Corticosteroid Therapy in Acute and Subacute Arachnoiditis - A Case Series.

Arachnoiditis is difficult to treat. Patients are often left frustrated after many failed trials of conservative therapies without symptom resolution. Surgery may provide symptom relief for a short period of time, but their pain often returned. Herein, we present three cases of acute arachnoiditis following three different pain procedures: epidural blood patch, IDDS implant, and epidural steroid injection. The patients were diagnosed and treated with corticosteroids within 10 days of the procedure. Two patients were treated with the same oral steroid regiment, while the third patient was treated with both oral and IV steroid. All three patients had good outcomes at the completion of their steroid therapy. This case series may provide insight into treating acute and subacute arachnoiditis from pain interventions.

Feasibility of Using a GENEActiv Accelerometer with Triaxial Acceleration and Temperature Sensors to Monitor Adherence to Shoulder Sling Wear Following Surgery.

BACKGROUND: Self-reported adherence to sling wear is unreliable due to recall bias. We aim to assess the feasibility and accuracy of quantifying sling wear and non-wear utilising slings pre-fitted with a GENEActiv accelerometer that houses triaxial acceleration and temperature sensors. METHODS: Ten participants were asked to wear slings for 480 min (8 h) incorporating 180 min of non-wear time in durations varying from 5-120 min. GENEActiv devices were fitted in sutured inner sling pockets and participants logged sling donning and doffing times. An algorithm based on variability in acceleration in three axes and temperature change was developed to identify sling wear and non-wear and compared to participants' logs. RESULTS: There was no significant difference between algorithm detected non-wear duration (mean ± standard deviation = 172.0 ± 6.8 min/participant) and actual non-wear (179.7 ± 1.0 min/participant). Minute-by-minute agreement of sensor-detected wear and non-wear with participant reported wear was 97.3 ± 1.5% (range = 93.9-99.0), with mean sensitivity 94.3 ± 3.5% (range = 86.1-98.3) and specificity 99.1 ± 0.8% (range = 93.7-100). CONCLUSION: An algorithm based on accelerometer-assessed acceleration and temperature can accurately identify shoulder sling wear/non-wear times. This method may have potential for assessing whether sling wear adherence after shoulder surgeries have any bearing on patient functional outcomes.

A NOTCH1 gene copy number gain is a prognostic indicator of worse survival and a predictive biomarker to a Notch1 targeting antibody in colorectal cancer.

Dysregulation of the Notch1 receptor has been shown to facilitate the development and progression of colorectal cancer (CRC) and has been identified as an independent predictor of disease progression and worse survival. Although mutations in the NOTCH1 receptor have not been described in CRC, we have previously discovered a NOTCH1 gene copy number gain in a portion of CRC tumor samples. Here, we demonstrated that a NOTCH1 gene copy number gain is significantly associated with worse survival and a high percentage of gene duplication in a cohort of patients with advanced CRC. In our CRC patient-derived tumor xenograft (PDTX) model, tumors harboring a NOTCH1 gain exhibited significant elevation of the Notch1 receptor, JAG1 ligand and cleaved Notch1 activity. In addition, a significant association was identified between a gain in NOTCH1 gene copy number and sensitivity to a Notch1-targeting antibody. These findings suggest that patients with metastatic CRC that harbor a gain in NOTCH1 gene copy number have worse survival and that targeting this patient population with a Notch1 antibody may yield improved outcomes.

A crucial role for Kupffer cell-derived galectin-9 in regulation of T cell immunity in hepatitis C infection.

Approximately 200 million people throughout the world are infected with hepatitis C virus (HCV). One of the most striking features of HCV infection is its high propensity to establish persistence (approximately 70-80%) and progressive liver injury. Galectins are evolutionarily conserved glycan-binding proteins with diverse roles in innate and adaptive immune responses. Here, we demonstrate that galectin-9, the natural ligand for the T cell immunoglobulin domain and mucin domain protein 3 (Tim-3), circulates at very high levels in the serum and its hepatic expression (particularly on Kupffer cells) is significantly increased in patients with chronic HCV as compared to normal controls. Galectin-9 production from monocytes and macrophages is induced by IFN-gamma, which has been shown to be elevated in chronic HCV infection. In turn, galectin-9 induces pro-inflammatory cytokines in liver-derived and peripheral mononuclear cells; galectin-9 also induces anti-inflammatory cytokines from peripheral but not hepatic mononuclear cells. Galectin-9 results in expansion of CD4(+)CD25(+)FoxP3(+)CD127(low) regulatory T cells, contraction of CD4(+) effector T cells, and apoptosis of HCV-specific CTLs. In conclusion, galectin-9 production by Kupffer cells links the innate and adaptive immune response, providing a potential novel immunotherapeutic target in this common viral infection.

Liver cyst cytokines promote endothelial cell proliferation and development.

Autosomal dominant polycystic kidney (ADPKD) is highly prevalent genetic disease. Liver cyst disease is the most common extrarenal manifestation in ADPKD and accounts for up to 10% of ADPKD morbidity and mortality. The clinical features of ADPKD liver disease arise from dramatic increases in liver cyst volumes. To identify mechanisms that promote liver cyst growth, the present study characterized the degree of vascularization of liver cyst walls and determined that cyst-specific cytokines and growth factors can drive endothelial cell proliferation and development. Microscopic techniques demonstrated liver cyst walls are well vascularized. A comparative analysis found the vascular density in free liver cyst walls was greater in mice than in humans. Treatment of human micro-vascular endothelial cells (HMEC-1) with human liver cyst fluid (huLCF) induced a rapid increase in vascular endothelium growth factor receptor 2 (VEGFR2) phosphorylation that persisted for 45-60 min and was blocked by 20 microM SU5416, a VEGFR tyrosine kinase inhibitor. Similarly, huLCF treatment of HMEC-1 cells induced an increase in the cell proliferation rate (131 +/- 6% of control levels; P > 0.05) and the degree of vascular development ('tube' diameter assay: 92 +/- 14 microm for huLCF vs. 12 +/- 7 microm for vehicle); P > 0.05). Both cell proliferation and vascular development were sensitive to SU5416. These studies indicate that factors secreted by liver cyst epithelia can activate VEGF signaling pathways and induce endothelial cell proliferation and differentiation. The present studies suggest that targeting VEGFR2-dependent angiogenesis may be an effective therapeutic strategy in blocking ADPKD liver cyst vascularization and growth.

Characterization of ionotrophic purinergic receptors in hepatocytes.

UNLABELLED: Ionotrophic purinergic (P2X) receptors function as receptor-gated cation channels, where agonist binding leads to opening of a nonselective cation pore permeable to both Na(+) and Ca(2+). Based on evidence that extracellular adenosine 5'-triphosphate (ATP) stimulates glucose release from liver, these studies evaluate whether P2X receptors are expressed by hepatocytes and contribute to ATP-dependent calcium signaling and glucose release. Studies were performed in isolated hepatocytes from rats and mice and hepatoma cells from humans and rats. Transcripts and protein for both P2X4 and P2X7 were detectable, and immunohistochemistry of intact liver revealed P2X4 in the basolateral and canalicular domains. In whole cell patch clamp studies, exposure to the P2X4/P2X7 receptor agonist 2'3'-O-(4-benzoyl-benzoyl)-adenosine 5'-triphosphate (BzATP; 10 microM) caused a rapid increase in membrane Na(+) conductance. Similarly, with Fluo-3 fluorescence, BzATP induced an increase in intracellular [Ca(2+)]. P2X4 receptors are likely involved because the calcium response to BzATP was inhibited by Cu(2+), and the P2X4 modulators Zn(2+) and ivermectin (0.3-3 microM) each increased intracellular [Ca(2+)]. Exposure to BzATP decreased cellular glycogen content; and P2X4 receptor messenger RNA increased in glycogen-rich liver samples. CONCLUSION: These studies provide evidence that P2X4 receptors are functionally important in hepatocyte Na(+) and Ca(2+) transport, are regulated by extracellular ATP and divalent cation concentrations, and may constitute a mechanism for autocrine regulation of hepatic glycogen metabolism.

Aerodynamics of saccate pollen and its implications for wind pollination.

Pollen grains of many wind-pollinated plants contain 1-3 air-filled bladders, or sacci. Sacci are thought to help orient the pollen grain in the pollination droplet. Sacci also increase surface area of the pollen grain, yet add minimal mass, thereby increasing dispersal distance; however, this aerodynamic hypothesis has not been tested in a published study. Using scanning electron and transmission electron microscopy, mathematical modeling, and the saccate pollen of three extant conifers with structurally different pollen grains (Pinus, Falcatifolium, Dacrydium), we developed a computational model to investigate pollen flight. The model calculates terminal settling velocity based on structural characters of the pollen grain, including lengths, widths, and depths of the main body and sacci; angle of saccus rotation; and thicknesses of the saccus wall, endoreticulations, intine, and exine. The settling speeds predicted by the model were empirically validated by stroboscopic photography. This study is the first to quantitatively demonstrate the adaptive significance of sacci for the aerodynamics of wind pollination. Modeling pollen both with and without sacci indicated that sacci can reduce pollen settling speeds, thereby increasing dispersal distance, with the exception of pollen grains having robust endoreticulations and those with thick saccus walls. Furthermore, because the mathematical model is based on structural characters and error propagation methods show that the model yields valid results when sample sizes are small, the flight dynamics of fossil pollen can be investigated. Several fossils were studied, including bisaccate (Pinus, Pteruchus, Caytonanthus), monosaccate (Gothania), and nonsaccate (Monoletes) pollen types.

Shank2E binds NaP(i) cotransporter at the apical membrane of proximal tubule cells.

Proteins expressing postsynaptic density (PSD)-95/Drosophila disk large (Dlg)/zonula occludens-1 (ZO-1) (PDZ) domains are commonly involved in moderating receptor, channel, and transporter activities at the plasma membrane in a variety of cell types. At the apical membrane of renal proximal tubules (PT), the type IIa NaP(i) cotransporter (NaP(i)-IIa) binds specific PDZ domain proteins. Shank2E is a spliceoform of a family of PDZ proteins that is concentrated at the apical domain of liver and pancreatic epithelial cell types and is expressed in kidney. In the present study, immunoblotting of enriched plasma membrane fractions and immunohistology found Shank2E concentrated at the brush border membrane of rat PT cells. Confocal localization of Flag-Shank2E and enhanced green fluorescent protein-NaP(i)-IIa in cotransfected OK cells showed these proteins colocalized in the apical microvilli of this PT cell model. Shank2E co-immunoprecipitated with NaP(i)-IIa from rat renal cortex tissue and HA-NaP(i)-IIa coprecipitated with Flag-Shank2E in cotransfected human embryonic kidney HEK cells. Domain analysis showed that the PDZ domain of Shank2E specifically bound NaP(i)-IIa and truncation of the COOH-terminal TRL motif from NaP(i)-IIa abolished this binding, and Far Western blotting showed that the Shank2E- NaP(i)-IIa interaction occurred directly between the two proteins. NaP(i)-IIa activity is regulated by moderating its abundance in the apical membrane. High-P(i) conditions induce NaP(i)-IIa internalization and degradation. In both rat kidney PT cells and OK cells, shifting to high-P(i) conditions induced an acute internal redistribution of Shank2E and, in OK cells, a significant degree of degradation. In sum, Shank2E is concentrated in the apical domain of renal PT cells, specifically binds NaP(i)-IIa via PDZ interactions, and undergoes P(i)-induced internalization.

Protein kinase C regulates the phosphorylation and oligomerization of ERM binding phosphoprotein 50.

Ezrin-Radixin-Moesin (ERM) binding phosphoprotein 50 (EBP50, a.k.a. NHERF-1) is a scaffold protein essential for the localization and coordinated activity of apical transporters, enzymes and receptors in epithelial cells. EBP50 acts via multiple protein binding interactions, including oligomerization through interactions of its PSD95-Dlg-ZO1 (PDZ) domains. EBP50 can be phosphorylated on multiple sites and phosphorylation of specific sites modulates the extent of oligomerization. The aim of the present study was to test the capacity of protein kinase C (PKC) to phosphorylate EBP50 and to regulate its oligomerization. In vitro experiments showed that the catalytic subunit of PKC directly phosphorylates EBP50. In HEK-293 cells transfected with rat EBP50 cDNA, a treatment with 12 myristate 13-acetate (PMA) induced a translocation of PKCalpha and beta isoforms to the membrane and increased 32P incorporation into EBP50. In co-transfection/co-precipitation studies, PMA treatment stimulated EBP50 oligomerization. Mass spectrometry analysis of full-length EBP50 and phosphorylation analyses of specific domains, and of mutated or truncated forms of EBP50, indicated that PKC-induced phosphorylation of EBP50 occurred on the Ser337/Ser338 residue within the carboxyl-tail domain of the protein. Truncation of Ser337/Ser338 also diminished PKC-induced oligomerization of EBP50. These results suggest the PKC signaling pathway can impact EBP50-dependent cellular functions by regulating EBP50 oligomerization.

Purinergic regulation of cholangiocyte secretion: identification of a novel role for P2X receptors.

The P2X family of ligand-gated cation channels is comprised of seven distinct isoforms activated by binding of extracellular purines. Although originally identified in neurons, there is increasing evidence for expression of P2X receptors in epithelia as well. Because ATP is released by both hepatocytes and cholangiocytes, these studies were performed to evaluate whether P2X receptors are present in cholangiocytes and contribute to local regulation of biliary secretion and bile formation. RT-PCR of cDNA from cultured normal rat cholangiocytes detected transcripts for P2X receptors 2, 3, 4, and 6; products from P2X3 and P2X4 were robust and always detectable. In cholangiocyte lysates, P2X4 protein was readily detected, and immunohistochemical staining of intact rat liver revealed P2X4 protein concentrated in intrahepatic bile ducts. To assess the functional significance of P2X4, isolated Mz-ChA-1 cells were exposed to the P2X4-preferring agonist 2',3'-O-(4-benzoyl-benzoyl)-ATP (BzATP), which activated inward currents of -18.2 + 3.0 pA/pF. In cholangiocyte monolayers, BzATP but not P2X3 agonists elicited robust Cl(-) secretory responses (short-circuit current) when applied to either the apical (DeltaI(sc) 22.1 +/- 3.3 microA) or basolateral (18.5 +/- 1.6 microA) chamber, with half-maximal stimulation at approximately 10 microM and approximately 1 microM, respectively. The response to BzATP was unaffected by suramin (not significant) and was inhibited by Cu(2+) (P < 0.01). These studies provide molecular and biochemical evidence for the presence of P2X receptors in cholangiocytes. Functional studies indicate that P2X4 is likely the primary isoform involved, representing a novel and functionally important component of the purinergic signaling complex modulating biliary secretion.

Characterization of an ankyrin repeat-containing Shank2 isoform (Shank2E) in liver epithelial cells.

Shank proteins are a family of multidomain scaffolding proteins best known for their role in organizing the postsynaptic density region in neurons. Unlike Shank1 and Shank3, Shank2 [also known as Pro-SAP1 (proline-rich synapse-associated protein 1), CortBP1 (cortactin binding protein 1) or Spank-3] has been described as a truncated family member without an N-terminal ankyrin repeat domain. The present study utilized bioinformatics to demonstrate the presence of exons encoding ankyrin repeats in the region preceding the previously described Shank2 gene. cDNA sequencing of mRNA from epithelial cells revealed a novel spliceoform of Shank2, termed Shank2E, that encodes a predicted 200 kDa protein with six N-terminal ankyrin repeats. Shank2 mRNA from epithelial tissues was larger than transcripts in brain. Likewise, the apparent mass of Shank2 protein was larger in epithelial tissues (230 kDa) when compared with brain (165/180 kDa). Immunofluorescence and membrane fractionation found Shank2E concentrated at the apical membrane of liver epithelial cells. In cultured cholangiocytes, co-immunoprecipitation and detergent solubility studies revealed Shank2E complexed with actin and co-distributed with actin in detergent-insoluble lipid rafts. These findings indicate epithelial cells express an ankyrin repeat-containing Shank2 isoform, termed Shank2E, that is poised to co-ordinate actin-dependent events at the apical membrane.