1型糖尿病大鼠左心室HCN2和HCN4重构

目的　探讨1型糖尿病大鼠左心室肌HCN2和HCN4的表达时空变化。 方法　60只大鼠随机分为正常对照组(N组, n=20)和1型糖尿病组(T1DM组, n=40)：腹腔注射链脲佐菌素制作大鼠1型糖尿病模型。90 d后,取左心室肌通过免疫荧光法检测HCN2、HCN4的定位;Western-blot、RT-PCR技术检测HCN2、HCN4在蛋白质及mRNA水平的变化。 结果　免疫荧光结果显示HCN2在心室肌细胞膜上呈点状或短线状不连续表达;HCN4未见表达。Western-blot、RT-PCR结果显示T1DM组大鼠　HCN2、HCN4在蛋白及mRNA水平表达升高。 结论　1型糖尿病大鼠左心室出现离子通道HCN2、HCN4表达升高,导致糖尿病心脏电重构,可能与糖尿病引起的室性心律失常有关。     

Probing S4 and S5 segment proximity in mammalian hyperpolarization-activated HCN channels by disulfide bridging and Cd2+ coordination

We explored the structural basis of voltage sensing in the HCN1 hyperpolarization-activated cyclic nucleotide-gated cation channel by examining the relative orientation of the voltage sensor and pore domains. The opening of channels engineered to contain single cysteine residues at the extracellular ends of the voltage-sensing S4 (V246C) and pore-forming S5 (C303) domains is inhibited by formation of disulfide or cysteine:Cd²⁺ bonds. As Cd²⁺ coordination is promoted by depolarization, the S4–S5 interaction occurs preferentially in the closed state. The failure of oxidation to catalyze dimer formation, as assayed by Western blotting, indicates the V246C:C303 interaction occurs within a subunit. Intriguingly, a similar interaction has been observed in depolarization-activated Shaker voltage-dependent potassium (Kv) channels at depolarized potentials but such an intrasubunit interaction is inconsistent with the X-ray crystal structure of Kv1.2, wherein S4 approaches S5 of an adjacent subunit. These findings suggest channels of opposite voltage-sensing polarity adopt a conserved S4–S5 orientation in the depolarized state that is distinct from that trapped upon crystallization. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Association of polymorphisms in HCN4 with mood disorders and obsessive compulsive disorder

Hyperpolarization activated cyclic nucleotide-gated (HCN) potassium channels are implicated in the control of neuronal excitability and are expressed widely in the brain. HCN4 is expressed in brain regions relevant to mood and anxiety disorders including specific thalamic nuclei, the basolateral amygdala, and the midbrain dopamine system. We therefore examined the association of HCN4 with a group of mood and anxiety disorders. We genotyped nine tag SNPs in the HCN4 gene using Sequenom iPLEX Gold technology in 285 Caucasian patients with DSM-IV mood disorders and/or obsessive compulsive disorder and 384 Caucasian controls. HCN4 polymorphisms were analyzed using single marker and haplotype-based association methods. Three SNPs showed nominal association in our population (rs12905211, rs3859014, rs498005). SNP rs12905211 maintained significance after Bonferroni correction, with allele T and haplotype CTC overrepresented in cases. These findings suggest HCN4 as a genetic susceptibility factor for mood and anxiety disorders; however, these results will require replication using a larger sample.     

Altered expression of signalling lymphocyte activation molecule (SLAM) family receptors CS1 (CD319) and 2B4 (CD244) in patients with systemic lupus erythematosus

CS1 (CRACC, CD319) and 2B4 (CD244), members of the signalling lymphocyte activation molecule (SLAM) family receptors, regulate various immune functions. Genes encoding SLAM family receptors are located at 1q23, implicated in systemic lupus erythematosus (SLE). In this study, we have investigated the expression and alternative splicing of CS1 and 2B4 in immune cells from SLE patients. The surface expression of CS1 and 2B4 on total peripheral blood mononuclear cells (PBMCs), T, B, natural killer (NK) cells and monocytes in 45 patients with SLE and 30 healthy individuals was analysed by flow cytometry. CS1‐positive B cell population was increased significantly in SLE patients. Because CS1 is a self‐ligand and homophilic interaction of CS1 induces B cell proliferation and autocrine cytokine secretion, this could account for autoreactive B cell proliferation in SLE. The proportion of NK cells and monocytes expressing 2B4 on their surface was significantly lower in patients with SLE compared to healthy controls. Our study demonstrated altered expression of splice variants of CS1 and 2B4 that mediate differential signalling in PBMC from patients with SLE.     

De novo expression of EphA2 in osteosarcoma modulates activation of the mitogenic signalling pathway

Fritsche‐Guenther R, Noske A, Ungethüm U, Kuban R‐J, Schlag PM, Tunn P‐U, Karle J, Krenn V, Dietel M & Sers C (2010) Histopathology 57, 836–850 De novo expression of EphA2 in osteosarcoma modulates activation of the mitogenic signalling pathway Aims: In osteosarcoma patients the development of metastases, often to the lungs, is the most frequent cause of death. The aim of this study was to elucidate the molecular mechanisms governing osteosarcoma development and dissemination and, thereby, to identify possible novel drug targets for improved treatment. Methods and results: Osteosarcoma samples were characterized using genome‐wide microarrays: increased expression of the EphA2 receptor and its ligand EFNA1 was detected. In addition, increased expression of EFNB1, EFNB3 and EphA3 was suggested. Immunohistochemistry revealed an absence of EphA2 in normal bone, and de novo expression in osteosarcomas. EFNA1 was expressed in normal bone, but was significantly elevated in tumours. Further in vitro investigations on the functional role of EphA2 and EFNA1 showed that EFNA1 ligand binding induced increased tyrosine phoshorylation, receptor degradation and downstream mitogen‐activated protein kinase (MAPK) activation. Interference with the MAPK pathway unravelled a potential autoregulatory loop governing mainly EFNA1 expression via the same pathway. Conclusion: Upregulation and de novo expression of ephrins in osteosarcomas are involved in oncogenic signalling and thus might stimulate osteosarcoma metastasis.     

Interleukin-8 differentially modulates interleukin-4- and interleukin-2-induced human B cell growth

The effect of interleukin-8 (IL)-8 on human B cell growth, as determined by thymidine uptake and viable cell numbers was studied. IL-8 inhibited IL-4-induced growth of B cells costimulated with anti-μ antibodies (Ab) or Staphylococcus aureus Cowan strain I (SAC) in a dose-dependent fashion. In contrast, IL-8 did not inhibit IL-2-induced growth of B cells. The IL-8-mediated inhibition was specific, since it was blocked by anti-IL-8 mAb but not by control IgG1. Moreover, anti-tumor necrosis factor-α (anti-TNF-α) Ab blocked IL-8-mediated inhibition. On the other hand, TNF-α, but not other cytokines including IL-1β, IL-3, IL-5, IL-6, interferon-α (IFN-α) or IFN-γ, inhibited IL-4-mediated growth, and inhibition by TNF-α was blocked by anti-TNF-α Ab but not by control IgG. IL-4 had no effect on TNF-α binding by B cells while it decreased TNF-α production by B cells. IL-8 had no effect in binding of IL-4, IL-2 or TNF-α by B cells, however, it enhanced TNF-α production by B cells. These results indicate that IL-8 inhibited IL-4-induced human B cell growth by enhancement of endogenous TNF-α production.     

FGF2 modulates the voltage-dependent K current and changes excitability of rat dentate gyrus granule cells

Fibroblast growth factor 2 (FGF2) is involved in hippocampus-dependent learning. In this study, the effects of FGF2 on the excitability were investigated in granule cells of rat dentate gyrus. Hippocampal slices were used to perform patch clamp recordings in granule cells. Extracellularly applied FGF2 early quenched the depolarization-induced repetitive firing, suggesting a decreased excitability under these conditions. Consistently, transient and sustained voltage-gated K⁺ currents decreased in a dose-dependent manner, repolarization phase of action potential was slowed down, afterhyperpolarization was reduced, and membrane resistance was decreased. These effects were not mediated by tyrosine kinase FGF2 receptors. Moreover, an involvement of G protein signaling was ruled out, as well as an intracellular action of FGF2. Considering the relationship between FGF2 and hippocampal functions, the modulation of neuron excitability by activity-driven FGF2 release may be regarded as a part of a homeostatic mechanism of self-regulation of hippocampal activity.     

Crosstalk between glucocorticoids and IL-4 modulates Ym1 expression in alternatively activated myeloid cells

Airway epithelial cells induce a tolerogenic microenvironment by modulating immune cells in the lung. We recently showed that the supernatant of airway epithelial cells induces two marker genes of alternative activation, Ym1 and Ms4a8a, in respiratory myeloid cells. This induction was partially mediated by glucocorticoids, secreted by airway epithelial cells. In this study, we further investigated Ym1 and Ms4a8a regulation in alternatively activated myeloid cells in the presence of the T_H2 cytokines IL-4 and IL-13. We show that Ym1 expression is boosted upon co-stimulation with airway epithelial cell supernatant and IL-4/IL-13, whereas Ms4a8a expression is down-regulated. This suggests that a crosstalk between IL-4/IL-13 and glucocorticoid signaling exists. Blocking protein synthesis indicated that dexamethasone-induced de novo protein synthesis is required for the interaction between glucocorticoid and IL-4 signaling regarding Ym1 regulation. Using reporter gene constructs, we demonstrate that the important regulatory region within the Ym1 promoter is found between −602 bp and −969 bp upstream of the start of translation. Bioinformatic analysis identified several glucocorticoid response elements (GREs) in this region. Further analysis identified overlapping but functionally active glucocorticoid receptor and STAT-6 binding sites, supporting the cooperative effect of glucocorticoids and IL-4 in the regulation of Ym1. These findings further prove the plasticity and complexity of alternatively activated myeloid cells and the importance of the local microenvironment. We believe that this regulation is of special importance in the pulmonary system, since both factors, glucocorticoids and IL-4/13, play a role in airway diseases such as asthma.     

T-cell costimulation blockade in immunologic diseases: role of CD28 family members

The destruction of many immune-mediated diseases is a result of T-cell responses against usually harmless antigens. Extensive research has been conducted to discover new mechanisms to specifically modulate harmful effector T cells while leaving normal immune responses intact. Since proteins of the CD28 family members are expressed on T cells, blockade of these proteins has become a possible target for potential therapies. The CD28 family contains proteins that have the ability to both enhance and diminish T-cell responses. Therefore, blockade of targets that enhance T-cell signaling may reduce destructive autoimmune responses, while blockade of targets that diminish T-cell signaling may enhance antitumor responses. In this article, the function of these proteins will be reviewed and a sample of clinical trials highlighting the potential efficacy and drawbacks of their use in humans will be described briefly. Finally, inducible costimulator and programmed death-1, two future targets of T-cell therapies, will be highlighted.     

4-Hydroxynonenal modulates the long-term potentiation induced by L-type Ca2+ channel activation in the rat dentate gyrus in vitro

Increased oxyradical production and membrane lipid peroxidation (MLP) occur under physiological and degenerative conditions in neurons. We investigated whether 4-hydroxynonenal (4HN), one of the membrane lipid peroxidation products, affects long-term potentiation (LTP) in the rat dentate gyrus in vitro. Treatment of hippocampal slices with 4HN (10 μM) enhanced LTP without affecting basal evoked potentials. The enhancement was completely inhibited by 2 μM nifedipine, a blocker of L-type Ca²⁺ channels. In cultured dentate gyrus neurons, treatment of the cells with 4HN for 24 h resulted in a significant amount of cell death that was detoxified by glutathione, whereas short-term treatment with 4HN (6 h) had no effect. Nifedipine partially but significantly suppressed the 4HN-induced cell death. These results suggest that 4HN modulates LTP and induces delayed cell death through L-type Ca²⁺ channel activation in the dentate gyrus. 4HN thereby plays an important role in both physiological and pathophysiological events in the hippocampus.     

Costimulatory TNFR family members in control of viral infection: Outstanding questions

Members of the TNFR family can play prominent roles in controlling the magnitude, duration and phenotype of the immune response to viruses. The importance of particular TNFRs in different viral infections and whether they contribute to viral control or pathology is dependent on the virus and the severity of the infection. TNFRs and their ligands are widely and differentially expressed on both adaptive and innate immune cell types. The cell types through which TNFRs exert their effects, the unique signals provided by each member of the family, and how these signals are ultimately integrated during an anti-viral immune response remain to be fully elucidated. Here we discuss the role of 4-1BB, OX40, CD27 and GITR and their ligands during viral infection and highlight some of the outstanding questions in the field.     

Regulation of 4-1BB expression by cell-cell interactions and the cytokines,interleukin-2 and interleukin-4

4-1BB expression increased gradually following T cell activation, and by day 3 post-stimulation with immobilized anti-CD3 (anti-CD3i) or concanavalin A (Con A), splenic T cells were routinely 35–45% 4-1BB⁺ by flow cytometric analysis. 4-1BB was expressed on activated CD8⁺, CD4⁺, CD28⁺ and CD45RB⁺ T cells. Optimal 4-1BB expression was seen by day 6 post-stimulation and was cell density dependent. When T cells were cultured for 6 days at 1 × 10⁶/well in a 24-well plate with anti-CD3i, 82% of the cells were 4-1BB⁺. In contrast, at lower cell densities (4 × 10⁵, 2 × 10⁵ and 1 × 10⁵), optimal 4-1BB expression was observed only if the cultures were supplemented with recombinant interleukin-2 (IL-2) or recombinant IL-4 (IL-4). In agreement, with these results, modes of inducing endogenous IL-2 production such as cross-linking the costimulatory molecule, CD28, or the addition of syngeneic accessory cells to T cells activated with anti-CD3i, resulted in high levels of 4-1BB expression. The addition of interleukin-1α(IL-1α) or interferon-γ (IFN-γ) did not increase 4-1BB expression on anti-CD3i-activated T cells. In addition, if T cells were incubated with IL-2, IL-4, IL-1α, IFN-γ or anti-CD28 alone, no 4-1BB expression was induced. T cells activated with soluble anti-CD3 (anti-CD3s) in the presence of IL-2, IL-4, or accessory cells, did not express higher levels of 4-1BB on their cell surface. These data suggest that initial events crucial for efficient T cell activation, such as signals delivered through the T cell receptor/CD3 complex and the CD28 molecule, are instrumental in regulating subsequent 4-1BB expression.     

Agaphelin modulates the activation of human bronchial epithelial cells induced by lipopolysaccharide and IL-4

Sand fly saliva presents molecules with potential to development of compounds for treatment of inflammatory diseases. Agaphelin, isolated from the saliva of the mosquito Anopheles gambiae, demonstrates anti-inflammatory properties such as neutrophils chemotaxis inhibition. Here, we extend these results and evaluated the role of agaphelin (0.1−100 nM) in an in vitro model consisting in the activation of human bronchial epithelial cells (BEAS-2B) by IL-4 (50 ng/mL) or lipopolysaccharide (LPS; 10 ng/mL). Agaphelin is non-cytotoxic for BEAS-2B cells. Notably, agaphelin markedly reduces CCL2 and IL-8 production induced by IL-4 or LPS, without altering the IL-10 production. The TLR4 expression and STAT1 phosphorylation induced by LPS were inhibited by agaphlin. In addition, agaphelin decreased the phosphorylation of STAT6 induce by IL-4, whose effect was independent of IL-4-binding activity. Taken together, these findings identify agaphelin as a potential anti-inflammatory therapeutic agent for airway inflammations.     

B7-H4 and HHLA2, members of B7 family,are aberrantly expressed in EGFR mutated lung adenocarcinoma

BackgroundIn order to find new immune targets for lung cancer with different EGFR mutant status, we describe differential expression profiles of checkpoint molecules of the new discovery B7 family member to find new immune targets for lung cancer with different EGFR statuses.MethodsWe performed immunohistochemistry with antibodies of B7-H3, B7-H4, VISTA, B7-H6, HHLA2, IDO-1, PD-L1 and CD8 in lung adenocarcinoma tissues constructed from 372 cases in the discovery cohort and 231 cases in the validation set. The differential expression profiles of these indices in EGFR mutant and wild-type lung adenocarcinoma was described and compared.ResultsIn the discovery cohort, the median IHC scores of B7-H4 and HHLA2 for the EGFR mutant group were significantly higher than those in the wild-type group (median score [interquartile range], mutant vs. wild type: 3.250 [0−7.000] vs. 5.000 [1.000−7.000], P = 0.045 for B7-H4; 8.000 [6.000−10.500] vs. 7.000 [5.000−8.630] P = 0.003 for HHLA2). Meanwhile, the median IHC scores of IDO-1 and PD-L1 in the wild-type group were significantly higher than those in the mutant group (median score [interquartile range], mutant vs. wild type: 1.000 [0−5.000] vs. 3.000 [0−8.500], P = 0.000 for IDO-1; 0 [0−3.500] vs. 3.000 [0−6.000], P = 0.000 for PD-L1). Results above was confirmed in the discovery cohort. The increased CD8 and decreased HHLA2 expression levels were associated with long disease-free survival in lung adenocarcinoma (P = 0.000 for CD8 expression and P = 0.004 for HHLA2 expression).ConclusionsB7-H4 and HHLA2 are promising immune targets for lung adenocarcinoma, especially for patients with EGFR mutation.     

Renoprotection: focus on TRPV1, TRPV4, TRPC6 and TRPM2

Members of the transient receptor potential (TRP) cation channel receptor family have unique sites of regulatory function in the kidney which enables them to promote regional vasodilatation and controlled Ca²⁺ influx into podocytes and tubular cells. Activated TRP vanilloid 1 receptor channels (TRPV1) have been found to elicit renoprotection in rodent models of acute kidney injury following ischaemia/reperfusion. Transient receptor potential cation channel, subfamily C, member 6 (TRPC6) in podocytes is involved in chronic proteinuric kidney disease, particularly in focal segmental glomerulosclerosis (FSGS). TRP vanilloid 4 receptor channels (TRPV4) are highly expressed in the kidney, where they induce Ca²⁺ influx into endothelial and tubular cells. TRP melastatin (TRPM2) non‐selective cation channels are expressed in the cytoplasm and intracellular organelles, where their inhibition ameliorates ischaemic renal pathology. Although some of their basic properties have been recently identified, the renovascular role of TRPV1, TRPV4, TRPC6 and TRPM2 channels in disease states such as obesity, hypertension and diabetes is largely unknown. In this review, we discuss recent evidence for TRPV1, TRPV4, TRPC6 and TRPM2 serving as potential targets for acute and chronic renoprotection in chronic vascular and metabolic disease.     

The influence of capsaicin on membrane currents in dorsal root ganglion neurones of guinea-pig and chicken

The effect of capsaicin on voltage-dependent membrane currents of isolated dorsal root ganglia (DRG) neurones of guinea-pig and chicken were investigated by the voltage-clamp technique and intracellular perfusion. In both species, administration of capsaicin (3·10^–5 M) to the outer surface of the cell membrane reduced the amplitude and accelerated the inactivation of the fast inactivating potassium current. In contrast, 3,4-diaminopyridine (3,4-DAP) reduced the fast potassium current without affecting the inactivation. Combined application of capsaicin and 3,4-DAP was more effective than either drug alone. The slow potassium current was diminished by capsaicin but not affected by 3,4-DAP. Capsaicin (3·10^–5 M) applied to the internal surface of the membrane had little effect on the fast outward current but primarily decreased the amplitude of the slow potassium current. Two subpopulations of sodium currents could be demonstrated in guinea-pig neurones according to their tetrodotoxin (TTX) sensitivity. In type I neurones the sodium current was completely blocked by TTX; type II neurones exhibited a TTX-sensitive as well as a TTX-resistant inward current. Capsaicin (3·10^–5 M) applied externally reduced the maximal amplitude of both current components. The time course of inactivation was delayed only in the TTX-resistant sodium current. The effect of capsaicin on Na-currents of DRG neurones was similar in guinea-pigs and chicken. In DRG neurones of chicken, only TTX-sensitive currents were observed. In both species the steady-state inactivation of the sodium currents was shifted by capsaicin to more negative potentials.     

Use-dependent effects of 4-aminopyridine on transient outward current in dog ventricular muscle

The use-dependent features of 4-aminopyridine-induced block of the transient outward current were investigated under voltage clamp conditions in dog ventricular myocardial bundles. The block depended strongly on the pattern of voltage clamp pulses. Its level (settled in rested membranes) became deeper at early time during depolarization. In contrast, prolonged depolarization (above 50 to 100 ms) produced relief of block. Our results suggest that the block strongly depends on the state of the channel gating system.This work was supported by the France-Czechoslovakia Scientific and Technical Cooperation Program (Annexe II, project B24).     

The effect of ethanol-induced CYP2E1 on proteasome activity: the role of 4-hydroxynonenal

Previous studies have shown that the induction of P450 cytochrome 2E1 (CYP2E1) is associated with the loss of proteasomal activities. To correlate the loss of proteasomal activity with CYP2E1 induction, ethanol was fed intragastrically for 1, 3, 7, and 15 days. The maximum induction of CYP2E1 (3.5-fold) occurred after 15 days of ethanol feeding. However, there was no significant decrease in the 26 S chymotrypsin-like and trypsin-like activity over this period of time. When ethanol was given to rats for 1 month, CYP2E1 was significantly induced, and the proteasomal activity was significantly decreased. These results indicate that proteasomal activity was not directly affected by ethanol or CYP2E1 induction. Since 4-hydroxynonenal (4-HNE) concentration was significantly increased at 1 month of ethanol feeding, it was suspected that 4-HNE adduct formation with proteasome subunits could be the mechanism of proteasome inhibition. Using an antibody to 4-HNE adducted proteins in Western blot analysis of the 26 S proteasome fraction isolated from the liver of alcohol fed rats, one extra band appeared around 44 kDa. When the antibody to an ATPase Rpt4 was used to stain the stripped membrane, the same band that was detected with the 4-HNE antibody was detected with the Rpt4 antibody. An adduct of 4-HNE formed with the Rpt4 subunit of 26 S could impede the association of 19 S and 20 S and thus account for the observed decrease of proteasomal activity.