Research Article
Abstract
Administration of the CD28 superagonistic antibody JJ316 is an efficient means to treat autoimmune diseases in rats, but the humanized antibody TGN1412 caused devastating side effects in healthy volunteers during a clinical trial. Here we show that JJ316 treatment of rats induced a dramatic redistribution of T lymphocytes from the periphery to the secondary lymphoid organs, resulting in severe T lymphopenia. Live imaging of secondary lymphoid organs revealed that JJ316 administration almost instantaneously (<2 minutes) arrested T cells in situ. This reduction in T cell motility was accompanied by profound cytoskeletal rearrangements and increased cell size. In addition, surface expression of lymphocyte function–associated antigen-1 was enhanced, endothelial differentiation sphingolipid G protein–coupled receptor 1 and L selectin levels were downregulated, and the cells lost their responsiveness to sphingosine 1–phosphate–directed migration. These proadhesive alterations were accompanied by signs of strong activation, including upregulation of CD25, CD69, CD134, and proinflammatory mediators. However, this did not lead to a cytokine storm similar to the clinical trial. While most of the early changes disappeared within 48 hours, we observed that CD4+CD25+FoxP3+ regulatory T cells experienced a second phase of activation, which resulted in massive cell enlargement, extensive polarization, and increased motility. These data suggest that CD28 superagonists elicit 2 qualitatively distinct waves of activation.
Authors
Nora Müller, Jens van den Brandt, Francesca Odoardi, Denise Tischner, Judith Herath, Alexander Flügel, Holger M. Reichardt
Abstract
The growth of normal cells is arrested when they come in contact with each other, a process known as contact inhibition. Contact inhibition is lost during tumorigenesis, resulting in uncontrolled cell growth. Here, we investigated the role of the tetraspanin transmembrane 4 superfamily member 5 (TM4SF5) in contact inhibition and tumorigenesis. We found that TM4SF5 was overexpressed in human hepatocarcinoma tissue. TM4SF5 expression in clinical samples and in human hepatocellular carcinoma cell lines correlated with enhanced p27Kip1 expression and cytosolic stabilization as well as morphological elongation mediated by RhoA inactivation. These TM4SF5-mediated effects resulted in epithelial-mesenchymal transition (EMT) via loss of E-cadherin expression. The consequence of this was aberrant cell growth, as assessed by S-phase transition in confluent conditions, anchorage-independent growth, and tumor formation in nude mice. The TM4SF5-mediated effects were abolished by suppressing the expression of either TM4SF5 or cytosolic p27Kip1, as well as by reconstituting the expression of E-cadherin. Our observations have revealed a role for TM4SF5 in causing uncontrolled growth of human hepatocarcinoma cells through EMT.
Authors
Sin-Ae Lee, Sung-Yul Lee, Ik-Hyun Cho, Min-A Oh, Eun-Sil Kang, Yong-Bae Kim, Woo Duck Seo, Suyong Choi, Ju-Ock Nam, Mimi Tamamori-Adachi, Shigetaka Kitajima, Sang-Kyu Ye, Semi Kim, Yoon-Jin Hwang, In-San Kim, Ki Hun Park, Jung Weon Lee
Abstract
Most patients (80%) with ovarian cancer (OvCa) present with metastatic disease. Attachment of OvCa cells to peritoneum and omentum represents the first rate-limiting step for metastatic spread. Therefore, identifying factors regulating cell attachment in the abdominal cavity is critical to the development of therapeutic agents. We show here that MMP-2 expression was upregulated in OvCa cells upon attachment to their microenvironment. Downregulation of MMP-2 mRNA or pharmacological inhibition of MMP-2 proteolytic function, in both human OvCa primary cells and cell lines, reduced attachment of OvCa cells to a 3D organotypic model of metastatic OvCa, full human omentum or peritoneum, and in vivo to mouse peritoneum and omentum. Absence of MMP-2 in the host did not alter OvCa adhesion, as determined utilizing mice harboring homozygous null mutations in either the Mmp2 or Mmp9 genes. Conversely, adhesion induced upregulation of MMP-2 mRNA in OvCa cells. MMP-2 inhibition in OvCa cells through pharmacological or antibody treatment prior to i.p. dissemination in nude mice significantly decreased tumor growth and metastasis and extended survival. MMP-2 enhanced peritoneal adhesion of OvCa cells through cleavage of ECM proteins fibronectin (FN) and vitronectin (Vn) into small fragments and increased binding of OvCa cells to these FN and Vn fragments and their receptors, α5β1 and αVβ3 integrin. These findings indicate that MMP-2 expressed by metastatic OvCa cells functionally regulates their attachment to peritoneal surfaces.
Authors
Hilary A. Kenny, Swayamjot Kaur, Lisa M. Coussens, Ernst Lengyel
Abstract
Priapism, abnormally prolonged penile erection in the absence of sexual excitation, is associated with ischemia-mediated erectile tissue damage and subsequent erectile dysfunction. It is common among males with sickle cell disease (SCD), and SCD transgenic mice are an accepted model of the disorder. Current strategies to manage priapism suffer from a poor fundamental understanding of the molecular mechanisms underlying the disorder. Here we report that mice lacking adenosine deaminase (ADA), an enzyme necessary for the breakdown of adenosine, displayed unexpected priapic activity. ADA enzyme therapy successfully corrected the priapic activity both in vivo and in vitro, suggesting that it was dependent on elevated adenosine levels. Further genetic and pharmacologic evidence demonstrated that A2B adenosine receptor–mediated (A2BR-mediated) cAMP and cGMP induction was required for elevated adenosine–induced prolonged penile erection. Finally, priapic activity in SCD transgenic mice was also caused by elevated adenosine levels and A2BR activation. Thus, we have shown that excessive adenosine accumulation in the penis contributes to priapism through increased A2BR signaling in both Ada–/– and SCD transgenic mice. These findings provide insight regarding the molecular basis of priapism and suggest that strategies to either reduce adenosine or block A2BR activation may prove beneficial in the treatment of this disorder.
Authors
Tiejuan Mi, Shahrzad Abbasi, Hong Zhang, Karen Uray, Janci L. Chunn, Ling Wei Xia, Jose G. Molina, Norman W. Weisbrodt, Rodney E. Kellems, Michael R. Blackburn, Yang Xia
Abstract
Becoming a phenotypic male is ultimately determined by androgen-induced masculinization. Disorders of fetal masculinization, resulting in hypospadias or cryptorchidism, are common, but their cause remains unclear. Together with the adult-onset disorders low sperm count and testicular cancer, they can constitute a testicular dysgenesis syndrome (TDS). Although masculinization is well studied, no unifying concept explains normal male reproductive development and its abnormalities, including TDS. We exposed rat fetuses to either anti-androgens or androgens and showed that masculinization of all reproductive tract tissues was programmed by androgen action during a common fetal programming window. This preceded morphological differentiation, when androgen action was, surprisingly, unnecessary. Only within the programming window did blocking androgen action induce hypospadias and cryptorchidism and altered penile length in male rats, all of which correlated with anogenital distance (AGD). Androgen-driven masculinization of females was also confined to the same programming window. This work has identified in rats a common programming window in which androgen action is essential for normal reproductive tract masculinization and has highlighted that measuring AGD in neonatal humans could provide a noninvasive method to predict neonatal and adult reproductive disorders. Based on the timings in rats, we believe the programming window in humans is likely to be 8–14 weeks of gestation.
Authors
Michelle Welsh, Philippa T.K. Saunders, Mark Fisken, Hayley M. Scott, Gary R. Hutchison, Lee B. Smith, Richard M. Sharpe
Abstract
Mobilization of endothelial progenitor cells (EPCs) from the bone marrow and their subsequent participation in neovessel formation are implicated in tumor growth and neovascularization. As the neurotransmitter dopamine (DA) modulates adult endothelial cell function, we hypothesized that DA might have a regulatory role in mobilization of EPCs from the bone marrow niche. We show that there was a significant decrease in bone marrow DA content and an increase in EPC mobilization in tumor-bearing mice associated with tumor neovascularization. DA treatment of tumor-bearing mice inhibited EPC mobilization and tumor growth through its D2 receptors, as DA treatment failed to inhibit EPC mobilization in tumor-bearing mice treated with a specific DA D2 receptor antagonist and in tumor-bearing mice lacking the D2 receptor. In addition, we found that DA, through D2 receptors, exerted its inhibitory effect on EPC mobilization through suppression of VEGFA-induced ERK1/ERK2 phosphorylation and MMP-9 synthesis. These findings reveal a new link between DA and EPC mobilization and suggest a novel use for DA and D2 agents in the treatment of cancer and other diseases involving neovessel formation.
Authors
Debanjan Chakroborty, Uttio Roy Chowdhury, Chandrani Sarkar, Rathindranath Baral, Partha Sarathi Dasgupta, Sujit Basu
Abstract
Axonal degeneration is an important determinant of progressive neurological disability in multiple sclerosis (MS). Thus, therapeutic approaches promoting neuroprotection could aid the treatment of progressive MS. Here, we used what we believe is a novel water-soluble fullerene derivative (ABS-75) attached to an NMDA receptor antagonist, which combines antioxidant and anti-excitotoxic properties, to block axonal damage and reduce disease progression in a chronic progressive EAE model. Fullerene ABS-75 treatment initiated after disease onset reduced the clinical progression of chronic EAE in NOD mice immunized with myelin-oligodendrocyte glycoprotein (MOG). Reduced disease progression in ABS-75–treated mice was associated with reduced axonal loss and demyelination in the spinal cord. Fullerene ABS-75 halted oxidative injury, CD11b+ infiltration, and CCL2 expression in the spinal cord of mice without interfering with antigen-specific T cell responses. In vitro, fullerene ABS-75 protected neurons from oxidative and glutamate-induced injury and restored glutamine synthetase and glutamate transporter expression in astrocytes under inflammatory insult. Glutamine synthetase expression was also increased in the white matter of fullerene ABS-75–treated animals. Our data demonstrate the neuroprotective effect of treatment with a fullerene compound combined with a NMDA receptor antagonist, which may be useful in the treatment of progressive MS and other neurodegenerative diseases.
Authors
Alexandre S. Basso, Dan Frenkel, Francisco J. Quintana, Frederico A. Costa-Pinto, Sanja Petrovic-Stojkovic, Lindsay Puckett, Alon Monsonego, Amnon Bar Shir, Yoni Engel, Michael Gozin, Howard L. Weiner
Abstract
A hallmark of SLE is the production of high-titer, high-affinity, isotype-switched IgG autoantibodies directed against nucleic acid–associated antigens. Several studies have established a role for both type I IFN (IFN-I) and the activation of TLRs by nucleic acid–associated autoantigens in the pathogenesis of this disease. Here, we demonstrate that 2 IFN-I signaling molecules, IFN regulatory factor 9 (IRF9) and STAT1, were required for the production of IgG autoantibodies in the pristane-induced mouse model of SLE. In addition, levels of IgM autoantibodies were increased in pristane-treated Irf9–/– mice, suggesting that IRF9 plays a role in isotype switching in response to self antigens. Upregulation of TLR7 by IFN-α was greatly reduced in Irf9–/– and Stat1–/– B cells. Irf9–/– B cells were incapable of being activated through TLR7, and Stat1–/– B cells were impaired in activation through both TLR7 and TLR9. These data may reveal a novel role for IFN-I signaling molecules in both TLR-specific B cell responses and production of IgG autoantibodies directed against nucleic acid–associated autoantigens. Our results suggest that IFN-I is upstream of TLR signaling in the activation of autoreactive B cells in SLE.
Authors
Donna L. Thibault, Alvina D. Chu, Kareem L. Graham, Imelda Balboni, Lowen Y. Lee, Cassidy Kohlmoos, Angela Landrigan, John P. Higgins, Robert Tibshirani, Paul J. Utz
Abstract
Deficiencies in the SBDS gene result in Shwachman-Diamond syndrome (SDS), an inherited bone marrow failure syndrome associated with leukemia predisposition. SBDS encodes a highly conserved protein previously implicated in ribosome biogenesis. Using human primary bone marrow stromal cells (BMSCs), lymphoblasts, and skin fibroblasts, we show that SBDS stabilized the mitotic spindle to prevent genomic instability. SBDS colocalized with the mitotic spindle in control primary BMSCs, lymphoblasts, and skin fibroblasts and bound to purified microtubules. Recombinant SBDS protein stabilized microtubules in vitro. We observed that primary BMSCs and lymphoblasts from SDS patients exhibited an increased incidence of abnormal mitoses. Similarly, depletion of SBDS by siRNA in human skin fibroblasts resulted in increased mitotic abnormalities and aneuploidy that accumulated over time. Treatment of primary BMSCs and lymphoblasts from SDS patients with nocodazole, a microtubule destabilizing agent, led to increased mitotic arrest and apoptosis, consistent with spindle destabilization. Conversely, SDS patient cells were resistant to taxol, a microtubule stabilizing agent. These findings suggest that spindle instability in SDS contributes to bone marrow failure and leukemogenesis.
Authors
Karyn M. Austin, Mohan L. Gupta Jr., Scott A. Coats, Asmin Tulpule, Gustavo Mostoslavsky, Alejandro B. Balazs, Richard C. Mulligan, George Daley, David Pellman, Akiko Shimamura
Abstract
DNA vaccines promote an immune response by providing antigen-encoding DNA to the recipient, but the efficacy of such vaccines needs improving. Many approaches have considerable potential but currently induce relatively weak immune responses despite multiple high doses of DNA vaccine. Here, we asked whether targeting vaccine antigens to DCs would increase the immunity and protection that result from DNA vaccines. To determine this, we generated a DNA vaccine encoding a fusion protein comprised of the vaccine antigen and a single-chain Fv antibody (scFv) specific for the DC-restricted antigen-uptake receptor DEC205. Following vaccination of mice, the vaccine antigen was expressed selectively by DCs, which were required for the increased efficacy of MHC class I and MHC class II antigen presentation relative to a control scFv DNA vaccine. In addition, a DNA vaccine encoding an HIV gag p41–scFv DEC205 fusion protein induced 10-fold higher antibody levels and increased numbers of IFN-γ–producing CD4+ and CD8+ T cells. After a single i.m. injection of the DNA vaccine encoding an HIV gag p41–scFv DEC205 fusion protein, mice were protected from an airway challenge with a recombinant vaccinia virus expressing the HIV gag p41, even with 1% of the dose of nontargeted DNA vaccine. The efficacy of DNA vaccines therefore may be enhanced by inclusion of sequences such as single-chain antibodies to target the antigen to DCs.
Authors
Godwin Nchinda, Janelle Kuroiwa, Margarita Oks, Christine Trumpfheller, Chae Gyu Park, Yaoxing Huang, Drew Hannaman, Sarah J. Schlesinger, Olga Mizenina, Michel C. Nussenzweig, Klaus Überla, Ralph M. Steinman
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ISSN: 0021-9738 (print), 1558-8238 (online)