Initial Diagnosis of Classic Hodgkin Lymphoma With Skin Biopsy: A Rare Case and Review of Diagnostic Considerations.

ABSTRACT: Classic Hodgkin lymphoma (CHL) is a B-cell-derived lymphoma that classically displays a bimodal age distribution. CHL typically involves the mediastinum, lymph nodes, and other visceral organs. CHL is characterized histologically by the presence of a relatively paucicellular neoplastic cell population composed of large atypical cells (including Hodgkin and Reed-Sternberg forms) in a reactive mixed inflammatory background, often with prominent necrosis. CHL rarely occurs in the skin, and the associated mixed inflammatory infiltrate or necrotic appearance can create diagnostic uncertainty. Herein, we report the case of a 31-year-old man presenting with a painful dendritic rash of the anterior chest wall with axillary lymphadenopathy. After multiple nondiagnostic biopsies that revealed largely necrotic material, a chest wall skin biopsy was obtained. The skin biopsy was diagnostic of CHL, based on the presence of large atypical dermal cells, including Hodgkin and Reed-Sternberg forms, which expressed CD15, CD30 and Fascin, in a typical mixed inflammatory and necrotic background. Through the lens of this case, we discuss the characteristics and mechanisms of skin involvement of CHL, and the histopathologic and immunohistochemical pitfalls when considering the rare diagnosis of CHL in the skin.

Therapeutic plasma exchange for exogenous insulin antibody syndrome in combined variable immunodeficiency: a case report.

A 32-year-old male with type I diabetes presented with profound hypoglycemia due to exogenous insulin antibody syndrome in the setting of newly-diagnosed common variable immunodeficiency. Immunomodulatory therapy was not initially effective, but after the initiation of plasma exchange hypoglycemia resolved, and glucose lability improved.

Dynamic regulation of ß1 subunit trafficking controls vascular contractility.

Ion channels composed of pore-forming and auxiliary subunits control physiological functions in virtually all cell types. A conventional view is that channels assemble with their auxiliary subunits before anterograde plasma membrane trafficking of the protein complex. Whether the multisubunit composition of surface channels is fixed following protein synthesis or flexible and open to acute and, potentially, rapid modulation to control activity and cellular excitability is unclear. Arterial smooth muscle cells (myocytes) express large-conductance Ca(2+)-activated potassium (BK) channel α and auxiliary ß1 subunits that are functionally significant modulators of arterial contractility. Here, we show that native BKα subunits are primarily (∼95%) plasma membrane-localized in human and rat arterial myocytes. In contrast, only a small fraction (∼10%) of total ß1 subunits are located at the cell surface. Immunofluorescence resonance energy transfer microscopy demonstrated that intracellular ß1 subunits are stored within Rab11A-postive recycling endosomes. Nitric oxide (NO), acting via cGMP-dependent protein kinase, and cAMP-dependent pathways stimulated rapid (≤1 min) anterograde trafficking of ß1 subunit-containing recycling endosomes, which increased surface ß1 almost threefold. These ß1 subunits associated with surface-resident BKα proteins, elevating channel Ca(2+) sensitivity and activity. Our data also show that rapid ß1 subunit anterograde trafficking is the primary mechanism by which NO activates myocyte BK channels and induces vasodilation. In summary, we show that rapid ß1 subunit surface trafficking controls functional BK channel activity in arterial myocytes and vascular contractility. Conceivably, regulated auxiliary subunit trafficking may control ion channel activity in a wide variety of cell types.

Local coupling of TRPC6 to ANO1/TMEM16A channels in smooth muscle cells amplifies vasoconstriction in cerebral arteries.

Anoctamin-1 [ANO1, also known as transmembrane protein 16A (TMEM16A)] is a Ca(2+)-activated Cl(-) channel expressed in arterial myocytes that regulates membrane potential and contractility. Signaling mechanisms that control ANO1 activity in arterial myocytes are poorly understood. In cerebral artery myocytes, ANO1 channels are activated by local Ca(2+) signals generated by plasma membrane nonselective cation channels, but the molecular identity of these proteins is unclear. Arterial myocytes express several different nonselective cation channels, including multiple members of the transient receptor potential receptor (TRP) family. The goal of this study was to identify localized ion channels that control ANO1 currents in cerebral artery myocytes. Coimmunoprecipitation and immunofluorescence resonance energy transfer microscopy experiments indicate that ANO1 and canonical TRP 6 (TRPC6) channels are present in the same macromolecular complex and localize in close spatial proximity in the myocyte plasma membrane. In contrast, ANO1 is not near TRPC3, TRP melastatin 4, or inositol trisphosphate receptor 1 channels. Hyp9, a selective TRPC6 channel activator, stimulated Cl(-) currents in myocytes that were blocked by T16Ainh-A01, an ANO1 inhibitor, ANO1 knockdown using siRNA, and equimolar replacement of intracellular EGTA with BAPTA, a fast Ca(2+) chelator that abolishes local Ca(2+) signaling. Hyp9 constricted pressurized cerebral arteries, and this response was attenuated by T16Ainh-A01. In contrast, T16Ainh-A01 did not alter depolarization-induced (60 mM K(+)) vasoconstriction. These data indicate that TRPC6 channels generate a local intracellular Ca(2+) signal that activates nearby ANO1 channels in myocytes to stimulate vasoconstriction.

Calcium/Ask1/MKK7/JNK2/c-Src signalling cascade mediates disruption of intestinal epithelial tight junctions by dextran sulfate sodium.

Disruption of intestinal epithelial tight junctions is an important event in the pathogenesis of ulcerative colitis. Dextran sodium sulfate (DSS) induces colitis in mice with symptoms similar to ulcerative colitis. However, the mechanism of DSS-induced colitis is unknown. We investigated the mechanism of DSS-induced disruption of intestinal epithelial tight junctions and barrier dysfunction in Caco-2 cell monolayers in vitro and mouse colon in vivo. DSS treatment resulted in disruption of tight junctions, adherens junctions and actin cytoskeleton leading to barrier dysfunction in Caco-2 cell monolayers. DSS induced a rapid activation of c-Jun N-terminal kinase (JNK), and the inhibition or knockdown of JNK2 attenuated DSS-induced tight junction disruption and barrier dysfunction. In mice, DSS administration for 4 days caused redistribution of tight junction and adherens junction proteins from the epithelial junctions, which was blocked by JNK inhibitor. In Caco-2 cell monolayers, DSS increased intracellular Ca(2+) concentration, and depletion of intracellular Ca(2+) by 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM) or thapsigargin attenuated DSS-induced JNK activation, tight junction disruption and barrier dysfunction. Knockdown of apoptosis signal-regulated kinase 1 (Ask1) or MKK7 blocked DSS-induced tight junction disruption and barrier dysfunction. DSS activated c-Src by a Ca2+ and JNK-dependent mechanism. Inhibition of Src kinase activity or knockdown of c-Src blocked DSS-induced tight junction disruption and barrier dysfunction. DSS increased tyrosine phosphorylation of occludin, zonula occludens-1 (ZO-1), E-cadherin and ß-catenin. SP600125 abrogated DSS-induced tyrosine phosphorylation of junctional proteins. Recombinant JNK2 induced threonine phosphorylation and auto-phosphorylation of c-Src. The present study demonstrates that Ca(2+)/Ask1/MKK7/JNK2/cSrc signalling cascade mediates DSS-induced tight junction disruption and barrier dysfunction.

Angiotensin II stimulates internalization and degradation of arterial myocyte plasma membrane BK channels to induce vasoconstriction.

Arterial smooth muscle cells (myocytes) express large-conductance Ca(2+)-activated K(+) (BK) channel α and auxiliary ß1 subunits that modulate arterial contractility. In arterial myocytes, ß1 subunits are stored within highly mobile rab11A-positive recycling endosomes. In contrast, BKα subunits are primarily plasma membrane-localized. Trafficking pathways for BKα and whether physiological stimuli that regulate arterial contractility alter BKα localization in arterial myocytes are unclear. Here, using biotinylation, immunofluorescence resonance energy transfer (immunoFRET) microscopy, and RNAi-mediated knockdown, we demonstrate that rab4A-positive early endosomes traffic BKα to the plasma membrane in myocytes of resistance-size cerebral arteries. Angiotensin II (ANG II), a vasoconstrictor, reduced both surface and total BKα, an effect blocked by bisindolylmaleimide-II, concanavalin A, and dynasore, protein kinase C (PKC), internalization, and endocytosis inhibitors, respectively. In contrast, ANG II did not reduce BKα mRNA, and sodium nitroprusside, a nitric oxide donor, did not alter surface BKα protein over the same time course. MG132 and bafilomycin A, proteasomal and lysosomal inhibitors, respectively, also inhibited the ANG II-induced reduction in surface and total BKα, resulting in intracellular BKα accumulation. ANG II-mediated BK channel degradation reduced BK currents in isolated myocytes and functional responses to iberiotoxin, a BK channel blocker, and NS1619, a BK activator, in pressurized (60 mmHg) cerebral arteries. These data indicate that rab4A-positive early endosomes traffic BKα to the plasma membrane in arterial myocytes. We also show that ANG II stimulates PKC-dependent BKα internalization and degradation. These data describe a unique mechanism by which ANG II inhibits arterial myocyte BK currents, by reducing surface channel number, to induce vasoconstriction.

Smooth muscle cell transient receptor potential polycystin-2 (TRPP2) channels contribute to the myogenic response in cerebral arteries.

Intravascular pressure-induced vasoconstriction is a smooth muscle cell-specific mechanism that controls systemic blood pressure and organ regional blood flow. Smooth muscle cell polycystin-1 and -2 (TRPP1 and -2) proteins modulate the myogenic response in mesenteric arteries, but involvement in other vascular beds is unclear. Here, we examined TRPP2 expression, cellular distribution, cation currents (ICat), and physiological functions in smooth muscle cells of rat and human cerebral arteries. We demonstrate that TRPP2 is the major TRPP isoform expressed in cerebral artery smooth muscle cells, with message levels higher than those of TRPP1. Arterial biotinylation and immunofluorescence indicated that TRPP2 is located primarily (∼88%) in the smooth muscle cell plasma membrane. RNA interference reduced TRPP2 expression by ∼55% compared to control, but did not alter levels of TRPP1, TRPC1, TRPC3, TRPC6, TRPM4, ANO1/TMEM16A, or voltage-dependent Ca(2+) (CaV1.2) channels, other ion channel proteins that modulate myogenic tone. Cell swelling induced by hyposmotic (250 osmol (l solution)(-1)) bath solution stimulated Gd(3+)-sensitive ICat in smooth muscle cells that were reduced by selective TRPP2 knockdown. TRPP2 knockdown did not alter myogenic tone at 20 mmHg but reduced tone between ∼28 and 39% over an intravascular pressure range between 40 and 100 mmHg. In contrast, TRPP2 knockdown did not alter depolarization-induced (60 mmol l K(+)) vasoconstriction. In summary, we show that TRPP2 is expressed in smooth muscle cells of resistance-size cerebral arteries, resides primarily in the plasma membrane, and contributes to the myogenic response. Data also suggest that TRPP2 differentially regulates the myogenic response in cerebral and mesenteric arteries.

The voltage-dependent L-type Ca2+ (CaV1.2) channel C-terminus fragment is a bi-modal vasodilator.

Voltage-dependent L-type Ca(2+) channels (CaV1.2) are the primary Ca(2+) entry pathway in vascular smooth muscle cells (myocytes). CaV1.2 channels control systemic blood pressure and organ blood flow and are pathologically altered in vascular diseases, which modifies vessel contractility. The CaV1.2 distal C-terminus is susceptible to proteolytic cleavage, which yields a truncated CaV1.2 subunit and a cleaved C-terminal fragment (CCt). Previous studies in cardiac myocytes and neurons have identified CCt as both a transcription factor and CaV1.2 channel inhibitor, with different signalling mechanisms proposed to underlie some of these effects. CCt existence and physiological functions in arterial myocytes are unclear, but important to study given the functional significance of CaV1.2 channels. Here, we show that CCt exists in myocytes of both rat and human resistance-size cerebral arteries, where it locates to both the nucleus and plasma membrane. Recombinant CCt expression in arterial myocytes inhibited CaV1.2 transcription and reduced CaV1.2 protein. CCt induced a depolarizing shift in the voltage dependence of both CaV1.2 current activation and inactivation, and reduced non-inactivating current in myocytes. Recombinant truncated CCt lacking a putative nuclear localization sequence (92CCt) did not locate to the nucleus and had no effect on arterial CaV1.2 transcription or protein. However, 92CCt shifted the voltage dependence of CaV1.2 activation and inactivation similarly to CCt. CCt and 92CCt both inhibited pressure- and depolarization-induced vasoconstriction, although CCt was a far more effective vasodilator. These data demonstrate that endogenous CCt exists and reduces both CaV1.2 channel expression and voltage sensitivity in arterial myocytes. Thus, CCt is a bi-modal vasodilator.

Regulation of interleukin-6 secretion by the two-pore-domain potassium channel Trek-1 in alveolar epithelial cells.

We recently proposed a role for the two-pore-domain K(+) (K2P) channel Trek-1 in the regulation of cytokine release from mouse alveolar epithelial cells (AECs) by demonstrating decreased interleukin-6 (IL-6) secretion from Trek-1-deficient cells, but the underlying mechanisms remained unknown. This study was designed to investigate the mechanisms by which Trek-1 decreases IL-6 secretion. We hypothesized that Trek-1 regulates tumor necrosis factor-α (TNF-α)-induced IL-6 release via NF-κB-, p38-, and PKC-dependent pathways. We found that Trek-1 deficiency decreased IL-6 secretion from mouse and human AECs at both transcriptional and translational levels. While NF-κB/p65 phosphorylation was unchanged, p38 phosphorylation was decreased in Trek-1-deficient cells, and pharmacological inhibition of p38 decreased IL-6 secretion in control but not Trek-1-deficient cells. Similarly, pharmacological inhibition of PKC also decreased IL-6 release, and we found decreased phosphorylation of the isoforms PKC/PKDµ (Ser(744/748)), PKCθ, PKCδ, PKCα/ßII, and PKCζ/λ, but not PKC/PKDµ (Ser(916)) in Trek-1-deficient AECs. Phosphorylation of PKCθ, a Ca(2+)-independent isoform, was intact in control cells but impaired in Trek-1-deficient cells. Furthermore, TNF-α did not elevate the intracellular Ca(2+) concentration in control or Trek-1-deficient cells, and removal of extracellular Ca(2+) did not impair IL-6 release. In summary, we report the expression of Trek-1 in human AECs and propose that Trek-1 deficiency may alter both IL-6 translation and transcription in AECs without affecting Ca(2+) signaling. The results of this study identify Trek-1 as a new potential target for the development of novel treatment strategies against acute lung injury.

Myeloid sarcoma with NPM1 mutation may be clinically and genetically distinct from AML with NPM1 mutation: a study from the Bone Marrow Pathology Group.

Myeloid sarcoma (MS) is currently considered equivalent to de novo acute myeloid leukemia (AML); however, the relationship between these entities is poorly understood. This retrospective multi-institutional cohort study compared 43 MS with NPM1 mutation to 106 AML with NPM1 mutation. Compared to AML, MS had more frequent cytogenetic abnormalities including complex karyotype (p = .009 and p = .007, respectively) and was enriched in mutations of genes involved in histone modification, including ASXL1 (p = .007 and p = .008, respectively). AML harbored a higher average number of gene mutations (p = .002) including more frequent PTPN11 mutations (p < .001) and mutations of DNA-methylating genes including DNMT3A and IDH1 (both p < .001). MS had significantly shorter overall survival (OS) than AML (median OS: 44.9 vs. 93.2 months, respectively, p = .037). MS with NPM1 mutation has a unique genetic landscape, and poorer OS, compared to AML with NPM1 mutation.

First study comparing genetic profiles of MS and AML with a common disease-defining lesion.NPM1^Mut MS may be genetically distinct from NPM1^Mut AML.NPM1^Mut MS may have inferior overall survival compared to NPM1^Mut AML.

CaV1.3 channels and intracellular calcium mediate osmotic stress-induced N-terminal c-Jun kinase activation and disruption of tight junctions in Caco-2 CELL MONOLAYERS.

We investigated the role of a Ca(2+) channel and intracellular calcium concentration ([Ca(2+)](i)) in osmotic stress-induced JNK activation and tight junction disruption in Caco-2 cell monolayers. Osmotic stress-induced tight junction disruption was attenuated by 1,2-bis(2-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-mediated intracellular Ca(2+) depletion. Depletion of extracellular Ca(2+) at the apical surface, but not basolateral surface, also prevented tight junction disruption. Similarly, thapsigargin-mediated endoplasmic reticulum (ER) Ca(2+) depletion attenuated tight junction disruption. Thapsigargin or extracellular Ca(2+) depletion partially reduced osmotic stress-induced rise in [Ca(2+)](i), whereas thapsigargin and extracellular Ca(2+) depletion together resulted in almost complete loss of rise in [Ca(2+)](i). L-type Ca(2+) channel blockers (isradipine and diltiazem) or knockdown of the Ca(V)1.3 channel abrogated [Ca(2+)](i) rise and disruption of tight junction. Osmotic stress-induced JNK2 activation was abolished by BAPTA and isradipine, and partially reduced by extracellular Ca(2+) depletion, thapsigargin, or Ca(V)1.3 knockdown. Osmotic stress rapidly induced c-Src activation, which was significantly attenuated by BAPTA, isradipine, or extracellular Ca(2+) depletion. Tight junction disruption by osmotic stress was blocked by tyrosine kinase inhibitors (genistein and PP2) or siRNA-mediated knockdown of c-Src. Osmotic stress induced a robust increase in tyrosine phosphorylation of occludin, which was attenuated by BAPTA, SP600125 (JNK inhibitor), or PP2. These results demonstrate that Ca(V)1.3 and rise in [Ca(2+)](i) play a role in the mechanism of osmotic stress-induced tight junction disruption in an intestinal epithelial monolayer. [Ca(2+)](i) mediate osmotic stress-induced JNK activation and subsequent c-Src activation and tyrosine phosphorylation of tight junction proteins. Additionally, inositol 1,4,5-trisphosphate receptor-mediated release of ER Ca(2+) also contributes to osmotic stress-induced tight junction disruption.

Caveolin-1 assembles type 1 inositol 1,4,5-trisphosphate receptors and canonical transient receptor potential 3 channels into a functional signaling complex in arterial smooth muscle cells.

Physical coupling of sarcoplasmic reticulum (SR) type 1 inositol 1,4,5-trisphosphate receptors (IP(3)R1) to plasma membrane canonical transient receptor potential 3 (TRPC3) channels activates a cation current (I(Cat)) in arterial smooth muscle cells that induces vasoconstriction. However, structural components that enable IP(3)R1 and TRPC3 channels to communicate locally are unclear. Caveolae are plasma membrane microdomains that can compartmentalize proteins. Here, we tested the hypothesis that caveolae and specifically caveolin-1 (cav-1), a caveolae scaffolding protein, facilitate functional IP(3)R1 to TRPC3 coupling in smooth muscle cells of resistance-size cerebral arteries. Methyl-ß-cyclodextrin (MßCD), which disassembles caveolae, reduced IP(3)-induced I(Cat) activation in smooth muscle cells and vasoconstriction in pressurized arteries. Cholesterol replenishment reversed these effects. Cav-1 knockdown using shRNA attenuated IP(3)-induced vasoconstriction, but did not alter TRPC3 and IP(3)R1 expression. A synthetic peptide corresponding to the cav-1 scaffolding domain (CSD) sequence (amino acids 82-101) also attenuated IP(3)-induced I(Cat) activation and vasoconstriction. A cav-1 antibody co-immunoprecipitated cav-1, TRPC3, and IP(3)R1 from cerebral artery lysate. ImmunoFRET indicated that cav-1, TRPC3 channels and IP(3)R1 are spatially co-localized in arterial smooth muscle cells. IP(3)R1 and TRPC3 channel spatial localization was disrupted by MßCD and a CSD peptide. Cholesterol replenishment re-established IP(3)R1 and TRPC3 channel close spatial proximity. Taken together, these data indicate that in arterial smooth muscle cells, cav-1 co-localizes SR IP(3)R1 and plasma membrane TRPC3 channels in close spatial proximity thereby enabling IP(3)-induced physical coupling of these proteins, leading to I(Cat) generation and vasoconstriction.

Inositol trisphosphate receptors in smooth muscle cells.

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are a family of tetrameric intracellular calcium (Ca(2+)) release channels that are located on the sarcoplasmic reticulum (SR) membrane of virtually all mammalian cell types, including smooth muscle cells (SMC). Here, we have reviewed literature investigating IP(3)R expression, cellular localization, tissue distribution, activity regulation, communication with ion channels and organelles, generation of Ca(2+) signals, modulation of physiological functions, and alterations in pathologies in SMCs. Three IP(3)R isoforms have been identified, with relative expression and cellular localization of each contributing to signaling differences in diverse SMC types. Several endogenous ligands, kinases, proteins, and other modulators control SMC IP(3)R channel activity. SMC IP(3)Rs communicate with nearby ryanodine-sensitive Ca(2+) channels and mitochondria to influence SR Ca(2+) release and reactive oxygen species generation. IP(3)R-mediated Ca(2+) release can stimulate plasma membrane-localized channels, including transient receptor potential (TRP) channels and store-operated Ca(2+) channels. SMC IP(3)Rs also signal to other proteins via SR Ca(2+) release-independent mechanisms through physical coupling to TRP channels and local communication with large-conductance Ca(2+)-activated potassium channels. IP(3)R-mediated Ca(2+) release generates a wide variety of intracellular Ca(2+) signals, which vary with respect to frequency, amplitude, spatial, and temporal properties. IP(3)R signaling controls multiple SMC functions, including contraction, gene expression, migration, and proliferation. IP(3)R expression and cellular signaling are altered in several SMC diseases, notably asthma, atherosclerosis, diabetes, and hypertension. In summary, IP(3)R-mediated pathways control diverse SMC physiological functions, with pathological alterations in IP(3)R signaling contributing to disease.

Mitochondria control functional CaV1.2 expression in smooth muscle cells of cerebral arteries.

RATIONALE: Physiological functions of mitochondria in contractile arterial myocytes are poorly understood. Mitochondria can uptake calcium (Ca(2+)), but intracellular Ca(2+) signals that regulate mitochondrial Ca(2+) concentration ([Ca(2+)](mito)) and physiological functions of changes in [Ca(2+)](mito) in arterial myocytes are unclear. OBJECTIVE: To identify Ca(2+) signals that regulate [Ca(2+)](mito), examine the significance of changes in [Ca(2+)](mito), and test the hypothesis that [Ca(2+)](mito) controls functional ion channel transcription in myocytes of resistance-size cerebral arteries. METHODS AND RESULTS: Endothelin (ET)-1 activated Ca(2+) waves and elevated global Ca(2+) concentration ([Ca(2+)](i)) via inositol 1,4,5-trisphosphate receptor (IP(3)R) activation. IP(3)R-mediated sarcoplasmic reticulum (SR) Ca(2+) release increased [Ca(2+)](mito) and induced mitochondrial depolarization, which stimulated mitochondrial reactive oxygen species (mitoROS) generation that elevated cytosolic ROS. In contrast, a global [Ca(2+)](i) elevation did not alter [Ca(2+)](mito), mitochondrial potential, or mitoROS generation. ET-1 stimulated nuclear translocation of nuclear factor (NF)-kappaB p50 subunit and ET-1-induced IP(3)R-mediated mitoROS elevated NF-kappaB-dependent transcriptional activity. ET-1 elevated voltage-dependent Ca(2+) (Ca(V)1.2) channel expression, leading to an increase in both pressure (myogenic tone)- and depolarization-induced vasoconstriction. Baseline Ca(V)1.2 expression and the ET-1-induced elevation in Ca(V)1.2 expression were both reduced by IP(3)R inhibition, mitochondrial electron transport chain block, antioxidant treatment, and NF-kappaB subunit knockdown, leading to vasodilation. CONCLUSIONS: IP(3)R-mediated SR Ca(2+) release elevates [Ca(2+)](mito), which induces mitoROS generation. MitoROS activate NF-kappaB, which stimulates Ca(V)1.2 channel transcription. Thus, mitochondria sense IP(3)R-mediated SR Ca(2+) release to control NF-kappaB-dependent Ca(V)1.2 channel expression in arterial myocytes, thereby modulating arterial contractility.

Isoform-selective physical coupling of TRPC3 channels to IP3 receptors in smooth muscle cells regulates arterial contractility.

RATIONALE: Inositol 1,4,5-trisphosphate (IP(3))-induced vasoconstriction can occur independently of intracellular Ca(2+) release and via IP(3) receptor (IP(3)R) and canonical transient receptor potential (TRPC) channel activation, but functional signaling mechanisms mediating this effect are unclear. OBJECTIVES: Study mechanisms by which IP(3)Rs stimulate TRPC channels in myocytes of resistance-size cerebral arteries. METHODS AND RESULTS: Immunofluorescence resonance energy transfer (immuno-FRET) microscopy using isoform-selective antibodies indicated that endogenous type 1 IP(3)Rs (IP(3)R1) are in close spatial proximity to TRPC3, but distant from TRPC6 or TRPM4 channels in arterial myocytes. Endothelin-1 (ET-1), a phospholipase C-coupled receptor agonist, elevated immuno-FRET between IP(3)R1 and TRPC3, but not between IP(3)R1 and TRPC6 or TRPM4. TRPC3, but not TRPC6, coimmunoprecipitated with IP(3)R1. TRPC3 and TRPC6 antibodies selectively inhibited recombinant channels, but only the TRPC3 antibody blocked IP(3)-induced nonselective cation current (I(Cat)) in myocytes. TRPC3 knockdown attenuated immuno-FRET between IP(3)R1 and TRPC3, IP(3)-induced I(Cat) activation, and ET-1 and IP(3)-induced vasoconstriction, whereas TRPC6 channel knockdown had no effect. ET-1 did not alter total or plasma membrane-localized TRPC3, as determined using surface biotinylation. RT-PCR demonstrated that C-terminal calmodulin and IP(3)R binding (CIRB) domains are present in myocyte TRPC3 and TRPC6 channels. A peptide corresponding to the IP(3)R N-terminal region that can interact with TRPC channels activated I(Cat). A TRPC3 CIRB domain peptide attenuated IP(3)- and ET-1-induced I(Cat) activation and vasoconstriction. CONCLUSIONS: IP(3) stimulates direct coupling between IP(3)R1 and membrane-resident TRPC3 channels in arterial myocytes, leading to I(Cat) activation and vasoconstriction. Close spatial proximity between IP(3)R1 and TRPC3 establishes this isoform-selective functional interaction.

A Case Study in Process Improvement to Minimize Delays from Obtaining Blood for Red Cell Exchange for a Patient with Sickle Cell Disease and Multiple Red Blood Cell Alloantibodies.

The process of procuring several units of red blood cells for red cell exchange can sometimes take several hours to days, especially for patients with multiple clinically significant red cell alloantibodies. This can introduce delays, inconveniences, and even health challenges for the patient. For most planned exchanges, these delays are preventable with some foresight and process modifications that are relatively minor yet high leverage. We report a case study of process improvement whereby the apheresis nurse sends an e-mail to the blood bank when the nurse makes the patient's next red cell exchange appointment as the signal to order blood about 6-8 weeks before the exchange.

TP53 mutation defines a unique subgroup within complex karyotype de novo and therapy-related MDS/AML.

A subset of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) show complex karyotype (CK), and these cases include a relatively high proportion of cases of therapy-related myeloid neoplasms and TP53 mutations. We aimed to evaluate the clinicopathologic features of outcome of 299 AML and MDS patients with CK collected from multiple academic institutions. Mutations were present in 287 patients (96%), and the most common mutation detected was in TP53 gene (247, 83%). A higher frequency of TP53 mutations was present in therapy-related cases (P = .008), with a trend for worse overall survival (OS) in therapy-related patients as compared with de novo disease (P = .08) and within the therapy-related group; the presence of TP53 mutation strongly predicted for worse outcome (P = .0017). However, there was no difference in survival between CK patients based on categorization of AML vs MDS (P = .96) or presence of absence of circulating blasts ≥1% (P = .52). TP53-mutated patients presented with older age (P = .06) and lower hemoglobin levels (P = .004) and marrow blast counts (P = .02) compared with those with CK lacking TP53 mutation. Multivariable analysis identified presence of multihit TP53 mutation as strongest predictor of worse outcome, whereas neither a diagnosis of AML vs MDS nor therapy-relatedness independently influenced OS. Our findings suggest that among patients with MDS and AML, the presence of TP53 mutation (in particular multihit TP53 mutation) in the context of CK identifies a homogeneously aggressive disease, irrespective of the blast count at presentation or therapy-relatedness. The current classification of these cases into different disease categories artificially separates a single biologic disease entity.

Smooth muscle cell alpha2delta-1 subunits are essential for vasoregulation by CaV1.2 channels.

RATIONALE: Voltage-dependent L-type (Ca(V)1.2) Ca(2+) channels are a heteromeric complex formed from pore-forming alpha(1) and auxiliary alpha(2)delta and beta subunits. Ca(V)1.2 channels are the principal Ca(2+) influx pathway in arterial myocytes and regulate multiple physiological functions, including contraction. The macromolecular composition of arterial myocyte Ca(V)1.2 channels remains poorly understood, with no studies having examined the molecular identity or physiological functions of alpha(2)delta subunits. OBJECTIVE: We investigated the functional significance of alpha(2)delta subunits in myocytes of resistance-size (100 to 200 mum diameter) cerebral arteries. METHODS AND RESULTS: alpha(2)delta-1 was the only alpha(2)delta isoform expressed in cerebral artery myocytes. Pregabalin, an alpha(2)delta-1/-2 ligand, and an alpha(2)delta-1 antibody, inhibited Ca(V)1.2 currents in isolated myocytes. Acute pregabalin application reversibly dilated pressurized arteries. Using a novel application of surface biotinylation, data indicated that >95% of Ca(V)1.2 alpha(1) and alpha(2)delta-1 subunits were present in the arterial myocyte plasma membrane. Alpha(2)delta-1 knockdown using short hairpin RNA reduced plasma membrane-localized Ca(V)1.2 alpha(1) subunits, caused a corresponding elevation in cytosolic Ca(V)1.2 alpha(1) subunits, decreased intracellular Ca(2+) concentration, inhibited pressure-induced vasoconstriction ("myogenic tone"), and attenuated pregabalin-induced vasodilation. Prolonged (24-hour) pregabalin exposure did not alter total alpha(2)delta-1 or Ca(V)1.2 alpha(1) proteins but decreased plasma membrane expression of each subunit, which reduced myogenic tone. CONCLUSIONS: alpha(2)delta-1 is essential for plasma membrane expression of arterial myocyte Ca(V)1.2 alpha(1) subunits. alpha(2)delta-1 targeting can block Ca(V)1.2 channels directly and inhibit surface expression of Ca(V)1.2 alpha(1) subunits, leading to vasodilation. These data identify alpha(2)delta-1 as a novel molecular target in arterial myocytes, the manipulation of which regulates contractility.