In This Issue
Scurvy: How a surgeon, a mariner, and a gentleman solved the greatest medical mystery of the age of sail
Platelets and metastasis revisited: a novel fatty link
Platelets have long been suspected of having a role in cancer progression and metastasis that has largely been attributed to platelet-mediated enhancement of tumor cell survival, extravasation, and angiogenesis. A study in this issue of the JCI suggests that platelet-derived lysophosphatidic acid is coopted by aggressive breast and ovarian cancer cells as a tumor cell mitogen and promoter of osteolysis during bone metastasis.
The Staphylococcus aureus “superbug”
There has been some debate about the disease-invoking potential of Staphylococcus aureus strains and whether invasive disease is associated with particularly virulent genotypes, or “superbugs.” A study in this issue of the JCI describes the genotyping of a large collection of nonclinical, commensal S. aureus strains from healthy individuals in a Dutch population. Extensive study of their genetic relatedness by amplified restriction fragment typing and comparison with strains that are associated with different types of infections revealed that the S. aureus population is clonal and that some strains have enhanced virulence. This is discussed in the context of growing interest in the mechanisms of bacterial colonization, antibiotic resistance, and novel vaccines.
Acid sensing in renal epithelial cells
The kidney adjusts net acid excretion to match production with exquisite precision, despite little or no change in the plasma bicarbonate concentration. The acid-sensing pathway that signals the kidney to increase acid secretion involves activation of the proto-oncogene c-Src. A new study in this issue shows that proline-rich tyrosine kinase 2 (Pyk2) is responsible for acid-induced activation of c-Src and is essential for acid sensing in renal epithelial cells. The findings implicate a broader role for Pyk2 in acid-base homeostasis in bone and other tissues beyond the kidney.
TB, or not TB: that is the question – does TLR signaling hold the answer?
Innate immunity critically depends on signaling by Toll-like receptors (TLRs) that rely heavily on an intracellular adapter protein called myeloid differentiation factor 88 (MyD88). Adaptive immune defenses are generally thought to be orchestrated by innate immune responses and so should require intact TLR-MyD88 signaling pathways. But a surprising new study in MyD88-null mice infected with Mycobacterium tuberculosis challenges this view and instead suggests that MyD88 may not be absolutely required for a normal adaptive immune response.
A crucial role of caspase-3 in osteogenic differentiation of bone marrow stromal stem cells
Masako Miura, Xiao-Dong Chen, Matthew R. Allen, Yanming Bi, Stan Gronthos, Byoung-Moo Seo, Saquib Lakhani, Richard A. Flavell, Xin-Hua Feng, Pamela Gehron Robey, Marian Young, Songtao ShiAbstract | Full text | PDF (Page 1704)
Caspase-3 is a critical enzyme for apoptosis and cell survival. Here we report delayed ossification and decreased bone mineral density in caspase-3–deficient (Casp3–/– and Casp3+/–) mice due to an attenuated osteogenic differentiation of bone marrow stromal stem cells (BMSSCs). The mechanism involved in the impaired differentiation of BMSSCs is due, at least partially, to the overactivated TGF-β/Smad2 signaling pathway and the upregulated expressions of p53 and p21 along with the downregulated expressions of Cdk2 and Cdc2, and ultimately increased replicative senescence. In addition, the overactivated TGF-β/Smad2 signaling may result in the compromised Runx2/Cbfa1 expression in preosteoblasts. Furthermore, we demonstrate that caspase-3 inhibitor, a potential agent for clinical treatment of human diseases, caused accelerated bone loss in ovariectomized mice, which is also associated with the overactivated TGF-β/Smad2 signaling in BMSSCs. This study demonstrates that caspase-3 is crucial for the differentiation of BMSSCs by influencing TGF-β/Smad2 pathway and cell cycle progression.
Platelet-derived lysophosphatidic acid supports the progression of osteolytic bone metastases in breast cancer
Ahmed Boucharaba, Claire-Marie Serre, Sandra Grès, Jean Sébastien Saulnier-Blache, Jean-Claude Bordet, Julien Guglielmi, Philippe Clézardin, Olivier PeyruchaudAbstract | Full text | PDF (Page 1714)
The role of lysophosphatidic acid (LPA) in cancer is poorly understood. Here we provide evidence for a role of LPA in the progression of breast cancer bone metastases. LPA receptors LPA1, LPA2, and LPA3 were expressed in human primary breast tumors and a series of human breast cancer cell lines. The inducible overexpression of LPA1 in MDA-BO2 breast cancer cells specifically sensitized these cells to the mitogenic action of LPA in vitro. In vivo, LPA1 overexpression in MDA-BO2 cells enhanced the growth of subcutaneous tumor xenografts and promoted bone metastasis formation in mice by increasing both skeletal tumor growth and bone destruction. This suggested that endogenous LPA was produced in the tumor microenvironment. However, MDA-BO2 cells or transfectants did not produce LPA. Instead, they induced the release of LPA from activated platelets which, in turn, promoted tumor cell proliferation and the LPA1-dependent secretion of IL-6 and IL-8, 2 potent bone resorption stimulators. Moreover, platelet-derived LPA deprivation in mice, achieved by treatment with the platelet antagonist Integrilin, inhibited the progression of bone metastases caused by parental and LPA1-overexpressing MDA-BO2 cells and reduced the progression of osteolytic lesions in mice bearing CHO-β3wt ovarian cancer cells. Overall, our data suggest that, at the bone metastatic site, tumor cells stimulate the production of LPA from activated platelets, which enhances both tumor growth and cytokine-mediated bone destruction.
Lentivector-mediated SMN replacement in a mouse model of spinal muscular atrophy
Mimoun Azzouz, Thanh Le, G. Scott Ralph, Lucy Walmsley, Umrao R. Monani, Debbie C.P. Lee, Fraser Wilkes, Kyriacos A. Mitrophanous, Susan M. Kingsman, Arthur H.M. Burghes, Nicholas D. MazarakisAbstract | Full text | PDF (Page 1726)
Spinal muscular atrophy (SMA) is a frequent recessive autosomal disorder. It is caused by mutations or deletion of the telomeric copy of the survival motor neuron (SMN) gene, leading to depletion in SMN protein levels. The treatment rationale for SMA is to halt or delay the degeneration of motor neurons, but to date there are no effective drug treatments for this disease. We have previously demonstrated that pseudotyping of the nonprimate equine infectious anemia virus (using the lentivector gene transfer system) with the glycoprotein of the Evelyn-Rokitnicki-Abelseth strain of the rabies virus confers retrograde axonal transport on these vectors. Here, we report that lentivector expressing human SMN was successfully used to restore SMN protein levels in SMA type 1 fibroblasts. Multiple single injections of a lentiviral vector expressing SMN in various muscles of SMA mice restored SMN to motor neurons, reduced motor neuron death, and increased the life expectancy by an average of 3 and 5 days (20% and 38%) compared with LacZ and untreated animals, respectively. Further extension of survival by SMN expression constructs will likely require a knowledge of when and/or where high levels of SMN are needed.
Natural population dynamics and expansion of pathogenic clones of Staphylococcus aureus
Damian C. Melles, Raymond F.J. Gorkink, Hélène A.M. Boelens, Susan V. Snijders, Justine K. Peeters, Michael J. Moorhouse, Peter J. van der Spek, Willem B. van Leeuwen, Guus Simons, Henri A. Verbrugh, Alex van BelkumAbstract | Full text | PDF | Supplemental material (Page 1732)
The population structure of Staphylococcus aureus carried by healthy humans was determined using a large strain collection of nonclinical origin (n = 829). High-throughput amplified fragment length polymorphism (AFLP) analysis revealed 3 major and 2 minor genetic clusters of S. aureus, which were corroborated by multilocus sequence typing. Major AFLP cluster I comprised 44.4% of the carriage isolates and showed additional heterogeneity whereas major AFLP groups II and III presented 2 homogeneous clusters, including 47.3% of all carriage isolates. Coanalysis of invasive S. aureus strains and epidemic methicillin-resistant S. aureus (MRSA) revealed that all major clusters contained invasive and multiresistant isolates. However, clusters and subclusters with overrepresentation of invasive isolates were also identified. Bacteremia in elderly adults, for instance, was caused by a IVa cluster–derived strain significantly more often than by strains from other AFLP clusters. Furthermore, expansion of multiresistant clones or clones associated with skin disease (impetigo) was detected, which suggests that epidemic potential is present in pathogenic strains of S. aureus. In addition, the virulence gene encoding Panton-Valentine leukocidin was significantly enriched in S. aureus strains causing abscesses and arthritis in comparison with the carriage group. We provide evidence that essentially any S. aureus genotype carried by humans can transform into a life-threatening human pathogen but that certain clones are more virulent than others.
Loss of pain perception in diabetes is dependent on a receptor of the immunoglobulin superfamily
Angelika Bierhaus, Karl-Matthias Haslbeck, Per M. Humpert, Birgit Liliensiek, Thomas Dehmer, Michael Morcos, Ahmed A.R. Sayed, Martin Andrassy, Stephan Schiekofer, Jochen G. Schneider, Jörg B. Schulz, Dieter Heuss, Bernhard Neundörfer, Stefan Dierl, Jochen Huber, Hans Tritschler, Ann-Marie Schmidt, Markus Schwaninger, Hans-Ulrich Haering, Erwin Schleicher, Michael Kasper, David M. Stern, Bernd Arnold, Peter P. NawrothAbstract | Full text | PDF (Page 1741)
Molecular events that result in loss of pain perception are poorly understood in diabetic neuropathy. Our results show that the receptor for advanced glycation end products (RAGE), a receptor associated with sustained NF-κB activation in the diabetic microenvironment, has a central role in sensory neuronal dysfunction. In sural nerve biopsies, ligands of RAGE, the receptor itself, activated NF-κBp65, and IL-6 colocalized in the microvasculature of patients with diabetic neuropathy. Activation of NF-κB and NF-κB–dependent gene expression was upregulated in peripheral nerves of diabetic mice, induced by advanced glycation end products, and prevented by RAGE blockade. NF-κB activation was blunted in RAGE-null (RAGE–/–) mice compared with robust enhancement in strain-matched controls, even 6 months after diabetes induction. Loss of pain perception, indicative of long-standing diabetic neuropathy, was reversed in WT mice treated with soluble RAGE. Most importantly, loss of pain perception was largely prevented in RAGE–/– mice, although they were not protected from diabetes-induced loss of PGP9.5-positive plantar nerve fibers. These data demonstrate, for the first time to our knowledge, that the RAGE–NF-κB axis operates in diabetic neuropathy, by mediating functional sensory deficits, and that its inhibition may provide new therapeutic approaches.
Increased oxidative stress in obesity and its impact on metabolic syndrome
Shigetada Furukawa, Takuya Fujita, Michio Shimabukuro, Masanori Iwaki, Yukio Yamada, Yoshimitsu Nakajima, Osamu Nakayama, Makoto Makishima, Morihiro Matsuda, Iichiro ShimomuraAbstract | Full text | PDF | Supplemental material (Page 1752)
Obesity is a principal causative factor in the development of metabolic syndrome. Here we report that increased oxidative stress in accumulated fat is an important pathogenic mechanism of obesity-associated metabolic syndrome. Fat accumulation correlated with systemic oxidative stress in humans and mice. Production of ROS increased selectively in adipose tissue of obese mice, accompanied by augmented expression of NADPH oxidase and decreased expression of antioxidative enzymes. In cultured adipocytes, elevated levels of fatty acids increased oxidative stress via NADPH oxidase activation, and oxidative stress caused dysregulated production of adipocytokines (fat-derived hormones), including adiponectin, plasminogen activator inhibitor–1, IL-6, and monocyte chemotactic protein–1. Finally, in obese mice, treatment with NADPH oxidase inhibitor reduced ROS production in adipose tissue, attenuated the dysregulation of adipocytokines, and improved diabetes, hyperlipidemia, and hepatic steatosis. Collectively, our results suggest that increased oxidative stress in accumulated fat is an early instigator of metabolic syndrome and that the redox state in adipose tissue is a potentially useful therapeutic target for obesity-associated metabolic syndrome.
EphB6-null mutation results in compromised T cell function
So far, there is very limited knowledge about the role of Eph kinases, the largest family of receptor tyrosine kinases, in the immune system. Here, using EphB6–/– mice, we demonstrated that in vitro and in vivo T cell responses such as lymphokine secretion, proliferation, and the development of delayed-type skin hypersensitivity and experimental autoimmune encephalitis in EphB6–/– mice were compromised. On the other hand, humoral immune responses, such as serum levels of different Ig isotypes and IgG response to tetanus toxoid, were normal in these mice. Mechanistically, we showed that EphB6 migrated to the aggregated TCRs and rafts after TCR activation. Further downstream, in the absence of EphB6, ZAP-70 activation, LAT phosphorylation, the association of PLCγ1 with SLP-76, and p44/42 MAPK activation were diminished. Thus, we have shown that EphB6 is pivotal in T cell function.
Adoptive immunotherapy of prostate cancer bone lesions using redirected effector lymphocytes
Jehonathan H. Pinthus, Tova Waks, Victoria Malina, Keren Kaufman-Francis, Alon Harmelin, Itzhak Aizenberg, Hannah Kanety, Jacob Ramon, Zelig EshharAbstract | Full text | PDF (Page 1774)
Prostate cancer is currently the most commonly diagnosed noncutaneous malignancy in American men. When metastatic, usually to the bone, the disease is no longer curable and is usually treated palliatively with androgen ablation. However, after conversion to androgen-independent disease, there is no effective therapy currently available. The “T body” approach, which uses genetically reprogrammed lymphocytes derived from the patient and expressing chimeric receptor genes, combines the effector functions of T lymphocytes and NK cells with the ability of antibodies to recognize predefined surface antigens with high specificity and in a non–MHC-restricted manner. We show here the therapeutic efficacy of human lymphocytes bearing erbB2-specific chimeric receptors on human prostate cancer BM lesions in a SCID mouse model after conditioning of the recipient to allow homing and persistent functioning of the adoptively transferred cells. Induction of stromal cell–derived factor-1 production within the BM using low-dose irradiation or cyclophosphamide combined with IL-2 administration enhanced the homing of systemically delivered T bodies, resulting in decreased tumor growth and prostate-specific antigen secretion, prolongation of survival, and even cure of the treated mice. These preclinical studies strongly support the idea that the T body approach has therapeutic potential in disseminated prostate cancer.
Pyk2 activation is integral to acid stimulation of sodium/hydrogen exchanger 3
Shaoying Li, Soichiro Sato, Xiaojing Yang, Patricia A. Preisig, Robert J. AlpernAbstract | Full text | PDF (Page 1782)
The present study examines the role of Pyk2 in acid regulation of sodium/hydrogen exchanger 3 (NHE3) activity in OKP cells, a kidney proximal tubule epithelial cell line. Incubation of OKP cells in acid media caused a transient increase in Pyk2 phosphorylation that peaked at 30 seconds and increased Pyk2/c-Src binding at 90 seconds. Pyk2 isolated by immunoprecipitation and studied in a cell-free system was activated and phosphorylated at acidic pH. Acid activation of Pyk2 (a) was specific for Pyk2 in that acid did not activate focal adhesion kinase, (b) required calcium, and (c) was associated with increased affinity for ATP. Transfection of OKP cells with dominant-negative pyk2K457A or small interfering pyk2 duplex RNA blocked acid activation of NHE3, while neither had an effect on glucocorticoid activation of NHE3. In addition, pyk2K457A blocked acid activation of c-Src kinase, which is also required for acid regulation of NHE3. The present results demonstrate that Pyk2 is directly activated by acidic pH and that Pyk2 activation is required for acid activation of c-Src kinase and NHE3. Given that partially purified Pyk2 can be activated by acid in a cell-free system, Pyk2 may serve as the pH sensor that initiates the acid-regulated signaling cascade involved in NHE3 regulation.
Fatal Mycobacterium tuberculosis infection despite adaptive immune response in the absence of MyD88
Cecile M. Fremond, Vladimir Yeremeev, Delphine M. Nicolle, Muazzam Jacobs, Valerie F. Quesniaux, Bernhard RyffelAbstract | Full text | PDF (Page 1790)
Toll-like receptors (TLRs) such as TLR2 and TLR4 have been implicated in host response to mycobacterial infection. Here, mice deficient in the TLR adaptor molecule myeloid differentiation factor 88 (MyD88) were infected with Mycobacterium tuberculosis (MTB). While primary MyD88–/– macrophages and DCs are defective in TNF, IL-12, and NO production in response to mycobacterial stimulation, the upregulation of costimulatory molecules CD40 and CD86 is unaffected. Aerogenic infection of MyD88–/– mice with MTB is lethal within 4 weeks with 2 log10 higher CFU in the lung; high pulmonary levels of cytokines and chemokines; and acute, necrotic pneumonia, despite a normal T cell response with IFN-γ production to mycobacterial antigens upon ex vivo restimulation. Vaccination with Mycobacterium bovis bacillus Calmette-Guérin conferred a substantial protection in MyD88–/– mice from acute MTB infection. These data demonstrate that MyD88 signaling is dispensable to raise an acquired immune response to MTB. Nonetheless, this acquired immune response is not sufficient to compensate for the profound innate immune defect and the inability of MyD88–/– mice to control MTB infection.
Utilizing the adjuvant properties of CD1d-dependent NK T cells in T cell–mediated immunotherapy
Jonathan D. Silk, Ian F. Hermans, Uzi Gileadi, Tsung Wen Chong, Dawn Shepherd, Mariolina Salio, Bini Mathew, Richard R. Schmidt, Sarah Jane Lunt, Kaye J. Williams, Ian J. Stratford, Adrian L. Harris, Vincenzo CerundoloAbstract | Full text | PDF | Supplemental material (Page 1800)
Activation of invariant CD1d-dependent NK T cells (iNKT cells) in vivo through administration of the glycolipid ligand α-galactosylceramide (α-GalCer) or the sphingosine-truncated α-GalCer analog OCH leads to CD40 signaling as well as the release of soluble molecules including type 1 and γ interferons that contribute to DC maturation. This process enhances T cell immunity to antigens presented by the DC. The adjuvant activity is further amplified if APCs are stimulated through Toll-like receptor 4, suggesting that iNKT cell signals can amplify maturation induced by microbial stimuli. The adjuvant activity of α-GalCer enhances both priming and boosting of CD8+ T cells to coadministered peptide or protein antigens, including a peptide encoding the clinically relevant, HLA-A2–restricted epitope of the human tumor antigen NY-ESO-1. Importantly, α-GalCer was used to induce CD8+ T cells to antigens delivered orally, despite the fact that this route of administration is normally associated with blunted responses. Only T cell responses induced in the presence of iNKT cell stimulation, whether by the i.v. or oral route, were capable of eradicating established tumors. Together these data highlight the therapeutic potential of iNKT cell ligands in vaccination strategies, particularly “heterologous prime-boost” strategies against tumors, and provide evidence that iNKT cell stimulation may be exploited in the development of oral vaccines.
T cell–dependent production of IFN-γ by NK cells in response to influenza A virus
Xiao-Song He, Monia Draghi, Kutubuddin Mahmood, Tyson H. Holmes, George W. Kemble, Cornelia L. Dekker, Ann M. Arvin, Peter Parham, Harry B. GreenbergAbstract | Full text | PDF (Page 1812)
The role of human NK cells in viral infections is poorly understood. We used a cytokine flow-cytometry assay to simultaneously investigate the IFN-γ response of NK and T lymphocytes to influenza A virus (fluA). When PBMCs from fluA-immune adult donors were incubated with fluA, IFN-γ was produced by both CD56dim and CD56bright subsets of NK cells, as well as by fluA-specific T cells. Purified NK cells did not produce IFN-γ in response to fluA, while depletion of T lymphocytes reduced to background levels the fluA-induced IFN-γ production by NK cells, which indicates that T cells are required for the IFN-γ response of NK cells. The fluA-induced IFN-γ production of NK cells was suppressed by anti–IL-2 Ab, while recombinant IL-2 replaced the helper function of T cells for IFN-γ production by NK cells. This indicates that IL-2 produced by fluA-specific T cells is involved in the T cell–dependent IFN-γ response of NK cells to fluA. Taken together, these results suggest that at an early stage of recurrent viral infection, NK-mediated innate immunity to the virus is enhanced by preexisting virus-specific T cells.
Stem cells: science, policy, and ethics
Nephrin strands contribute to a porous slit diaphragm scaffold as revealed by electron tomography