Nature Medicine

Published by Springer Nature

Online ISSN: 1546-170X


Print ISSN: 1078-8956


Gene Expression in Space
  • Article
  • Full-text available

May 1999


746 Reads


F C Lewis





J H Kaysen

Cells in space

April 1997


17 Reads

How does one treat in a seriously injured astronaut in outer space or even another planet? To answer such a question, the US National Aeronautical Space Administration (NASA) has embarked on a program of growing tissues--and possibly whole organs--in space. NASA has developed a unique rotating bioreactor that allow cells to be grown in a microgravity environment that eliminates almost all shear forces placed upon a cell culture system while entering space. Back on earth, this novel bioreactor has led to exciting discoveries and applications by scientists trying to get cells to differentiate and form their natural three-dimensional tissue matrices--the holy grail of tissue engineers. NASA's bioreactor has allowed various labs to culture cells and even viruses previously impossible to grow using traditional methods. These successes are attributed to the bioreactor's ability to provide an unique environment that closely resembles tissue differentiation during embryogenesis, and thus allowing cellular expression of surface epitopes similar to that of intact tissues. It also appears that cells grown in a microgravity, low-shear environment allows for greater chemical signaling, probably as a result of more surface contact between cells. Realizing the bioreactor's commercial potential, Santa Monica, California-based VivoRx licensed exclusive rights from NASA for both therapeutic and diagnostic commercial applications. VivoRx has, in the past, successfully transplanted encapsulated islet cells from cadavers and porcine pancreas into insulin-dependent diabetics, perhaps a major breakthrough in the treatment of diabetes. However, pancreas from cadavers are in very short supply. The bioreactor may be the answer; VivoRx hopes the bioreactor will allow them to propagate enough human islet cells to use their cell-based approach to treat a large diabetic population. The company has already successfully grown islet cells generated from the bioreactors, and is beginning FDA-approved Phase I/II clinical trials.

Khuri FR, Nemunaitis J, Ganly I, Arseneau J, Tannock IF, Romel L, Gore M, Ironside J, MacDougall RH, Heise C, Randlev B, Gillenwater AM, Bruso P, Kaye SB, Hong WK, Kirn DHA controlled trial of intratumoral ONYX-015, a selectively replicating adenovirus, in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer. Nat Med 6(8): 879-885

September 2000


48 Reads

ONYX-015 is an adenovirus with the E1B 55-kDa gene deleted, engineered to selectively replicate in and lyse p53-deficient cancer cells while sparing normal cells. Although ONYX-015 and chemotherapy have demonstrated anti-tumoral activity in patients with recurrent head and neck cancer, disease recurs rapidly with either therapy alone. We undertook a phase II trial of a combination of intratumoral ONYX-015 injection with cisplatin and 5-fluorouracil in patients with recurrent squamous cell cancer of the head and neck. There were substantial objective responses, including a high proportion of complete responses. By 6 months, none of the responding tumors had progressed, whereas all non-injected tumors treated with chemotherapy alone had progressed. The toxic effects that occurred were acceptable. Tumor biopsies obtained after treatment showed tumor-selective viral replication and necrosis induction.

Table 1 Replication of dl1520 in tumor cells in relation to p14 ARF and p53 status a
Loss of p14ARF in tumor cells facilitates replication of the adenovirus mutant dl1520 (ONYX-015)

November 2000


104 Reads

The adenovirus mutant dl1520 (ONYX-015) does not express the E1B-55K protein that binds and inactivates p53. This virus replicates in tumor cells with mutant p53, but not in normal cells with functional p53. Although intra-tumoral injection of dl1520 shows promising responses in patients with solid tumors, previous in vitro studies have not established a close correlation between p53 status and dl1520 replication. Here we identify loss of p14ARF as a mechanism that allows dl1520 replication in tumor cells retaining wild-type p53. We demonstrate that the re-introduction of p14ARF into tumor cells with wild-type p53 suppresses replication of dl1520 in a p53-dependent manner. Our study supports the therapeutic use of dl1520 in tumors with lesions within the p53 pathway other than mutation of p53.

MUC1 cross-reactive Gal alpha(1,3)Gal antibodies in humans switch immune responses from cellular to humoral

March 1998


52 Reads

Successful tumor immunotherapy with peptides requires the induction of cytotoxic T lymphocytes (CTLs) rather than antibodies. Mice immunized with mannan conjugated to MUC1, a peptide found in large amounts in breast cancer, develop CTL responses. In contrast, immunized patients produce high antibodies with poor CTL responses to MUC1. Here, we provide evidence that this "switch" in the immune response is due to the fact that antibodies against the Gal alpha(1,3)Gal epitope, which are normally present in humans but not mice, cross-react with MUC1 peptides. In particular, mice that lack the gene for the epitope (and that produce anti-Gal antibodies) (Gal-/- mice) are like humans in their response to MUC1 immunization in that they develop antibody rather than CTL responses. After we exposed macrophages from Gal-/- mice in vitro to MUC1, in the absence of Gal antibody, and adoptively transferred them into the mice, Gal-/- mice produced a predominantly CTL response. The findings are of relevance for immunotherapy studies in humans and emphasize the differences seen in preclinical testing in rodents before clinical trials.

Table 1 Peripheral opioid analgesia: comparison of experimental and clinical studies 
Stein C, Schafer M, Machelska HAttacking pain at its source: new perspectives on opioids. Nat Med 9:1003-1008

September 2003


1,998 Reads

The treatment of severe pain with opioids has thus far been limited by their unwanted central side effects. Recent research promises new approaches, including opioid analgesics acting outside the central nervous system, targeting of opioid peptide-containing immune cells to peripheral damaged tissue, and gene transfer to enhance opioid production at sites of injury.

Lewis DA, Gonzalez-Burgos G. Pathophysiologically based treatment interventions in schizophrenia. Nat Med 12: 1016-1022

October 2006


1,163 Reads

Identifying the molecular alterations that underlie the pathophysiology of critical clinical features of schizophrenia is an essential step in the rational development of new therapeutic interventions for this devastating illness. Cognitive deficits, such as the impairments in working memory that arise from dysfunction of the dorsolateral prefrontal cortex, are a major determinant of functional outcome in schizophrenia. Here we consider the contributions of disturbances in glutamate, dopamine and GABA neurotransmission to the pathophysiology of working memory impairments in schizophrenia, suggest a cascade of molecular events that might link these disturbances, and argue that the molecular alterations most proximal to the pathophysiology of prefrontal dysfunction offer the most promise as targets for new drug development.

Figure 1: Localization of tumor cells in the subpleural pulmonary microvessels. a, Structure of the subpleural vasculature in the mouse lung. A, arteriole; V, post-capillary venule; C, capillaries; ALV, alveolus. b, Fluorescence overlay image of endothelium stained with DiI-acetylated LDL (red) and a GFP-expressing tumor cell (green). c and d, Endothelium-attached GFP-expressing tumor cells in mouse lung pre-capillary arterioles (c) and in a capillary (d), 4−6 h after intravenous injection. e and f, Endothelium-attached GFP-expressing tumor cells in a pre-capillary arteriole (e) and in a capillary ( f), 10 min after perfusate administration in isolated rat lung. Scale bars represent 50 m (a) and 32 m (b−f, shown in b).
Table 1 Fate of tumor cells in the pulmonary circulation after intravenous injection
Al-Mehdi AB, Tozawa K, Fisher AB, Shientag L, Lee A, Mushel RJIntravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metasasis. Nat Med 6: 100-102

January 2000


130 Reads

Metastasis is a frequent complication of cancer, yet the process through which circulating tumor cells form distant colonies is poorly understood. We have been able to observe the steps in early hematogenous metastasis by epifluorescence microscopy of tumor cells expressing green fluorescent protein in subpleural microvessels in intact, perfused mouse and rat lungs. Metastatic tumor cells attached to the endothelia of pulmonary pre-capillary arterioles and capillaries. Extravasation of tumor cells was rare, and it seemed that the transmigrated cells were cleared quickly by the lung, leaving only the endothelium-attached cells as the seeds of secondary tumors. Early colonies were entirely within the blood vessels. Although most models of metastasis include an extravasation step early in the process, here we show that in the lung, metastasis is initiated by attachment of tumor cells to the vascular endothelium and that hematogenous metastasis originates from the proliferation of attached intravascular tumor cells rather than from extravasated ones. Intravascular metastasis formation would make early colonies especially vulnerable to intravascular drugs, and this possibility has potential for the prevention of tumor cell attachment to the endothelium.

Fahmy RG, Dass CR, Sun LQ, Chesterman CN, Khachigian LM.. Transcription factor Egr-1 supports FGF-dependent angiogenesis during neovascularization and tumor growth. Nat Med 9: 1026-1032

September 2003


52 Reads

Current understanding of key transcription factors regulating angiogenesis is limited. Here we show that RNA-cleaving phosphodiester-linked DNA-based enzymes (DNAzymes), targeting a specific motif in the 5' untranslated region of early growth response (Egr-1) mRNA, inhibit Egr-1 protein expression, microvascular endothelial cell replication and migration, and microtubule network formation on basement membrane matrices. Egr-1 DNAzymes blocked angiogenesis in subcutaneous Matrigel plugs in mice, an observation that was independently confirmed by plug analysis in Egr-1-deficient animals, and inhibited MCF-7 human breast carcinoma growth in nude mice. Egr-1 DNAzymes suppressed tumor growth without influencing body weight, wound healing, blood coagulation or other hematological parameters. These agents inhibited endothelial expression of fibroblast growth factor (FGF)-2, a proangiogenic factor downstream of Egr-1, but not that of vascular endothelial growth factor (VEGF). Egr-1 DNAzymes also repressed neovascularization of rat cornea. Thus, microvascular endothelial cell growth, neovascularization, tumor angiogenesis and tumor growth are processes that are critically dependent on Egr-1.

Figure 1: Overview of wound repair and fibrosis.Epithelial and/or endothelial damage caused by various insults triggers complex interconnected wound-healing programs to quickly restore homeostasis. The coagulation pathway, which functions to stem blood loss, is triggered first, followed by acute inflammation and activation of innate immune mediators such as resident macrophages, neutrophils and dendritic cells. Epithelial and innate immune cell–derived cytokines subsequently influence the activation of the adaptive immune response. The tissue damage can also directly activate the adaptive immune response. Inflammatory and immune mediators (cytokines, chemokines and free radicals) attempt to eliminate the inciting factor while activating the resident quiescent fibroblasts into myofibroblasts that orchestrate angiogenesis and production of ECM components. Failure to adequately contain or eliminate the inciting factors can exacerbate the inflammatory response and lead to a chronic wound-healing response, with continued tissue damage, repair and regeneration, ultimately resulting in fibrosis. TSLP, thymic stromal lymphopoietin; Ab, antibody; PMN, polymorphonuclear leukocyte; EOS, eosinophil; Baso, basophil; Mast, mast cell.
Figure 2: Innate immune cells in fibrosis.The macrophage is the prototypical innate immune cell involved in chronic inflammation and fibrosis. Macrophages are generated from blood monocytes that differentiate into macrophages as they enter tissues or, in some cases, from the local proliferation of tissue-resident macrophages. Depending on their etiology, macrophages are activated by a variety of triggers. IFN-γ and/or Toll-like receptor ligands such as lipopolysaccharide (LPS) and low molecular weight hyaluronic acid (LMWHA) lead to classical activation, which is characterized by the production of reactive oxygen and nitrogen species; IL-4, IL-13 and granulocyte-macrophage colony–stimulating factor (GM-CSF) mediate alternative activation, leading to the production of polyamines and L-proline by L-arginine catabolism. Certain triggers (such as extracellular bacteria and tissue damage) also elicit persistent or recurrent neutrophil infiltration mediated by IL-17 and other neutrophil-recruiting and/or neutrophil-activating signals that can substantially augment the microbicidal and tissue-damaging activities by free radicals. Likewise, helminth antigens and allergens mediate infiltration of eosinophils that assist in parasite killing that can result in substantial collateral damage to host tissues if not tightly confined inside fibrotic granulomata. EPO, erythropoietin; MBP, myelin basic protein; EDN, eosinophil-derived neurotoxin; AAM, alternatively activated macrophage; MMP2, metalloproteinase-2; MMP9, metalloproteinase-9; CAM, classically activated macrophage; iNOS, inducible nitric oxide synthase; ROS, reactive oxygen species; RNS, reactive nitrogen species.
Figure 3: Adaptive immune pathways in fibrosis.Naive CD4+ T cells differentiate into various distinct functional lineages driven by cues produced by injured epithelial cells and activated antigen presenting cells (dendritic cells and macrophages). Intracellular infections trigger IL-12–driven TH1 responses that produce IFN-γ, which activates microbicidal and cytotoxic activities that aid in pathogen clearance. Extracellular bacteria and certain fungi can lead to inflammasome activation and IL-6 production and can, in the presence of TGF-β1, drive TH17 differentiation. IL-17 from TH17 cells helps recruit neutrophils to clear the infection and exacerbates inflammation. Infection with extracellular, tissue-dwelling helminth parasites drives TH2 differentiation, with IL-4, IL-25, IL-33 and thymic stromal lymphopoietin (TSLP) from innate and epithelial sources guiding the differentiation of CD4+ TH2 cells. IL-13, when not competed for by the higher-affinity decoy receptor IL-13Rα2, binds its signaling receptor IL-13Rα1, leading to alternative activation of macrophages as well as epithelial apoptosis and myofibroblast activation. Treg cells are crucial in limiting the magnitude of TH cell responses and thereby ensure proper regulation of the wound-healing response. There is also a great deal of cross-regulation among TH cell subsets. For example, IL-13 suppresses TH17 differentiation, whereas IFN-γ can suppress IL-13–induced fibrosis by inducing classical macrophage activation and suppressing IL-13 and TGF-β1–induced collagen synthesis in myofibroblasts. Rx, pathways being targeted for therapy, either preclinically or in clinical trials; iNOS, inducible nitric oxide synthase; ROS, reactive oxygen species; PMN, polymorphonuclear leukocyte; MUC5AC, mucin-5AC.
Figure 4: EMT in fibrosis.In mouse models of renal and hepatic fibrosis, up to 40% of α-SMA–positive, collagen-secreting myofibroblasts have been shown to arise from the differentiation of local epithelial progenitors via EMT. Repression of the transcription factors Snail1, Snail2, Zeb1 and Zeb2 are important for the maintenance of epithelial morphology. Several factors that are upregulated in the context of inflammation, including nuclear factor-κB (NF-κB), TGF-β1, bone morphogenetic proteins (BMPs), Wnt and Notch signaling proteins, can activate the Snail-Zeb pathway, leading to mesenchymal differentiation in these cells. Rx, pathways being targeted for therapy, either preclinically or in clinical trials; HA, hyaluronic acid; FSP-1, fibroblast-specific protein-1.
Wynn TA, Ramalingam TR.Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat Med 18:1028-1040
Fibrosis is a pathological feature of most chronic inflammatory diseases. Fibrosis, or scarring, is defined by the accumulation of excess extracellular matrix components. If highly progressive, the fibrotic process eventually leads to organ malfunction and death. Fibrosis affects nearly every tissue in the body. Here we discuss how key components of the innate and adaptive immune response contribute to the pathogenesis of fibrosis. We also describe how cell-intrinsic changes in important structural cells can perpetuate the fibrotic response by regulating the differentiation, recruitment, proliferation and activation of extracellular matrix-producing myofibroblasts. Finally, we highlight some of the key mechanisms and pathways of fibrosis that are being targeted as potential therapies for a variety of important human diseases.

Masuzaki H, Ogawa Y, Sagawa N, Hosoda K, Matsumoto T, Mise H, Nishimura H, Yoshimasa Y, Tanaka I, Mori T, and Nakao K. Nonadipose tissue production of leptin: Leptin as a novel placenta-derived hormone in humans. Nat Med 3: 1029-1033

October 1997


133 Reads

Leptin is a circulating hormone that is expressed abundantly and specifically in the adipose tissue. It is involved in the regulation of energy homeostasis, as well as the neuroendocrine and reproductive systems. Here, we demonstrate production of leptin by nonadipose tissue, namely, placental trophoblasts and amnion cells from uteri of pregnant women. We show that pregnant women secrete a considerable amount of leptin from the placenta into the maternal circulation as compared with nonpregnant obese women. Leptin production was also detected in a cultured human choriocarcinoma cell line, BeWo cells, and was augmented during the course of forskolin-induced differentiation of cytotrophoblasts into syncytiotrophoblasts. Plasma leptin levels were markedly elevated in patients with hydatidiform mole or choriocarcinoma and were reduced after surgical treatment or chemotherapy. Leptin is also produced by primary cultured human amnion cells and is secreted into the amniotic fluid. The present study provides evidence for leptin as a novel placenta-derived hormone in humans and suggests the physiologic and pathophysiologic significance of leptin in normal pregnancy and gestational trophoblastic neoplasms.

Figure 1: Effects of LPS on Mrp14-/- mouse bone marrow cells (BMCs).(a) The expression of 15 representative genes as analyzed by microarray analysis of RNA from BMCs of wild-type mice and Mrp14-/- mice in the absence (n = 5) or presence (n = 3) of 1 g/ml LPS for 4 h. Error bars represent fold changes after LPS stimulation compared to controls (mean values s.d.). (b) Surface expression of TLR4 in the phagocyte fraction of BMCs treated with 1 g/ml LPS (right) for 4 h or left untreated (left), as assessed by flow cytometry. Specific profiles for wild-type cells (thick lines) show an identical surface expression of TLR4 as compared to that in Mrp14-/- cells (broken lines; isotype controls are presented as thin lines). (c) TNF- concentrations in the supernatants of BMCs treated with 1 g/ml LPS. Graphs show the means and s.d. of quadruplicates of one of three representative experiments (*P 0.05). (d) Stability of TNF- mRNA in cells that were treated with LPS (1 g/ml) for 2 h and then subjected to transcriptional block with 4 M actinomycin D. RNA was extracted at 0, 1, 2 and 4 h after addition of actinomycin D. TNF- mRNA abundance as detected by PCR. RNA loading onto blots was normalized to the amounts of TNF- mRNA at time point 0 (100%) in wild-type (black circles) and Mrp14-/- cells (open circles). (e) Transcription factor DNA-binding activity to the TNF- promoter in BMCs (5 106 cells per sample) after 2-h treatment with LPS (1 g/ml), as analyzed by ChIP (using CREM-specific antibody, c-Jun–specific antibody, p50-specific antibody and p65-specific antibody). The TNF- promoter binding sites of transcription factors NF-B, AP1 and CRE are indicated below. PCR products were run on a 1.5% agarose gel including a DNA input control (top) and quantified densitometrically (middle). Shown are the mean densitometric values from three independent ChIP assays on the NF-B factors p50 (0.51 0.11) and p65 (0.56 0.08). Each value is adjusted to the quotient of the mean densitometric values of input DNA from wild-type and Mrp14-/- cells (set as 1) to account for the small differences in input DNAs.
Figure 5: Binding of Mrp8 to the TLR4 signaling complex.(a) Mrp8 and LPS induce NF-B promoter activity in a HEK293 cell line stably transfected with TLR4, CD14 and MD2 and transiently transfected with the 3X NF-B promoter luciferase plasmid. Twenty-four hours after transfection, cells were stimulated with 5 g/ml Mrp8 or 100 ng/ml LPS for 4 h (in the presence or absence of 25 g/ml polymyxin B) and assayed for luciferase activity. Data represent mean values s.d. of three independent transfections and are given as the ratio of luciferase activity relative to unstimulated cells (**P 0.01). (b) TNF- mRNA (left) and IL8 mRNA (right) abundance in cells treated with either 5 g/ml Mrp8 or 100 ng/ml LPS for 2 h, as detected by RT-PCR using GAPDH, RPL and B2M as housekeeping gene internal controls. Bars represent fold induction compared with controls (mean values s.d., *P 0.05). (c) Binding of TLR4-MD2 with immobilized Mrp8 as determined by surface plasmon resonance analysis (BIAcore). Sensorgrams show association and dissociation of TLR4-MD2 to Mrp8 (solid lines). The sensorgrams of Rage-Fc or sample buffer to immobilized Mrp8 are shown as controls (solid lines). No unspecific binding of TLR4-MD2 to immobilized mouse serum albumin was observed (long dashed line). One representative out of three independent experiments is shown. Response difference is the difference between experimental and control flow cells in response units (RU). Time is shown in seconds. (d) TNF- abundance, as determined by ELISA, of human monocytes after preincubation for 30 min with 1 g/ml of TLR4 antagonist or 0.5 g/ml antibody to human TLR4 (HTA125) or isotype control antibody (IgG2a), followed by 4-h stimulation with LPS or Mrp8 as indicated. Data represent mean values s.d. from three independent experiments (*P 0.05 and **P 0.01).
Figure 6: Mrp8-Mrp14 and Mrp8 promote LPS-induced shock.(a) Survival of wild-type (n = 25, five independent experiments) and Mrp14-/- mice (n = 25, five independent experiments) treated with LPS and D-gal. In parallel experiments, 30 min after treatment of Mrp14-/- mice with LPS and D-gal, either Mrp8-Mrp14 (300 g per mouse, n = 18, three independent experiments) or Mrp8 (300 g per mouse, n = 12, two independent experiments) was given in a single intravenous dose. The figure shows a summary of the survival times of the different groups over 16 h (presented as percentage of survivors) (*P 0.05; **P 0.01 for significant differences in survival rates between individual groups). (b) Serum concentrations of IL-6, TNF- and IL-12p40 in Mrp14-/- and wild-type mice as determined by ELISA 120 min after injection of LPS and D-gal. Data represent mean values s.d. of five individual mice (**P 0.01). (c) Survival of wild-type (n = 16, two independent experiments) and Mrp14-/- mice (n = 16, two independent experiments) treated intraperitoneally with 1 104 CFU live E. coli 018:K1 per mouse. The figure shows a summary of the survival times of both groups over 72 h (presented as percentage of survivors, *P 0.05 for significant differences in survival rates between both groups).
Vogl T, Tenbrock K, Ludwig S, Leukert N, Ehrhardt C, van Zoelen MA, Nacken W, Foell D, van der Poll T, Sorg C, Roth JMrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock. Nat Med 13:1042-1049

October 2007


560 Reads

To identify new components that regulate the inflammatory cascade during sepsis, we characterized the functions of myeloid-related protein-8 (Mrp8, S100A8) and myeloid-related protein-14 (Mrp14, S100A9), two abundant cytoplasmic proteins of phagocytes. We now demonstrate that mice lacking Mrp8-Mrp14 complexes are protected from endotoxin-induced lethal shock and Escherichia coli-induced abdominal sepsis. Both proteins are released during activation of phagocytes, and Mrp8-Mrp14 complexes amplify the endotoxin-triggered inflammatory responses of phagocytes. Mrp8 is the active component that induces intracellular translocation of myeloid differentiation primary response protein 88 and activation of interleukin-1 receptor-associated kinase-1 and nuclear factor-kappaB, resulting in elevated expression of tumor necrosis factor-alpha (TNF-alpha). Using phagocytes expressing a nonfunctional Toll-like receptor 4 (TLR4), HEK293 cells transfected with TLR4, CD14 and MD2, and by surface plasmon resonance studies in vitro, we demonstrate that Mrp8 specifically interacts with the TLR4-MD2 complex, thus representing an endogenous ligand of TLR4. Therefore Mrp8-Mrp14 complexes are new inflammatory components that amplify phagocyte activation during sepsis upstream of TNFalpha-dependent effects.

Sonoshita M, Takaku K, Sasaki N, Sugimoto Y, Ushikubi F, Narumiya S, Oshima M, Taketo MMAcceleration of intestinal polyposis through prostaglandin receptor EP2 in Apc(Delta 716) knockout mice. Nat Med 7: 1048-1051

October 2001


76 Reads

Arachidonic acid is metabolized to prostaglandin H(2) (PGH(2)) by cyclooxygenase (COX). COX-2, the inducible COX isozyme, has a key role in intestinal polyposis. Among the metabolites of PGH(2), PGE(2) is implicated in tumorigenesis because its level is markedly elevated in tissues of intestinal adenoma and colon cancer. Here we show that homozygous deletion of the gene encoding a cell-surface receptor of PGE(2), EP2, causes decreases in number and size of intestinal polyps in Apc(Delta 716) mice (a mouse model for human familial adenomatous polyposis). This effect is similar to that of COX-2 gene disruption. We also show that COX-2 expression is boosted by PGE(2) through the EP2 receptor via a positive feedback loop. Homozygous gene knockout for other PGE(2) receptors, EP1 or EP3, did not affect intestinal polyp formation in Apc(Delta 716) mice. We conclude that EP2 is the major receptor mediating the PGE2 signal generated by COX-2 upregulation in intestinal polyposis, and that increased cellular cAMP stimulates expression of more COX-2 and vascular endothelial growth factor in the polyp stroma.

Figure 6: Potential sources of cPR-3(105−201)-like proteins.(a) Identification of in vivo PRTN3 antisense transcripts in PR-3-ANCA patient leukocytes (lanes 1−3 and 5), as determined by RT-PCR. Lane 4, negative patient; + and -, with and without reverse transcriptase (RT), respectively; C1, positive control RNA; C2, buffer only. Sequences of upper and lower bands corresponded to cPR-3(105−201). Additional flanking sequences of upper band could not be identified by BLAST. Bar graph shows individuals tested for antisense PRTN3 transcripts. Non-ANCA (age 50) denotes a patient group with renal disease that is ANCA-negative and age-matched to the ANCA patient group. (b) Examples of proteins that mimic cPR-3(105−201). Shown is antisense complementarity sequence alignment of PR-3 with potential cPR-3(105−201)-like proteins of microbial or viral origin. Blue residues are complementary to corresponding sense residues of PR-3; pink residues are mismatches. Hits are in the C- to N-terminal orientation.
Autoimmunity is triggered by cPR-3(105–201), a protein complementary to human autoantigen proteinase-3

February 2004


269 Reads

It remains unclear how and why autoimmunity occurs. Here we show evidence for a previously unrecognized and possibly general mechanism of autoimmunity. This new finding was discovered serendipitously using material from patients with inflammatory vascular disease caused by antineutrophil cytoplasmic autoantibodies (ANCA) with specificity for proteinase-3 (PR-3). Such patients harbor not only antibodies to the autoantigen (PR-3), but also antibodies to a peptide translated from the antisense DNA strand of PR-3 (complementary PR-3, cPR-3) or to a mimic of this peptide. Immunization of mice with the middle region of cPR-3 resulted in production of antibodies not only to cPR-3, but also to the immunogen's sense peptide counterpart, PR-3. Both human and mouse antibodies to PR-3 and cPR-3 bound to each other, indicating idiotypic relationships. These findings indicate that autoimmunity can be initiated through an immune response against a peptide that is antisense or complementary to the autoantigen, which then induces anti-idiotypic antibodies (autoantibodies) that cross-react with the autoantigen.

Lefebvre AM, Chen I, Desreumaux P, Najib J, Fruchart JC, Geboes K, Briggs M, Heyman R, Auwerx JActivation of the peroxisome proliferator-activated receptor gamma promotes the development of colon tumors in C57BL/6J-APCMin/+ mice. Nat Med 4: 1053-1057

October 1998


43 Reads

The development of colorectal cancer, one of the most frequent cancers, is influenced by prostaglandins and fatty acids. Decreased prostaglandin production, seen in mice with mutations in the cyclooxygenase 2 gene or in animals and humans treated with cyclooxygenase inhibitors, prevents or attenuates colon cancer development. There is also a strong correlation between the intake of fatty acids from animal origin and colon cancer. Therefore, the peroxisome proliferator-activated receptor gamma (PPARgamma), a downstream transcriptional mediator for prostaglandins and fatty acids which is highly expressed in the colon may be involved in this process. Activation of PPARgamma by two different synthetic agonists increased the frequency and size of colon tumors in C57BL/6J-APCMin/+ mice, an animal model susceptible to intestinal neoplasia. Tumor frequency was only increased in the colon, and did not change in the small intestine, coinciding with the colon-restricted expression of PPARgamma. Treatment with PPARgamma agonists increased beta-catenin levels both in the colon of C57BL/61-APCMin/+ mice and in HT-29 colon carcinoma cells. Genetic abnormalities in the Wnt/wingless/APC pathway, which enhance the transcriptional activity of the beta-catenin-T-cell factor/lymphoid enhancer factor 1 transcription complex, often underly the development of colon tumors. Our data indicate that PPARgamma activation modifies the development of colon tumors in C57BL/61-APCMin/+ mice.

Figure 4 AP-1 is necessary for activation of TGFB1 promoter. (a) AP-1 decoy ODN blocks TGFB1 promoter activity in THP-1 cells. TGF-b 1 luciferase assay was performed in THP-1 cells after transfection of AP-1 decoy ODN. White bars, AP-1 decoy ODN; black bars, scrambled ODN. (b) AP-1 decoy ODN blocks TGFB1 promoter activation in MM6 cells that express IL-13Ra 2. TGF-b 1 luciferase assay was performed in MM6 cells after transfection with a plasmid encoding full-length IL-13Ra 2 and AP-1 decoy ODN. White bars, AP-1 decoy ODN; black bars, scrambled ODN. (c) AP-1 decoy ODN blocks TGFB1 promoter activation independent of IL-13Ra 1 in MM6 cells that express IL-13Ra 2. TGF-b 1 luciferase assay was performed in MM6 cells after transfection with a plasmid encoding fulllength IL-13Ra 2 and AP-1 decoy ODN. Cells were preincubated with IL-13Ra 1-specific antibody. White bars, IL-13Ra 1-specific antibody +AP-1 decoy ODN; black bars, IL-13Ra 1-specific antibody + scrambled ODN. (d) IL-13 induces activation of AP-1 in MM6 cells expressing IL-13Ra 2. MM6 cells were transfected with IL-13Ra 2 and stimulated for 3 h with IL-13, after which they were subjected to nuclear extraction. EMSAs with supershift analyses targeting AP-1 family members were performed.
Figure 5 Role of IL-13Ra 2 expression in oxazolone-induced colitis and bleomycin-induced lung fibrosis. (a) Blocking TNF-a signaling with etanercept does not ameliorate oxazolone-induced colitis. Weight loss is expressed as percentage of starting weight. Data represent mean of three or more challenged mice per group. (b) IL-13 is present in mesenteric lymph nodes (MLN) on day 3 in oxazolone-induced colitis. IL-13 was measured by ELISA after stimulation of MLN cells. (c) Blocking TNF-a signaling downregulates expression of IL-13Ra 2 in oxazolone-induced colitis; expression of IL-13Ra 1 remains unchanged. Immunoblot for IL-13Ra 2 and b-actin, and RT-PCR for Il13ra1 and Gapdh on day 3. (d) Blocking TNF-a signaling reduces production of TGF-b 1 by lamina propria mononuclear cells on day 3 after induction of oxazolone-induced colitis. TGF-b 1 was measured by ELISA after stimulation of LPMC. (e) Blocking TNF-a signaling induces pancolonic disease. H&E-stained cross-sections and scoring on day 3. (f) IL-13 is elevated after administration of bleomycin. IL-13 was measured in BAL by ELISA on day 24. Data represent mean ± s.d. of five mice per group. (g) Blocking TNF-a signaling with etanercept reduces production of TGF-b 1 in bleomycin-induced lung fibrosis. Total TGF-b 1 was measured by ELISA in BAL on day 24. (h) Blocking TNF-a signaling with etanercept decreases IL-13Ra 2 expression in the lung; expression of IL-13Ra 2 remained unchanged. Immunoblot for IL-13Ra 2 and b-actin and RT-PCR for Il13ra1 and Gapdh on day 24. (i) Blocking TNF-a signaling with etanercept reduces lung collagen content in bleomycin-induced lung fibrosis. Collagen content was measured by Sircol assay. (j) Blocking TNF-a signaling with etanercept prevents bleomycin-induced lung fibrosis. Lung sections stained by Masson trichrome technique on day 24.  
Fichtner-Feigl, S, Strober, W, Kawakami, K, Puri, RK and Kitani, A. IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and fibrosis. Nat Med 12: 99-106

February 2006


917 Reads

Interleukin (IL)-13 is a major inducer of fibrosis in many chronic infectious and autoimmune diseases. In studies of the mechanisms underlying such induction, we found that IL-13 induces transforming growth factor (TGF)-beta(1) in macrophages through a two-stage process involving, first, the induction of a receptor formerly considered to function only as a decoy receptor, IL-13Ralpha(2). Such induction requires IL-13 (or IL-4) and tumor necrosis factor (TNF)-alpha. Second, it involves IL-13 signaling through IL-13Ralpha(2) to activate an AP-1 variant containing c-jun and Fra-2, which then activates the TGFB1 promoter. In vivo, we found that prevention of IL-13Ralpha(2) expression reduced production of TGF-beta(1) in oxazolone-induced colitis and that prevention of IL-13Ralpha(2) expression, Il13ra2 gene silencing or blockade of IL-13Ralpha(2) signaling led to marked downregulation of TGF-beta(1) production and collagen deposition in bleomycin-induced lung fibrosis. These data suggest that IL-13Ralpha(2) signaling during prolonged inflammation is an important therapeutic target for the prevention of TGF-beta(1)-mediated fibrosis.

Zhou J, Payen JF, Wilson DA, Traystman RJ, van Zijl PCUsing the amide proton signals of intracellular proteins and peptides to detect pH effects in MRI. Nat Med 9:1085-1090

September 2003


116 Reads

In the past decade, it has become possible to use the nuclear (proton, 1H) signal of the hydrogen atoms in water for noninvasive assessment of functional and physiological parameters with magnetic resonance imaging (MRI). Here we show that it is possible to produce pH-sensitive MRI contrast by exploiting the exchange between the hydrogen atoms of water and the amide hydrogen atoms of endogenous mobile cellular proteins and peptides. Although amide proton concentrations are in the millimolar range, we achieved a detection sensitivity of several percent on the water signal (molar concentration). The pH dependence of the signal was calibrated in situ, using phosphorus spectroscopy to determine pH, and proton exchange spectroscopy to measure the amide proton transfer rate. To show the potential of amide proton transfer (APT) contrast for detecting acute stroke, pH effects were noninvasively imaged in ischemic rat brain. This observation opens the possibility of using intrinsic pH contrast, as well as protein- and/or peptide-content contrast, as diagnostic tools in clinical imaging.

Figure 1: Altered intracellular signaling in mice with targeted gp130 mutations. a, Targeting strategy for the introduction of the gp130757F knock-in mutation. Cytoplasmic domain of mouse gp130 is depicted alongside the corresponding genomic structure with exons numbered13 and the structure of the mutation in gp130STAT mice12. The targeting vector contains Y757RHQ760 to FRHA substitutions and the endogenous translational stop codon at position 918 followed by a ribosomal re-entry site and the sequence encoding neomycin resistance (IRESneo). A diagnostic digest with HindIII (H) yields a fragment of 4.5-kb from the wild-type (wt) allele and a 7.5-kb fragment from the targeted allele when hybridized with a probe external to the targeting vector (*)12. S, SalI. TM, transmembrane domain. b, Biochemical analysis of the intracellular signaling pathway in the liver of gp130 mutant mice. Mice were injected with IL-6 (5 g, i.v.) and liver lysates were subsequently analyzed for phosphorylated species of SHP2, the MAP kinases ERK1/2 and STAT3 by western-blot analysis. The membranes were reprobed for SHP2, ERK1/2 and STAT3, respectively, to assess equality of protein loading. c, Sustained activation of intracellular signaling proteins in the gastrointestinal tract of gp130 mutant mice. For the analysis of ERK activation in the colon, mice were injected with IL-6 (5 g, i.v.), whereas analysis of STAT3 activation in the antrum was done on IL-11-treated (5 g, i.v.) mice. Lysates were analyzed by western blot for phosphorylated ERK1/2 or phosphorylated STAT3 and total ERK1/2 or STAT3, respectively.
Figure 3: Increased susceptibility to DSS-mediated intestinal injury in 12−16-wk-old mice. a, Weight loss (expressed as percent of initial body weight) and associated death is greater after DSS treatment in gp130STAT mice (blue lines and symbols) compared with wild-type (black) and gp130757F mice (red). b, Treatment with 3.5% DSS for 5 d results inincreased morbidity of gp130STAT mice as assessed by weight loss, colonic blood content and crypt damage. This response is phenocopied in IL-6-/- and IL-6-/-IL-11R1-/- compound mice, while the susceptibility of IL-11R1-/- mice is comparable with that of wild-type mice. In contrast, gp130757F mice are completely resistant to DSS-induced colonic injury. c−h, Representative images of the distal colon following 5 d treatment with 3.5% DSS of gp130STAT (d), IL-6-/- (f) and IL-6-/-IL-11R1-/- mice (h) showing extensive mucosal injury, loss of surface epithelium and inflammation (arrows). Less severe damage is observed in sections of wild-type (c) and IL-11R1-/- mice (g) with areas of preserved microvillus architecture (arrowheads). Note absence of histological damage in colon of gp130757F mice (e). Magnification, 20.
Tebbutt NC, Giraud AS, Inglese M, Jenkins B, Waring P, Clay FJ, Malki S, Alderman BM, Grail D, Hollande F, Heath JK, Ernst MReciprocal regulation of gastrointestinal homeostasis by SHP2 and STAT-mediated trefoil gene activation in gp130 mutant mice. Nat Med 8:1089-1097

November 2002


266 Reads

The intracellular signaling mechanisms that specify tissue-specific responses to the interleukin-6 (IL-6) family of cytokines are not well understood. Here, we evaluated the functions of the two major signaling pathways, the signal transducers and activators of transcription 1 and 3 (STAT1/3) and the Src-homology tyrosine phosphatase 2 (SHP2)-Ras-ERK, emanating from the common signal transducer, gp130, in the gastrointestinal tract. Gp130(757F) mice, with a 'knock-in' mutation abrogating SHP2-Ras-ERK signaling, developed gastric adenomas by three months of age. In contrast, mice harboring the reciprocal mutation ablating STAT1/3 signaling (gp130(Delta STAT)), or deficient in IL-6-mediated gp130 signaling (IL-6(-/-) mice), showed impaired colonic mucosal wound healing. These gastrointestinal phenotypes are highly similar to the phenotypes exhibited by mice deficient in trefoil factor 1 (pS2/TFF1) and intestinal trefoil factor (ITF)/TFF3, respectively, and corresponded closely with the capacity of the two pathways to stimulate transcription of the genes encoding pS2/TFF1 and ITF/TFF3. We propose a model whereby mucosal wound healing depends solely on activation of STAT1/3, whereas gastric hyperplasia ensues when the coordinated activation of the STAT1/3 and SHP2-Ras-ERK pathways is disrupted.

Chen J, Somanath PR, Razorenova O, Chen WS, Hay N, Bornstein P, Byzova TVAkt1 regulates pathological angiogenesis, vascular maturation and permeability in vivo. Nat Med 11: 1188-1196

December 2005


147 Reads

Akt kinases control essential cellular functions, including proliferation, apoptosis, metabolism and transcription, and have been proposed as promising targets for treatment of angiogenesis-dependent pathologies, such as cancer and ischemic injury. But their precise roles in neovascularization remain elusive. Here we show that Akt1 is the predominant isoform in vascular cells and describe the unexpected consequences of Akt1 knockout on vascular integrity and pathological angiogenesis. Angiogenic responses in three distinct in vivo models were enhanced in Akt1(-/-) mice; these enhanced responses were associated with impairment of blood vessel maturation and increased vascular permeability. Although impaired vascular maturation in Akt1(-/-) mice may be attributed to reduced activation of endothelial nitric oxide synthase (eNOS), the major phenotypic changes in vascular permeability and angiogenesis were linked to reduced expression of two endogenous vascular regulators, thrombospondins 1 (TSP-1) and 2 (TSP-2). Re-expression of TSP-1 and TSP-2 in mice transplanted with wild-type bone marrow corrected the angiogenic abnormalities in Akt1(-/-) mice. These findings establish a crucial role of an Akt-thrombospondin axis in angiogenesis.

Figure 1: R-15a and miR-16 downregulation is associated with oncogenic activities.(a) miR-15a and miR-16 expression, as analyzed by real-time PCR in primary cells isolated from prostate cancer samples of 20 individuals diagnosed with prostatic adenocarcinomas. miRNA levels were evaluated as the ratio between the values obtained with normal (dotted line) and cancer prostate cells from each subject. Data are mean values of four independent experiments performed in duplicate. (b) H&E staining (left) and detection of miR-15a and miR-16 by in situ hybridization (middle and right) in serial sections from prostatic adenocarcinoma. Tumor (red arrows) and non-neoplastic prostate epithelial cell areas (black arrows) are indicated. Scale bars, 50 m. (c) Real time PCR evaluation of miR-15a and miR-16 levels in wild-type or decoy15-16–transduced RWPE-1 cells, LNCaP cells, and primary tumor cells with normal levels of miR-15a and miR-16 (T) transduced with control (TW3) or decoy15-16 vectors. The untransformed prostate cell line RWPE-1 was used as expression reference. (d) Cell growth of RWPE-1 and primary tumor cells with normal miRNA expression transduced with decoy15-16 or the empty vector TW3. (e) Cell cycle analysis by cytofluorimetric profiling of BrdU-7AAD–stained RWPE-1 cells transduced with TW3 or decoy15-16 vector. P < 0.001 for the two groups. Data are means s.d. of five experiments evaluating the relative percentage of cells at S and G2/M phase of the cell cycle. (f,g) Soft agar colony formation assay for RWPE-1 cells transduced with TW3 or decoy15-16. Data are means s.d. of three independent experiments. (h,i) Migration assay for RWPE-1 cells transduced with TW3 or decoy15-16 and maintained in standard culture medium (Control) or in prostate cancer fibroblast–conditioned medium (Conditioned). **P < 0.01 and ***P < 0.001. Data are means s.d. of four independent experiments in c,d and i. Scale bars, 100 m.
Figure 2: Restoration of miR-15a and miR-16 induces growth arrest and apoptosis in defective prostate cancer cells.(a) Schematic depiction of TWmiR-15-16. The miR-15a-miR-16-1 cluster was subcloned in the TWEEN vector under the control of the cytomegalovirus (CMV) promoter. PGK, phosphoglycerate kinase promoter. (b) Real-time PCR evaluation of miR-15a and miR-16 expression in LNCaP cells transduced with TWmiR-15-16 vector. Transduced cells were compared with cells infected with empty vector (TW) and with the RWPE-1 cell line. (c) Cell growth of LNCaP cells infected with empty vector (TW) or TWmiR-15-16. Data are means s.d. of four independent experiments. (d) Cell death of normal (RWPE-1) and tumor (LNCaP) cells transduced with TWmiR-15-16 or control vector, as evaluated 48 h after lentiviral infection. (e) Flow cytometry profiles of LNCaP and RWPE-1 cells infected with TW and TWmiR-15-16 viruses. GFP expression is shown 24 h (Day 1) and 11 d after infection. The percentages represent GFP-positive cells. (f) Real-time PCR analysis of miR-15a and miR-16 expression in miR-15– and miR-16–defective prostate tumor primary cells after transduction with TWmiR-15-16 or empty TW vector. Primary normal prostate cells were used as expression reference. (g) Cell death evaluation in tumor and normal prostate primary cells infected as in f. Data are means s.d. of three independent experiments for all panels except c.
Bonci D, Coppola V, Musumeci M, Addario A, Giuffrida R, Memeo L, D’Urso L, Pagliuca A, Biffoni M, Labbaye C, Bartucci M, Muto G, Peschle C, De Maria RThe miR-15a-miR-16-1 cluster controls prostate cancer by targeting multiple oncogenic activities. Nat Med 14(11): 1271-1277

November 2008


466 Reads

MicroRNAs (miRNAs) are noncoding small RNAs that repress protein translation by targeting specific messenger RNAs. miR-15a and miR-16-1 act as putative tumor suppressors by targeting the oncogene BCL2. These miRNAs form a cluster at the chromosomal region 13q14, which is frequently deleted in cancer. Here, we report that the miR-15a and miR-16-1 cluster targets CCND1 (encoding cyclin D1) and WNT3A, which promotes several tumorigenic features such as survival, proliferation and invasion. In cancer cells of advanced prostate tumors, the miR-15a and miR-16 level is significantly decreased, whereas the expression of BCL2, CCND1 and WNT3A is inversely upregulated. Delivery of antagomirs specific for miR-15a and miR-16 to normal mouse prostate results in marked hyperplasia, and knockdown of miR-15a and miR-16 promotes survival, proliferation and invasiveness of untransformed prostate cells, which become tumorigenic in immunodeficient NOD-SCID mice. Conversely, reconstitution of miR-15a and miR-16-1 expression results in growth arrest, apoptosis and marked regression of prostate tumor xenografts. Altogether, we propose that miR-15a and miR-16 act as tumor suppressor genes in prostate cancer through the control of cell survival, proliferation and invasion. These findings have therapeutic implications and may be exploited for future treatment of prostate cancer.

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