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BackgroundMapping of allele-specific DNA methylation (ASM) can be a post-GWAS strategy for localizing regulatory sequence polymorphisms (rSNPs). However, the advantages of this approach, and the mechanisms underlying ASM in normal and neoplastic cells, remain to be clarified.ResultsWe performed whole genome methyl-seq on diverse normal cells and tissues and three types of cancers (multiple myeloma, lymphoma, glioblastoma multiforme). After excluding imprinting, the data pinpointed 15,114 high-confidence ASM differentially methylated regions (DMRs), of which 1,842 contained SNPs in strong linkage disequilibrium or coinciding with GWAS peaks. ASM frequencies were increased 5 to 9-fold in cancers vs. matched normal tissues, due to widespread allele-specific hypomethylation and focal allele-specific hypermethylation in poised chromatin. Cancers showed increased allele switching at ASM loci, but disruptive SNPs in specific classes of CTCF and transcription factor (TF) binding motifs were similarly correlated with ASM in cancer and non-cancer. Rare somatic mutations affecting these same motif classes tracked with de novo ASM in the cancers. Allele-specific TF binding from ChIP-seq was enriched among ASM loci, but most ASM DMRs lacked such annotations, and some were found in otherwise uninformative “chromatin deserts”.ConclusionsASM is increased in cancers but occurs by a shared mechanism involving disruptive SNPs in CTCF and TF binding sites in both normal and neoplastic cells. Dense ASM mapping in normal plus cancer samples reveals candidate rSNPs that are difficult to find by other approaches. Together with GWAS data, these rSNPs can nominate specific transcriptional pathways in susceptibility to autoimmune, neuropsychiatric, and neoplastic diseases. Custom genome browser tracks with annotated ASM loci can be viewed at a UCSC browser session hosted by our laboratory (https://bit.ly/tycko-asm)

Interactions of toll-like receptors (TLRs) with nonmicrobial factors play a major role in the pathogenesis of early trauma-hemorrhagic shock (T/HS)-induced organ injury and inflammation. Thus, we tested the hypothesis that TLR4 mutant (TLR4 mut) mice would be more resistant to T/HS-induced gut injury and polymorphonuclear neutrophil (PMN) priming than their wild-type littermates and found that both were significantly reduced in the TLR4 mut mice. In addition, the in vivo and ex vivo PMN priming effect of T/HS intestinal lymph observed in the wild-type mice was abrogated in TLR4 mut mice as well the TRIF mut-deficient mice and partially attenuated in Myd88 mice, suggesting that TRIF activation played a more predominant role than MyD88 in T/HS lymph-induced PMN priming. Polymorphonuclear neutrophil depletion studies showed that T/HS lymph-induced acute lung injury was PMN dependent, because lung injury was totally abrogated in PMN-depleted animals. Because the lymph samples were sterile and devoid of endotoxin or bacterial DNA, we investigated whether the effects of T/HS lymph was related to endogenous nonmicrobial TLR4 ligands. High-mobility group box 1 protein 1, heat shock protein 70, heat shock protein 27, and hyaluronic acid all have been implicated in ischemia-reperfusion-induced tissue injury. None of these "danger" proteins appeared to be involved, because their levels were similar between the sham and shock lymph samples. In conclusion, TLR4 activation is important in T/HS-induced gut injury and in T/HS lymph-induced PMN priming and lung injury. However, the T/HS-associated effects of TLR4 on gut barrier dysfunction can be uncoupled from the T/HS lymph-associated effects of TLR4 on PMN priming.

Gut injury and loss of normal intestinal barrier function are key elements in the paradigm of gut-origin systemic inflammatory response syndrome, acute lung injury, and multiple organ dysfunction syndrome (MODS). As hypoxia-inducible factor (HIF-1) is a critical determinant of the physiological and pathophysiological response to hypoxia and ischemia, we asked whether HIF-1 plays a proximal role in the induction of gut injury and subsequent lung injury. Using partially HIF-1α-deficient mice in an isolated superior mesenteric artery occlusion (SMAO) intestinal ischemia reperfusion (I/R) injury model (45 min SMAO followed by 3 h of reperfusion), we showed a direct relationship between HIF-1 activation and intestinal I/R injury. Specifically, partial HIF-1α deficiency attenuated SMAO-induced increases in intestinal permeability, lipid peroxidation, mucosal caspase-3 activity, and IL-1β mRNA levels. Furthermore, partial HIF-1α deficiency prevented the induction of ileal mucosal inducible nitric oxide synthase (iNOS) protein levels after SMAO and iNOS deficiency ameliorated SMAO-induced villus injury. Resistance to SMAO-induced gut injury was also associated with resistance to lung injury, as reflected by decreased levels of myeloperoxidase, IL-6 and IL-10 in the lungs of HIF-1α+/− mice. In contrast, a short duration of SMAO (15 min) followed by 3 h of reperfusion neither induced mucosal HIF-1α protein levels nor caused significant gut and lung injury in wild-type or HIF-1α+/− mice. This study indicates that intestinal HIF-1 activation is a proximal regulator of I/R-induced gut mucosal injury and gut-induced lung injury. However, the duration and severity of the gut I/R insult dictate whether HIF-1 plays a gut-protective or deleterious role.

Acute lung injury (ALI) and the development of the multiple organ dysfunction syndrome (MODS) are major causes of death in trauma patients. Gut inflammation and loss of gut barrier function as a consequence of splanchnic ischemia-reperfusion (I/R) have been implicated as the initial triggering events that contribute to the development of the systemic inflammatory response, ALI, and MODS. Since hypoxia-inducible factor (HIF-1) is a key regulator of the physiological and pathophysiological response to hypoxia, we asked whether HIF-1 plays a proximal role in the induction of gut injury and subsequent lung injury. Utilizing partially HIF-1α-deficient mice in a global trauma hemorrhagic shock (T/HS) model, we found that HIF-1 activation was necessary for the development of gut injury and that the prevention of gut injury was associated with an abrogation of lung injury. Specifically, in vivo studies demonstrated that partial HIF-1α deficiency ameliorated T/HS-induced increases in intestinal permeability, bacterial translocation, and caspase-3 activation. Lastly, partial HIF-1α deficiency reduced TNF-α, IL-1β, cyclooxygenase-2, and inducible nitric oxide synthase levels in the ileal mucosa after T/HS whereas IL-1β mRNA levels were reduced in the lung after T/HS. This study indicates that prolonged intestinal HIF-1 activation is a proximal regulator of I/R-induced gut mucosal injury and gut-induced lung injury. Consequently, these results provide unique information on the initiating events in trauma-hemorrhagic shock-induced ALI and MODS as well as potential therapeutic insights.

Intestinal ischemia after trauma-hemorrhagic shock (T/HS) results in gut barrier dysfunction and the production/release of biologically active and tissue injurious factors in the mesenteric lymph, which, in turn, causes acute lung injury and a systemic inflammatory state. Since T/HS-induced lung injury is associated with pulmonary endothelial and epithelial cell programmed cell death (PCD) and was abrogated by mesenteric lymph duct ligation, we sought to investigate the cellular pathways involved. Compared with trauma-sham shock (T/SS) rats, a significant increase in caspase-3 and M30 expression was detected in the pulmonary epithelial cells undergoing PCD, whereas apoptosis-inducing factor (AIF), but not caspase-3, was detected in endothelial cells undergoing PCD. This AIF-mediated pulmonary endothelial PCD response was validated in an in situ femoral vein assay where endothelial cells were found to express AIF but not caspase-3. To complement these studies, human umbilical vein endothelial cell (HUVEC), human lung microvascular endothelial cell (HLMEC), and human alveolar type II epithelial cell (A549) lines were used as in vitro models. T/HS lymph induced the nuclear translocation of AIF in HUVEC and HLMEC, and caspase inhibition in these cells did not afford any cytoprotection. For proof of principle, AIF silencing in HUVEC reversed the cytotoxic effects of T/HS on cell viability and DNA fragmentation. In A549 cells, T/HS lymph activated caspase-3-mediated apoptosis, which was partially abrogated by N-benzyloxycarbonyl-Val-Ala-Asp (zVAD). Additionally, T/HS lymph did not cause the nuclear translocation of AIF in A549 cells. Collectively, T/HS-induced pulmonary endothelial PCD occurs via an AIF-dependent caspase-independent pathway, whereas epithelial cells undergo apoptosis by a caspase-dependent pathway.

The etiology of trauma-hemorrhagic shock (T/HS)-induced acute lung injury has been difficult to elucidate because of, at least in part, the inability of in vivo studies to separate the noninjurious pulmonary effects of trauma-hemorrhage from the tissue-injurious ones. To circumvent this in vivo limitation, we used a model of T/HS in which T/HS lung injury was abrogated by dividing the mesenteric lymph duct. In this way, it was possible to separate the pulmonary injurious response from the noninjurious systemic response to T/HS by comparing the pulmonary molecular responses of rats subjected to T/HS, which did and did not develop lung injury, with those of nonshocked rats. Using high-density oligonucleotide arrays and treatment group comparisons of whole lung tissue collected at 3 h after the end of the shock or sham-shock period, 139 of 8,799 assessed genes were identified by significant analysis of microarrays. Hemorrhage without the secondary effects of lung injury modulated the expression of 21 genes such as interleukin 1beta, metallothionein-2, and myeloctomatosis oncogene (c-myc). In response to injury, 42 genes were identified to be differentially expressed. Upregulated genes included the L1 retroposon and guanine deaminase, whereas downregulated genes included catalase and superoxide dismutase 1. Real-time polymerase chain reaction confirmed the differential expression for selected genes. PathwayAssist analysis identified interleukin 1beta as a central regulator of two subpathways of stress response-related genes (c-myc and superoxide dismutase 1/catalase) as well as several unrelated genes such as lipoprotein lipase. Our model system provided a unique opportunity to distinguish the molecular changes associated with T/HS-induced acute lung injury from the systemic molecular response to T/HS.

In both animal models of hemorrhagic shock and clinical settings, shock-induced gut ischemia has been implicated in the development of the systemic inflammatory response syndrome and distant organ injury, yet the factors transducing these events remain to be fully determined. Because hypoxia-inducible factor (HIF-1), a transcription factor composed of oxygen-labile HIF-1alpha and constitutive HIF-1beta subunits, regulates the physiologic/pathophysiologic response to hypoxia and ischemia, we examined the HIF-1 response in two rat models of gut ischemia-reperfusion. We found that ileal nuclear HIF-1alpha protein levels were induced in rats subjected to trauma (laparotomy) plus hemorrhagic shock for 90 min relative to their trauma sham-shock and naïve counterparts and that this trauma hemorrhagic shock-induced mucosal HIF-1alpha protein response persisted after 1 h and 3 h of reperfusion. Likewise, in a model of isolated gut ischemia-reperfusion injury, where the superior mesenteric artery was occluded for 45 min, nuclear HIF-1alpha were induced in the gut mucosa relative to their sham counterparts and persisted after 1 h and 3 h or reperfusion. Similar to the in vivo response, in vitro hypoxia induced HIF-alpha expression in three different enterocyte cell lines (rat IEC-6 and human Caco-2 and HT-29 cell lines). However, in contrast to the in vivo response, HIF-1 expression rapidly disappeared on subsequent reoxygenation. Because in vivo enterocytes are exposed to bacteria, we tested whether the in vitro HIF-1alpha response would persist on reoxygenation if the enterocytes were cocultured with bacteria. P. aeruginosa, an enteric bacterium, markedly induced enterocyte HIF-1alpha protein levels under normoxic conditions. Furthermore, the addition of P. aeruginosa during either the hypoxic or reoxygenation phase prevented the degradation of HIF-1alpha protein levels. Moreover, the observation that lipopolysaccharide induced HIF-1alpha expression in a time-dependent manner in IEC-6 cells indicated that the induction of HIF-1 by exposure to P. aeruginosa is not dependent on bacterial viability. In conclusion, these results suggest that HIF-1alpha activation is an early reperfusion-independent event in models of gut ischemia-reperfusion and that this HIF-1alpha response is potentiated by the presence of P. aeruginosa or lipopolysaccharide.

Purpose: Cytotoxic chemotherapy has been used to treat patients with metastatic colorectal cancer with limited success. Therefore novel chemotherapeutic approaches are needed. Based on encouraging preclinical data, there has been an interest in developing derivatives of butyrate as clinically applicable agents. The purpose of this study was to investigate the effects of phenylbutyrate (PB), a butyrate analogue, on the cell growth and apoptosis in a colon cancer cell model. Methods: Growth curves, flow cytometric studies, Western blotting, DNA binding assays and transient transfection experiments were performed in vitro using the colon cancer cell line HT-29 after exposure to PB. Results: Exposure of HT-29 colon cancer cells to PB resulted in growth inhibition and induction of apoptosis as measured by annexin V staining. This increase in apoptosis was associated with a decrease in mitochondrial membrane potential, an increase in caspase-3 activity and a decrease in intact PARP protein levels. Since NF-κB plays a pivotal role in the regulation of apoptosis, we explored the effects of PB on the DNA binding and transcriptional activity of this transcription factor. After PB treatment, NF-κB-DNA binding was markedly decreased and specifically, this decreased DNA binding was observed in the p50:p65 heterodimer. The decreased NF-κB DNA binding was observed as early as 3 h after PB treatment, while no apparent changes in annexin V binding were detected until 12 h after PB treatment. Untreated HT-29 cells transfected with a κB-luciferase reporter plasmid demonstrated significant constitutive activity of the κB binding site, which was markedly decreased after treating the cells with PB. Conclusion: These results suggest that PB-induced apoptosis may be partly regulated through the inactivation of NF-κB. PB, an oral butyrate analogue, may have therapeutic potential in colon cancer.

The etiology of graft-versus-host disease (GVHD) is rooted in the alloreactive response of donor T cells present in the hematopoietic graft resulting in the destruction of the patient's tissues, particularly the gastrointestinal tract. Gut GVHD affects up to 50% of patients, is a leading cause of death, and has overlapping features with inflammatory bowel disease (IBD). Increased gut permeability, alterations of the gut microbiome and intestinal stem cell niche damage predispose the gut to the local and systemic effects of GVHD, and is exacerbated by the inability of the gut to adequately regenerate. Severe shifts in metabolism and reduced oxygen (O2) availability in the inflamed gut are major underlying factors in the pathogenesis of IBD. Two related transcription factors, hypoxia-inducible factor-1 (HIF-1) and HIF-2, originally discovered as master regulators of the adaptive response to hypoxia, have been recently shown to be gut protective and promote mucosal healing in response to injury. Using a MHC mismatched B10.BR→B6 bone marrow transplant (BMT) model, we previously found that loss of intestinal epithelial (IE) HIF-1α or HIF-2α worsened survival compared to wild-type mice and exhibited increased GVHD-induced-histopathology. Thus, we hypothesized that HIF-1 and HIF-2 protects and repairs the gut from conditioning and GVHD-related damage. HIF-1 and HIF-2 are heterodimers consisting of an O2-labile HIF-1α or HIF-2α subunit respectively and a constitutively expressed HIF-1β subunit. The recent discovery that iron-dependent prolyl hydroxylase enzymes (PHD1-3) can trigger the O2-dependent proteasomal degradation of the HIF-1α subunit has led to the development of pan-PHD inhibitors (PHDi) that activate HIF-1 and HIF-2. PHDi such as dimethyloxallyl glycine (DMOG) and AKB-4924 have been shown to attenuate colitis and radiation-induced gut toxicity in animal models. We thus sought to determine whether PHDi could also ameliorate gut GVHD in the B10.BR→B6 BMT model. B6 mice were lethally irradiated (10Gy, split dose) and transplanted with 5x106 T-cell depleted bone marrow (BM) cells from B10.BR donors with 2x106 enriched T cells from spleens and lymph nodes (BM+T). B6 mice transplanted with only T cell depleted BM cells served as our non-GVHD control group. B6 mice were treated with AKB-4924 (AKB, 5 mg/kg) or vehicle (β-cyclodextrin) via intraperitoneal delivery, beginning 1 day prior to BMT and for 6 consecutive days post-BMT. Treatment with AKB prevented significant weight loss, compared to vehicle-treated mice, 7 days post-BMT (n=6, p Disclosures Peters: Aerpio Therapeutics: Employment, Equity Ownership.

The molecular mechanisms by which multiple myeloma (MM) cells evade glucocorticoid-induced apoptosis have not been delineated. Using a human IgAκ MM cell line (ARP-1), we found that dexamethasone (Dex)-induced apoptosis is associated with decreased NF-κB DNA binding and κB-dependent transcription. Both nuclear p50:p50 and p50:p65 NF-κB complexes are detected in ARP-1 cells by supershift electrophoretic mobility shift assay (EMSA). Dex-mediated inhibition of NF-κB DNA binding precedes a notable increase in annexin V binding, thereby indicating that diminished NF-κB activity is an early event in Dex-induced apoptosis. Overexpression of bcl-2 in ARP-1 cells prevents Dex-mediated repression of NF-κB activity and apoptosis. Sustained NF-κB DNA binding is also observed in two previously characterized Dex-resistant MM cell lines (RPMI8226 and ARH-77) that express moderate levels of endogenous bcl-2 and IκB proteins. In addition, enforced bcl-2 expression in ARP-1 cells did not prevent the augmentation of IκB protein by Dex. We also noted a possible association between Dex-mediated downregulation of NF-κB in freshly obtained primary myeloma cells and the patients’ responsiveness to glucocorticoid-based chemotherapy. Collectively, our data suggest that the protective effects of bcl-2 in MM cells act upstream in the NF-κB activation–signaling pathway and the potential use of NF-κB as a biomarker in progressive MM.

A tripartite domain of the immunoglobulin mu heavy-chain gene enhancer that activates transcription in B cells contains binding sites for PU.1, Ets-1, and a leucine zipper-containing basic helix-loop-helix factor. Because PU.1 is expressed only in B cells and macrophages, we tested the activity of a minimal mu enhancer fragment in macrophages by transient transfections. The minimal mu enhancer activated transcription in macrophages, and the activity was dependent on all three sites. Analysis of mutated enhancers, in which spacing and orientation of the ETS protein binding sites had been changed, suggested that the mechanisms of enhancer activation were different in B cells and macrophages. Thus, ETS protein binding sites may be combined in different ways to generate tissue-specific transcription activators. Despite the activity of the minimal enhancer in macrophages, a larger mu enhancer fragment was inactive in these cells. We propose that formation of the nucleoprotein complex that is formed on the minimal enhancer in macrophages cannot be helped by the neighboring muE elements that are essential for activity of the monomeric enhancer.

The failure to control both innate and adaptive immune responses in the gut has recently been implicated as a major pathogenic mechanism in the development of graft-versus-host disease (GVHD). Reduced oxygen availability in the intestine has been causally linked to gastrointestinal disease. During intestinal inflammation, increased metabolic activity of resident and infiltrating immune cells, bacteria and reduced blood flow may lead to a sharp decrease of oxygen, resulting in "inflammatory" hypoxia. The transcription factor family, hypoxia-inducible factor (HIF) originally discovered as a master regulator of the adaptive response to hypoxia, has recently emerged as a key regulator of the innate and adaptive immune responses. The HIF heterodimer consists of an oxygen-labile α subunit (HIFα) and a constitutively expressed HIF-1β subunit. Both HIF-1α and HIF-2α expression are markedly elevated in intestinal epithelial cells of patients with inflammatory bowel disease (IBD) and intestinal epithelial HIF-1 attenuates colitis in preclinical mouse models. Although HIF-2 has not been studied extensively in intestinal inflammation, it has emerged as a key regulator in intestinal iron homeostasis. Given that IBD and GVHD share many pathogenic mechanisms, we hypothesized that a sustained HIF response will protect the host intestinal epithelium from conditioning- and alloreactive T cell-induced gut damage. To determine the functional significance of intestinal epithelial HIF-1 and HIF-2 in gut GVHD, we generated conditional intestinal epithelial HIF-1α (HIF-1αΔIE) and HIF-2αΔIE vil-cre knockout (KO) mice on a C57BL/6 (B6) background lacking HIF-1α or HIF-2α in the host intestinal epithelium. Using a fully MHC mismatched B10.BR (H2k)→B6 (H2b) bone marrow transplant (BMT) model, loss of intestinal epithelial HIF-2 reduced the median survival time (43d) compared to wild-type (WT) recipients (58d, log-rank test, P < 0.005). Although intestinal epithelial HIF-1 deficiency shortened the median survival time (48.5d), it did not reach statistical difference. Loss of intestinal epithelial HIF-1 or HIF-2 worsened GVHD-induced histopathologic crypt damage compared to WT mice transplanted with T cell depleted bone marrow (BM) and enriched T cells (BM+T), 8d post- BMT. Pronounced subepithelial lifting, mucosal edema and sloughing were more evident in the villus tips of HIF-2αΔIE mice than HIF-1αΔIE mice. Hyperplastic crypts that are characteristic of regenerating crypts after radiation-induced damage were observed in Ki67-stained ileal/jejunal sections of WT mice post-BMT whereas fewer regenerating Ki67-labeled crypts were found in both HIF-1αΔIE and HIF-2αΔIE mice. In control T cell depleted BM groups (WT, HIF-1αΔIE and HIF-2αΔIE), Ki67+ -proliferating cells resided at the crypt base. Using quantitative real-time PCR analysis, we determined whether intestinal epithelial HIF-1 and HIF-2 differentially regulated the expression of Paneth cells and intestinal stem cell markers in the jejunum, 8d post-BMT. A 5-fold and 2-fold decrease in lysozyme (Lyz) mRNA levels occurred in WT (p Disclosures No relevant conflicts of interest to declare.

Expression of the low-affinity Fc receptor for IgG (murine Fc gamma RIIIA) is restricted to cells of myelomonocytic origin. We report here the promoter structure, the proximal DNA sequences responsible for transcription of Fc gamma RIIIA in macrophages and the protein factors which interact with these sequences. A 51 bp sequence, termed the myeloid restricted region (MRR), was both necessary and sufficient for conferring cell type-specific expression in macrophages. Reporter constructs containing mutations in this sequence result in the loss of MRR activity upon transfection into the macrophage cell line, RAW264.7. Two cis-acting elements have been identified and are required for full promoter function. These same elements analyzed by EMSA define two binding sites recognized by nuclear factors derived from macrophages. A 3' purine tract (-50 to -39) within the MRR binds the macrophage and B cell-specific factor, PU.1, and a second E box-like element, termed MyE, upstream of the PU.1 box (-88 to -78) binds the HLH factors TFE3 and USF. EMSA studies using RAW cell extracts suggest that both PU.1 and MyE factors may bind simultaneously to the MRR resulting in a ternary complex that is responsible, in part, for the myeloid-specific activity of the Fc gamma RIIIA promoter.

BackgroundInjurious non-microbial factors released from the stressed gut during shocked states contribute to the development of acute lung injury (ALI) and multiple organ dysfunction syndrome (MODS). Since Toll-like receptors (TLR) act as sensors of tissue injury as well as microbial invasion and TLR4 signaling occurs in both sepsis and noninfectious models of ischemia/reperfusion (I/R) injury, we hypothesized that factors in the intestinal mesenteric lymph after trauma hemorrhagic shock (T/HS) mediate gut-induced lung injury via TLR4 activation.Methods/principal findingsThe concept that factors in T/HS lymph exiting the gut recreates ALI is evidenced by our findings that the infusion of porcine lymph, collected from animals subjected to global T/HS injury, into naïve wildtype (WT) mice induced lung injury. Using C3H/HeJ mice that harbor a TLR4 mutation, we found that TLR4 activation was necessary for the development of T/HS porcine lymph-induced lung injury as determined by Evan's blue dye (EBD) lung permeability and myeloperoxidase (MPO) levels as well as the induction of the injurious pulmonary iNOS response. TRIF and Myd88 deficiency fully and partially attenuated T/HS lymph-induced increases in lung permeability respectively. Additional studies in TLR2 deficient mice showed that TLR2 activation was not involved in the pathology of T/HS lymph-induced lung injury. Lastly, the lymph samples were devoid of bacteria, endotoxin and bacterial DNA and passage of lymph through an endotoxin removal column did not abrogate the ability of T/HS lymph to cause lung injury in naïve mice.Conclusions/significanceOur findings suggest that non-microbial factors in the intestinal mesenteric lymph after T/HS are capable of recreating T/HS-induced lung injury via TLR4 activation.

Objective: To test the hypothesis that gut-derived factors carried in trauma-hemorrhagic shock (T/HS) lymph is sufficient to induce lung injury. Additionally, because our previous studies showed that T/HS-induced nitric oxide production was associated with lung injury, we examined whether T/HS lymph-induced lung injury occurs via an inducible nitric oxide synthase (iNOS)-dependent pathway. Background: We have previously shown that T/HS-induced lung injury is mediated by gut-derived humoral factors carried in the mesenteric lymph. However, it remains unclear whether T/HS lymph itself is sufficient to induce lung injury, or requires the activation of other factors during the T/HS period to exert its effect. Methods: Mesenteric lymph collected from T/HS or trauma-sham shock (T/SS) animals was injected intravenously into male rats at a rate of 1 mL/h for 3 hours. At the end of infusion, lung injury was assessed by lung permeability and lung histology. The effect of iNOS inhibition on T/HS lymph-induced lung injury was studied and this was further confirmed in iNOS knockout mice. Finally, iNOS immunohistochemistry was performed to identify the cells of origin of iNOS. Results: The injection of T/HS lymph, but not sham shock lymph, caused lung injury. This was associated with increased plasma nitrite/nitrate levels as well as induction of iNOS protein in the lung, liver, and gut. Treatment with the selective iNOS inhibitor aminoguanidine prevented T/HS lymph-induced lung injury. iNOS knockout mice, but not their wild-type controls, were resistant to T/HS lymph-induced lung injury. By immunohistochemistry, neutrophils and macrophages, rather than parenchymal cells, were the source of T/HS lymph-induced lung iNOS. Conclusions: These results indicate that T/HS lymph is sufficient to induce acute lung injury and that lymph-induced lung injury occurs via an iNOS-dependent pathway.

Acute graft-versus-host disease (GVHD) is a major cause of nonrelapse morbidity and mortality following allogeneic blood and marrow transplantation (BMT). The majority of studies delineating the mechanisms involved in GVHD have focused on the alloreactive T cell response and their downstream cellular and inflammatory effector phases. While these efforts have provided important mechanistic insights that have been translated into therapies that target the T cell compartment, they ignore the role that the target organ plays in both initiating and propagating this disease. Acute gut GVHD affects more than 60% of patients and is a leading cause of death. The mechanisms for the early, conditioning-related, intestinal injuries that are the precipitating event leading to GVHD or how subsequent gut-specific GVHD actually disrupts intestinal homeostasis have not been fully explored. The transcription factor, hypoxia inducible factor-1 (HIF-1) has emerged as a common denominator for hypoxia and inflammation. Given that inflammatory bowel disease (IBD) and GVHD share many pathogenic mechanisms and intestinal epithelial HIF-1α afforded protection in IBD models, we hypothesize that the persistent activation of HIF-1 will protect the intestinal epithelium from radiation/chemotherapy and alloreactive T cell-induced damage. Since crypt damage is a hallmark of gut GVHD, we first determined whether loss of intestinal epithelial HIF-1α impaired intestinal regeneration after GVHD damage using conditional HIF-1α (HIF-1αIE) knockout mice that lack HIF-1α in the intestinal epithelium. In a major MHC mismatched B10.BR (H2k) into B6 (H2b) GVHD model, 8 days post-BMT, histopathologic assessment of H&E stained ileums of HIF-1αIE mice showed more severe crypt damage, loss of Paneth cells and fewer regenerating crypts as compared to wild-type (WT) mice. A two-fold increase of aberrant mitoses (p Disclosures No relevant conflicts of interest to declare.

Having documented that proestrus female rats are more resistant to shock-induced acute gut and hence lung injury than male rats, we tested the hypothesis that the female gut is more resistant to injury and produces less of an inflammatory response than the male gut when exposed to conditions associated with shock states (hypoxia and acidosis) utilizing the ex vivo Ussing chamber system. Ileal mucosal membranes harvested from normal male and female rats mounted in Ussing chamber systems were exposed to normoxia or 40 min of hypoxia at a normal pH (pH 7.3) or acidosis (pH 6.8). Cytokine and nitric oxide levels in the serosal compartment of the Ussing chamber were measured at the end of the 3-h experimental period to assess the immunoinflammatory response, whereas FITC-dextran (mol wt 4,300) was employed to assess barrier function. Histomorphological changes were used to quantitate gut mucosal injury. Hypoxia, acidosis, or hypoxia plus acidosis was associated with a significant increase in proinflammatory cytokine production [interleukin (IL)-6, tumor necrosis factor, and macrophage inflammatory protein (MIP)-2] by the male compared with the female intestinal segments. In contrast, the female gut manifested a higher anti-inflammatory response (nitric oxide and IL-10) and improved intestinal barrier function as well as less evidence of mucosal injury than the male intestinal segments. Administration of estradiol or the testosterone receptor antagonist, flutamide, to male rats abrogated the increase in gut injury and the increased IL-6 and MIP-2 response observed after hypoxia plus acidosis. These results suggest that gender differences in the ex vivo intestinal response to stresses, such as hypoxia and acidosis, exist and that the administration of estradiol or blockade of the testosterone receptor to male rats mitigates these gender differences.

The purpose of this study was to investigate the anti-proliferative and pro-apoptotic effects of the butyrate analogues, tributyrin (TB) and phenylbutyrate (PB), in a colon cancer model. We demonstrate that HT-29 colon cancer cells exposed to PB and TB result in growth inhibition associated with an induction of apoptosis mediated through the activation of caspase-3 activity. A block in the G1/S cell cycle traverse associated with a decrease in CDK2 (cyclin dependent kinase) protein levels and retinoblastoma protein hypophosphorylation was also noted after PB and TB exposure. Importantly, TB proved to be the most potent agent in its ability to induce these phenotypic changes, and potentially may represent a novel therapy for patients with advanced colorectal cancer.

Specific cytogenetic abnormalities have been identified in multiple myeloma that confer a poor prognosis, even with intensive chemotherapy and autotransplants. The identification and characterization of potential genes involved in these different chromosomal changes and their interplay with oncogenes and tumor suppressor genes controlling cellular growth and apoptosis is the major focus of this review.

The intestinal epithelium is a primary target of graft-versus-host disease (GVHD). The hypoxia-signaling pathway has been implicated as an adaptive response in the intestinal epithelium in numerous models of inflammatory bowel disease, such as colitis and T-cell induced diarrhea. The transcription factor family, hypoxia-inducible factor (HIF) has emerged as master regulators of the transcriptional response to hypoxic stress in normal and transformed cells. HIF heterodimers consist of an oxygen-labile α subunit (HIF-1α, HIF-2α) and a constitutively expressed HIF-1β subunit that mediate a wide spectrum of physiological and cellular adaptive responses, including angiogenesis, metabolic adaption, and erythropoiesis. HIF-1 has recently been implicated as a gut-protective factor in inflammatory bowel disease models by maintaining intestinal homeostasis. HIF-1 can also skew the differentiation of T cells to regulatory T cells (Treg) via the induction of FoxP3, thereby attenuating T-cell driven colitis. Although HIF-1 and HIF-2 share many overlapping functions, HIF-1 has been implicated in the inflammatory phenotype of M1 macrophages via inducible nitric oxide synthase (iNOS) whereas HIF-2 is involved in the anti-inflammatory phenotype of M2 macrophages via arginase-1 (Arg1). Based on these findings and that mucosal inflamed tissues are hypoxic, we hypothesized that the induction of the hypoxia-signaling pathway may limit GVHD-induced mucosal inflammation and injury. To determine the adaptive roles of HIF-1 and HIF-2 in GVHD, we first tested the major histocompatibility complex (MHC)-haploidentical C567BL/6 (B6,H2b) -> B6xDBA/2 (B6D2)F1 (H2b/d) model in which donor spenocytes (2x107) and anti-Thy1 + C’ treated (T- cell depleted) bone marrow (ATBM) cells (5x106) are transplanted into B6D2F1 recipients after exposure to lethal irradiation (11Gy, split dose). B6 ATBM cells transplanted alone into B6D2F1 mice served as a negative control for all comparisons. Realtime PCR analysis demonstrated a modest increase in ileal mucosal HIF-2α expression 8 days (d) post-transplant (pBALB.B and B6->CXB-2 models. However, after 20d post-transplant, a 4- and 3-fold decrease in Arg1 mRNA levels occurred in both models. Likewise, two anti-inflammatory, Treg-associated cytokines, interleukin-10 (IL-10) and transforming growth factor beta (TGFβ) mRNA expression were elevated by 2-and 7-fold, respectively, after d8 in B6->BALB.B mice. TGFβ levels returned to baseline (p0.001) in a B6 anti-B6D2F1 mixed lymphocyte reaction. Taken together, our data suggest that HIF-2/Arg1 axis confers an anti-inflammatory response in the ileum after 8d of GVHD. However, after 20d, this response is inversely correlated with the lethality of the GVHD response. The amelioration of alloreactivity by DMOG suggests that the persistent activation of HIF may be necessary to dampen GVHD. Further studies will delineate the contribution of the HIF-2 response in the maintenance of intestinal homeostasis and limiting T cell alloreactivity. Disclosures: Zilberberg: Onyx Pharmaceuticals: Research Funding. Dziopa:Onyx Pharmaceuticals: Research Funding.

A 39-nt DNA sequence, the interferon gamma (IFN-gamma) response region (GRR), is necessary for the IFN-gamma-induced transcription of the high-affinity Fc receptor for IgG (Fc gamma RI) and sufficient for the IFN-gamma-induced transcription of transfected plasmids. By using extracts from IFN-gamma-treated cells, three protein complexes will assemble in vitro on a 9-nt core region in the 3' domain of the GRR. The sequence of this core resembles the IFN-gamma-activated sequence (GAS) described for the GBP gene. Mutations in this GAS core region prevent complex assembly and result in the loss of IFN-gamma induction of reporter constructs containing the mutation. In addition to the GAS core region, a 5' region of the GRR is necessary for optimal IFN-gamma induction and for formation of one of the DNA-protein complexes. By antibody reactivity, we show that a 91-kDa protein, first identified as a component of ISGF3, the IFN-alpha-induced transcription complex, is present in at least two of the DNA-protein complexes. IFN-alpha can induce the formation of the faster-migrating 91-kDa protein-GAS complex but not the slower-migrating complex. Furthermore, IFN-alpha does not result in appreciable transcriptional activation of Fc gamma RI or constructs containing the GRR. Thus, these data demonstrate that the IFN-gamma-activated 91-kDa protein is required for IFN-gamma induction of Fc gamma RI and suggest that an additional complex may be required for optimal expression and specificity.

Expression of the high-affinity receptor for IgG (Fc gamma RI) is restricted to cells of myeloid lineage and is induced by gamma-interferon (IFN-gamma) but not by IFN-alpha/beta. The organization of the human Fc gamma RI gene has been determined and the DNA elements governing its cell type-restricted transcription and IFN-gamma induction are reported here. A 39-nucleotide sequence (IFN-gamma response region, or GRR) is defined that is both necessary and sufficient for IFN-gamma inducibility. Sequence analysis of the GRR reveals the presence of promoter elements initially defined for the major histocompatibility complex class II genes: i.e., X, H, and gamma-IRE sequences. Comparison of a number of genes whose expression is induced selectively by IFN-gamma indicates that the presence of these elements is a general feature of IFN-gamma-responsive genes. Our studies suggest that the combination of X, H, and gamma-IRE elements is a common motif in the pathway of transcriptional induction by this lymphokine.

Given that hypoxia and insulin-like growth factor-1 (IGF-1) induce hypoxia inducible factor (HIF-1)-dependent vascular endothelial growth factor (VEGF) expression in different cancer models, we began to explore the possibility that HIF-1α may act as a proliferative and survival factor in multiple myeloma cells. In this study, we demonstrate that MM cells express constitutive HIF-1α protein levels and that hypoxia and cobalt chloride, a hypoxia mimetic, increase HIF-1α and VEGF expression in several MM cell lines (ARP-1, MM1.S, RPMI8266). In the presence of IGF-1, maximal induction of HIF-1α expression in MM cells under normoxic conditions occurred after 12 hr of IGF-1 and declined thereafter. In contrast to hypoxia which inhibits the proteasomal degradation of HIF-1α, IGF-1 requires de novo protein synthesis of HIF-1α. Pharmacologic inhibition of PI3K/Akt or MAPK signaling pathways prevented IGF-1 induced HIF-1α and VEGF expression in ARP-1 cells. Since 2-methoxyestradiol (2-ME) has been shown to inhibit MM growth and associated angiogenesis, we examined whether 2-ME would inhibit the accumulation of IGF-1 induced HIF-1α expression. 2-ME markedly reduced constitutive and IGF-1-induced HIF-1α protein levels in ARP-1 cells. To further demonstrate that HIF-1α plays a role in the growth and survival of MM cells, RNA interference was used to inhibit HIF-1α expression. in ARP-1 cells. Nucleofection of ARP-1 cells with small interfering RNA (siRNA) targeting HIF-1α inhibited constitutive HIF-1α as well as IGF-1-induced HIF-1α protein levels. HIF-1β protein levels were unaffected. Consequently, the suppression of HIF-1α inhibited the proliferation of untreated and IGF-1 treated ARP-1 cells. More interestingly, IGF-1 did not fully overcome the growth inhibitory effect of dexamethasone in ARP-1 cells that were nucleofected with HIF-1 siRNA. Taken together, our results suggest that HIF-1 plays a role in the pathophysiology of MM.

Tumor necrosis factor (TNF) is a monocyte-derived protein cytotoxic or cytostatic for some tumor cell lines. Here we show that highly purified E. coli-derived recombinant human TNF stimulated the growth of human FS-4 diploid fibroblasts. Stimulation of cell growth was demonstrable at a TNF concentration of 10 pg/ml (3 X 10(-13) M). Maximal stimulation was attained at TNF concentrations of 10 ng/ml (3 X 10(-10) M) or higher. Growth-stimulatory activity of TNF was inhibited by an mAb neutralizing the cytotoxic activity of TNF. Growth stimulation was not inhibited by another mAb specific for TNF, lacking neutralizing activity for the cytotoxic activity of TNF. Growth stimulation by TNF was more marked and more sustained in the presence of greater than or equal to 10% FCS than in medium with less than or equal to 5% FCS. Addition of TNF to confluent FS-4 cultures also produced a marked stimulation of cell growth in the presence of fresh FCS, while a much less marked stimulation was seen in the absence of FCS. Stimulation of confluent cultures by TNF in serum-free medium was enhanced by insulin, suggesting that insulin or insulin-like growth factor(s) in the serum can act synergistically with TNF in producing growth stimulation. While the growth-stimulatory effects of TNF and insulin were synergistic, the actions of TNF and epidermal growth factor (EGF) were less than additive, suggesting that TNF and EGF may activate identical or similar pathways. We conclude that stimulation of cell growth is probably a physiological function of TNF, and that the cytotoxic and cytostatic actions of TNF may be the result of an anomalous growth signal transduction in neoplastic cells lacking the constraints of normal growth control mechanisms.

Receptors for tumor necrosis factor (TNF) were characterized in the U-937 human histiocytic lymphoma cell line with the aid of highly purified recombinant human TNF, radiolabeled with 125I. Saturation binding to specific cell surface receptors occurred with less than 15% nonspecific binding. Analysis of the equilibrium binding data obtained at 4 degrees C revealed a single class of noninteracting binding sites. The mean number of binding sites per cell was calculated to be 12,000, and the apparent dissociation constant (Kd) was 2 X 10(-10) M. Crosslinking of 125I-TNF to the cell surface receptor with disuccinimidyl suberate, followed by NaDodSO4-polyacrylamide gel electrophoresis of the cell lysate, revealed a TNF-receptor complex with a molecular weight of approximately 100,000. Binding to concanavalin A-Sepharose suggested that the TNF receptor is a glycoprotein.

Allogeneic blood and marrow transplantation (allo-BMT) is an effective immunotherapeutic treatment that can provide partial or complete remission for patients with hematological malignancies. Mature donor T cells in the donor inoculum play a central role in mediating graft-versus-tumor (GVT) responses by destroying residual tumor cells that persist after conditioning regimens. Alloreactivity towards minor histocompatibility antigens (miHA), which are varied tissue-related self-peptides presented in the context of major histocompatibility complex (MHC) molecules on recipient cells, some of which may be shared on tumor cells, is a dominant factor for the development of GVT. Potentially, GVT can also be directed to tumor-associated antigens or tumor-specific antigens that are more specific to the tumor cells themselves. The full exploitation of allo-BMT, however, is greatly limited by the development of graft-versus-host disease (GVHD), which is mediated by the donor T cell response against the miHA expressed in the recipient's cells of the intestine, skin, and liver. Because of the significance of GVT and GVHD responses in determining the clinical outcome of patients, miHA and tumor antigens have been intensively studied, and one active immunotherapeutic approach to separate these two responses has been cancer vaccination after allo-BMT. The combination of these two strategies has an advantage over vaccination of the patient without allo-BMT because his or her immune system has already been exposed and rendered unresponsive to the tumor antigens. The conditioning for allo-BMT eliminates the patient's existing immune system, including regulatory elements, and provides a more permissive environment for the newly developing donor immune compartment to selectively target the malignant cells. Utilizing recent technological advances, the identities of many human miHA and tumor antigenic peptides have been defined and are currently being evaluated in clinical and basic immunological studies for their ability to produce effective T cell responses. The first step towards this goal is the identification of targetable tumor antigens. In this review, we will highlight some of the technologies currently used to identify tumor antigens and anti-tumor T cell clones in hematological malignancies.

The mechanism of human peripheral blood monocyte-mediated cytotoxicity was investigated using the HT-29 human colon adenocarcinoma line, A673 human rhabdomyosarcoma line, and A375 human melanoma line as target cells. Pretreatment of these target cells with 100 U/ml of recombinant human interferon (IFN)-gamma for 48 hr increased their susceptibility to monocyte killing. Increased susceptibility to the lytic action was particularly pronounced at low effector/target cell ratios. Unlike IFN-gamma human IFN-alpha did not potentiate monocyte cytotoxicity, and pretreatment of HT-29 with IFN-alpha also had virtually no effect on their susceptibility to monocyte killing. However, IFN-gamma appeared to prime either monocytes or target cells to become responsive to IFN-alpha. Our data suggest that IFN-gamma can promote the killing of tumor cells by monocytes through two separate actions, one on the monocyte and one on the target cell.