胃肠动力影响的实验研究

目的:探讨烫伤后胃肠动力障碍发生机制.方法:豚鼠30只,随机分为烫伤、内毒素及对照组,在烫伤及内毒素腹腔注射1小时后测定豚鼠胃肠推进距离、肠道组织降钙素基因相关肽(CGRP)、Na -K 、Mg2 、Ca2 、及Ca2 -Mg2 -ATP酶含量.结果:①烫伤及内毒素组肠道推进功能受到抑制,烫伤组抑制更明显.②烫伤及内毒素组CGRP含量较对照组增多,烫伤组增高幅度更明显.③烫伤及内毒素组豚鼠肠道组织中各ATP酶的含量较对照组明显减少,两组ATP酶减少程度无明显差异.结论:①烫伤及内毒素抑制豚鼠的肠道推进功能,烫伤比内毒素组抑制更明显.②烫伤及内毒素组CGRP含量较对照组明显增多,烫伤组CGRP增高幅度更明显.③烫伤及内毒素组各ATP酶的含量较对照组减少,烫伤组、内毒素组各ATP酶减少程度无明显差异.     

Lipopolysaccharide-induced ischemic tolerance is associated with increased levels of ceramide in brain and in plasma

Intravenous administration of lipopolysaccharide (LPS) (0.9 mg/kg) has been shown to induce ischemic tolerance in spontaneously hypertensive rats (SHR). TNF-alpha is believed to play a crucial role in preconditioning as its inhibition with TNF-alpha-binding protein abolished tolerance. Our recent studies (Liu et al., Am. J. Physiol. 278 C144, 2000) have demonstrated that ceramide, a downstream messenger in TNF-alpha signaling, is a mediator of hypoxia-induced tolerance in neuronal cells. To test the hypothesis that ceramide contributes to LPS-induced tolerance in vivo, SHR were injected intravenously with either LPS or saline and the levels of ceramide in brain and in plasma were determined by reversed phase HPLC. LPS injection resulted in a significant increase of ceramide in plasma with a maximum at 24 h (8.32+/-1.14 pmol/microl (LPS) vs. 2.65+/-0.62 pmol/microl (saline)). LPS also induced ceramide upregulation in brain cortex, which started between 6 and 12 h and remained elevated up to 48 h after LPS injection. Fluorescent NBD-C6 ceramide was able to cross blood-brain barrier and was found in brain vessels, perivascular cells and in brain parenchyma 30 min after intravenous injection. These findings demonstrate that LPS preconditioning leads to elevation of ceramide in brain and plasma and, in conjunction with previous work, suggests that ceramide plays a role in LPS-induced protection against brain ischemic injury in vivo.     

Protein tyrosine kinase inhibitors suppress the production of nitric oxide in mixed glia, microglia-enriched or astrocyte-enriched cultures

Nitric oxide (NO) produced by glial cells has been implicated in the neuropathogenesis of various diseases. However, the signaling transduction pathway(s) for the production of NO in these cells is not well understood. To test whether protein tyrosine kinases (PTKs) are required for signaling events of NO production in glial cells, this study examined the effects of genistein and tyrphostin A25, two potent inhibitors of PTKs, on the production of NO in mouse primary mixed glia, microglia-enriched or astrocyte-enriched cultures exposed to lipopolysaccharide (LPS) or a combination of LPS and interferon-γ (IFNγ). LPS induced a dose-dependent increase in NO production from the mixed glia cultures. The LPS-induced NO production was significantly enhanced by stimulating the cells with IFNγ. Genistein or tyrphostin A25 inhibited the production of NO in both LPS- and IFNγ/LPS-stimulated mixed glia cultures. The production of NO in the stimulated microglia-enriched or astrocyte-enriched cultures was also inhibited by tyrphostin A25. To verify the cellular sources of NO, immunocytochemical staining of inducible NO synthase (iNOS) was followed by staining with the microglia marker Mac-1 or the astrocyte marker glial fibrillary acid protein (GFAP) in microglia-enriched or astrocyte-enriched cultures. The expression of iNOS and the production of NO in microglia-enriched cultures were significantly higher than those in the identically stimulated astrocyte-enriched cultures. These results demonstrate that PTKs are involved in the signaling events of LPS-induced NO production in microglia and astrocytes, and that microglia are more responsive than astrocytes to stimuli which induce NO. These results may provide insights into therapeutic interventions in the pathway for NO production in the brain.     

Cytogenetic analysis of the spontaneous murine B-cell leukemia (BCL1

Cytogenetic studies were carried out on the spontaneous transplantable murine B cell leukemia (BCL₁) of BALB/c mice, a recently discovered model of chronic lympholytic leukemia. BCL₁ tumor cells appear as medium to large size mature lymphocytes located predominantly in the blood (up to 500,000/mm³) and in the x50 enlarged spleen. BCL₁ cells obtained from the spleen show a certain degree of spontaneous proliferation unlike peripheral blood lymphocytes (PBL), but both show a good in vitro response to the B-cell mitogen lipopolysaccharide (LPS). Out of 213 spontaneously dividing spleen cells and LPS stimulated PBL studied, the majority (75.5%) showed a rather stable and complex female karyotype consisting of 36 chromosomes. No normal representative of chromosome Nos. 6, 8, 12 and 15 was present and only one normal representative was found for chromosome Nos. 4, 5, 9, 14 and X. Two deleted chromosomes and seven characteristic marker chromosomes, originating from complex chromosomal rearrangements could be identified in each cell. Of special interest was the t(12:15) translocation, which had been found in several murine plasmacytomas. No direct correlation between the chromosomal changes and the phenotypic characteristics of BCL₁ tumor cells could be made. However, the highly specific karyotype may serve as a useful marker in identifying single BCL₁ tumor cells.     

No phenotype associated with established lipopolysaccharide model for cerebral palsy

OBJECTIVE: Cerebral palsy (CP) is associated with childhood spasticity, seizures, and paralysis. Oligodendrocyte damage resulting in periventicular leukomalacia (PVL) in the developing brain has been implicated. Animal models of CP have used prenatal hypoxia and infection with histopathology of PVL as the end point. To evaluate whether this histologic end point is associated with a CP phenotype, we reproduced a lipopolysaccharide (LPS) model for PVL, 1 and evaluated developmental, behavioral, and motor outcomes. STUDY DESIGN: On gestational day 15, Fischer 344 rats were intracervically injected with .1 mg/kg LPS (n = 5) or saline (n = 4). After delivery, evaluation for developmental milestones was performed on days 1 to 21 (LPS = 45; control = 30 pups). Males were also tested at 2.5 months using open-field, rotarod, and anxiety tests. On day 21, 2 pups/litter were perfused for immunohistochemistry, and stained with 2 oligodendrocyte antibodies: 2', d'-cyclic nucleotide phosphodiesterase (CNP), and myelin proteolipid protein (PLP) with relative densities of staining assessed using NIH Image software. Statistical analysis included Mann-Whitney U and analysis of variance (ANOVA). RESULTS: LPS pups demonstrated decreased CNP (P = .04) and PLP (P = .06) staining, replicating the model. There was no difference seen in neonatal weight, righting, negative geotaxis, cliff aversion, rooting, forelimb grasp, audio startle, air righting, eye opening, and activity. Surprisingly, LPS-exposed neonatal rats mastered forelimb placement (P < .01) and surface righting (P = .02) earlier than control rats. There were no differences between adult groups in open field distance traveled (P = .8), open-field locomotion time (P = .6), rotarod (P = .6), or anxiety (P = .7). CONCLUSION: Histologic evidence of white matter damage can be replicated using an LPS model for intrauterine inflammation. Significant phenotypic differences consistent with the motor and cognitive damage sequelae of such lesions (ie, CP) were not demonstrated. When evaluating animal models, it is important to assess not only biochemical markers for human disease, but also clinically relevant phenotypes.     

Induction of FOXP3-expressing regulatory CD4pos T cells by human mature autologous dendritic cells

Current literature suggests that T cells recognizing antigen on mature dendritic cells (DC) differentiate into effector T cells whereas tolerance is induced when antigen is presented by immature DC. We investigated the consequences of the interactions between immature or lipopolysaccharide-matured DC and CD4(pos) T lymphocytes in absence of foreign antigen. While immature DC did not induce significant CD4(pos) T cell activation, we observed that a significant fraction of CD4(pos) T cells cultured with mature autologous DC displayed phenotypic features of activation and produced IL-2, IFN-gamma, IL-10 and TGF-beta. Furthermore, CD4(pos) T lymphocytes primed by mature, but not immature, autologous DC acquired regulatory properties. Indeed, when added to an allogeneic mixed leukocyte reaction, they suppressed the response of alloreactive T lymphocytes to the priming DC while responses to third-party stimulators were spared. The generation of CD4(pos) T cells with regulatory function by autologous stimulation did not require the presence of natural CD4(pos)CD25(pos) regulatory T cells. In addition, the acquisition of regulatory function by CD4(pos)CD25(neg) T cells stimulated by autologous mature DC was accompanied by the induction of FOXP3 expression. Our data suggest that during inflammatory conditions, presentation of self antigens by mature DC to autologous T lymphocytes could contribute to the generation of regulatory mechanisms.     

Abnormal production of tumor necrosis factor (TNF) -- alpha and clinical efficacy of the TNF inhibitor etanercept in a patient with PAPA syndrome [corrected

We report a family with pyogenic sterile arthritis, pyoderna and acne syndrome (PAPA). The proband presented several episodes of sterile pyogenic arthritis and became unresponsive to glucocorticoids. After treatment with the tumor necrosis factor inhibitor etanercept, the disease underwent rapid and sustained clinical remission. Production of tumor necrosis factor-alpha by mononuclear cells of the proband and of the affected relatives was abnormally elevated.