Impaired insulin secretion in type 2 diabetes (T2D) is linked to reduced insulin granule docking, disorganization of the exocytotic site, and an impaired glucose-dependent facilitation of insulin exocytosis. We show in β-cells from 80 human donors that the glucose-dependent amplification of exocytosis is disrupted in T2D. Spatial analyses of granule fusion, visualized by total internal reflection fluorescence (TIRF) microscopy in 24 of these donors, demonstrate that these are non-random across the surface of β-cells from donors with no diabetes (ND). The compartmentalization of events occurs within regions defined by concurrent or recent membrane-resident secretory granules. This organization, and the number of membrane-associated granules, is glucose-dependent and notably impaired in T2D β-cells. Mechanistically, multi-channel Kv2.1 clusters contribute to maintaining the density of membrane-resident granules and the number of fusion ‘hotspots’, while SUMOylation sites at the channel N- (K145) and C-terminus (K470) determine the relative proportion of fusion events occurring within these regions. Thus, a glucose-dependent compartmentalization of fusion, regulated in part by a structural role for Kv2.1, is disrupted in β-cells from donors with type 2 diabetes.
Jianyang Fu, John Maringa Githaka, Xiaoqing Dai, Gregory Plummer, Kunimasa Suzuki, Aliya F. Spigelman, Austin Bautista, Ryekjang Kim, Dafna Greitzer-Antes, Jocelyn E. Manning Fox, Herbert Y. Gaisano, Patrick E. MacDonald
Background: The lymphocyte-depleting antibody alemtuzumab is a highly effective treatment of relapsing-remitting multiple sclerosis (RRMS); however 50% of patients develop novel autoimmunity post-treatment. Most at risk are individuals who reconstitute their T-cell pool by proliferating residual cells, rather than producing new T-cells in the thymus; raising the possibility that autoimmunity might be prevented by increasing thymopoiesis. Keratinocyte growth factor (palifermin) promotes thymopoiesis in non-human primates. Methods: Following a dose-tolerability sub-study, individuals with RRMS (duration ≤10 years; expanded disability status scale ≤5·0; with ≥2 relapses in the previous 2 years) were randomised to placebo or 180mcg/kg/day palifermin, given for 3 days immediately prior to and after each cycle of alemtuzumab, with repeat doses at M1 and M3. The interim primary endpoint was naïve CD4+ T-cell count at M6. Exploratory endpoints included: number of recent thymic-emigrants (RTEs) and signal-joint T-cell receptor excision circles (sjTRECs)/mL of blood. The trial primary endpoint was incidence of autoimmunity at M30. Findings: At M6, individuals receiving palifermin had fewer naïve CD4+T-cells (2.229x107/L vs. 7.733x107/L; p=0.007), RTEs (16% vs. 34%) and sjTRECs/mL (1100 vs. 3396), leading to protocol-defined termination of recruitment. No difference was observed in the rate of autoimmunity between the two groups Conclusion: In contrast to animal studies, palifermin reduced thymopoiesis in our patients. These results offer a note of caution to those using palifermin to promote thymopoiesis in other settings, particularly in the oncology/haematology setting where alemtuzumab is often used as part of the conditioning regime. Trial Registration: ClinicalTrials.gov NCT01712945 Funding: MRC and Moulton Charitable Foundation
Alasdair J. Coles, Laura Azzopardi, Onajite Kousin-Ezewu, Harpreet Kaur Mullay, Sara A.J. Thompson, Lorna Jarvis, Jessica Davies, Sarah Howlett, Daniel Rainbow, Judith Babar, Timothy J. Sadler, J. William L. Brown, Edward Needham, Karen May, Zoya G. Georgieva, Adam E. Handel, Stefano Maio, Mary Deadman, Ioanna Rota, Georg Holländer, Sarah Dawson, David Jayne, Ruth Seggewiss-Bernhardt, Daniel C. Douek, John D. Isaacs, Joanne L. Jones
Traumatic brain injury (TBI) causes cortical dysfunction and can lead to post-traumatic epilepsy. Multiple studies demonstrate that GABAergic inhibitory network function is compromised following TBI, which may contribute to hyperexcitability and motor, behavioral, and cognitive deficits. Preserving the function of GABAergic interneurons, therefore, is a rational therapeutic strategy to preserve cortical function after TBI and prevent long-term clinical complications. Here, we explored an approach based on the ketogenic diet, a neuroprotective and anticonvulsant dietary therapy which results in reduced glycolysis and increased ketosis. Utilizing a pharmacologic inhibitor of glycolysis (2-deoxyglucose, or 2-DG), we found that acute in vitro application of 2-DG decreased the excitability of excitatory neurons, but not inhibitory interneurons, in cortical slices from naïve mice. Employing the controlled cortical impact (CCI) model of TBI in mice, we found that in vitro 2-DG treatment rapidly attenuated epileptiform activity seen in acute cortical slices 3 to 5 weeks after TBI. One week of in vivo 2-DG treatment immediately after TBI prevented the development of epileptiform activity, restored excitatory and inhibitory synaptic activity, and attenuated the loss of parvalbumin-expressing inhibitory interneurons. In summary, 2-DG may have therapeutic potential to restore network function following TBI.
Jenny B. Koenig, David Cantu, Cho S. Low, Mary E. Sommer, Farzad Noubary, Danielle Croker, Michael Whalen, Dong Kong, Chris G. Dulla
BACKGROUND. Cutaneous neurofibromas (cNF) are physically disfiguring, painful, and cause extensive psychologic harm in patients with neurofibromatosis type 1 (NF1). There is currently no effective medical treatment and surgical procedures are inaccessible to most NF1 patients globally. OBJECTIVE. While research is underway to find an effective medical treatment for cNF, there is an urgent need to develop surgical approach that is accessible to all NF1 patients in the world with the skill set and equipment found in most general medical office settings. Here, we present a robust surgical approach to remove cNF that does not require sterile surgical field, utilizes accessible clinical equipment, and can be performed by any health care providers including family practitioners, and physician assistants. METHODS. In a prospective case-series, patients with NF1 underwent this surgical procedure which removes multiple cutaneous neurofibromas. The Dermatology Life Quality Index was given to subjects before and after the procedure as surrogate for patient satisfaction. RESULTS. 83 tumors were removed throughout the body from twelve individuals. Examination at follow-up visits revealed well-healed scars without infection or adverse events including aberrant scarring. Patient satisfaction with the procedure was high with significant improvements in symptoms, daily activities, leisure, personal relationships, and treatment experience (P = 0.00062). CONCLUSION. This study demonstrates a robust surgical approach to management cutaneous neurofibromas which can be accessed world-wide to individuals with NF1 and performed by a wide-variety of medical specialists with high clinical efficacy and patient satisfaction.
Bahir H. Chamseddin, La’Nette Hernandez, Dezehree Solorzano, Juan Vega, Lu Q. Le
Chimeric antigen receptor (CAR) T cell therapies have achieved promising outcomes in several cancers, however more challenging oncology indications may necessitate advanced antigen receptor designs and functions. Here we describe a bipartite receptor system comprised of separate antigen targeting and signal transduction polypeptides, each containing an extracellular dimerization domain. We demonstrate that T cell activation remains antigen dependent but can only be achieved in the presence of a dimerizing drug, rapamycin. Studies performed in vitro and in xenograft mouse models illustrate equivalent to superior anti-tumor potency compared to currently used CAR designs, and at rapamycin concentrations well below immunosuppressive levels. We further show that the extracellular positioning of the dimerization domains enables the administration of recombinant re-targeting modules, potentially extending antigen targeting. Overall, this novel regulatable CAR design has exquisite drug sensitivity, provides robust anti-tumor responses, and is uniquely flexible for multiplex antigen targeting or retargeting, which may further assist the development of safe, potent and durable T cell therapeutics.
Wai-Hang Leung, Joel Gay, Unja Martin, Tracy E. Garrett, Holly M. Horton, Michael T. Certo, Bruce R. Blazar, Richard A. Morgan, Philip D. Gregory, Jordan Jarjour, Alexander Astrakhan
Enchondroma and chondrosarcoma are the most common benign and malignant cartilaginous neoplasms. Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are present in the majority of these tumors. We performed RNA-seq analysis on chondrocytes from Col2a1Cre;Idh1LSL/+ animals and found that genes implied in cholesterol synthesis pathway were significantly upregulated in the mutant chondrocytes. We examined the phenotypic effect of inhibiting intracellular cholesterol biosynthesis on enchondroma formation by conditionally deleting SCAP (sterol regulatory element-binding protein cleavage-activating protein), a protein activating intracellular cholesterol synthesis, in IDH1 mutant mice. We found fewer enchondromas in animals lacking SCAP. Furthermore, in chondrosarcomas, pharmacological inhibition of intracellular cholesterol synthesis significantly reduced chondrosarcoma cell viability in vitro and suppressed tumor growth in vivo. Taken together, these data suggest that intracellular cholesterol synthesis is a potential therapeutic target for enchondromas and chondrosarcomas.
Hongyuan Zhang, Qingxia Wei, Hidetoshi Tsushima, Vijitha Puviindran, Yuning J. Tang, Sinthu Pathmanapan, Raymond Poon, Eyal Ramu, Mushriq Al-Jazrawe, Jay S. Wunder, Benjamin A. Alman
In the current preclinical study, we demonstrate the therapeutic potential of sarcospan (SSPN) overexpression to alleviate cardiomyopathy associated with Duchenne muscular dystrophy (DMD) utilizing dystrophin-deficient mdx mice with utrophin haploinsufficiency that more accurately represent the severe disease course of human DMD. SSPN interacts with dystrophin, the DMD disease gene product, and its autosomal paralog utrophin, which is upregulated in DMD as a partial compensatory mechanism. SSPN transgenic mice have enhanced abundance of fully glycosylated α-dystroglycan, which may further protect dystrophin-deficient cardiac membranes. Baseline echocardiography reveals SSPN improves systolic function and hypertrophic indices in mdx and mdx:utr-heterozygous mice. Assessment of SSPN transgenic mdx mice by hemodynamic pressure-volume methods highlights enhanced systolic performance compared to mdx controls. SSPN restores cardiac sarcolemma stability, the primary defect in DMD disease, reduces fibrotic response and improves contractile function. We demonstrate that SSPN ameliorates more advanced cardiac disease in the context of diminished sarcolemma expression of utrophin and β1D integrin that mitigate disease severity and partially restores responsiveness to β-adrenergic stimulation. Overall, our current and previous findings suggest SSPN overexpression in DMD mouse models positively impacts skeletal, pulmonary and cardiac performance by addressing the stability of proteins at the sarcolemma that protect the heart from injury, supporting SSPN and membrane stabilization as a therapeutic target for DMD.
Michelle S. Parvatiyar, Alexandra J. Brownstein, Rosemeire M. Kanashiro-Takeuchi, Judd R. Collado, Karissa M. Dieseldorff Jones, Jay Gopal, Katherine G. Hammond, Jamie L. Marshall, Abel Ferrel, Aaron M. Beedle, Jeffrey S. Chamberlain, Jose Renato Pinto, Rachelle H. Crosbie
Monosomy 7 or deletion of 7q (del(7q)) are common clonal cytogenetic abnormalities associated with high grade myelodysplastic syndrome (MDS) arising in inherited and acquired bone marrow failure. Current non-transplant approaches to treat marrow failure may be complicated by stimulation of clonal outgrowth. To study the biological consequences of del(7q) within the context of a failing marrow, we generated induced pluripotent stem cells (iPSCs) derived from patients with Shwachman Diamond Syndrome (SDS), a bone marrow failure disorder with MDS predisposition, and genomically engineered a 7q deletion. The TGFβ pathway was the top differentially regulated pathway in transcriptomic analysis of SDS versus SDSdel(7q) iPSCs. SMAD2 phosphorylation was increased in SDS relative to wild type cells consistent with hyperactivation of the TGFbeta pathway in SDS. Phospho-SMAD2 levels were reduced following 7q deletion in SDS cells and increased upon restoration of 7q diploidy. Inhibition of the TGFbeta pathway rescued hematopoiesis in SDS-iPSCs and in bone marrow hematopoietic cells from SDS patients while it had no impact on the SDSdel(7q) cells. These results identified a potential targetable vulnerability to improve hematopoiesis in an MDS-predisposition syndrome, and highlight the importance of the germline context of somatic alterations to inform precision medicine approaches to therapy.
Melisa Ruiz-Gutierrez, Özge Vargel Bölükbaşi, Gabriela Alexe, Andriana G. Kotini, Kaitlyn Ballotti, Cailin E. Joyce, David W. Russell, Kimberly Stegmaier, Kasiani Myers, Carl D. Novina, Eirini P. Papapetrou, Akiko Shimamura
Atrial fibrillation (AF) is the most common cardiac arrhythmia and accounts for substantial morbidity and mortality. Recently, we created a mouse model with spontaneous and sustained AF caused by a mutation in the NaV1.5 channel (F1759A) that enhances persistent Na+ current, thereby enabling the investigation of molecular mechanisms that cause AF and the identification of novel treatment strategies. The mice have regional heterogeneity of action potential duration of the atria similar to observations in patients with AF. In these mice, we found that the initiation and persistence of the rotational reentrant AF arrhythmias, known as spiral waves or rotors, were dependent upon action potential duration heterogeneity. The centers of the rotors were localized to regions of greatest heterogeneity of the action potential duration. Pharmacologically attenuating the action potential duration heterogeneity reduced both spontaneous and pacing-induced AF. Computer-based simulations also demonstrated that the action potential duration heterogeneity is sufficient to generate rotors that manifest as AF. Taken together, these findings suggest that action potential duration heterogeneity in mice and humans is one mechanism by which AF is initiated and that reducing action potential duration heterogeneity can lessen the burden of AF.
Uma Mahesh R. Avula, Jeffrey Abrams, Alexander Katchman, Sergey Zakharov, Sergey Mironov, Joseph Bayne, Daniel Roybal, Anirudh Gorti, Lin Yang, Vivek Iyer, Marc Waase, Deepak Saluja, Edward J. Ciaccio, Hasan Garan, Andrew R. Marks, Steven O. Marx, Elaine Y. Wan
Right ventricular dysfunction is highly prevalent across cardiopulmonary diseases and independently predicts death in both heart failure (HF) and pulmonary hypertension (PH). Progression towards right ventricular failure (RVF) can occur in spite of optimal medical treatment of HF or PH, highlighting current insufficient understanding of RVF molecular pathophysiology. To identify molecular mechanisms that may distinctly underlie RVF, we investigated the cardiac ventricular transcriptome of advanced HF patients, with and without RVF. Using an integrated systems genomic and functional biology approach, we identified an RVF-specific gene module, for which WIPI1 served as a hub and HSPB6 and MAP4 as drivers, and confirmed the ventricular specificity of Wipi1, Hspb6, and Map4 transcriptional changes in adult murine models of pressure overload induced RV- versus LV- failure. We uncovered a shift towards non-canonical autophagy in the failing RV that correlated with RV-specific Wipi1 upregulation. In vitro siRNA silencing of Wipi1 in neonatal rat ventricular myocytes limited non-canonical autophagy and blunted aldosterone-induced mitochondrial superoxide levels. Our findings suggest that Wipi1 regulates mitochondrial oxidative signaling and non-canonical autophagy in cardiac myocytes. Together with our human transcriptomic analysis and corroborating studies in an RVF mouse model, these data render Wipi1 a potential target for RV-directed HF therapy.
Christos Tzimas, Christoph D. Rau, Petra E. Buergisser, Gaston Jean-Louis Jr., Katherine Lee, Jeffrey Chukwuneke, Wen Dun, Yibin Wang, Emily J. Tsai
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