Gill, Roop MD; Gribar, Steven C. MD; Kaczorowski, David J. MD; Kohut, Lauryn; Williams, Debra; Scott, Melanie MD, PhD; Hackam, David J. MD, PhD, FACS; Billiar, Timothy R. MD, FACS Author Information

Background: Torulopsis glabrata and Candida krusei are both resistant to fluconazole therefore rapid identification of these species may prevent unnecessary prescribing of this commonly used antifungal agent.Methods: A total of 290 organisms were examined for their fluorescent appearance using epi-illumination with ultra-violet excitation and their fluorescence was compared.Results: Incorporating the fluorochrome calcofluor white at a concentration of 0.0025% in a peptone/glucose medium, and in sera for the germ tube test with Candida albicans enhances the recognition of germ tubes. Several other species of yeast-like organisms were found to have characteristic fluorescent appearances in these and in aqueous solutions at this concentration.Conclusion. The use of these appearances as a rapid (in minutes) identification criteria for Torulopsis glabrata and Candida krusei is described. This may be of value as infections with these 2 species are often not treatable with fluconazole.

The liver is the primary site for LPS clearance. We have shown that hepatocytes (HC) take up LPS in vitro in a CD14- dependent manner. Here we developed the hypothesis that CD14 would be required for liver and specifically, HC LPS uptake in vivo. Methods: C57BL/6 (WT) and CD14 −/− mice were anesthetized and underwent laparotomy. Green fluorescent Alexa-488 LPS (5 mg/kg) or saline control was injected directly into the hepatic portal vein and livers were harvested and fixed after 15/45/60/90 mins (n = 2 per group/time point). Livers were sectioned and visualized by fluorescent microscope to assess LPS uptake. Results: In WT mice, LPS was taken up into HC after 15 mins and uptake increased to 90 mins. There was little or no uptake detected in livers of CD14−/− mice over the time points tested. (Figure)FigureConclusions: WT mice rapidly internalize LPS in HC in vivo in a CD14-dependent manner. The rapid CD14-dependent uptake of LPS by HC in vivo provides a previously unknown function for CD14 expressed by HC.

Overhaus, Marcus MD; Gallo, David J.; Togel, Sandra; Williams, Debra L.; Billiar, Timothy R. MD; Bauer, Anthony J. PhD Author Information

BACKGROUND:Idiopathic pneumonia syndrome (IPS) is a frequently fatal complication of allogeneic bone marrow transplantation (BMT). IPS is associated with elevated bronchoalveolar lavage (BAL) fluid levels of tumor necrosis factor-alpha and lipopolysaccharide, both of which are potent activators of endothelial cells (ECs). EC expression of the adhesion molecule CD54 (intercellular adhesion molecule [ICAM]-1) has been shown to be a major regulator of pulmonary inflammation in various experimental models.METHODS:Using a well-established murine BMT system in which lung injury and graft-versus-host disease (GvHD) are induced by minor histocompatibility antigenic differences between donor and host, the RNase Protection Assay, mice deficient in ICAM-1 expression, and a monoclonal blocking antibody to ICAM, we evaluated the role of the pulmonary vascular expression of CD54 in the development of IPS.RESULTS:Enhanced pulmonary vascular expression of ICAM-1 coincided with the development of IPS. When ICAM-1 -/- mice were used as allogeneic BMT recipients, IPS severity (measured by lung histopathology, BAL cellularity, and cytokine expression) was significantly reduced compared with wild-type controls. Similar results were also observed when wild-type recipients were treated with a monoclonal blocking antibody to ICAM-1. Surprisingly, ICAM-1 had differential effects on leukocyte infiltration into GvHD target organs; ICAM-1 deficiency had no impact on intestinal histopathology, whereas ICAM-1-/- BMT recipients had significantly enhanced hepatic injury.CONCLUSIONS:These data demonstrate that although the expression of ICAM-1 is critical for the development of IPS, different mechanisms of leukocyte recruitment are operative in other GvHD target organs.

Animal models with impaired thymic negative selection do not always cause autoimmune diseases despite the development of an autoreactive T-cell repertoire. We investigated the requirements for the de velopment of systemic autoimmune disease by using bone marrow chimeras that lacked expression of major histocompatibility complex (MHC) class II on thymic antigen-presenting cells (APCs), leading to impaired negative selection. We found that impaired negative selection mediated by absence of MHC class II, but not MHC class I, permitted the development of systemic autoimmune disease that is indistinguishable from acute graft-versus-host disease (GVHD). Thymectomy prevented disease, confirming the causal association of the thymus with its development. Adoptive transfer of CD4+ T cells caused GVHD in secondary hosts only when they were irradiated, and cotransfer of peripheral CD4+ and CD8+ T cells from naive mice prevented the disease. These results demonstrate that impaired thymic negative selection can cause lethal autoimmune disease indistinguishable from acute GVHD in the context of a proinflammatory milieu when peripheral regulatory mechanisms are absent.

The liver, skin, and gastrointestinal tract are major target organs of acute graft-versus-host disease (GVHD), the major complication of allogeneic bone marrow transplantation (BMT). In order to gain a better understanding of acute GVHD in the liver, we compared the gene expression profiles of livers after experimental allogeneic and syngeneic BMT using oligonucleotide microarray. At 35 days after allogeneic BMT when hepatic GVHD was histologically evident, genes related to cellular effectors and acute-phase proteins were up-regulated, whereas genes largely related to metabolism and endocrine function were down-regulated. At day 7 after BMT before the development of histologic changes in the liver, interferon gamma (IFN-gamma)-inducible genes, major histocompatibility (MHC) class II molecules, and genes related to leukocyte trafficking had been up-regulated. Immunohistochemistry demonstrated that expression of IFN-gamma protein itself was increased in the spleen but not in hepatic tissue. These results suggest that the increased expression of genes associated with the attraction and activation of donor T cells induced by IFN-gamma early after BMT is important in the initiation of hepatic GVHD in this model and provide new potential molecular targets for early detection and intervention of acute GVHD.

Objective. The chemokine receptor CXCR3 has an important role in the migration of effector T cells. To investigate the role of CXCR3 on donor cells in acute graft vs host disease (GVHD) we used a well-defined experimental bone marrow transplantation (BMT) model where acute GVHD is mediated by donor CD8(+) T cells against minor histocompatibility antigens. Methods. Lethally irradiated C3H.SW recipients were transplanted from either wild-type B6 or CXCR3(-/-) B6 donors. Donor T-cell expansion was analyzed in the spleen and small intestine of recipients by FACS. Donor T-cell function was analyzed by cytokine secretion. The severity of acute GVHD was assessed by histopathological analysis of intestine and liver, GVHD clinical scores, and survival after BMT.Results. Significantly higher numbers of donor CD8(+) CXCR3(-/-) T cells were found in the spleen on days +7 and +14 compared to donor wild-type T cells. By contrast, the number of CD8+ T cells in the small bowel of BMT recipients from CXCR3(-/-) donors was sevenfold lower than from wild-type donors. Systemic concentrations of INF-gamma and TNF-alpha were equivalent between groups. Animals that received CXCR3(-/-) donor T cells demonstrated diminished GI tract and liver damage and showed improved survival after BMT compared to recipients of wild-type donor cells (43% vs 0%, p < 0.001).Conclusion. The migration of donor CD8(+) T cells to GVHD target organs such as the intestine depends on the expression of CXCR3 and contributes significantly to GVHD damage and overall mortality. (C) 2003 International Society for Experimental Hematology. Published by Elsevier Inc.

Idiopathic pneumonia syndrome (IPS) is a major complication after allogeneic bone marrow transplantation (allo-BMT) and involves the infiltration of donor leukocytes and the secretion of inflammatory cytokines. We hypothesized that leukocyte recruitment during IPS is dependent in part upon interactions between chemokine receptor 2 (CCR2) and its primary ligand monocyte chemoattractant protein-1 (MCP-1). To test this hypothesis, IPS was induced in a lethally irradiated parent --> F1 mouse BMT model. Compared with syngeneic controls, pulmonary expression of MCP-1 and CCR2 mRNA was significantly increased after allo-BMT. Transplantation of CCR2-deficient (CCR2-/-) donor cells resulted in a significant reduction in IPS severity compared with transplantation of wild-type (CCR2+/+) cells and in reduced bronchoalveolar lavage (BAL) fluid cellularity and BAL fluid levels of tumor necrosis factor-alpha (TNF-alpha) and soluble p55 TNF receptor (sTNFRI). In addition, neutralization of MCP-1 resulted in significantly decreased lung injury compared with control-treated allogeneic recipients. Experimental data correlated with preliminary clinical findings; patients with IPS have elevated levels of MCP-1 in the BAL fluid at the time of diagnosis. Collectively, these data demonstrate that CCR2/MCP-1 interactions significantly contribute to the development of experimental IPS and suggest that interventions blocking these receptor-ligand interactions may represent novel strategies to prevent or treat this lethal complication after allo-BMT.

The liver is an important site of host-microbe interaction. Although hepatocytes have been reported to be responsive to lipopolysaccharide (LPS), the global gene expression changes by LPS and mechanism(s) by which LPS stimulates cultured hepatocytes remain uncertain. Cultures of primary mouse hepatocytes were incubated with LPS to assess its effects on the global gene expression, hepatic transcription factors, and mitogen-activated protein (MAP) kinase activation. DNA microarray analysis indicated that LPS modulates the selective expression of more than 80 genes and expressed sequence tags. We have shown previously that hepatocytes express CD14, which is required both for uptake and responsiveness to LPS. In other cells, responsiveness to microbial products requires expression of Toll-like receptors (TLR) and their associated accessory molecules. Hepatocytes expressed TLR1 through TLR9 as well as MyD88 and MD-2 transcripts, as shown by reverse transcriptase PCR analysis, indicating that hepatocytes express all known microbe recognition molecules. The MAP kinase extracellular signal-regulated kinase 1/2 was phosphorylated in response to LPS in mouse hepatocytes, and the levels of phosphorylation were lower in hepatocytes from TLR4-null mice. NF-kappa B activation was reduced in TLR4-mutant or -null hepatocytes compared to control hepatocytes, and this defect was partially restored by adenoviral transduction of mouse TLR4. Thus, hepatocytes respond to nanogram concentrations of LPS through a TLR4 response pathway.

Vodovotz, Yoram; Liu, Shubing; Green, Angela; Williams, Debra; Shapiro, Rick; Billiar, Timothy R. Author Information

Inducible nitric oxide synthase (iNOS) can be coexpressed with acute phase reactants in hepatocytes; however, it is unknown if NO can regulate the acute phase response. We tested the hypothesis that iNOS-derived nitric oxide (NO) attenuates the acute phase response by inhibiting IL-6-enhanced Stat3 DNA-binding activity and type II acute phase mRNA expression. iNOS was overexpressed in cultured rat hepatocytes via transduction with a replication defective adenovirus containing cDNA for human iNOS (AdiNOS), and Stat3 DNA-binding activity was determined by electrophoretic mobility shift assay (EMSA). EMSAs demonstrated that AdiNOS inhibits IL-6-induced Stat3 activation and that this inhibition is reversible in the presence of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMA). The induction of beta-fibrinogen mRNA by IL-6, a Stat3 dependent process, is attenuated in AdiNOS-transduced cells and partially reversed by L-NMA. Thus, iNOS overexpression suppresses IL-6-induced Stat3 activation and type II acute phase mRNA expression in cultured hepatocytes. This suppression may represent a mechanism by which NO down-regulates the acute phase response.

Nitric oxide (NO) derived from the inducible isoform of NO synthase (iNOS) is an inflammatory product implicated both in secondary damage and in recovery from brain injury. To address the role of iNOS in experimental traumatic brain injury (TBI), we used 2 paradigms in 2 species. In a model of controlled cortical impact (CCI) with secondary hypoxemia, rats were treated with vehicle or with 1 of 2 iNOS inhibitors (aminoguanidine and L-N-iminoethyl-lysine), administered by Alzet pump for 5 days and 1. 5 days after injury, respectively. In a model of CCI, knockout mice lacking the iNOS gene (iNOS(-/-)) were compared with wild-type (iNOS(+/+)) mice. Functional outcome (motor and cognitive) during the first 20 days after injury, and histopathology at 21 days, were assessed in both studies. Treatment of rats with either of the iNOS inhibitors after TBI significantly exacerbated deficits in cognitive performance, as assessed by Morris water maze (MWM) and increased neuron loss in vulnerable regions (CA3 and CA1) of hippocampus. Uninjured iNOS(+/+) and iNOS(-/-) mice performed equally well in both motor and cognitive tasks. However, after TBI, iNOS(-/-) mice showed markedly worse performance in the MWM task than iNOS(+/+) mice. A beneficial role for iNOS in TBI is supported.

We have designed a drug that protects the liver from apoptotic cell death by organ-selective pharmacological generation of the bioregulatory agent, nitric oxide (NO). The discovery strategy involved three steps: identifying a diazeniumdiolate ion (R2N[N(O)NO]-, where R2N = pyrrolidinyl) that spontaneously decomposes to NO with a very short half-life (3 s) at physiological pH; converting this ion to a series of potential prodrug derivatives by covalent attachment of protecting groups that we postulated might be rapidly removed by enzymes prevalent in the liver; and screening the prodrug candidates in vitro and in vivo to select a lead and to confirm the desired activity. Of five cell types examined, only cultured hepatocytes metabolized O2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) to NO, triggering cyclic guanosine 3',5'-monophosphate (cGMP) synthesis and protecting the hepatocytes from apoptotic cell death induced by treatment with tumor necrosis factor-alpha (TNF alpha) plus actinomycin D. In vivo, V-PYRRO/NO increased liver cGMP levels while minimally affecting systemic hemodynamics, protecting rats dosed with TNF alpha plus galactosamine from apoptosis and hepatotoxicity. The results illustrate the potential utility of diazeniumdiolates for targeting NO delivery in vivo and suggest a possible therapeutic strategy for hepatic disorders such as fulminant liver failure.

Tetrahydrobiopterin (BH4) is an important cofactor for two hepatic enzymes, inducible nitric oxide synthase (iNOS) and phenylalanine hydroxylase (PAH), and competition for BH4 between the two enzymes might limit hepatic iNOS or PAH activity. To test this hypothesis, we determined whether conversion of phenylalanine to tyrosine was modified by changes in NO synthase activity, and conversely whether NO synthesis was limited by the rate of phenylalanine conversion to tyrosine in rat hepatocytes and perfused livers. NO production was decreased only slightly, when flux through PAH was maximized in isolated perfused livers, and in isolated hepatocytes only when BH4 synthesis was inhibited. Increases in NO synthesis did not reduce tyrosine formation from phenylalanine. Phenylalanine markedly increased biopterin synthesis, whereas arginine had no effect. Thus, basal BH4 synthesis appears to be adequate to support iNOS activity, whereas BH4 synthesis is increased to support PAH activity.

It has been postulated that Kupffer cells provide signals that regulate hepatocyte responses in sepsis and inflammation. Although in vitro data support such a hypothesis, to our knowledge, no in vivo evidence has been reported. We injected rats with lipopolysaccharide intraperitoneally to simulate sepsis or turpentine intramuscularly to mimic localized inflammation. Both treatments are known to induce the hepatic acute-phase response. Liver nonparenchymal cells and hepatocytes were isolated and placed in culture. Hepatocyte fibrinogen synthesis was measured as an indication of interleukin 6 exposure, while nonparenchymal interleukin 6 production was measured directly. Both lipopolysaccharide and turpentine stimulated a sharp increase in hepatocyte fibrinogen synthesis (turpentine greater than lipopolysaccharide). However, only lipopolysaccharide injection was associated with increased nonparenchymal cell interleukin 6 synthesis. Increased circulating levels of interleukin 6 could be found only after lipopolysaccharide injection. In addition, tumor necrosis factor synthesis was enhanced by lipopolysaccharide but not turpentine. Our data show that nonparenchymal cells are stimulated to provide the interleukin 6 signal to hepatocytes in endotoxemia but not in remote localized inflammation, even though both treatments stimulate the hepatic acute-phase response. Our findings support paracrine functions for liver sinusoidal cells in certain septic states.

The gracilis muscle was excised from cold-acclimated rats, placed in vitro, and simultaneously perfused via its artery by high pO2 medium and superfused by low pO2 medium. With a doubling of the perfusion rate (from 50 to 100 μl/min) phosphocreatine and ATP increased by 39% and 44%, respectively.