Anti-inflammatory activity and neutrophil reductions mediated by the JAK1/JAK3 inhibitor, CP-690,550, in rat adjuvant-induced arthritis

<p>Abstract</p> <p>Background</p> <p>The Janus kinase (JAK) family of tyrosine kinases includes JAK1, JAK2, JAK3 and TYK2, and is required for signaling through Type I and Type II cytokine receptors. CP-690,550 is a potent and selective JAK inhibitor currently in clinical trials for rheumatoid arthritis (RA) and other autoimmune disease indications. In RA trials, dose-dependent decreases in neutrophil counts (PBNC) were observed with CP-690,550 treatment. These studies were undertaken to better understand the relationship between JAK selectivity and PBNC decreases observed with CP-690,550 treatment.</p> <p>Methods</p> <p>Potency and selectivity of CP-690,550 for mouse, rat and human JAKs was evaluated in a panel of <it>in vitro </it>assays. The effect of CP-690,550 on granulopoiesis from progenitor cells was also assessed <it>in vitro </it>using colony forming assays. <it>In vivo </it>the potency of orally administered CP-690,550 on arthritis (paw edema), plasma cytokines, PBNC and bone marrow differentials were evaluated in the rat adjuvant-induced arthritis (AIA) model.</p> <p>Results</p> <p>CP-690,550 potently inhibited signaling through JAK1 and JAK3 with 5-100 fold selectivity over JAK2 in cellular assays, despite inhibiting all four JAK isoforms with nM potency in <it>in vitro </it>enzyme assays. Dose-dependent inhibition of paw edema was observed <it>in vivo </it>with CP-690,550 treatment. Plasma cytokines (IL-6 and IL-17), PBNC, and bone marrow myeloid progenitor cells were elevated in the context of AIA disease. At efficacious exposures, CP-690,550 returned all of these parameters to pre-disease levels. The plasma concentration of CP-690,550 at efficacious doses was above the <it>in vitro </it>whole blood IC50 of JAK1 and JAK3 inhibition, but not that of JAK2.</p> <p>Conclusion</p> <p>Results from this investigation suggest that CP-690,550 is a potent inhibitor of JAK1 and JAK3 with potentially reduced cellular potency for JAK2. In rat AIA, as in the case of human RA, PBNC were decreased at efficacious exposures of CP-690,550. Inflammatory end points were similarly reduced, as judged by attenuation of paw edema and cytokines IL-6 and IL-17. Plasma concentration at these exposures was consistent with inhibition of JAK1 and JAK3 but not JAK2. Decreases in PBNC following CP-690,550 treatment may thus be related to attenuation of inflammation and are likely not due to suppression of granulopoiesis through JAK2 inhibition.</p

Acute flaccid myelitis:cause, diagnosis, and management

Acute flaccid myelitis (AFM) is a disabling, polio-like illness mainly affecting children. Outbreaks of MM have occurred across multiple global regions since 2012, and the disease appears to be caused by non-polio enterovirus infection, posing a major public health challenge. The clinical presentation of flaccid and often profound muscle weakness (which can invoke respiratory failure and other critical complications) can mimic several other acute neurological illnesses. There is no single sensitive and specific test for MM, and the diagnosis relies on identification of several important clinical, neuroimaging, and cerebrospinal fluid characteristics. Following the acute phase of AFM, patients typically have substantial residual disability and unique long-term rehabilitation needs. In this Review we describe the epidemiology, clinical features, course, and outcomes of AFM to help to guide diagnosis, management, and rehabilitation. Future research directions include further studies evaluating host and pathogen factors, including investigations into genetic, viral, and immunological features of affected patients, host-virus interactions, and investigations of targeted therapeutic approaches to improve the long-term outcomes in this population

Autophagy and Cell Autonomous Neurodegeneration in Niemann-Pick type C Disease.

Niemann-Pick type C disease (NPC) is an autosomal recessive lysosomal storage disorder characterized by liver dysfunction and neurodegeneration causing diverse neurologic symptoms such as ataxia, cognitive decline, and seizures, and finally leading to premature death. It is caused by mutations in the NPC1 or NPC2 genes, leading to accumulation of cholesterol and glycosphingolipids in late endosomes and lysosomes of all tissues. At present, the link between lipid storage and neurodegeneration is unknown, representing a major impediment to the development of effective therapies for NPC disease. In chapter 2, I characterize a novel conditional knockout mouse model for NPC disease and use it to determine the extent to which the degeneration of cerebellar Purkinje cells is cell autonomous. Deletion of Npc1 only in Purkinje cells was sufficient to cause their degeneration, and lead to symptoms of ataxia and tremors. However, these mice did not demonstrate the weight loss or premature death that is characteristic of global Npc1 mutants, demonstrating that these phenotypes arise from other cell types. I also noted a marked differential vulnerability to degeneration among Purkinje cell subpopulations. In chapter 3, I use bioinformatic methods to identify 16 genes whose expression patterns correlated strongly with this pattern of Purkinje cell death. One of these genes, Hsp27, promotes the survival of neurons in an in vitro model of NPC neurodegeneration. In chapter 4, I identify autophagy as a contributor to neurodegeneration in NPC. Autophagy is a pathway for delivering cytoplasmic cargoes to the lysosome via double-membrane bound organelles known as autophagosomes. It has been closely linked to the process of neurodegeneration, and the induction of autophagy along with accumulation of autophagosomes has been documented in NPC disease. Here I show that autophagy induction lies downstream of Toll-like receptor signaling through the adapter protein TRIF. Further, autophagy is a major source for stored cholesterol in NPC lysosomes. Finally, lipid storage impairs the maturation of autolysosomes via inhibition of lysosomal cathepsin activity. Inhibition of autophagy by wortmannin reduced cholesterol storage, restored lysosomal proteolysis, and rescued neurodegeneration in vitro, thus demonstrating that autophagy plays a detrimental role in NPC pathogenesis.PHDNeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/97896/1/melrick_1.pd

Fining of Pinot noir wine with egg white: potential for residual protein and early sediment formation

In recent years consumer demands have been for wine to be rapidly processed and available in the market place earlier than would have traditionally occurred. This has lead to the introduction of various winemaking techniques for the stabilisation and clarification of wine so as to be palatable immediately, and free from hazes and sediments. This study was undertaken to examine the effect of egg white fining on changes in protein concentrations in Pinot noir wines, and to relate this to a potential instability problem, which manifests as a haze/sediment soon after bottling. Accelerated aging was used to indicate the potential of the Pinot noir wines fined with spray dried egg white, to form haze/sediment. The addition of spray dried egg white to two different styles of Pinot noir wine, a 'heavy' style and a 'light' style (less colour and less phenolics than 'heavy' style) from two different vintages (1998 and 2000 vintages respectively), resulted in residual protein and specifically egg albumen, remaining in solution following clarification by fining. Furthermore, it was shown that the wines fined with spray dried egg white were susceptible to haze/sediment formation following accelerated aging. Two techniques, silica sol co-fining and cold stabilisation were investigated as possible methods for reducing the potential of the wines to form haze/sediment. Co-fining with silica sol was partially effective in reducing the potential for haze/sediment formation following accelerated aging in 'light' style (2000) Pinot noir wine, whereas cold stabilisation was effective in preventing haze/sediment formation following accelerated aging in both 'light' style (2000) and 'heavy' style (1998) Pinot noir wine. The potential of wine to form haze/sediments is discussed in relation to proteins and phenolics, and their complex formation and equilibrium. A simple model explaining protein and phenolic interactions that can lead to haze/sediment formation is discussed

Sphingosine, a Product of Ceramide Hydrolysis, Influences the Formation of Ceramide Channels

Ceramides are known to have a regulatory function in apoptosis, including the release of cytochrome c and other proapoptotic factors from the mitochondrial intermembrane space. Ceramides can form large, stable channels in the outer mitochondrial membrane, leading to the proposal that ceramide channels are the pathway through which these proteins are released. Here, we report that sphingosine, a product of ceramide hydrolysis by ceramidase, is capable of destabilizing ceramide channels, leading to their disassembly. Sphingosine is directly responsible for the disassembly of ceramide channels in planar membrane experiments and markedly reduces the ability of ceramide to induce the release of intermembrane space proteins from mitochondria in vitro. Low concentrations of both L and D sphingosine potentiate the release of intermembrane space proteins by long-chain ceramide and channel formation in liposomes. These results provide evidence for a mechanism by which the disassembly of ceramide channels, as initiated by ceramidase, could be accelerated by the direct interaction of the hydrolysis product with the ceramide channels themselves. This mechanism therefore could form a positive feedback loop for rapid shut-down of ceramide channels. However, potentiation of ceramide channel formation is also possible and thus both effects could influence the propensity for mitochondria-mediated apoptosis

Generation of hepatocytes expressing functional cytochromes P450 from a pancreatic progenitor cell line in vitro.

The proliferating AR42J-B13 pancreatic cell line is known to respond to glucocorticoid treatment by producing foci of cells that express the liver-specific albumin gene. We demonstrate that this cell line also expresses liver-specific or liver-enriched functional cytochrome P450 proteins when stimulated to trans-differentiate into hepatocytes by glucocorticoid. These data suggest that this cell line has an unusual ability to trans-differentiate into functional hepatocytes and that it could be possible to generate a limitless supply of functional hepatocyte-like cells in vitro

Heat Shock Protein Beta-1 Modifies Anterior to Posterior Purkinje Cell Vulnerability in a Mouse Model of Niemann-Pick Type C Disease

<div><p>Selective neuronal vulnerability is characteristic of most degenerative disorders of the CNS, yet mechanisms underlying this phenomenon remain poorly characterized. Many forms of cerebellar degeneration exhibit an anterior-to-posterior gradient of Purkinje cell loss including Niemann-Pick type C1 (NPC) disease, a lysosomal storage disorder characterized by progressive neurological deficits that often begin in childhood. Here, we sought to identify candidate genes underlying vulnerability of Purkinje cells in anterior cerebellar lobules using data freely available in the Allen Brain Atlas. This approach led to the identification of 16 candidate neuroprotective or susceptibility genes. We demonstrate that one candidate gene, heat shock protein beta-1 (<i>HSPB1</i>), promoted neuronal survival in cellular models of NPC disease through a mechanism that involved inhibition of apoptosis. Additionally, we show that over-expression of wild type <i>HSPB1</i> or a phosphomimetic mutant in NPC mice slowed the progression of motor impairment and diminished cerebellar Purkinje cell loss. We confirmed the modulatory effect of Hspb1 on Purkinje cell degeneration <i>in vivo</i>, as knockdown by <i>Hspb1</i> shRNA significantly enhanced neuron loss. These results suggest that strategies to promote HSPB1 activity may slow the rate of cerebellar degeneration in NPC disease and highlight the use of bioinformatics tools to uncover pathways leading to neuronal protection in neurodegenerative disorders.</p></div

HSPB1 promotes survival in cellular models of NPC1 disease.

<p><b>(A)</b> Expression of Hspb1 <i>(left panel)</i> and calbindin <i>(right panel</i>, Purkinje cells) in cerebellar midline of <i>Npc1 flox/-</i>, <i>Pcp2-Cre</i> mice 7 weeks. Scale bar = 200 μm. <b>(B)</b> (<i>Upper panel</i>) HeLa cells were transfected with non-targeted (NT, lanes 1 and 3) or <i>HSPB1</i> siRNA (lanes 2 and 4), then treated with vehicle (lanes 1–2) or 1 mg/ml U18666A (lanes 3–4) for 24 hr. HSPB1 expression was determined by western blot. GAPDH controls for loading. (<i>Lower panel</i>) Caspase-3 in HeLa cell lysates. Data are mean ± SEM. *<i>p</i><0.05. <b>(C)</b> NPC1 patient fibroblasts were transfected with non-targeted or <i>HSPB1</i> siRNA. Cells were stained with Hoechst, and the percentage of cells with condensed chromatin was scored. Data are mean ± SEM. **<i>p</i><0.01. <b>(D)</b> Primary mouse cortical neurons were transduced with wild type <i>HSPB1</i>, <i>HSPB1</i>^<i>3A</i>, <i>HSPB1</i>^<i>3E</i>, or empty vector, and then treated with 2.5 μg/ml U18666. XTT assay was performed 72 hrs post U18666A. Neuron survival is reported relative to vehicle treated cells. Data are mean ± SEM. ***<i>p</i><0.001.</p