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Peptidoglycan (PGN), a component of bacterial cell wall and belonging to "Microbe-Associated Molecular Patterns" (MAMP) triggers host reactions contributing to the pathophysiology of infectious disease. Host cell responses to PGN exposure include apoptosis. Bacterial infections may result in activation of blood platelets and thrombocytopenia. The present study explored, whether HPLC-purified fractions of PGNs from Staphylococcus aureus 113 triggers apoptosis of platelets. To this end platelets were exposed to PGN fractions and annexin-V binding determined to depict cell membrane scrambling, DiOC6 fluorescence to estimate depolarization of mitochondrial potential, Fluo-3AM staining for intracellular Ca(2+) activity ([Ca(2+)](i)) and immunofluorescence to quantify protein abundance of active caspase-3. As a result, a 30 min exposure to monomeric fraction (mPGN) (≥50 ng/ml) was followed by annexin-V binding, paralleled by increase of [Ca(2+)](i), mitochondrial depolarization, caspase-3 activation and integrin α(IIb)β(3) upregulation. The annexin-V binding was significantly blunted by anti-TLR-2 antibodies, in absence of extracellular Ca(2+), and by pancaspase inhibitor zVAD-FMK (1 μM). In conclusion, PGN triggers apoptosis of platelets in activation-dependent manner, characterized by mitochondrial depolarization, caspase-3 activation and cell membrane scrambling.

Activation of platelets by thrombin opens pore forming channel protein Orai1 with subsequent store operated Ca(2+) entry (SOCE) and Ca(2+) dependent platelet granule release, integrin α(IIb)β(3) activation, adhesion, aggregation and thrombus formation. Orai1 and thus SOCE as well as platelet activation are up-regulated by the serum- and glucocorticoid-inducible kinase-1 (SGK1), which transcriptionally regulates Orai1 expression in megakaryocytes and thus determines Orai1 protein abundance in mature, circulating platelets. As platelets are devoid of nuclei, they are unable to modify protein abundance by regulation of transcription. However, they contain mRNA and thus could express novel protein by stimulation of protein translation. Translation is sensitive to actin polymerization and phosphoinositide-3-kinase (PI3K). Translational regulation of SGK1 expression has never been described before. The present study thus explored whether thrombin regulates SGK1 expression in platelets. As a result, according to RT-PCR mRNA encoding SGK1 is present in circulating platelets and significantly decreased by activation of platelets with thrombin (1 U/ml). The protein abundance of SGK1 is significantly enhanced by thrombin treatment, an effect significantly decreased by inhibition of translation with puromycin (100 nM) but not by inhibition of transcription with actinomycin (4 μg/ml). The increase of SGK1 protein abundance is blunted by inhibition of PI3K with wortmannin (100 nM) or LY294002 (25 μM), and by disruption of the cytoskeleton with cytochalasin B (1 μM). In conclusion, activation of platelets with thrombin stimulates the translation of SGK1., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The CTLA-4 pathway is a key regulator of T cell activation and a critical failsafe against autoimmunity. Although early models postulated that CTLA-4 transduced a negative signal, in vivo evidence suggests that CTLA-4 functions in a cell-extrinsic manner. That multiple cell-intrinsic mechanisms have been attributed to CTLA-4, yet its function in vivo appears to be cell-extrinsic, has been an ongoing paradox in the field. Although CTLA-4 expressed on conventional T cells (Tconv) can mediate inhibitory function, it is unclear why this fails to manifest as an intrinsic effect. In this study, we show that Tconv-expressed CTLA-4 can function in a cell-extrinsic manner in vivo. CTLA-4(+/+) T cells, from DO11/rag(-/-) mice that lack regulatory T cells, were able to regulate the response of CTLA-4(-/-) T cells in cotransfer experiments. This observation provides a potential resolution to the above paradox and suggests CTLA-4 function on both Tconv and regulatory T cells can be achieved through cell-extrinsic mechanisms.

Extracellular cyclophilin A (CyPA) and its receptor Extracellular Matrix Metalloproteinase Inducer (EMMPRIN, CD147) modulate inflammatory processes beyond metalloproteinase (MMP) activity. Recently, we have shown that CyPA and CD147 are upregulated in patients with inflammatory cardiomyopathy. Here we investigate the role of CyPA and CD147 in murine coxsackievirus B3 (CVB3)-induced myocarditis. CVB3-infected CyPA(-/-) mice (129S6/SvEv) revealed a significantly reduced T-cell and macrophage recruitment at 8 days p.i. compared to wild-type mice. In A.BY/SnJ mice, treatment with the cyclophilin-inhibitor NIM811 was associated with a reduction of inflammatory lesions and MMP-9 expression but with enhanced virus replication 8 days p.i. At 28 days p.i. the extent of lesion areas was not affected bei NIM811, whereas the collagen content was reduced. Initiation of NIM811-treatment on day 12 (after an effective virus defense) resulted in an even more pronounced reduction of myocardial fibrosis. In conclusion, in CVB3-induced myocarditis CyPA is important for macrophage and T cell recruitment and effective virus defense and may represent a pharmacological target to modulate myocardial remodeling in myocarditis., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

An exceptionally easy to assemble source for ambient mass spectrometry is described. Based on Venturi easy ambient sonic-spray ionization (V-EASI), the source was further simplified by the use of a can of compressed air which simultaneously provides solution or solvent Venturi self-pumping and continuous, stable and abundant low-noise ion signal via voltage-free sonic-spraying. Further simplification was also attained by the use of inexpensive and readily commercially available parts: a surgical 2-way catheter, an aerosol can of compressed air, a 30 cm long fused-silica capillary and a hypodermic needle. This "Spartan" V-EASI source seems to offer one of the easiest and cheapest ways to make ions for ambient desorption/ionization mass spectrometry analysis of both liquid and solid samples.

Background: Surgical resection is the mainstay of treatment for colorectal cancer with curative intent., Aim: To evaluate the postoperative results of laparoscopic and laparotomic colorectal resections for colorectal cancer., Methods: A retrospective study of a series of 189 patients. The descriptive variables were age and gender, and for outcome were type of resection, number of lymph nodes resected, free margins, the need for colostomy, complications, operative time and hospital stay. They were analyzed using the chi-square, Student t and Mann-Whitney test, with significance level <0.05., Results: Of the 189 operated patients, 110 met the inclusion criteria, 75 (68.2%) operated by open surgery and 35 (31.8%) by laparoscopic. The sigmoid colon was the most common site presented by neoplasia and rectosigmoidectomy was performed more by open colorectal resection (p = 0.042). The conversion rate was 7.9% (3/38). The patients were operated by open surgery in 81.5% of time less than 180 minutes (p <0.001). In both pathways, the average number of removed lymph nodes was greater than 12, but laparotomy enabled, more frequently, the resection of 12 or more nodes (p = 0.012). No patient had surgical margins involved, but laparotomy allowed a greater number of patients with a margin greater than 5 cm from the tumor (p = 0.036). Increased number of patients treated by open surgery were hospitalized for more than seven days (p <0.001). There were no statistically significant differences regarding the need for ostomies, complications and mortality., Conclusions: The laparoscopic approach was as safe and effective as laparotomy in the treatment of colorectal cancer, and was associated with increased operative time, shorter hospital stay and less morbidity.

The serum- and glucocorticoid-dependent kinases 1-3 (SGK1-3) are downstream effectors of phosphatidylinositol 3-kinases, implicated in various cell responses including colon cancer tumorigenesis in mice. Here, we investigated the role of SGK1 in the regulation of cell motility. Using Caco-2 colon tumor and HEK293 embryonic kidney cells, we report that transfection with the constitutively active SGK1 mutant (SGK1-SD) significantly enhanced cell motility. The cell-adhesion protein vinculin was effectively dephosphorylated in SGK1-SD-transfected cells. Treatment of the cells with phosphatase inhibitors restored vinculin phosphorylation and inhibited cell migration, indicating a significant role for vinculin phosphorylation in SGK1-induced motility. SGK1-SD-enhanced cell motility was inhibited by activation of membrane androgen-binding sites (mAR) via testosterone-conjugates in both cell lines, whereas intracellular androgen receptor (iAR)-silencing and flutamide treatment revealed that these effects were clearly independent of the interaction of SGK1 with the classical androgen receptors (iAR). More importantly, mAR activation restored vinculin phosphorylation in SGK1-SD-transfected cells, whereas silencing of vinculin fully reversed the mAR-induced inhibition of the migratory capacity, implying that this protein is directly involved in cell motility regulation by SGK1 and mAR. This study indicates for the first time that SGK1 regulates cell migration via vinculin dephosphorylation, a mechanism that is controlled by mAR function., (© 2012 The Authors Journal compilation © 2012 FEBS.)

Chorea-acanthocytosis is an inevitably lethal genetic disease characterized by a progressive hyperkinetic movement disorder and cognitive and behavioral abnormalities as well as acanthocytosis. The disease is caused by loss-of-function mutations of the gene encoding vacuolar protein sorting-associated protein 13A (VPS13A) or chorein, a protein with unknown function expressed in various cell types. How chorein deficiency leads to the pathophysiology of chorea-acanthocytosis remains enigmatic. Here we show decreased phosphoinositide-3-kinase (PI3K)-p85-subunit phosphorylation, ras-related C3 botulinum toxin substrate 1 (Rac1) activity, and p21 protein-activated kinase 1 (PAK1) phosphorylation as well as depolymerized cortical actin in erythrocytes from patients with chorea-acanthocytosis and in K562-erythrocytic cells following chorein silencing. Pharmacological inhibition of PI3K, Rac1, or PAK1 similarly triggered actin depolymerization. Moreover, in K562 cells, both chorein silencing and PAK1 inhibition with IPA-3 decreased phosphorylation of Bad, a Bcl2-associated protein, promoting apoptosis by forming mitochondrial pores, followed by mitochondrial depolarization, DNA fragmentation, and phosphatidylserine exposure at the cell surface, all hallmarks of apoptosis. Our observations reveal chorein as a novel powerful regulator of cytoskeletal architecture and cell survival, thus explaining erythrocyte misshape and possibly neurodegeneration in chorea-acanthocytosis.

The serum and glucocorticoid-inducible kinase SGK1 increases the activity of Orai1, the pore forming unit of store-operated Ca(2+) entry, and thus influences Ca(2+)-dependent cellular functions such as migration. SGK1 further regulates transcription factor nuclear factor κB (NF-κB). This study explored whether SGK1 influences transcription of Orai1 and/or STIM1, the Orai1-activating Ca(2+) sensor. Orai1 and STIM1 transcript levels were decreased in mast cells from SGK1 knock-out mice and increased in HEK293 cells transfected with active (S422D)SGK1 but not with inactive (K127N)SGK1 or in (S422D)SGK1-transfected cells treated with the NF-κB inhibitor Wogonin (100 μm). Treatment with the stem cell factor enhanced transcript levels of STIM1 and Orai1 in sgk1(+/+) but not in sgk1(-/-) mast cells and not in sgk1(+/+) cells treated with Wogonin. Orai1 and STIM1 transcript levels were further increased in sgk1(+/+) and sgk1(-/-) mast cells by transfection with active NF-κB subunit p65 as well as in HEK293 cells by transfection with NF-κB subunits p65/p50 or p65/p52. They were decreased by silencing of NF-κB subunits p65, p50, or p52 or by NF-κB inhibitor Wogonin (100 μm). Luciferase assay and chromatin immunoprecipitation defined NF-κB-binding sites in promoter regions accounting for NF-κB sensitive genomic regulation of STIM1 and Orai1. Store-operated Ca(2+) entry was similarly increased by overexpression of p65/p50 or p65/p52 and decreased by treatment with Wogonin. Transfection of HEK293 cells with p65/p50 or p65/p52 further augmented migration. The present observations reveal powerful genomic regulation of Orai1/STIM1 by SGK1-dependent NF-κB signaling.

Background: Chronic kidney disease (CKD) is a condition associated with inflammation and high levels of uremic toxins and reactive oxygen species. As a counterregulation to systemic stress heat shock proteins (HSP) are increased expressed to minimize cell death and preserve cell integrity by inhibiting apoptotic pathways. The aim of this study was to determine HSP27 and HSP70 concentrations in sera and urine of patients suffering from CKD., Methods: Concentrations of HSP27 and HSP70 in urine and serum were determined in 119 patients with CKD stages 1 to 5 and 23 healthy volunteers by using ELISA technique., Results: HSP27 serum levels were significantly elevated in patients suffering from CKD stages 3 to 5 as well as fractional HSP27 excretion in stages 2-5 versus healthy controls. Absolute HSP70 urinary values were significantly elevated in stages 4 and 5 and fractional HSP70 excretion was increased in stage 5 compared to controls. Moreover, ROC curve analysis showed the potential of urine and especially serum HSP levels to identify various stages of CKD., Conclusion: We provide evidence for elevated HSP27 concentrations in serum and urine and increased HSP70 excretion levels in patients suffering from CKD. Moreover, our results show that HSP levels might offer potential to examine the stages of CKD as well as the disease course which could further promote individually adjusted treatment planning., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Platelets are activated on increase of cytosolic Ca2+ activity ([Ca2+](i)), accomplished by store-operated Ca2+ entry (SOCE) involving the pore-forming ion channel subunit Orai1. Here, we show, for the first time, that the serum- and glucocorticoid-inducible kinase 1 (SGK1) is expressed in platelets and megakaryocytes. SOCE and agonist-induced [Ca2+](i) increase are significantly blunted in platelets from SGK1 knockout mice (sgk1(-/-)). Similarly, Ca2+ -dependent degranulation, integrin α(IIb)β3 activation, phosphatidylserine exposure, aggregation, and in vitro thrombus formation were significantly impaired in sgk1(-/-) platelets, whereas tail bleeding time was not significantly enhanced. Platelet and megakaryocyte Orai1 transcript levels and membrane protein abundance were significantly reduced in sgk1(-/-) mice. In human megakaryoblastic cells (MEG-01), transfection with constitutively active (S422D)SGK1 but not with inactive (K127N)SGK1 significantly enhanced Orai1 expression and SOCE, while effects reversed by the SGK1 inhibitor GSK650394 (1μM). Transfection of MEG-01 cells with (S422D)SGK1 significantly increased phosphorylation of IκB kinase α/β and IκBα resulting in nuclear translocation of NF-κB subunit p65. Treatment of (S422D)SGK1-transfected MEG-01 cells with the IκB kinase inhibitor BMS-345541 (10μM) abolished SGK1-induced increase of Orai1 expression and SOCE. The present observations unravel SGK1 as novel regulator of platelet function, effective at least in part by NF-κB-dependent transcriptional up-regulation of Orai1 in megakaryocytes and increasing platelet SOCE.

Recent observations pointed to the ability of platelets to migrate and thus to invade the inflamed vascular wall. Platelet migration could be stimulated by stromal cell-derived factor-1 (SDF-1), an effect dependent on phosphatidylinositide-3-kinase (PI3K) and paralleled by activation and phosphorylation of Wiskott-Aldrich syndrome protein (WASP). Migration is inhibited by vinculin, which is similarly regulated by phosphorylation. PI3K-sensitive kinases include the serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored whether SGK1 modifies WASP and vinculin phosphorylation in murine platelets and participates in the regulation of platelet migration. Platelets were isolated from gene-targeted mice lacking SGK1 (sgk1(-/-)) and from their wild type littermates (sgk1(+/+)). Platelet migration stimulated with SDF-1 was significantly less pronounced in sgk1(-/-)platelets than in sgk1(+/+) platelets. Moreover, SDF-1 significantly induced WASP phosphorylation, an effect again reduced in platelets lacking SGK1. Phosphorylation of vinculin was significantly enhanced in sgk1(-/-)platelets and was significantly reduced following treatment of platelets with Ca(2+) chelator BAPTA. Immunohistochemical analysis of in vivo experiments in intestinal vessels after vascular inflammation revealed that transmigration of platelets into inflamed vessel walls was significantly less pronounced in sgk1(-/-)than in sgk1(+/+) mice. In conclusion, SGK1 is a powerful regulator of platelet migration., (Copyright © 2012 S. Karger AG, Basel.)

An acetate anion bearing an imidazolium cation as its charge tag was reacted with M(OAc)(2) complexes (where M = Ni, Cu, and Pd; in situ reaction) to form members of a new class of charge-tagged metal complexes. The formation of these unprecedented precatalysts with potential for cross-coupling reactions was confirmed by electrospray ionization (and tandem) mass spectrometry. The catalytic performance of the palladium complex was tested in Heck and Suzuki cross-coupling reactions, often with superior activity and yields as compared with Pd(OAc)(2).

Objective: To test the implementation of a novel structured panel process in the evaluation of quality indicators., Data Source: National panel of 64 clinicians rating usefulness of indicator applications in 2008-2009., Study Design: Hybrid panel combined Delphi Group and Nominal Group (NG) techniques to evaluate 81 indicator applications., Principal Findings: The Delphi Group and NG rated 56 percent of indicator applications similarly. Group assignment (Delphi versus Nominal) was not significantly associated with mean ratings, but specialty and research interests of panelists, and indicator factors such as denominator level and proposed use were. Rating distributions narrowed significantly in 20.8 percent of applications between review rounds., Conclusions: The hybrid panel process facilitated information exchange and tightened rating distributions. Future assessments of this method might include a control panel., (© Health Research and Educational Trust.)

Platelets have been shown to migrate and thus to invade the vascular wall. Platelet migration is stimulated by SDF-1. In other cell types, migration is dependent on Ca(2+) entry via Ca(2+) channels. Ca(2+) influx is sensitive to cell membrane potential which is maintained by K(+) channel activity and/or Cl(-) channel activity. The present study explored the role of ion channels in the regulation of SDF-1 induced migration. Platelets were isolated from human volunteers as well as from gene targeted mice lacking the Ca(2+) activated K(+) channel SK4 (sk4(-/-)) and their wild type littermates (sk4(+/+)). According to confocal microscopy human platelets expressed the Ca(2+) channel Orai1 and the Ca(2+)-activated K(+) channel K(Ca)3.1 (SK4). SDF-1 (100 ng/ml) stimulated migration in human platelets, an effect blunted by Orai1 inhibitors 2-aminoethoxydiphenyl borate 2-APB (10 μM) and SKF-96365 (10 μM), by unspecific K(+) channel inhibitor TEA (30 mM), by SK4 specific K(+) channel blocker clotrimazole (10 μM), but not by Cl(-) channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid NPPB (100 μM). Significant stimulation of migration by SDF-1 was further observed in sk4(+/+) platelets but was virtually absent in sk4(-/-) platelets. In conclusion, platelet migration requires activity of the Ca(2+) channel Orai1 and of the Ca(2+) activated K(+) channel SK4, but not of NPPB-sensitive Cl(-) channels., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Thymoquinone (TQ) is a nutrient with anticarcinogenic activity that stimulates suicidal death of tumor cells. Moreover, TQ triggers suicidal death of erythrocytes or eryptosis, an effect at least partially due to increase in cytosolic Ca(2+) activity and ceramide formation. The present experiments explored whether TQ influences apoptosis of blood platelets. Cell membrane scrambling was determined utilizing Annexin V binding to phosphatidylserine exposing platelets, cytosolic Ca(2+) activity utilizing Fluo 3-AM fluorescence, caspase activity utilizing immunofluorescence and Western blotting of active caspase-3 and inactive procaspase-3, mitochondrial potential utilizing DiOC(6) fluorescence and ceramide by FACS analysis of ceramide-binding antibodies. A 30 min exposure to TQ (≥5 µM) was followed by Annexin V binding, paralleled by caspase activation, increase of cytosolic Ca(2+) activity, mitochondrial depolarization, and ceramide formation. P-selectin exposure and integrin α(IIb) β(3) activation did not increase in response to TQ. Nominal absence of extracellular Ca(2+) blunted but did not fully abolish the TQ-induced activation of caspase-3. The effects of TQ on platelets are significantly abolished with phosphoinositide-3 kinase (PI3K) inhibitor wortmannin and G-protein coupled receptor (GPCR) inhibitor pertussis toxin treatment prior to TQ stimulation. In conclusion, TQ triggers suicidal death of blood platelets in a PI3K-dependent manner, possibly through a GPCR family receptor; an effect paralleled by increase of cytosolic Ca(2+) activity, ceramide formation, mitochondrial depolarization, and caspase-3 activation., (Copyright © 2011 Wiley Periodicals, Inc.)

Background: The importance of cytotoxic T lymphocyte antigen-4 (CTLA-4) in immune regulation is unquestioned, yet a precise understanding of which cells express it, and how it mediates immune inhibitory function, is lacking. Regulatory T cells are known to constitutively express CTLA-4 intracellularly, whereas conventional T cells require activation to trigger CTLA-4 expression. However comparative analysis of CTLA-4 trafficking in regulatory and conventional subsets has not been performed., Methods: Here we assess CTLA-4 expression in antigen-specific conventional and regulatory cells responding to immunizing antigen in vivo and analyse the membrane trafficking of CTLA-4 using an in vitro recycling assay. We assess the expression of CTLA-4 on Treg infiltrating the pancreas in the DO11×RIP-mOVA diabetes model and the role of CTLA-4 in Treg function., Results: Regulatory T cells show an enhanced capacity to traffic CTLA-4 following stimulation compared with conventional T cells. Treg infiltrating the pancreas in DO11×RIP-mOVA mice show high expression of CTLA-4. Furthermore CTLA-4-deficient Treg fail to control diabetes in an adoptive transfer model of diabetes, even in situations where they outnumber the disease-inducing conventional T cells., Conclusions: These data show that not only do regulatory T cells express higher levels of intracellular CTLA-4 than conventional T cells, but they also show an increased capacity to traffic CTLA-4 to the cell surface following stimulation. CTLA-4 is strongly upregulated in regulatory T cells infiltrating the target tissue in a mouse model of type 1 diabetes and expression of this protein is critical for effective regulation., (Copyright © 2011 John Wiley & Sons, Ltd.)

Low-magnification high-numerical aperture objectives maximize the collection efficiency for scattered two-photon excited fluorescence (2PEF), but non-descanned detection schemes for such objectives demand optical components much bigger than standard microscope optics. Fiber coupling offers the possibility of removing bulky multi-channel detectors from the collection site, but coupling and transmission losses are generally believed to outweigh the benefits of optical fibers. We present here two new developments based on large-core fiber-optic fluorescence detection that illustrate clear advantages over conventional air-coupled 2PEF detection schemes. First, with minimal modifications of a commercial microscope, we efficiently couple the output of a 20×/NA0.95 objective to a large-core liquid light guide and we obtain a 7-fold collection gain when imaging astrocytes at 100 μm depth in acute brain slices of adult ALDH1L1-GFP mice. Second, combining 2PEF microscopy and 4-color detection on a custom microscope, mode scrambling inside a 2-mm plastic optical fiber is shown to cancel out the spatially non-uniform spectral sensitivity observed with air-coupled detectors. Spectral unmixing of images of brainbow mice taken with a fiber-coupled detector revealed a uniform color distribution of hippocampal neurons across a large field of view. Thus, fiber coupling improves both the efficiency and the homogeneity of 2PEF collection., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is an essential negative regulator of T cell immune responses whose mechanism of action is the subject of debate. CTLA-4 shares two ligands (CD80 and CD86) with a stimulatory receptor, CD28. Here, we show that CTLA-4 can capture its ligands from opposing cells by a process of trans-endocytosis. After removal, these costimulatory ligands are degraded inside CTLA-4-expressing cells, resulting in impaired costimulation via CD28. Acquisition of CD86 from antigen-presenting cells is stimulated by T cell receptor engagement and observed in vitro and in vivo. These data reveal a mechanism of immune regulation in which CTLA-4 acts as an effector molecule to inhibit CD28 costimulation by the cell-extrinsic depletion of ligands, accounting for many of the known features of the CD28-CTLA-4 system.

The entry of autoreactive T cells into the pancreas is a critical checkpoint in the development of autoimmune diabetes. In this study, we identify a role for B1 cells in this process using the DO11 x RIP-mOVA mouse model. In transgenic mice with islet-specific T cells, but no B cells, T cells are primed in the pancreatic lymph node but fail to enter the pancreas. Reconstitution of the B1 cell population by adoptive transfer permits extensive T cell pancreas infiltration. Reconstituted B1 cells traffic to the pancreas and modify expression of adhesion molecules on pancreatic vasculature, notably VCAM-1. Despite substantial pancreas infiltration, islet destruction is minimal unless regulatory T cells are depleted. These data identify a role for B1 cells in permitting circulating islet-specific T cells to access their Ag-bearing tissue and emphasize the existence of multiple checkpoints to regulate autoimmune disease.

Patients with type 2 diabetes mellitus and early diabetic nephropathy have a poor disease-related prognosis; furthermore these patients are often also mentally stressed. We investigated an acceptance- and mindfulness-based group intervention for these patients in addition to regular medical therapy. Both intervention program and descriptive outcomes of patients' evaluation are presented. A total of 51 patients attended the groups. Patients reported developing a mindfulness attitude towards life during the group process as well as an improvement in pain, sleep and worrying.

Preliminary reference intervals for hematologic and total plasma protein profiles were determined for nine adult Red-capped parrots (Pionopsitta pileata) (six males and three females) and six Vinaceous Amazon parrots (Amazona vinacea) (two adult males, two adult females, one juvenile, and one nonsexed) from the Curitiba Zoo, Paraná, Brazil. For both Red-capped parrots and Vinaceous Amazon parrots, adult males had higher red blood cell counts than adult females. Regarding white blood cell distribution, differences due to gender were also found for both species of parrots.

The CTLA-4 pathway is recognized as a major immune inhibitory axis and is a key therapeutic target for augmenting antitumor immunity or curbing autoimmunity. CTLA-4-deficient mice provide the archetypal example of dysregulated immune homeostasis, developing lethal lymphoproliferation with multiorgan inflammation. In this study, we show that surprisingly these mice have an enlarged population of Foxp3(+) regulatory T cells (Treg). The increase in Treg is associated with normal thymic output but enhanced proliferation of Foxp3(+) cells in the periphery. We confirmed the effect of CTLA-4 deficiency on the Treg population using OVA-specific Treg which develop normally in the absence of CTLA-4, but show increased proliferation in response to peripheral self-Ag. Functional analysis revealed that Ag-specific Treg lacking CTLA-4 were unable to regulate disease in an adoptive transfer model of diabetes. Collectively, these data suggest that the proliferation of Treg in the periphery is tuned by CTLA-4 signals and that Treg expression of CTLA-4 is required for regulation of pancreas autoimmunity.

The activity of regulatory T cells (Treg) is widely accepted to play a central role in preventing pathogenic immune responses against self-Ags. However, it is not clear why such regulation breaks down during the onset of autoimmunity. We have studied self-Ag-specific Treg during the induction of spontaneous diabetes. Our data reveal a shift in the balance between regulatory and pathogenic islet-reactive T cells in the pancreas-draining lymph nodes during disease onset. Treg function was not compromised during disease initiation, but instead conventional T cells showed reduced susceptibility to Treg-mediated suppression. Release from Treg suppression was associated with elevated levels of IL-21 in vivo, and provision of this cytokine abrogated Treg suppression in vitro and in vivo. These data suggest that immunological protection of a peripheral tissue by Treg can be subverted by IL-21, suggesting new strategies for intervention in autoimmunity.

Objective: To describe the normal bony orbital structure of the large fruit-eating bat (Artibeus lituratus) with emphasis on a unique intraorbital bony structure previously not described in the literature., Procedures: The bony anatomy of the orbital cavity was studied on dissected skulls of large fruit-eating bats. The anatomic description of a unique intraorbital spine was made while studying the bony orbit of macerated skulls. Additional observations were made on dissected formalin-fixed whole heads. Both procedures were performed under a stereo dissecting microscope, using x2-4-magnification. A histologic analysis of soft tissues surrounding this cylindrical bony structure was performed using cross and longitudinal oblique sections from decalcified whole heads, which had been fixed in formalin. Additionally, biometric measurements and a histomorphometric analysis were performed., Results and Conclusions: An intraorbital cylindrical osseous structure measuring 3.96 +/- 0.68 mm in length and 155.62 +/- 14.03 microm in diameter was observed in the large fruit-eating bat (A. lituratus), creating a unique orbital structural design among mammals. We suggest the name optic spine of the alisphenoid bone. The anatomic, biometric and histologic characterization of this element might contribute to a further understanding of the dynamics of bat vision and the sort of factors that influenced evolution of the visual system of microbats. The authors hope that the documentation of this distinctive anatomic feature will also expand the debate about the phylogenetic analysis of the relationship among bat species in the near future.

The RNA-binding protein CHLAMY1 from the green alga Chlamydomonas reinhardtii consists of two subunits. One (named C1) contains three lysine homology motifs and the other (named C3) has three RNA recognition motifs. CHLAMY1 binds specifically to uridine-guanine-repeat sequences and its circadian-binding activity is controlled at the posttranslational level, presumably by time-dependent formation of protein complexes consisting of C1 and C3 or C1 alone. Here we have characterized the role of the two subunits within the circadian system by measurements of a circadian rhythm of phototaxis in strains where C1 or C3 are either up- or down-regulated. Further, we have measured the rhythm of nitrite reductase activity in strains with reduced levels of C1 or C3. In case of changes in the C3 level (both increases and decreases), the acrophase of the phototaxis rhythm and of the nitrite reductase rhythm (C3 decrease) was shifted by several hours from subjective day (maximum in wild-type cells) back towards the night. In contrast, both silencing and overexpression of C1 resulted in disturbed circadian rhythms and arrhythmicity. Interestingly, the expression of C1 is interconnected with that of C3. Our data suggest that CHLAMY1 is involved in the control of the phase angle and period of the circadian clock in C. reinhardtii.

Objective: This text presents an anatomical study of the normal bony orbital structure of a sample of different bird species belonging to the order Psittaciformes., Procedures: The bony anatomy of Psittaciformes' skulls was examined and described using cadavers of birds that were presented already dead to the Federal University of Paraná, Brazil or had been euthanized for humane reasons. Dissections of the orbital cavity were performed under 2-4 x magnification, and descriptions of the orbital bones were made from observations of macerated skulls that had been boiled and cleaned. The present paper discusses the main features of the bony orbit of psittaciform birds, describing known anatomical information but also bringing new information, mainly concerning species differences that might help not only veterinary anatomists but also zoologists, clinicians, researchers, and students of veterinary ophthalmology to better comprehend this order of birds., Results and Conclusions: Variations in the anatomic conformation of the bony elements of the orbit were observed in different species of Psittaciformes. Based on these differences, Psittaciformes were classified into two different groups. The first group of Psittaciformes shows an enclosed (complete) bony orbit formed by the junction of the orbital with the postorbital processes, creating a suborbital arch. The second group of Psittaciformes essentially lacked a suborbital arch, presenting an open (incomplete) bony orbit, typical of most modern birds. In the latter group, orbital and postorbital processes are present.

We report on our efforts to establish an animal model for the development and testing of a cortical visual prostheses. One-hundred-fifty-two electrodes were implanted in the primary visual cortex of a rhesus monkey. The electrodes were made from iridium with an activated iridium oxide film, which has a large charge capacity for a given surface area, and insulated with parylene-C. One-hundred-fourteen electrodes were functional after implantation. The activity of small (2-3) neuronal clusters was first recorded to map the visually responsive region corresponding to each electrode. The animal was then trained in a memory (delayed) saccade task, first with a visual target, then to a target defined by direct cortical stimulation with coordinates specified by the stimulating electrode's mapped receptive field. The SD of saccade endpoints was approximately 2.5 larger for electrically stimulated versus visual saccades; nevertheless, when trial-to-trial scatter was averaged out, the correlation between saccade end points and receptive field locations was highly significant and approached unity after several months of training. Five electrodes were left unused until the monkey was fully trained; when these were introduced, the receptive field-saccade correlations were high on the first day of use (R = 0.85, P = 0.03 for angle, R = 0.98, P < 0.001 for eccentricity), indicating that the monkey had not learned to perform the task empirically by memorizing reward zones. The results of this experiment suggest the potential for rigorous behavioral testing of cortical visual prostheses in the macaque.

The RNA-binding protein CHLAMY 1 from Chlamydomonas reinhardtii binds specifically to UG> or =7 repeat sequences situated in the 3' untranslated regions of several mRNAs. Its binding activity is controlled by the circadian clock. The biochemical purification and characterization of CHLAMY 1 revealed a novel type of RNA-binding protein. It includes two different subunits (named C1 and C3), whose interaction appears necessary for RNA binding. One of them (C3) belongs to the proteins of the CELF (CUG-BP-ETR-3-like factors) family and thus bears three RNA recognition motif domains. The other is composed of three lysine homology domains and a protein-protein interaction domain (WW). The subunits C1 and C3 have theoretical molecular masses of 45 and 52 kDa, respectively, and are present in nearly equal amounts during the circadian cycle. At the beginning of the subjective night, both can be found in protein complexes of 100 to 160 kDa. However, during subjective day when binding activity of CHLAMY 1 is low, the C1 subunit in addition is present in a high-molecular-mass protein complex of more than 680 kDa. These data indicate posttranslational control of the circadian binding activity of CHLAMY 1. Notably, the C3 subunit shows significant homology to the rat CUG-binding protein 2. Anti-C3 antibodies can recognize the rat homologue, which can also be found in a protein complex in this vertebrate., (Copyright 2004 American Society for Microbiology)

The synthesis of 1-alkyl and 1-aryl-1-azacyclotetradeca-3,5,10,12-tetraynes was achieved in a stepwise approach. The key intermediate was 1,13-dibromotrideca-2,4,9,11-tetrayne (18). Reaction with methyl- (19 a), ethyl- (19 b), isopropyl- (19 c), n-butyl- (19 d), and tert-butylamine (19 e) as well as aniline (19 f) and p-methoxyaniline (19 g) gave the corresponding 14-membered tetraynes 20 a-20 g. The ring inversion process of 20 b was studied by variable temperature (1)H NMR spectroscopy. From these measurements a value of 10.6 kcal mol(-1) was calculated for DeltaG(not equal). X-ray investigations on single crystals of 20 b, 20 c, and 20 f revealed the axial position for the substituent at each nitrogen atom. For 20 b we encountered the chair conformation, for 20 c both chair and boat conformations, and for 20 f the boat conformation in the solid state. The reaction of 20 c with concentrated HCl in ethanol yielded 2,10-dichloro-6-isopropyl-6-azatricyclo[9.3.0.0(4,8)]tetradeca-1(11),2,4(8),9-tetraene (25 c). Compound 25 c was oxidized by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) to 27 c. The structure of the latter was confirmed by X-ray investigations. The reaction of 20 c in aqueous HCl lead to the formation of 10-chloro-2-isopropyl-1,3,4,6,7,8-hexahydro-2H-benzo[g]isoquinolin-9-one (37 c). The structure of 37 c was verified by X-ray studies on single crystals.

The feasibility of producing a visual prosthesis for the blind using intracortical microstimulation (ICMS) of the visual cortex was studied in a 42-year-old woman who had been totally blind for 22 years secondary to glaucoma. Thirty-eight microelectrodes were implanted in the right visual cortex, near the occipital pole, for a period of 4 months. Percepts reported as small spots of light, called phosphenes, were produced with 34 of the 38 implanted microelectrodes. Threshold currents for phosphene generation with trains of biphasic pulses were as low as 1.9 microA, and most of the microelectrodes had thresholds below 25 microA. Phosphene brightness could be modified with stimulus amplitude, frequency and pulse duration. Repeated stimulation over a period of minutes produced a gradual decrease in phosphene brightness. Phosphenes did not flicker. The apparent size of phosphenes ranged from a "pin-point' to a "nickel' (20 mm diameter coin) held at arm's length. Phosphene size usually decreased as stimulation current was increased but increased slightly as the train length (TL) was increased. At levels of stimulation near threshold, the phosphenes were often reported to have colours. As the stimulation level was increased, the phosphenes generally became white, greyish or yellowish. Individual phosphenes appeared at different distances from the subject. When two phosphenes were simultaneously generated, the apparent distances of the individual phosphenes sometimes changed to make them appear to be at about the same distance. When three or more phosphenes were simultaneously generated, they became coplanar. Except for rare occasions, phosphenes extinguished rapidly at the termination of the stimulation train. When stimulation TLs were increased beyond 1 s, phosphenes usually disappeared before the end of the train. The duration of phosphene perception could be increased by interrupting a long stimulation train with brief pauses in stimulation. Intracortical microelectrodes spaced 500 microns apart generated separate phosphenes, but microelectrodes spaced 250 microns typically did not. This two-point resolution was about five times closer than has typically been achieved with surface stimulation. With some individual microelectrodes, a second closely spaced phosphene was sometimes produced by increasing the stimulation current. Phosphenes moved with eye movements. When up to six phosphenes were simultaneously elicited, they all moved with the same relative orientation during eye movements. All phosphenes were located in the left hemi-field with the majority above the horizontal meridian. There was a clustering of most of the phosphenes within a relatively small area of visual space. The potentially greater microelectrode density and lower power requirements of ICMS compared with surface stimulation appears encouraging for a visual prosthesis. However, further studies with blind subjects are required to optimize stimulation parameters and test complex image recognition before the feasibility of a visual prosthesis based on ICMS can be established.

The polymer, Parylene-C, has proven to be a biocompatible insulation for microelectrodes. However, due to its inert nature, the removal of the insulation from the tips of microelectrodes is difficult. This paper describes the use of an ultraviolet laser system to micromachine Parylene-C insulation with photoablation to precisely expose an arbitrary shape recording or stimulating surface.

It has been postulated that the supplementary motor area (SMA) is involved in the initiation of movement and in gating of afferent input to motor cortex (MI) from peripheral receptors. We studied the responses of 119 neurons in MI to imposed disturbances of wrist-movement performance generated by the introduction of torque pulses before, during and after localized cooling of the SMA in conscious monkeys. The cooling of SMA did not prevent monkeys from making these simple movements. Eighty-two neurons responded to the wrist perturbations. Only 7 of these neurons changed their responsiveness with unilateral or bilateral cooling of SMA. From the data we have obtained on MI neuronal firing patterns, the SMA does not appear to modulate the long-latency trans-cortical stretch reflex during the periods in a task that we have investigated. Nor does it prevent animals from performing these simple movements to a visual target.

1. The precentral cortex of three Macaca mulatta monkeys were mapped with intracortical microstimulation (ICMS) while the monkeys performed alternating wrist flexion and extension movements. A forearm cocontraction task was also employed with one monkey. Electromyogram (EMG) recordings from forearm muscles were used to evaluate the results of ICMS. 2. We have found that the results of ICMS can be misleading if EMG activity is not recorded from the responding muscle. Inhibition can be interpreted as excitation if muscle palpation or joint movement are the only response criteria. 3. Movement of the stimulating electrode by as little as 200 microns in a single radial column sometimes changed EMG responses from inhibition to excitation or vice versa, indicating that cortical inhibitory areas for a muscle can be located very close to excitatory zones. 4. Both excitation and inhibition of muscles could be produced with ICMS of precentral cortex when the animal was performing a task involving the muscles being mapped. EMG responses to ICMS were stable, provided that the stimulation was applied at the same time during a repetitive task such that the motoneurons were at a given level of excitability. 5. Zones where ICMS produced inhibition of a particular forearm muscle were interspersed among zones that produced excitation for that muscle. 6. Regions exist in precentral cortex where ICMS activates antagonistic wrist muscles producing cocontraction. 7. The extensive cortical region from which any individual muscle can be activated or suppressed with ICMS and the various combinations of muscles that are activated from within this region suggest that different types of movements involving a single muscle are represented at different locations within this region. 8. At a few locations in precentral cortex, the EMG responses to ICMS were not just a function of the level of excitation of the motoneuron pool at the time of stimulation but were also dependent on the specific task the monkey was performing at the time of stimulation.

Abstracted records of all patients discharged from New York State acute care hospitals from January 1, 1983, through October 1, 1987, containing a diagnosis of HIV infection (N = 36,664) were linked into a longitudinal file of 20,005 patient-specific case histories. A validation study utilizing Medicaid patient-specific discharge information for calendar year 1985 showed that, on the average, each case history of the longitudinal file contained 85% (+/- 5%) of the expected discharges. The number of patients present in the longitudinal file was 10% (+/- 4%)--too large a percentage, due to a failure to link all the discharges to the appropriate case histories. The number of patients with a diagnosis of Pneumocystis carinii pneumonia (PCP) found in the longitudinal file was found to be consistent with the number reported to the New York State AIDS Registry. The longitudinal file appears to be sufficiently accurate and complete to use in evaluating HIV-related acute care in hospitals.

Histochemical staining methods were applied to selected superficial forearm muscles of Macaca mulatta monkeys. The muscles were analyzed with regard to relative percentage distribution of different fiber types. In extensor carpi radialis brevis, extensor carpi radialis longus, and palmaris longus there was an even dispersion of each fiber type from the superficial to the deep part of the muscle. Extensor digiti communis showed a slightly higher percentage of type I fibers and correspondingly lower percentage of type II fibers in its central as compared to its superficial area. Three muscles, bracioradialis, extensor carpi ulnaris, and flexor carpi radialis, displayed marked differences between their superficial and deep areas. All of them contained a higher proportion of type I fibers (and correspondingly lower percentage of type II fibers) in their deep parts than in their superficial areas. Flexor carpi ulnaris (FCU) differed from the other muscles studied in that it showed distinctly different fiber proportions on either side of a central tendon. While the ulnar head of FCU was dominated by type II fibers (71% compared to 27% type I fibers), the humeral head contained a larger proportion of type I fibers (58% vs. 40% type II fibers). This difference in fiber type distribution suggests different functional demands for the two heads of FCU, with the possibility of more sustained activity in the humeral head.