Issue published February 1, 2007

M uch has changed in the 5 years since the responsibility for editing the JCI was transferred to Columbia University. Wars and a hurricane have conspired with other factors to overwhelm the national treasury. Support for investigator-initiated research at the NIH is now at a level that jeopardizes the nation’s ability to adequately train future scientists to maintain the country’s leadership in biomedical research. Indeed, there is insufficient support for even the best and brightest biomedical scientists to pursue the frontiers of the sciences at a time of unprecedented opportunities. Human embryonic stem cell research is still being suppressed in the United States. Economic models that enable academic health centers to flourish in the face of increasing challenges and the rising costs of health care have for the most part remained elusive. Translational research has become the buzzword, but there is widespread confusion and disagreement about how to do it. Despite all of these and other challenges to the biomedical research enterprise, the JCI remains vibrant, with record numbers of submissions and a loyal and enthusiastic readership.

S cientific discovery occasionally occurs as a sudden and dramatic leap ahead but more often proceeds at a subtler and steadier pace. Each small step forward may escape public notice but is ultimately vital to the journey’s success. Indeed, such gradual advancement represents the collective contributions of many workers in the field, some new to the journey. While the notion of combined effort and multiple contributors is honorable, it poses an inherent danger. In our society, unproven, unorthodox, or unnoticed researchers may not receive the funding or support needed to make their contributions. Furthermore, even if they have the potential to make a leap, a hostile environment may preclude their doing so. This article concentrates on the looming crisis in diabetes research, but the principles pertain to all fields of clinical and biomedical science.

C ytokine production by the immune system contributes importantly to both health and disease. The nervous system, via an inflammatory reflex of the vagus nerve, can inhibit cytokine release and thereby prevent tissue injury and death. The efferent neural signaling pathway is termed the cholinergic antiinflammatory pathway. Cholinergic agonists inhibit cytokine synthesis and protect against cytokine-mediated diseases. Stimulation of the vagus nerve prevents the damaging effects of cytokine release in experimental sepsis, endotoxemia, ischemia/reperfusion injury, hemorrhagic shock, arthritis, and other inflammatory syndromes. Herein is a review of this physiological, functional anatomical mechanism for neurological regulation of cytokine-dependent disease that begins to define an immunological homunculus.

T he exact role(s) of the cytokine IFN-γ in the demyelinating disease multiple sclerosis remain controversial, with evidence suggesting both detrimental and protective effects of the cytokine in MS and MS models such as EAE. The study by Lin and coworkers in this issue of the JCI produces evidence that protective effects of IFN-γ on mature oligodendrocytes during EAE induction are mediated via activation of the pancreatic ER kinase (PERK), resulting in induction of the endoplasmic reticular stress response pathway (see the related article beginning on page 448). Modulation of this stress pathway has what we believe to be novel therapeutic potential for MS.

T he way in which multiple cell types organize themselves into a carefully sculpted, 3D labyrinth of vessels that regulate blood flow throughout the body has been a longstanding mystery. Clinicians familiar with congenital cardiovascular disease recognize how genetic variants and modest perturbations in this complex set of spatiotemporal interactions and stochastic processes can result in life-threatening anomalies. Although the mystery is not yet fully solved, we are poised at an exciting juncture, as insights from murine disease models are converging with advances in human genetics to shed new light on puzzling clinical phenotypes of vascular disease. The study by High et al. in this issue of the JCI establishes a model system that mimics clinical features of congenital cardiovascular disease and further defines the role of the Notch signaling pathway in the neural crest as an essential determinant of cardiovascular structure (see the related article beginning on page 353).

H SCs are one of only a few cell types that resist HIV-1 infection despite the presence of HIV-1 receptors. An increasing number of genes have been identified that can reduce the sensitivity of cultured cells to retrovirus infection, and in this issue of the JCI, Zhang et al. identify p21^{Waf1/Cip1/Sdi1} (p21) as a gene product that can influence the sensitivity of HSCs to HIV-1 infection (see the related article beginning on page 473). Strikingly, p21 appears to alter the fate of nuclear HIV-1 DNA, promoting the formation of circular viral DNA forms rather than functional proviruses.

I t has become increasingly obvious that the notion of a terminally differentiated cell is likely a simplified concept. Epithelial-mesenchymal transition (EMT), during which epithelial cells assume a mesenchymal phenotype, is a key event occurring during normal development and pathological processes. Multiple extracellular stimuli and transcriptional regulators can trigger EMT, but how such distinct signaling pathways orchestrate the complex cellular events that facilitate EMT is not well understood. In this issue of the JCI, Venkov et al. report on their examination of fibroblasts resulting from EMT and describe a novel protein-DNA complex that is essential for transcription of fibroblast-specific protein 1 (FSP1) and sufficient to induce early EMT events (see the related article beginning on page 482). Collectively, their results suggest that this complex is an important regulator of the EMT transcriptome.

T he combination of the induction of lymphopenia and vaccination and/or T cell transfer is garnering much attention for cancer treatment. Preclinical studies have shown that the induction of lymphopenia by chemotherapy or radiation can enhance the antitumor efficacy of several distinct, cell-based immunotherapeutic approaches. The mechanism(s) by which such enhancement is achieved are being intensively studied, yet there is much opportunity for improvement. The animal studies reported by Wrzesinski and colleagues in this issue of the JCI are a promising and timely step in this direction (see the related article beginning on page 492). The authors have evaluated both the effect of increasing the intensity of lymphodepletion and the influence of HSC transfer on the in vivo function of adoptively transferred CD8⁺ T cells. We discuss their results in light of the evolving field and their implications for advancing cell-based immunotherapies for cancer.

N utrient overload induces obesity, a primary risk factor for type 2 diabetes. Ribosomal biogenesis and protein synthesis, which are controlled by the mammalian target of rapamycin (mTOR), are primary energy-consuming processes in cells. mTOR phosphorylates and inactivates members of the eukaryotic translation initiation factor 4E–binding (eIF4E-binding) protein (4E-BP) family, which are translational repressors of 5′ cap–dependent protein synthesis. In this issue of the JCI, Le Bacquer et al. report that simultaneous deletion of both 4E-BP1 and 4E-BP2 in mice results in insulin resistance, decreased energy expenditure, and increased adipogenesis (see the related article beginning on page 387). These findings link protein synthesis, insulin sensitivity, and body weight.

A lthough ras is a potent mitogenic oncogene, its tumorigenicity depends on cellular context and cooperative events. Here we show that low-level expression of a constitutively active Ha-ras in mouse urothelium induces simple urothelial hyperplasia that is resistant to progression to full-fledged bladder tumors even in the absence of Ink4a/Arf. In stark contrast, doubling of the gene dosage of the activated Ha-ras triggered early-onset, rapidly growing, and 100% penetrant tumors throughout the urinary tract. Tumor initiation required superseding a rate-limiting step between simple and nodular hyperplasia, the latter of which is marked by the emergence of mesenchymal components and the coactivation of AKT and STAT pathways as well as PTEN inactivation. These results indicate that overactivation of Ha-ras is both necessary and sufficient to induce bladder tumors along a low-grade, noninvasive papillary pathway, and they shed light on the recent findings that ras activation, via point mutation, overexpression, or intensified signaling from FGF receptor 3, occurs in 70%–90% of these tumors in humans. Our results highlight the critical importance of the dosage/strength of Ha-ras activation in dictating its tumorigenicity — a mechanism of oncogene activation not fully appreciated to date. Finally, our results have clinical implications, as inhibiting ras and/or its downstream effectors, such as AKT and STAT3/5, could provide alternative means to treat low-grade, superficial papillary bladder tumors, the most common tumor in the urinary system.

A utophagy is a lysosome-dependent degradative pathway frequently activated in tumor cells treated with chemotherapy or radiation. Whether autophagy observed in treated cancer cells represents a mechanism that allows tumor cells to survive therapy or a mechanism for initiating a nonapoptotic form of programmed cell death remains controversial. To address this issue, the role of autophagy in a Myc-induced model of lymphoma generated from cells derived from p53ER^TAM/p53ER^TAM mice (with ER denoting estrogen receptor) was examined. Such tumors are resistant to apoptosis due to a lack of nuclear p53. Systemic administration of tamoxifen led to p53 activation and tumor regression followed by tumor recurrence. Activation of p53 was associated with the rapid appearance of apoptotic cells and the induction of autophagy in surviving cells. Inhibition of autophagy with either chloroquine or ATG5 short hairpin RNA (shRNA) enhanced the ability of either p53 activation or alkylating drug therapy to induce tumor cell death. These studies provide evidence that autophagy serves as a survival pathway in tumor cells treated with apoptosis activators and a rationale for the use of autophagy inhibitors such as chloroquine in combination with therapies designed to induce apoptosis in human cancers.

T he ability to proliferate independently of signals from other cell types is a fundamental characteristic of tumor cells. Using a 3D culture model of human breast cancer progression, we have delineated a protease-dependent autocrine loop that provides an oncogenic stimulus in the absence of proto-oncogene mutation. Targeting this protease, TNF-α–converting enzyme (TACE; also referred to as a disintegrin and metalloproteinase 17 [ADAM17]), with small molecular inhibitors or siRNAs reverted the malignant phenotype in a breast cancer cell line by preventing mobilization of 2 crucial growth factors, TGF-α and amphiregulin. We show that TACE-dependent ligand shedding was prevalent in a series of additional breast cancer cell lines and, in all cases examined, was amenable to inhibition. Using existing patient outcome data, we demonstrated a strong correlation between TACE and TGFA expression in human breast cancers that was predictive of poor prognosis. Tumors resulting from inappropriate activation of the EGFR were common in multiple tissues and were, for the most part, refractory to current targeted therapies. The data presented here delineate the molecular mechanism by which constitutive EGFR activity may be achieved in tumor progression without mutation of the EGFR itself or downstream pathway components and suggest that this important oncogenic pathway might usefully be targeted upstream of the receptor.

A ctivating EGFR mutations occur in human non–small cell lung cancer (NSCLC), with 5% of human lung squamous cell carcinomas having EGFRvIII mutations and approximately 10%–30% of lung adenocarcinomas having EGFR kinase domain mutations. An EGFR-targeting monoclonal antibody, mAb806, recognizes a conformational epitope of WT EGFR as well as the truncated EGFRvIII mutant. To explore the anticancer spectrum of this antibody for EGFR targeted cancer therapy, mAb806 was used to treat genetically engineered mice with lung tumors that were driven by either EGFRvIII or EGFR kinase domain mutations. Our results demonstrate that mAb806 is remarkably effective in blocking EGFRvIII signaling and inducing tumor cell apoptosis, resulting in dramatic tumor regression in the EGFRvIII-driven murine lung cancers. Another EGFR-targeting antibody, cetuximab, failed to show activity in these lung tumors. Furthermore, treatment of murine lung tumors driven by the EGFR kinase domain mutation with mAb806 also induced significant tumor regression, albeit to a less degree than that observed in EGFRvIII-driven tumors. Taken together, these data support the hypothesis that mAb806 may lead to significant advancements in the treatment of the population of NSCLC patients with these 2 classes of EGFR mutations.

T he cardiac outflow tract develops as a result of a complex interplay among several cell types, including cardiac neural crest cells, endothelial cells, and cardiomyocytes. In both humans and mice, mutations in components of the Notch signaling pathway result in congenital heart disease characterized by cardiac outflow tract defects. However, the specific cell types in which Notch functions during cardiovascular development remain to be defined. In addition, in vitro studies have provided conflicting data regarding the ability of Notch to promote or inhibit smooth muscle differentiation, while the physiological role for Notch in smooth muscle formation during development remains unclear. In this study, we generated mice in which Notch signaling was specifically inactivated in derivatives of the neural crest. These mice exhibited cardiovascular anomalies, including aortic arch patterning defects, pulmonary artery stenosis, and ventricular septal defects. We show that Notch plays a critical, cell-autonomous role in the differentiation of cardiac neural crest precursors into smooth muscle cells both in vitro and in vivo, and we identify specific Notch targets in neural crest that are implicated in this process. These results provide a molecular and cellular framework for understanding the role of Notch signaling in the etiology of congenital heart disease.

T he role of the cystic fibrosis transmembrane conductance regulator (CFTR) as a cAMP-dependent chloride channel on the apical membrane of epithelia is well established. However, the processes by which CFTR is regulated on the cell surface are not clear. Here we report the identification of a protein-protein interaction between CFTR and the cytoskeletal filamin proteins. Using proteomic approaches, we identified filamins as proteins that associate with the extreme CFTR N terminus. Furthermore, we identified a disease-causing missense mutation in CFTR, serine 13 to phenylalanine (S13F), which disrupted this interaction. In cells, filamins tethered plasma membrane CFTR to the underlying actin network. This interaction stabilized CFTR at the cell surface and regulated the plasma membrane dynamics and confinement of the channel. In the absence of filamin binding, CFTR was internalized from the cell surface, where it prematurely accumulated in lysosomes and was ultimately degraded. Our data demonstrate what we believe to be a previously unrecognized role for the CFTR N terminus in the regulation of the plasma membrane stability and metabolic stability of CFTR. In addition, we elucidate the molecular defect associated with the S13F mutation.

O besity and type 2 diabetes are associated with chronic inflammation. Adiponectin is an adipocyte-derived hormone with antidiabetic and antiinflammatory actions. Here, we demonstrate what we believe to be a previously undocumented activity of adiponectin, facilitating the uptake of early apoptotic cells by macrophages, an essential feature of immune system function. Adiponectin-deficient (APN-KO) mice were impaired in their ability to clear apoptotic thymocytes in response to dexamethasone treatment, and these animals displayed a reduced ability to clear early apoptotic cells that were injected into their intraperitoneal cavities. Conversely, adiponectin administration promoted the clearance of apoptotic cells by macrophages in both APN-KO and wild-type mice. Adiponectin overexpression also promoted apoptotic cell clearance and reduced features of autoimmunity in lpr mice whereas adiponectin deficiency in lpr mice led to a further reduction in apoptotic cell clearance, which was accompanied by exacerbated systemic inflammation. Adiponectin was capable of opsonizing apoptotic cells, and phagocytosis of cell corpses was mediated by the binding of adiponectin to calreticulin on the macrophage cell surface. We propose that adiponectin protects the organism from systemic inflammation by promoting the clearance of early apoptotic cells by macrophages through a receptor-dependent pathway involving calreticulin.

T he most common pathology associated with obesity is insulin resistance, which results in the onset of type 2 diabetes mellitus. Several studies have implicated the mammalian target of rapamycin (mTOR) signaling pathway in obesity. Eukaryotic translation initiation factor 4E–binding (eIF4E-binding) proteins (4E-BPs), which repress translation by binding to eIF4E, are downstream effectors of mTOR. We report that the combined disruption of 4E-BP1 and 4E-BP2 in mice increased their sensitivity to diet-induced obesity. Increased adiposity was explained at least in part by accelerated adipogenesis driven by increased expression of CCAAT/enhancer-binding protein δ (C/EBPδ), C/EBPα, and PPARγ coupled with reduced energy expenditure, reduced lipolysis, and greater fatty acid reesterification in the adipose tissue of 4E-BP1 and 4E-BP2 double KO mice. Increased insulin resistance in 4E-BP1 and 4E-BP2 double KO mice was associated with increased ribosomal protein S6 kinase (S6K) activity and impairment of Akt signaling in muscle, liver, and adipose tissue. These data clearly demonstrate the role of 4E-BPs as a metabolic brake in the development of obesity and reinforce the idea that deregulated mTOR signaling is associated with the development of the metabolic syndrome.

S H2B1 (previously named SH2-B), a cytoplasmic adaptor protein, binds via its Src homology 2 (SH2) domain to a variety of protein tyrosine kinases, including JAK2 and the insulin receptor. SH2B1-deficient mice are obese and diabetic. Here we demonstrated that multiple isoforms of SH2B1 (α, β, γ, and/or δ) were expressed in numerous tissues, including the brain, hypothalamus, liver, muscle, adipose tissue, heart, and pancreas. Rat SH2B1β was specifically expressed in neural tissue in SH2B1-transgenic (SH2B1^Tg) mice. SH2B1^Tg mice were crossed with SH2B1-knockout (SH2B1^KO) mice to generate SH2B1^TgKO mice expressing SH2B1 only in neural tissue but not in other tissues. Systemic deletion of the SH2B1 gene resulted in metabolic disorders in SH2B1^KO mice, including hyperlipidemia, leptin resistance, hyperphagia, obesity, hyperglycemia, insulin resistance, and glucose intolerance. Neuron-specific restoration of SH2B1β not only corrected the metabolic disorders in SH2B1^TgKO mice, but also improved JAK2-mediated leptin signaling and leptin regulation of orexigenic neuropeptide expression in the hypothalamus. Moreover, neuron-specific overexpression of SH2B1 dose-dependently protected against high-fat diet–induced leptin resistance and obesity. These observations suggest that neuronal SH2B1 regulates energy balance, body weight, peripheral insulin sensitivity, and glucose homeostasis at least in part by enhancing hypothalamic leptin sensitivity.

W iskott-Aldrich syndrome protein (WASp) is essential for optimal T cell activation. Patients with WAS exhibit both immunodeficiency and a marked susceptibility to systemic autoimmunity. We investigated whether alterations in Treg function might explain these paradoxical observations. While WASp-deficient (WASp^–/–) mice exhibited normal thymic Treg generation, the competitive fitness of peripheral Tregs was severely compromised. The total percentage of forkhead box P3–positive (Foxp3⁺) Tregs among CD4⁺ T cells was reduced, and WASp^–/– Tregs were rapidly outcompeted by WASp⁺ Tregs in vivo. These findings correlated with reduced expression of markers associated with self-antigen–driven peripheral Treg activation and homing to inflamed tissue. Consistent with these findings, WASp^–/– Tregs showed a reduced ability to control aberrant T cell activation and autoimmune pathology in Foxp3^–/– Scurfy (sf) mice. Finally, WASp⁺ Tregs exhibited a marked selective advantage in vivo in a WAS patient with a spontaneous revertant mutation, indicating that altered Treg fitness likely explains the autoimmune features in human WAS.

< i>Pseudomonas aeruginosa, an important cause of opportunistic infections in humans, delivers bacterial cytotoxins by type III secretion directly into the host cell cytoplasm, resulting in disruption of host cell signaling and host innate immunity. However, little is known about the fate of the toxins themselves following injection into the host cytosol. Here, we show by both in vitro and in vivo studies that the host ubiquitin ligase Cbl-b interacts with the type III–secreted effector exotoxin T (ExoT) and plays a key role in vivo in limiting bacterial dissemination mediated by ExoT. We demonstrate that, following polyubiquitination, ExoT undergoes regulated proteasomal degradation in the host cell cytosol. ExoT interacts with the E3 ubiquitin ligase Cbl-b and Crk, the substrate for the ExoT ADP ribosyltransferase (ADPRT) domain. The efficiency of degradation is dependent upon the activity of the ADPRT domain. In mouse models of acute pneumonia and systemic infection, Cbl-b is specifically required to limit the dissemination of ExoT-producing bacteria whereas c-Cbl plays no detectable role. To the best of our knowledge, this represents the first identification of a mammalian gene product that is specifically required for in vivo resistance to disease mediated by a type III–secreted effector.

E nteropathogenic E. coli (EPEC) is a major cause of infantile diarrhea, but the pathophysiology underlying associated diarrhea is poorly understood. We examined the role of the luminal membrane Cl^–/OH^– exchange process in EPEC pathogenesis using in vitro and in vivo models. Cl^–/OH^– exchange activity was measured as OH^– gradient–driven ³⁶Cl^– uptake. EPEC infection (60 minutes–3 hours) inhibited apical Cl^–/OH^– exchange activity in human intestinal Caco-2 and T84 cells. This effect was dependent upon the bacterial type III secretory system (TTSS) and involved secreted effector molecules EspG and EspG2, known to disrupt the host microtubular network. The microtubule-disrupting agent colchicine (100 μM, 3 hours) also inhibited ³⁶Cl^– uptake. The plasma membrane expression of major apical anion exchanger DRA (SLC26A3) was considerably reduced in EPEC-infected cells, corresponding with decreased Cl^–/OH^– exchange activity. Confocal microscopic studies showed that EPEC infection caused a marked redistribution of DRA from the apical membrane to intracellular compartments. Interestingly, infection of cells with an EPEC mutant deficient in espG significantly attenuated the decrease in surface expression of DRA protein as compared with treatment with wild-type EPEC. EPEC infection in vivo (1 day) also caused marked redistribution of surface DRA protein in the mouse colon. Our data demonstrate that EspG and EspG2 play an important role in contributing to EPEC infection–associated inhibition of luminal membrane chloride transport via modulation of surface DRA expression.

H eme oxygenase–1 (HO-1, encoded by HMOX1) dampens inflammatory reactions via the catabolism of heme into CO, Fe, and biliverdin. We report that expression of HO-1 dictates the pathologic outcome of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). Induction of EAE in Hmox1^–/– C57BL/6 mice led to enhanced CNS demyelination, paralysis, and mortality, as compared with Hmox1^+/+ mice. Induction of HO-1 by cobalt protoporphyrin IX (CoPPIX) administration after EAE onset reversed paralysis in C57BL/6 and SJL/J mice and disease relapse in SJL/J mice. These effects were not observed using zinc protoporphyrin IX, which does not induce HO-1. CoPPIX protection was abrogated in Hmox1^–/– C57BL/6 mice, indicating that CoPPIX acts via HO-1 to suppress EAE progression. The protective effect of HO-1 was associated with inhibition of MHC class II expression by APCs and inhibition of Th and CD8 T cell accumulation, proliferation, and effector function within the CNS. Exogenous CO mimicked these effects, suggesting that CO contributes to the protective action of HO-1. In conclusion, HO-1 or exposure to its end product CO counters autoimmune neuroinflammation and thus might be used therapeutically to treat MS.

I n response to ER stress, the pancreatic endoplasmic reticulum kinase (PERK) coordinates an adaptive program known as the integrated stress response (ISR) by phosphorylating the α subunit of eukaryotic translation initiation factor 2 (eIF2α). IFN-γ, which activates the ER stress response in oligodendrocytes, is believed to play a critical role in the immune-mediated CNS disorder multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE). Here we report that CNS delivery of IFN-γ before EAE onset ameliorated the disease course and prevented demyelination, axonal damage, and oligodendrocyte loss. The beneficial effects of IFN-γ were accompanied by PERK activation in oligodendrocytes and were abrogated in PERK-deficient animals. Our results indicate that IFN-γ activation of PERK in mature oligodendrocytes attenuates EAE severity and suggest that therapeutic approaches to activate the ISR could prove beneficial in MS.

I diopathic hypogonadotropic hypogonadism (IHH) due to defects of gonadotropin-releasing hormone (GnRH) secretion and/or action is a developmental disorder of sexual maturation. To date, several single-gene defects have been implicated in the pathogenesis of IHH. However, significant inter- and intrafamilial variability and apparent incomplete penetrance in familial cases of IHH are difficult to reconcile with the model of a single-gene defect. We therefore hypothesized that mutations at different IHH loci interact in some families to modify their phenotypes. To address this issue, we studied 2 families, one with Kallmann syndrome (IHH and anosmia) and another with normosmic IHH, in which a single-gene defect had been identified: a heterozygous FGF receptor 1 (FGFR1) mutation in pedigree 1 and a compound heterozygous gonadotropin-releasing hormone receptor (GNRHR) mutation in pedigree 2, both of which varied markedly in expressivity within and across families. Further candidate gene screening revealed a second heterozygous deletion in the nasal embryonic LHRH factor (NELF) gene in pedigree 1 and an additional heterozygous FGFR1 mutation in pedigree 2 that accounted for the considerable phenotypic variability. Therefore, 2 different gene defects can synergize to produce a more severe phenotype in IHH families than either alone. This genetic model could account for some phenotypic heterogeneity seen in GnRH deficiency.

I nhalation of iloprost, a stable prostacyclin (PGI₂) analog, is a well-accepted and safe treatment for pulmonary arterial hypertension. Although iloprost mainly acts as a vasodilator by binding to the I prostanoid (IP) receptor, recent evidence suggests that signaling via this receptor also has antiinflammatory effects through unclear mechanisms. Here we show in a murine model of asthma that iloprost inhalation suppressed the cardinal features of asthma when given during the priming or challenge phase. As a mechanism of action, iloprost interfered with the function of lung myeloid DCs, critical antigen-presenting cells of the airways. Iloprost treatment inhibited the maturation and migration of lung DCs to the mediastinal LNs, thereby abolishing the induction of an allergen-specific Th2 response in these nodes. The effect of iloprost was DC autonomous, as iloprost-treated DCs no longer induced Th2 differentiation from naive T cells or boosted effector cytokine production in primed Th2 cells. These data should pave the way for a clinical effectiveness study using inhaled iloprost for the treatment of asthma.

H ematopoietic stem cells are resistant to HIV-1 infection. Here, we report a novel mechanism by which the cyclin-dependent kinase inhibitor (CKI) p21^{Waf1/Cip1/Sdi1} (p21), a known regulator of stem cell pool size, restricts HIV-1 infection of primitive hematopoietic cells. Modifying p21 expression altered HIV-1 infection prior to changes in cell cycling and was selective for p21 since silencing the related CKIs, p27^Kip1 and p18^INK4C, had no effect on HIV-1. We show that p21 blocked viral infection by complexing with HIV-1 integrase and aborting chromosomal integration. A closely related lentivirus with a distinct integrase, SIVmac-251, and the other cell-intrinsic inhibitors of HIV-1, Trim5α, PML, Murr1, and IFN-α, were unaffected by p21. Therefore, p21 is an endogenous cellular component in stem cells that provides a unique molecular barrier to HIV-1 infection and may explain how these cells remain an uninfected “sanctuary” in HIV disease.

E pithelial-mesenchymal transition (EMT) is an important mechanism for phenotypic conversion in normal development and disease states such as tissue fibrosis and metastasis. While this conversion of epithelia is under tight transcriptional control, few of the key transcriptional proteins are known. Fibroblasts produced by EMT express a gene encoding fibroblast-specific protein 1 (FSP1), which is regulated by a proximal cis-acting promoter element called fibroblast transcription site–1 (FTS-1). In mass spectrometry, chromatin immunoprecipitation, and siRNA studies, we used FTS-1 as a unique probe for mediators of EMT and identified a complex of 2 proteins, CArG box–binding factor–A (CBF-A) and KRAB-associated protein 1 (KAP-1), that bind this site. Epithelial cells engineered to conditionally express recombinant CBF-A (rCBF-A) activate the transcription of FSP1 and undergo EMT. The FTS-1 response element also exists in the promoters modulating a broader EMT transcriptome, including Twist, and Snail, as well as E-cadherin, β-catenin, ZO 1, vimentin, α1(I) collagen, and α–smooth muscle actin, and the induction of rCBF-A appropriately alters their expression as well. We believe formation of the CBF-A/KAP-1/FTS-1 complex is sufficient for the induction of FSP1 and a novel proximal activator of EMT.

D epleting host immune elements with nonmyeloablative regimens prior to the adoptive transfer of tumor-specific CD8⁺ T cells significantly enhances tumor treatment. In the current study, superior antitumor efficacy was achieved by further increasing the intensity of lymphodepletion to a level that required HSC transplantation. Surprisingly, the HSC transplant and not the increased lymphodepletion caused a robust expansion of adoptively transferred tumor-specific CD8⁺ T cells. The HSC-driven cell expansion of effector, but not of naive, CD8⁺ T cells was independent of in vivo restimulation by MHC class I–expressing APCs. Simultaneously, HSCs also facilitated the reconstitution of the host lymphoid compartment, including inhibitory elements, not merely via the production of progeny cells but by enhancing the expansion of cells that had survived lymphodepletion. Profound lymphodepletion, by myeloablation or by genetic means, focused the nonspecific HSC boost preferentially toward the transferred tumor-specific T cells, leading to successful tumor treatment. These findings indicate that CD8⁺ T cell–mediated tumor responses can be efficiently driven by HSCs in the myeloablative setting and have substantial implications for the design of new antitumor immunotherapies.