Altered Cell Responses Research Articles

A Review of the Combination of Experimental Measurements and Fibril-Reinforced Modeling for Investigation of Articular Cartilage and Chondrocyte Response to Loading

The function of articular cartilage depends on its structure and composition, sensitively impaired in disease (e.g. osteoarthritis, OA). Responses of chondrocytes to tissue loading are modulated by the structure. Altered cell responses as an effect of OA may regulate cartilage mechanotransduction and cell biosynthesis. To be able to evaluate cell responses and factors affecting the onset and progression of OA, local tissue and cell stresses and strains in cartilage need to be characterized. This is extremely challenging with the presently available experimental techniques and therefore computational modeling is required. Modern models of articular cartilage are inhomogeneous and anisotropic, and they include many aspects of the real tissue structure and composition. In this paper, we provide an overview of the computational applications that have been developed for modeling the mechanics of articular cartilage at the tissue and cellular level. We concentrate on the use of fibril-reinforced models of cartilage. Furthermore, we introduce practical considerations for modeling applications, including also experimental tests that can be combined with the modeling approach. At the end, we discuss the prospects for patient-specific models when aiming to use finite element modeling analysis and evaluation of articular cartilage function, cellular responses, failure points, OA progression, and rehabilitation.

Metabolic monosaccharides altered cell responses to anticancer drugs

Metabolic glycoengineering has been used to manipulate the glycochemistry of cell surfaces and thus the cell/cell interaction, cell adhesion, and cell migration. However, potential application of glycoengineering in pharmaceutical sciences has not been studied until recently. Here, we reported that Ac4ManNAc, an analog of N-acetyl-d-mannosamine (ManNAc), could affect cell responses to anticancer drugs. Although cells from different tissues and organs responded to Ac4ManNAc treatment differently, treated cells with increased sialic acid contents showed dramatically reduced sensitivity (up to 130 times) to anti-cancer drugs as tested on various drugs with distinct chemical structures and acting mechanisms. Neither increased P-glycoprotein activity nor decreased drug uptake was observed during the course of Ac4ManNAc treatment. However, greatly altered intracellular drug distributions were observed. Most intracellular daunorubicin was found in the perinuclear region, but not the expected nuclei in the Ac4ManNAc treated cells. Since sialoglycoproteins and gangliosides were synthesized in the Golgi, intracellular glycans affected intracellular signal transduction and drug distributions seem to be the main reason for Ac4ManNAc affected cell sensitivity to anticancer drugs. It was interesting to find that although Ac4ManNAc treated breast cancer cells (MDA-MB-231) maintained the same sensitivity to 5-Fluorouracil, the IC50 value of 5-Fluorouracil to the same Ac4ManNAc treated normal cells (MCF-10A) was increased by more than 20times. Thus, this Ac4ManNAc treatment enlarged drug response difference between normal and tumor cells provides a unique opportunity to further improve the selectivity and therapeutic efficiency of anticancer drugs.

Vitreous IGFBP-3 Effects on Müller Cell Proliferation and Tractional Force Generation

Previous studies from this laboratory revealed that vitreous insulin-like growth factor binding protein-3 (IGFBP-3) is a biologically active fragment of the intact protein. The goal of this study was to characterize its effects on Müller cell proliferation and tractional force generation, activities relevant to proliferative diabetic retinopathy (PDR). Müller cells were isolated from normal porcine retina. The vitreous-type IGFBP-3 fragment was isolated from normal human plasma and compared with intact recombinant protein for the ability to modulate Müller cell proliferation and tractional force generation in tissue culture models. Müller cells were stimulated to proliferate by serum and platelet-derived growth factor (PDGF), but not insulin-like growth factor (IGF)-I or IGF-II. The cells were similarly unresponsive to IGFBP-3 or the IGFBP-3 fragment alone or in combination with IGF-I or IGF-II. In contrast, Müller cells demonstrated robust extracellular matrix contraction in response to IGF-I, IGF-II, and PDGF. Intact IGFBP-3 attenuated extracellular matrix contraction in response to IGF-I and IGF-II while the IGFBP-3 fragment modulated cell responses to IGF-II only. Neither binding protein altered cell responses to PDGF. Intact IGFBP-3 modulates Müller cell tractional force generation stimulated by IGF-I and IGF-II while the effects of the vitreous-type fragment are limited to IGF-II. Porcine Müller cells proliferate in response to PDGF, but not IGF-I or IGF-II. Both forms of IGFBP-3 are also without mitogenic effects alone or in combination with IGFs. It appears that Müller cell tractional force generation in PDR is driven by vitreous IGF activity and proliferation is stimulated by growth factors outside of the IGF system.

Integrin organization: Linking adhesion ligand nanopatterns with altered cell responses

Integrin receptors bind to adhesion ligand (e.g. arginine–glycine–aspartic acid or RGD containing peptides) on extracellular matrix and organize into high-density complexes which mediate many cell behaviors. Biomaterials with RGD nanopatterned into multivalent “islands” (∼30–70nm diameter) have been shown to alter cell responses, although the length scale of pattern features is orders of magnitude smaller than adhesion complexes. In this work, we employ together for the first time an extensive data set on osteoblast responses as a function of ligand nanopatterns, a computational model of integrin binding to ligand nanopatterns, and new measures of integrin organization on the cell surface. We quantify, at multiple length scales, integrin organization generated in silico as a function of RGD nanopattern parameters. We develop a correlative model relating these measures of in silico integrin organization and in vitro MC3T3 preosteoblast cell responses as functions of the same RGD nanopatterns: cell spreading correlates with the number of bound integrins, focal adhesion kinase (FAK) phosphorylation correlates with small, homogeneously distributed clusters of integrins, and osteogenic differentiation correlates with large, heterogeneously distributed integrin clusters. These findings highlight the significance of engineering biomaterials at the nanolevel and suggest new approaches to understanding the mechanisms linking integrin organization to cell responses.

Triggering of toll-like receptor-4 in human multiple myeloma cells promotes proliferation and alters cell responses to immune and chemotherapy drug attack

Multiple myeloma (MM) is an incurable B-cell malignancy characterized by accumulation of malignant plasma cells in the bone marrow and by recurrent or persistent infections. Toll-like receptors (TLRs) are essential in the host defense against infections. The aim of this study was to investigate TLR initiated responses in MM cells including proliferation, anti-apoptosis and immune escape. Myeloma cell lines gene transcription, cell cycle and protein expression were detected by RT-PCR, real-time PCR, western blot, ELISA and flow cytometry analysis. 3H-thymidine was used for measuring cell proliferation, and Annexin V-PI flow cytometry for the detection of cell apoptosis. We show that human myeloma cell lines expressed TLRs，and LPS induced the proliferation and partially protected MM.1S and ARP-1 cells from adriamycin-induced apoptosis. LPS appears to induce proliferation via MyD88 and MAPKs signaling. In addition, LPS treatment upregulated myeloma cell secretion of cytokine IL-18 and expression of immunoregulatory factors B7-H1, B7-H2 and CD40 mRNA and helped myeloma cells to escape immune surveillance. Our results show that TLRs are functional on myeloma tumor cells, and the ligands to these TLRs have a functional role in affecting myeloma cell proliferation, survival, and response to chemotherapy and immune attacks.See commentary: The TOLL of inflammation in multiple myeloma

Triggering of Toll-Like Receptor-4 In Human Multiple Myeloma Cells Promotes Proliferation and Alters Cell Responses to Immune and Chemotherapy Drug Attack

AbstractAbstract 1905Multiple myeloma (MM) is a fatal plasma cell malignancy mainly localized in the bone marrow. The clonal expansion of tumor cells is associated with the disappearance of normal plasma cells and with a marked depression in the production of normal immunoglobulin (Ig). This makes MM patients highly vulnerable to bacterial, fungal and viral infections and recurrent infections remain to be a major cause of death in MM patients. It has been shown that most primary myeloma cells and cell lines express multiple Toll-like receptors (TLRs). Among them, TLR4 is most frequently expressed. To investigate TLR-initiated responses in MM cells including proliferation, anti-apoptosis and immune escape, we first screened four commonly used human myeloma cell line (HMCL) for the expression of major TLRs by RT-PCR. Surprisingly, all the HMCL expressed multiple TLRs. We also examined primary myeloma cells from 4 patients with MM and our results showed that TLR4 was expressed by all the tumor cells. We incubated myeloma cells with LPS, the natural ligand for TLR4, and found that cell proliferation increased significantly. Targeting TLRs on malignant B cells can induce resistance to chemotherapeutic agents but can also be exploited for combined therapeutic approaches. As mechanisms involved in the resistance to apoptosis play a major role in MM escape to therapies, we sought to determine the capacity of TLR4 ligand to promote the survival of HMCL cells. Myeloma cells were pretreated for four hours with LPS before being induced apoptosis by adriamycin. Results showed that LPS pretreatment partially protected the cells from adriamycin-induced apoptosis. The TLR signaling pathway activates several signaling elements, including NF-kB and ERK/JNK/p38 MAPKs, which regulate many immunologically relevant proteins. Time-dependent MAPK phosphorylation was measured to assess the activation of these kinases upon treatment with LPS in cell lines. ERK1/2, p38, and JNK phosphorylation and NF-kB were significantly up-regulated following LPS treatment. Moreover, our findings demonstrated that LPS-induced cell proliferation was dependent on JNK, ERK and p38 signaling. IL-18, a recently described member of the IL-1 cytokine superfamily, is now recognized as an important regulator of innate and acquired immune responses. In this study, we found that LPS induced IL-18 secretion and activated MAPK and NF-kB signaling simultaneously. Therefore, our results suggest that activation of the MAPK signaling and secretion of IL-18 are interconnected. Tumors evade immune surveillance by multiple mechanisms, including the production of factors such as TGF-β and VEGF, which inhibit and impair tumor-specific T cell immunity. Our study also showed that T cell proliferation induced by allostimulatory cells decreased when the HMCL were pre-treated with LPS. Moreover, immunoregulatory molecules on HMCL, such as B7-H1, B7-H2 and CD40, were upregulated after treatment with LPS, suggesting that TLR4 ligand LPS facilitates tumor cell evasion of the immune system. Our results show that TLRs are functional on myeloma tumor cells, and the ligands to these TLRs have a functional role in affecting myeloma cell proliferation, survival, and response to chemotherapy and immune attacks. Disclosures:No relevant conflicts of interest to declare.

Activation of p38 mitogen-activated protein kinase by norepinephrine in T-lineage cells

The catecholamine norepinephrine (NE) stimulates T lymphocytes through a beta-adrenergic receptor (βAR)/adenylyl cyclase (AC)/cyclic AMP (cAMP)/protein kinase A (PKA) pathway, leading to altered cell responsiveness and apoptosis. p38 Mitogen-activated protein kinase (MAPK), a major intracellular signalling mediator for cellular and environmental stressors, is involved in the production of immune modulators and in the regulation of T-cell development, survival and death. In these studies we investigated the relationship among NE signalling, p38 MAPK activity and T-cell death. We showed that NE stimulation of BALB/c mouse thymocytes and S49 thymoma cells selectively increases the dual phosphorylation and activity of p38α MAPK. p38 MAPK activation involves the βAR, Gs protein, AC, cAMP and PKA, as determined through the use of a βAR antagonist, activators of AC and cAMP, and S49 clonal mutants deficient in Gs and PKA. Dual phosphorylation of p38 MAPK is also dependent on its own catalytic activity. Inhibition of p38 MAPK activity revealed its involvement in cAMP-mediated activating transcription factor-2 (ATF-2) phosphorylation, Fas ligand messenger RNA (mRNA) up-regulation, and cell death. These results identify a mechanism through which NE stimulation of the βAR/Gs/PKA pathway activates p38 MAPK, which can be potentiated by autophosphorylation, and leads to changes in T-cell dynamics, in part through the regulation of Fas ligand mRNA expression.

Abstract 1651: The use of ‘X-MAN’ isogenic cell lines to define PI3-kinase inhibitor activity profiles

Abstract The tumour microenvironment promotes selective outgrowth of cells carrying mutations in oncogenes or tumour suppressors, enabling the activated pathways to drive tumour growth. Two such pathways are the PI3-K/AKT and RAS/RAF/MEK signaling pathways, which demonstrate a high frequency of mutations in human cancer. These pathways represent important therapeutic targets, and a number of targeted agents are now in clinical trials. Despite being pertinent to the clinical situation, the role that the tumour microenvironment plays in modifying responsiveness to therapeutic agents targeted against these pathways is poorly understood. A major challenge faced during the development of PI3-K/AKT and RAS/RAF/MEK targeted agents is to demonstrate inhibitor selectivity and efficacy in a patient-relevant cell based system. Isogenic ‘X-MAN’ paired cell lines help address this by providing a system in which the endogenous wild-type or mutant alleles of the parental cell line are selectively activated or silenced via targeted homologous integration, providing a pair of human cell-lines in which the only difference between the cells is the mutation of interest. This approach therefore overcomes the issues that may occur when over-expression methods are employed. We therefore set out to investigate how changes in the tumour microenvironment altered cell responses to PI3-K and MEK inhibitors. We employed isogenic HCT116 human colon cancer cells where the parental cells harbor pre-existing H1047R PIK3CA and G13D KRAS alleles and compared these to isogenic clones that retain either the PIK3CA or KRAS wild-type alleles. Inhibitors were profiled under a range of defined conditions chosen to model the tumour microenvironment, including low glucose, low serum and hypoxia, and the activity of the compounds investigated. Under standard culture conditions, we found that PI3-K and MEK inhibitors showed minimal selectivity for cells with activating mutations compared to wild-type cells. When grown under defined culture conditions which more closely mimic the tumour microenvironment, the effects of the driving mutations became dominant and a differential between wild-type and mutant cell responses to inhibitors was observed. The selective PI3-K inhibitors GDC-0941 and PI-103 showed a >10-fold increase in potency when tested in anti-proliferative assays in mutant vs, wild-type cell lines. However, the dual PI3-K/mTOR inhibitor BEZ-235 did not show selectivity in this system. We show that by using isogenic cell lines under conditions which model the tumour microenvironment, differences in activity profiles between specific inhibitors can be quickly and definitively identified, highlighting the advantages of using this system in profiling molecularly targeted agents. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1651.

Circulating Cytokines as Biomarkers of Alcohol Abuse and Alcoholism

There are currently no consistent objective biochemical markers of alcohol abuse and alcoholism. Development of reliable diagnostic biomarkers that permit accurate assessment of alcohol intake and patterns of drinking is of prime importance to treatment and research fields. Diagnostic biomarker development in other diseases has demonstrated the utility of both open, systems biology, screening for biomarkers and more rational focused efforts on specific biomolecules or families of biomolecules. Long-term alcohol consumption leads to altered inflammatory cell and adaptive immune responses with associated pathologies and increased incidence of infections. This has led researchers to focus attention on identifying cytokine biomarkers in models of alcohol abuse. Alcohol is known to alter cytokine levels in plasma and a variety of tissues including lung, liver, and very importantly brain. A number of cytokine biomarker candidates have been identified, including: tumor necrosis factor-alpha, interleukin (IL)-1-alpha, IL-1-beta, IL-6, IL-8, IL-12, and monocyte chemoattractant protein-1. This is an emerging and potentially exciting avenue of research in that circulating cytokines may contribute to diagnostic biomarker panels, and a combination of multiple biomarkers may significantly increase the sensitivity and specificity of the biochemical tests aiding reliable and accurate detection of excessive alcohol intake.

Low concentrations of LL-37 alter IL-8 production by keratinocytes and bronchial epithelial cells in response to proinflammatory stimuli

The immunomodulatory cationic host defence peptide LL-37 plays an important role in epithelial innate immunity; at higher concentrations (20-50 microg mL(-1)) associated with inflammation, LL-37 elicits the production of cytokines and chemokines. It was demonstrated here that lower, physiologically relevant LL-37 concentrations (2-3 microg mL(-1)) altered epithelial cell responses to proinflammatory stimuli. In combination with interleukin-1beta (IL-1beta) and the Toll-like receptor-5 (TLR5) agonist flagellin, these low concentrations of LL-37 synergistically increased IL-8 production by both proliferating and differentiated keratinocytes and by bronchial epithelial cells. In combination with the TLR2/1 agonist PAM3CSK4, LL-37 synergistically induced transcription and the release of both IL-8 and IL-6 from primary bronchial epithelial cells; the IL-8 response was demonstrated to be regulated by epidermal growth factor receptor signalling. Treatment of bronchial epithelial cells with LL-37 and the TLR3 agonist polyI:C resulted in synergistic increases in IL-8 release and cytotoxicity. These data indicate that low concentrations of LL-37 may alter epithelial responses to infecting microorganisms in vivo.

Maternal mortality from systemic illness: unraveling the contribution of the immune response

Maternal morbidity and/or mortality (MM) is increased in pyelonephritis and influenza. Alterations in the immune response could account for the increase MM. We sought to determine whether the immune response is functionally different during pregnant and nonpregnant (NP) states. Mouse model of systemic and localized inflammation was used. Maternal serum was assessed for expression of T-helper cell type 1 and 2 cytokines. Maternal spleens were harvested for immunohistochemistry. Systemic administration of lipopolysaccharides resulted in no mortality to NP mice compared with 88% in preterm and 100% in term mice. A potent cytokine response was present in both NP and pregnancy. Systemic inflammation in pregnancy results in increased CD8 and CD11c expression in spleens. Differences in cytokine response to systemic inflammation is unlikley to modulate the increased MM during pregnancy. Altered T-cell and dendritic cell responses in pregnancy may be responsible for the increase in MM.

A recent perspective on alcohol, immunity, and host defense.

Multiple line of clinical and experimental evidence demonstrates that both acute, moderate, and chronic, excessive alcohol use result in various abnormalities in the functions of the immune system. Medline and PubMed databases were used to identify published reports with particular interest in the period of 2000-2008 in the subject of alcohol use, infection, inflammation, innate, and adaptive immunity. This review article summarizes recent findings relevant to acute or chronic alcohol use-induced immunomodulation and its consequences on host defense against microbial pathogens and tissue injury. Studies with in vivo and in vitro alcohol administration are both discussed. The effects of alcohol on lung infections, trauma and burn injury, liver, pancreas, and cardiovascular diseases are evaluated with respect to the role of immune cells. Specific changes in innate immune response and abnormalities in adaptive immunity caused by alcohol intake are detailed. Altered inflammatory cell and adaptive immune responses after alcohol consumption result in increased incidence and poor outcome of infections and other organ-specific immune-mediated effects.

E. coli nitroreductase/CB1954 gene-directed enzyme prodrug therapy: role of arylamine N-acetlytransferase 2

Gene-directed enzyme prodrug therapy is a promising approach to the local management of cancer and a number of gene prodrug combinations have entered clinical trials. The antitumor activity of Escherichia coli nitroreductase (NTR) in combination with the prodrug CB1954 relies on the reduction of the nitro groups to reactive N-hydroxylamine intermediates that are toxic in proliferating and nonproliferating cells. We examined whether secondary metabolic activation of the N-hydroxylamines by sulfotransferases or acetyltransferases altered cell responsiveness to the drug. We evaluated the coexpression of NTR with the human cytosolic sulfotransferases SULT1A1, 1A2, 1A3, 1E1 and 2A1, or the human arylamine N-acetyltransferases NAT1 and NAT2 on SKOV3 cell survival. Only NAT2 significantly altered the toxicity of CB1954, decreasing the IC(50) 16-fold from 0.61 to 0.04 microM. These results suggest that one or more of the N-hydroxyl metabolites are a substrate for O-acetylation by NAT2. We also examined the bystander effect of SKOV3 cells expressing NTR or NTR plus NAT2. Addition of the acetyltransferase resulted in a significant decreased bystander effect (P>0.01), possibly due to a lower concentration of reactive metabolites in the culture medium. These results suggest that a combination of bacterial NTR and NAT2 may provide a greater clinical response at therapeutic concentrations of CB1954 provided the reduction in bystander effect is not clinically significant. Moreover, endogenous NAT2, which is localized predominantly in the liver and gut, may be involved in the dose-limiting hepatic toxicity and gastrointestinal side effects seen in patients treated with the higher doses of CB1954.

Mechanical loading prevents decreased bone formation responses of osteoblasts by a bone morphogenic protein‐dependent mechanism

Bone loss due to osteoporosis, disuse, and microgravity is a significant health problem. A low magnitude and high frequency (LMHF) mechanical loading has been shown to prevent bone loss in animals and humans. However, the underlying mechanisms and the target cells through which LMHF loading alleviates bone loss are not known. We hypothesized that direct application of LMHF loading to osteoblasts alters cell response, preventing decreased bone formation induced by disuse. To test our hypothesis, pre‐osteoblast 2T3 cells were exposed to the Random Positioning Machine (RPM) and intervened with LMHF loading (0.3g, 30Hz, 10 min). The RPM decreased bone formation markers as determined by alkaline phosphatase (ALP) activity and mineralization even in the presence of BMP4 (20ng/ml). However, LMHF loading prevented the RPM‐induced decrease in both markers. Mineralization induced by LMHF loading was enhanced by treatment with BMP4 and blocked by the BMP antagonist noggin, suggesting a role for BMPs. LMHF loading also rescued the RPM‐induced decrease in ALP, runx2, osteomodulin, parathyroid hormone receptor 1, and osteoglycin. These findings show that osteoblasts respond directly to LMHF loading, providing mechanistic insight into how LMHF loading prevented bone loss in vivo. The genes identified here provide potential targets for pharmaceutical treatments that may be used with LMHF loading to better treat bone loss.

Engineering RGD nanopatterned hydrogels to control preosteoblast behavior: A combined computational and experimental approach

The adhesion ligand arginine–glycine–aspartic acid (RGD) has been coupled to various materials to be used as tissue culture matrices or cell transplantation vehicles, and recent studies indicate that nanopatterning RGD into high-density islands alters key cell behaviors. Previous studies have failed, however, to conclusively decouple the effects of RGD bulk density and individual pattern parameters (i.e. RGDs/island and island distribution) on these altered cell responses. Using a nanopatterned RGD-coupled alginate hydrogel matrix, this work combines computational, statistical and experimental approaches to elucidate the effects of RGD patterns on four key cell responses. This study shows that in MC3T3 preosteoblasts focal adhesion kinase (FAK) Y397 phosphorylation, cell spreading, and osteogenic differentiation can be controlled by RGD nanopatterning, with the distribution of islands throughout the hydrogel (i.e. how closely spaced the islands are) being the most significant pattern parameter. More closely spaced islands favor FAK Y397 phosphorylation and cell spreading, while more widely spaced islands favor differentiation. Proliferation, in contrast, is primarily a function of RGD bulk density. Nanopatterning of cell adhesion ligands has tremendous potential as a simple tool to gain significant control over multiple cell behaviors in engineered extracellular matrix (ECM).

160: Cell surface LPS binding protein mediates an altered Kupffer cell response following common bile duct ligation and LPS challenge

160: Cell surface LPS binding protein mediates an altered Kupffer cell response following common bile duct ligation and LPS challenge

Endothelial barrier function in preeclampsia

Impaired endothelial barrier function resulting in increased vascular permeability is a characteristic vascular response in preeclampsia, a hypertensive and multiple systemic disorder of human pregnancy. During the last two decades, endothelial function in preeclampsia has been intensively studied and significant progress has been made in understanding the cellular and molecular bases of the altered endothelial cell response. In this review, we address the nature and mechanisms that are proposed to underlie the disturbed endothelial barrier function in preeclampsia and discuss the potential relevance of the endothelial cell responses to the initiation and/or progression of this vascular syndrome. Insights gained from the characterization of the endothelial cell phenotype assumed by the preeclamptic microvasculature may lead to novel therapeutic strategies for the management of this syndrome.

A Role for TLRs in the Regulation of Immune Cell Migration by First Trimester Trophoblast Cells

Normal pregnancy is characterized by the presence of innate immune cells at the maternal-fetal interface. Originally, it was postulated that the presence of these leukocytes was due to an immune response toward paternal Ags expressed by the invading trophoblasts. Instead, we and others postulate that these innate immune cells are necessary for successful implantation and pregnancy. However, elevated leukocyte infiltration may be an underlying cause of pregnancy complications, such as preterm labor or preeclampsia. Furthermore, such conditions have been attributed to an intrauterine infection. Therefore, we hypothesize that first trimester trophoblast cells, upon recognition of microbes through TLRs, may coordinate an immune response by recruiting cells of the innate immune system to the maternal-fetal interface. In this study, we have demonstrated that human first trimester trophoblast cells constitutively secrete the chemokines growth-related oncogene, growth-related oncogene alpha, IL-8, and MCP-1 and are able to recruit monocytes and NK cells, and to a lesser degree, neutrophils. Following the ligation of TLR-3 by the viral ligand, poly(I:C), or TLR-4 by bacterial LPS, trophoblast secretion of chemokines is significantly increased and this in turn results in elevated monocyte and neutrophil chemotaxis. In addition, TLR-3 stimulation also induces trophoblast cells to secrete RANTES. These results suggest a novel mechanism by which first trimester trophoblast cells may differentially modulate the maternal immune system during normal pregnancy and in the presence of an intrauterine infection. Such altered trophoblast cell responses might contribute to the pathogenesis of certain pregnancy complications.

TRAIL (TNF‐Related Apoptosis‐Inducing Ligand) Induces Necrosis‐Like Cell Death in Tumor Cells at Acidic Extracellular pH

How tumor microenvironment, more specifically low extracellular pH (6.5), alters cell response to TNF-related apoptosis-inducing ligand (TRAIL)-based cancer therapy has yet to be determined. The aim of the current work was to test the effect of acidic extracellular pH on TRAIL-induced cell death in human HT29 colon carcinoma and HepG2 hepatocarcinoma cell lines as well as in human primary hepatocytes. We found an increase in TRAIL sensitivity at low extracellular pH, which is partially inhibited by Bcl-2 expression in HT29 cells. At low extracellular pH, TRAIL induced a new form of cell death, sharing necrotic and apoptotic features in tumor cells. By contrast, human primary hepatocytes were resistant to TRAIL-induced cell death even at acidic extracellular pH.

Altered Kupffer cell function in biliary obstruction

An altered Kupffer cell (KC) response is thought to be responsible for the characteristic phenotype observed after biliary obstruction: a phenotype marked by a defect in the hepatic reticuloendothelial system and a hypersensitivity to endotoxin. Few studies, however, have directly examined KC function. We have sought to define the specific alterations in function and phenotype that occur in the KC after biliary obstruction. KCs were isolated from female C57BL/6 mice 4 days after a sham or common bile duct ligation (CBDL) operation. Phagocytosis, oxidative burst potential, and intracellular bacterial killing were measured as markers of reticuloendothelial system function. The KC response to endotoxin was assessed by measuring tumor necrosis factor alpha and interleukin 6 levels in the media after stimulation with lipopolysaccharide (LPS) or with LPS plus LPS-binding protein (LBP). CBDL KCs demonstrated a significant increase in phagocytic ability and significantly decreased baseline oxidative stress, compared with Shams. The oxidative burst potential, however, was equivalent or higher for CBDL KCs. CBDL KCs also demonstrated increased numbers of viable intracellular bacteria after infection; however, it is unclear if this finding represents impaired intracellular bacterial killing or increased phagocytosis of bacteria. With respect to the KC response to endotoxin, CBDL KCs were found to be less sensitive to the stimulatory effects of LPS alone but were exquisitely sensitive to the effects of LBP. LBP levels were found to be significantly elevated in CBDL animals, and CBDL KCs demonstrated a dose-dependent, exaggerated tumor necrosis factor alpha and interleukin 6 response to LPS administered with LBP. KC function is clearly altered after biliary obstruction. Phagocytic ability is actually increased, although the ability of CBDL KCs to kill bacteria within the phagosome remains ill defined. CBDL KCs are exquisitely sensitive to the effects of LBP, and LBP levels are elevated after biliary obstruction. LBP may be responsible for the increased proinflammatory response observed after endotoxin challenge in animals with biliary obstruction.