Early transcriptional and epigenetic regulation of CD8+ T cell differentiation revealed by single-cell RNA sequencing.

During microbial infection, responding CD8+ T lymphocytes differentiate into heterogeneous subsets that together provide immediate and durable protection. To elucidate the dynamic transcriptional changes that underlie this process, we applied a single-cell RNA-sequencing approach and analyzed individual CD8+ T lymphocytes sequentially throughout the course of a viral infection in vivo. Our analyses revealed a striking transcriptional divergence among cells that had undergone their first division and identified previously unknown molecular determinants that controlled the fate specification of CD8+ T lymphocytes. Our findings suggest a model for the differentiation of terminal effector cells initiated by an early burst of transcriptional activity and subsequently refined by epigenetic silencing of transcripts associated with memory lymphocytes, which highlights the power and necessity of single-cell approaches.

Early specification of CD8+ T lymphocyte fates during adaptive immunity revealed by single-cell gene-expression analyses.

T lymphocytes responding to microbial infection give rise to effector cells that mediate acute host defense and memory cells that provide long-lived immunity, but the fundamental question of when and how these cells arise remains unresolved. Here we combined single-cell gene-expression analyses with 'machine-learning' approaches to trace the transcriptional 'roadmap' of individual CD8(+) T lymphocytes throughout the course of an immune response in vivo. Gene-expression signatures predictive of eventual fates could be discerned as early as the first T lymphocyte division and may have been influenced by asymmetric partitioning of the receptor for interleukin 2 (IL-2Rα) during mitosis. Our findings emphasize the importance of single-cell analyses in understanding fate determination and provide new insights into the specification of divergent lymphocyte fates early during an immune response to microbial infection.

Integrin Activation Controls Regulatory T Cell-Mediated Peripheral Tolerance.

Maintenance of the regulatory T (Treg) cell pool is essential for peripheral tolerance and prevention of autoimmunity. Integrins, heterodimeric transmembrane proteins consisting of α and ß subunits that mediate cell-to-cell and cell-to-extracellular matrix interactions, play an important role in facilitating Treg cell contact-mediated suppression. In this article, we show that integrin activation plays an essential, previously unappreciated role in maintaining murine Treg cell function. Treg cell-specific loss of talin, a ß integrin-binding protein, or expression of talin(L325R), a mutant that selectively abrogates integrin activation, resulted in lethal systemic autoimmunity. This dysfunction could be attributed, in part, to a global dysregulation of the Treg cell transcriptome. Activation of integrin α4ß1 led to increased suppressive capacity of the Treg cell pool, suggesting that modulating integrin activation on Treg cells may be a useful therapeutic strategy for autoimmune and inflammatory disorders. Taken together, these results reveal a critical role for integrin-mediated signals in controlling peripheral tolerance by virtue of maintaining Treg cell function.

Talin Plays a Critical Role in the Maintenance of the Regulatory T Cell Pool.

Talin, a cytoskeletal protein essential in mediating integrin activation, has been previously shown to be involved in the regulation of T cell proliferation and function. In this study, we describe a role for talin in maintaining the homeostasis and survival of the regulatory T (Treg) cell pool. T cell-specific deletion of talin in Tln1fl/flCd4Cre mice resulted in spontaneous lymphocyte activation, primarily due to numerical and functional deficiencies of Treg cells in the periphery. Peripheral talin-deficient Treg cells were unable to maintain high expression of IL-2Rα, resulting in impaired IL-2 signaling and ultimately leading to increased apoptosis through downregulation of prosurvival proteins Bcl-2 and Mcl-1. The requirement for talin in maintaining high IL-2Rα expression by Treg cells was due, in part, to integrin LFA-1-mediated interactions between Treg cells and dendritic cells. Collectively, our data suggest a critical role for talin in Treg cell-mediated maintenance of immune homeostasis.

Regulation of asymmetric division and CD8+ T lymphocyte fate specification by protein kinase Cζ and protein kinase Cλ/ι.

During an immune response against a microbial pathogen, activated naive T lymphocytes give rise to effector cells that provide acute host defense and memory cells that provide long-lived immunity. It has been shown that T lymphocytes can undergo asymmetric division, enabling the daughter cells to inherit unequal amounts of fate-determining proteins and thereby acquire distinct fates from their inception. In this study, we show that the absence of the atypical protein kinase C (PKC) isoforms, PKCζ and PKCλ/ι, disrupts asymmetric CD8(+) T lymphocyte division. These alterations were associated with aberrant acquisition of a pre-effector transcriptional program, detected by single-cell gene expression analyses, in lymphocytes that had undergone their first division in vivo and enhanced differentiation toward effector fates at the expense of memory fates. Together, these results demonstrate a role for atypical PKC in regulating asymmetric division and the specification of divergent CD8(+) T lymphocyte fates early during an immune response.

Immunodeficiency and autoimmune enterocolopathy linked to NFAT5 haploinsufficiency.

The link between autoimmune diseases and primary immunodeficiency syndromes has been increasingly appreciated. Immunologic evaluation of a young man with autoimmune enterocolopathy and unexplained infections revealed evidence of immunodeficiency, including IgG subclass deficiency, impaired Ag-induced lymphocyte proliferation, reduced cytokine production by CD8(+) T lymphocytes, and decreased numbers of NK cells. Genetic evaluation identified haploinsufficiency of NFAT5, a transcription factor regulating immune cell function and cellular adaptation to hyperosmotic stress, as a possible cause of this syndrome. Inhibition or deletion of NFAT5 in normal human and murine cells recapitulated several of the immune deficits identified in the patient. These results provide evidence of a primary immunodeficiency disorder associated with organ-specific autoimmunity linked to NFAT5 deficiency.

BMP9-regulated angiogenic signaling plays an important role in the osteogenic differentiation of mesenchymal progenitor cells.

Mesenchymal stromal progenitor cells (MSCs) are multipotent progenitors that can be isolated from numerous tissues. MSCs can undergo osteogenic differentiation under proper stimuli. We have recently demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most osteogenic BMPs. As one of the least studied BMPs, BMP9 has been shown to regulate angiogenesis in endothelial cells. However, it is unclear whether BMP9-regulated angiogenic signaling plays any important role in the BMP9-initiated osteogenic pathway in MSCs. Here, we investigate the functional role of hypoxia-inducible factor 1α (HIF1α)-mediated angiogenic signaling in BMP9-regulated osteogenic differentiation of MSCs. We find that BMP9 induces HIF1α expression in MSCs through Smad1/5/8 signaling. Exogenous expression of HIF1α potentiates BMP9-induced osteogenic differentiation of MSCs both in vitro and in vivo. siRNA-mediated silencing of HIF1α or HIF1α inhibitor CAY10585 profoundly blunts BMP9-induced osteogenic signaling in MSCs. HIF1α expression regulated by cobalt-induced hypoxia also recapitulates the synergistic effect between HIF1α and BMP9 in osteogenic differentiation. Mechanistically, HIF1α is shown to exert its synergistic effect with BMP9 by inducing both angiogenic signaling and osteogenic signaling in MSCs. Thus, our findings should not only expand our understanding of the molecular basis behind BMP9-regulated osteoblastic lineage-specific differentiation, but also provide an opportunity to harness the BMP9-induced synergy between osteogenic and angiogenic signaling pathways in regenerative medicine.

Cross-talk between EGF and BMP9 signalling pathways regulates the osteogenic differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent progenitors, which give rise to several lineages, including bone, cartilage and fat. Epidermal growth factor (EGF) stimulates cell growth, proliferation and differentiation. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein tyrosine kinase activity of its receptor, which initiates a signal transduction cascade causing a variety of biochemical changes within the cell and regulating cell proliferation and differentiation. We have identified BMP9 as one of the most osteogenic BMPs in MSCs. In this study, we investigate if EGF signalling cross-talks with BMP9 and regulates BMP9-induced osteogenic differentiation. We find that EGF potentiates BMP9-induced early and late osteogenic markers of MSCs in vitro, which can be effectively blunted by EGFR inhibitors Gefitinib and Erlotinib or receptor tyrosine kinase inhibitors AG-1478 and AG-494 in a dose- and time-dependent manner. Furthermore, EGF significantly augments BMP9-induced bone formation in the cultured mouse foetal limb explants. In vivo stem cell implantation experiment reveals that exogenous expression of EGF in MSCs can effectively potentiate BMP9-induced ectopic bone formation, yielding larger and more mature bone masses. Interestingly, we find that, while EGF can induce BMP9 expression in MSCs, EGFR expression is directly up-regulated by BMP9 through Smad1/5/8 signalling pathway. Thus, the cross-talk between EGF and BMP9 signalling pathways in MSCs may underline their important roles in regulating osteogenic differentiation. Harnessing the synergy between BMP9 and EGF should be beneficial for enhancing osteogenesis in regenerative medicine.

Viatorr Stent Migration and Retrieval during Transjugular Intrahepatic Portosystemic Shunt Revision.

Transjugular intrahepatic portosystemic shunt (TIPS) is an important interventional option for the treatment of complications related to cirrhosis and portal hypertension. Emergent TIPS placement can be a life-saving measure in patients with uncontrolled variceal hemorrhage. After TIPS placement, patients may benefit from additional interventions for clinical optimization including stent dilation, stent extension, and embolization of varices. Here, we describe a case of emergent TIPS placement and revision which resulted in TIPS stent migration requiring stent removal and replacement. We discuss our technique and review previously reported methods for the management of migrated TIPS stents.

Use of partial AUC to demonstrate bioequivalence of Zolpidem Tartrate Extended Release formulations.

PURPOSE: FDA's bioequivalence recommendation for Zolpidem Tartrate Extended Release Tablets is the first to use partial AUC (pAUC) metrics for determining bioequivalence of modified-release dosage forms. Modeling and simulation studies were performed to aid in understanding the need for pAUC measures and also the proper pAUC truncation times. METHODS: Deconvolution techniques, In Vitro/In Vivo Correlations, and the CAT (Compartmental Absorption and Transit) model were used to predict the PK profiles for zolpidem. Models were validated using in-house data submitted to the FDA. Using dissolution profiles expressed by the Weibull model as input for the CAT model, dissolution spaces were derived for simulated test formulations. RESULTS: The AUC(0-1.5) parameter was indicative of IR characteristics of early exposure and effectively distinguished among formulations that produced different pharmacodynamic effects. The AUC(1.5-t) parameter ensured equivalence with respect to the sustained release phase of Ambien CR. The variability of AUC(0-1.5) is higher than other PK parameters, but is reasonable for use in an equivalence test. CONCLUSIONS: In addition to the traditional PK parameters of AUCinf and Cmax, AUC(0-1.5) and AUC(1.5-t) are recommended to provide bioequivalence measures with respect to label indications for Ambien CR: onset of sleep and sleep maintenance.

Tetrandrine inhibits Wnt/ß-catenin signaling and suppresses tumor growth of human colorectal cancer.

As one of the most common malignancies, colon cancer is initiated by abnormal activation of the Wnt/ß-catenin pathway. Although the treatment options have increased for some patients, overall progress has been modest. Thus, there is a great need to develop new treatments. We have found that bisbenzylisoquinoline alkaloid tetrandrine (TET) exhibits anticancer activity. TET is used as a calcium channel blocker to treat hypertensive and arrhythmic conditions in Chinese medicine. Here, we investigate the molecular basis underlying TET's anticancer activity. We compare TET with six chemotherapy drugs in eight cancer lines and find that TET exhibits comparable anticancer activities with camptothecin, vincristine, paclitaxel, and doxorubicin, and better than that of 5-fluorouracil (5-FU) and carboplatin. TET IC50 is ≤5 µM in most of the tested cancer lines. TET exhibits synergistic anticancer activity with 5-FU and reduces migration and invasion capabilities of HCT116 cells. Furthermore, TET induces apoptosis and inhibits xenograft tumor growth of colon cancer. TET treatment leads to a decrease in ß-catenin protein level in xenograft tumors, which is confirmed by T-cell factor/lymphocyte enhancer factor and c-Myc reporter assays. It is noteworthy that HCT116 cells with allelic oncogenic ß-catenin deleted are less sensitive to TET-mediated inhibition of proliferation, viability, and xenograft tumor growth. Thus, our findings strongly suggest that the anticancer effect of TET in colon cancer may be at least in part mediated by targeting ß-catenin activity. Therefore, TET may be used alone or in combination as an effective anticancer agent.

TGFbeta/BMP type I receptors ALK1 and ALK2 are essential for BMP9-induced osteogenic signaling in mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are bone marrow stromal cells that can differentiate into multiple lineages. We previously demonstrated that BMP9 is one of the most potent BMPs to induce osteogenic differentiation of MSCs. BMP9 is one of the least studied BMPs. Whereas ALK1, ALK5, and/or endoglin have recently been reported as potential BMP9 type I receptors in endothelial cells, little is known about type I receptor involvement in BMP9-induced osteogenic differentiation in MSCs. Here, we conduct a comprehensive analysis of the functional role of seven type I receptors in BMP9-induced osteogenic signaling in MSCs. We have found that most of the seven type I receptors are expressed in MSCs. However, using dominant-negative mutants for the seven type I receptors, we demonstrate that only ALK1 and ALK2 mutants effectively inhibit BMP9-induced osteogenic differentiation in vitro and ectopic ossification in MSC implantation assays. Protein fragment complementation assays demonstrate that ALK1 and ALK2 directly interact with BMP9. Likewise, RNAi silencing of ALK1 and ALK2 expression inhibits BMP9-induced BMPR-Smad activity and osteogenic differentiation in MSCs both in vitro and in vivo. Therefore, our results strongly suggest that ALK1 and ALK2 may play an important role in mediating BMP9-induced osteogenic differentiation. These findings should further aid us in understanding the molecular mechanism through which BMP9 regulates osteogenic differentiation of MSCs.

Defective osteogenic differentiation in the development of osteosarcoma.

Osteosarcoma (OS) is associated with poor prognosis due to its high incidence of metastasis and chemoresistance. It often arises in areas of rapid bone growth in long bones during the adolescent growth spurt. Although certain genetic conditions and alterations increase the risk of developing OS, the molecular pathogenesis is poorly understood. Recently, defects in differentiation have been linked to cancers, as they are associated with high cell proliferation. Treatments overcoming these defects enable terminal differentiation and subsequent tumor inhibition. OS development may be associated with defects in osteogenic differentiation. While early regulators of osteogenesis are unable to bypass these defects, late osteogenic regulators, including Runx2 and Osterix, are able to overcome some of the defects and inhibit tumor propagation through promoting osteogenic differentiation. Further understanding of the relationship between defects in osteogenic differentiation and tumor development holds tremendous potential in treating OS.

Surveillance Imaging following Endovascular Aneurysm Repair: State of the Art.

Endovascular aneurysmal repair (EVAR) has become a prominent modality for the treatment of abdominal aortic aneurysm. Surveillance imaging is important for the detection of device-related complications, which include endoleak, structural abnormalities, and infection. Currently used modalities include ultrasound, X-ray, computed tomography, magnetic resonance imaging, and angiography. Understanding the advantages and drawbacks of each modality, as well available guidelines, can guide selection of the appropriate technique for individual patients. We review complications following EVAR and advances in surveillance imaging modalities.

miR-142 controls metabolic reprogramming that regulates dendritic cell activation.

DCs undergo metabolic reprogramming from a predominantly oxidative phosphorylation (OXPHOS) to glycolysis to mount an immunogenic response. The mechanism underpinning the metabolic reprogramming remains elusive. We demonstrate that miRNA-142 (miR-142) is pivotal for this shift in metabolism, which regulates the tolerogenic and immunogenic responses of DCs. In the absence of miR-142, DCs fail to switch from OXPHOS and show reduced production of proinflammatory cytokines and the ability to activate T cells in vitro and in in vivo models of sepsis and alloimmunity. Mechanistic studies demonstrate that miR-142 regulates fatty acid (FA) oxidation, which causes the failure to switch to glycolysis. Loss- and gain-of-function experiments identified carnitine palmitoyltransferase -1a (CPT1a), a key regulator of the FA pathway, as a direct target of miR-142 that is pivotal for the metabolic switch. Thus, our findings show that miR-142 is central to the metabolic reprogramming that specifically favors glycolysis and immunogenic response by DCs.

SNARE protein SEC22B regulates early embryonic development.

The highly conserved SNARE protein SEC22B mediates diverse and critical functions, including phagocytosis, cell growth, autophagy, and protein secretion. However, these characterizations have thus far been limited to in vitro work. Here, we expand our understanding of the role Sec22b plays in vivo. We utilized Cre-Lox mice to delete Sec22b in three tissue compartments. With a germline deletion of Sec22b, we observed embryonic death at E8.5. Hematopoietic/endothelial cell deletion of Sec22b also resulted in in utero death. Notably, mice with Sec22b deletion in CD11c-expressing cells of the hematopoietic system survive to adulthood. These data demonstrate Sec22b contributes to early embryogenesis through activity both in hematopoietic/endothelial tissues as well as in other tissues yet to be defined.

von Hippel-Lindau disease-associated hemangioblastomas are derived from embryologic multipotent cells.

BACKGROUND: To determine the origin of the neoplastic cell in central nervous system (CNS) hemangioblastomas in von Hippel-Lindau disease (VHL) and its role in tumor formation and distribution, we characterized and differentiated neoplastic cells from hemangioblastomas removed from VHL patients. METHODS AND FINDINGS: A total of 31 CNS hemangioblastomas from 25 VHL patients were resected and analyzed. Tumor cells from the hemangioblastomas were characterized, grown, and differentiated into multiple lineages. Resected hemangioblastomas were located in the cerebellum (11 tumors), brainstem (five tumors), and spinal cord (15 tumors). Consistent with an embryologically derived hemangioblast, the neoplastic cells demonstrated coexpression of the mesodermal markers brachyury, Flk-1 (vascular endothelial growth factor-2), and stem cell leukemia (Scl). The neoplastic cells also expressed hematopoietic stem cell antigens and receptors including CD133, CD34, c-kit, Scl, erythropoietin, and erythropoietin receptor. Under specific microenvironments, neoplastic cells (hemangioblasts) were expanded and differentiated into erythrocytic, granulocytic, and endothelial progenitors. Deletion of the wild-type VHL allele in the hematopoietic and endothelial progeny confirmed their neoplastic origin. CONCLUSIONS: The neoplastic cell of origin for CNS hemangioblastomas in VHL patients is the mesoderm-derived, embryologically arrested hemangioblast. The hematopoietic and endothelial differentiation potential of these cells can be reactivated under suitable conditions. These findings may also explain the unique tissue distribution of tumor involvement.

Assessment of subconjunctival and intrascleral drug delivery to the posterior segment using dynamic contrast-enhanced magnetic resonance imaging.

PURPOSE: Sustained-release intravitreal drug implants for posterior segment diseases are associated with significant complications. As an alternative, subconjunctival infusions of drug to the episclera of the back of the eye have been performed, but results in clinical trials for macular diseases showed mixed RESULTS: To improve understanding of transscleral drug delivery to the posterior segment, the distribution and clearance of gadolinium-diethylene-triamino-penta-acetic acid (Gd-DTPA) infused in the subconjunctival or intrascleral space was investigated by means of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). METHODS: In anesthetized rabbits, catheters were placed anteriorly in the subconjunctival or intrascleral space and infused with Gd-DTPA at 1 and 10 muL/min. Distribution and clearance of Gd-DTPA were measured using DCE-MRI. Histologic examination was performed to assess ocular toxicity of the delivery system. results. Subconjunctival infusions failed to produce detectable levels of Gd-DTPA in the back of the eye. In contrast, intrascleral infusions expanded the suprachoroidal layer and delivered Gd-DTPA to the posterior segment. Suprachoroidal clearance of Gd-DTPA followed first-order kinetics with an average half-life of 5.4 and 11.8 minutes after intrascleral infusions at 1 and 10 muL/min, respectively. Histologic examination demonstrated expansion of the tissues in the suprachoroidal space that normalized after infusion termination. CONCLUSIONS: An intrascleral infusion was successful in transporting Gd-DTPA to the posterior segment from an anterior infusion site with limited anterior segment exposure. The suprachoroidal space appears to be an expandible conduit for drug transport to the posterior segment. Further studies are indicated to explore the feasibility of clinical applications.

Transport barriers in transscleral drug delivery for retinal diseases.

Transscleral delivery has emerged as an attractive method for treating retinal disorders because it offers localized delivery of drugs as a less invasive method compared to intravitreal administration. Numerous novel transscleral drug delivery systems ranging from microparticles to implants have been reported. However, transscleral delivery is currently not as clinically effective as intravitreal delivery in the treatment of retinal diseases. Transscleral drug delivery systems require drugs to permeate through several layers of ocular tissue (sclera, Bruch's membrane-choroid, retinal pigment epithelium) to reach the neuroretina. As a result, a steep drug concentration gradient from the sclera to the retina is established, and very low concentrations of drug are detected in the retina. This steep gradient is created by the barriers to transport that hinder drug molecules from successfully reaching the retina. A review of the literature reveals 3 types of barriers hindering transscleral drug delivery: static, dynamic and metabolic. While static barriers have been examined in detail, the literature on dynamic and metabolic barriers is lacking. These barriers must be investigated further to gain a more complete understanding of the transport barriers involved in transscleral drug delivery.