Interferon-α antagonizes IL-3-mediated immunoregulatory functions of human basophils

Human basophils are major inflammatory cells in maintaining chronic allergic asthma. It has been published that interferon-α (IFN-α) improves clinical symptoms of asthma patients. In contrast, IL-3 exacerbates airway inflammation by inducing IL-4, IL-8 and IL-13 secretion from human basophils thus regulating their immunoregulatory functions. Furthermore, IL-3 exceptionally promotes survival of basophils. Here, we assessed cellular response of human basophils treated with IFN-α alone or in combination with IL-3. Our data show that IFN-α enhances apoptosis in purified human blood basophils compared to spontaneous apoptosis of controls or IFN-γ treated cells. Furthermore, we demonstrate that both IFN-α and FasL enhance apoptosis in human basophils with similar efficiency in a rather additive than synergistic way. IFN-α inhibits IL-3-induced survival to a minor degree. Particularly however, it suppresses IL-3-induced de-novo production of IL-8 and IL-13 up to 80%. In contrast, the production of IL-4 is not affected. Analyses of signaling pathways reveal that IFN-α promotes prolonged phosphorylation of STAT1/STAT2. By using a pan-JAK inhibitor the phosphorylation of STAT1/STAT2 is inhibited and most importantly the pro-apoptotic effect of IFN-α is abolished. Although the phosphorylation of p38-MAPK in IFN-α-treated cells is comparable to non-treated cells, inhibition of p-p38 activity abrogates IFN-α-enhanced apoptosis as well. We conclude that IFN-α-enhanced apoptosis is tightly regulated by the cooperation of JAK/STAT and p38-MAPK pathways. Our study identifies IFN-α as a novel inhibitor of IL-3-induced IL-8 and IL-13 production of human basophils. Taken together our study may explain the improved clinical symptoms of asthma patients treated with IFN-α.

Adenoviral expression of IKs contributes to wavebreak and fibrillatory conduction in neonatal rat ventricular cardiomyocyte monolayers

Previous studies have shown that the gating kinetics of the slow component of the delayed rectifier K(+) current (I(Ks)) contribute to postrepolarization refractoriness in isolated cardiomyocytes. However, the impact of such kinetics on arrhythmogenesis remains unknown. We surmised that expression of I(Ks) in rat cardiomyocyte monolayers contributes to wavebreak formation and facilitates fibrillatory conduction by promoting postrepolarization refractoriness. Optical mapping was performed in 44 rat ventricular myocyte monolayers infected with an adenovirus carrying the genomic sequences of KvLQT1 and minK (molecular correlates of I(Ks)) and 41 littermate controls infected with a GFP adenovirus. Repetitive bipolar stimulation was applied at increasing frequencies, starting at 1 Hz until loss of 1:1 capture or initiation of reentry. Action potential duration (APD) was significantly shorter in I(Ks)-infected monolayers than in controls at 1 to 3 Hz (P<0.05), whereas differences at higher pacing frequencies did not reach statistical significance. Stable rotors occurred in both groups, with significantly higher rotation frequencies, lower conduction velocities, and shorter action potentials in the I(Ks) group. Wavelengths in the latter were significantly shorter than in controls at all rotation frequencies. Wavebreaks leading to fibrillatory conduction occurred in 45% of the I(Ks) reentry episodes but in none of the controls. Moreover, the density of wavebreaks increased with time as long as a stable source sustained the fibrillatory activity. These results provide the first demonstration that I(Ks)-mediated postrepolarization refractoriness can promote wavebreak formation and fibrillatory conduction during pacing and sustained reentry and may have important implications in tachyarrhythmias.

Norepinephrine to increase blood pressure in endotoxaemic pigs is associated with improved hepatic mitochondrial respiration

ABSTRACT: INTRODUCTION: Low blood pressure, inadequate tissue oxygen delivery and mitochondrial dysfunction have all been implicated in the development of sepsis-induced organ failure. This study evaluated the effect on liver mitochondrial function of using norepinephrine to increase blood pressure in experimental sepsis. METHODS: Thirteen anaesthetized pigs received endotoxin (Escherichia coli lipopolysaccharide B0111:B4; 0.4 mug/kg per hour) and were subsequently randomly assigned to norepinephrine treatment or placebo for 10 hours. Norepinephrine dose was adjusted at 2-hour intervals to achieve 15 mmHg increases in mean arterial blood pressure up to 95 mmHg. Systemic (thermodilution) and hepatosplanchnic (ultrasound Doppler) blood flow were measured at each step. At the end of the experiment, hepatic mitochondrial oxygen consumption (high-resolution respirometry) and citrate synthase activity (spectrophotometry) were assessed. RESULTS: Mean arterial pressure (mmHg) increased only in norepinephrine-treated animals (from 73 [median; range 69 to 81] to 63 [60 to 68] in controls [P = 0.09] and from 83 [69 to 93] to 96 [86 to 108] in norepinephrine-treated animals [P = 0.019]). Cardiac index and systemic oxygen delivery (DO2) increased in both groups, but significantly more in the norepinephrine group (P < 0.03 for both). Cardiac index (ml/min per.kg) increased from 99 (range: 72 to 112) to 117 (110 to 232) in controls (P = 0.002), and from 107 (84 to 132) to 161 (147 to 340) in norepinephrine-treated animals (P = 0.001). DO2 (ml/min per.kg) increased from 13 (range: 11 to 15) to 16 (15 to 24) in controls (P = 0.028), and from 16 (12 to 19) to 29 (25 to 52) in norepinephrine-treated animals (P = 0.018). Systemic oxygen consumption (systemic VO2) increased in both groups (P < 0.05), whereas hepatosplanchnic flows, DO2 and VO2 remained stable. The hepatic lactate extraction ratio decreased in both groups (P = 0.05). Liver mitochondria complex I-dependent and II-dependent respiratory control ratios were increased in the norepinephrine group (complex I: 3.5 [range: 2.1 to 5.7] in controls versus 5.8 [4.8 to 6.4] in norepinephrine-treated animals [P = 0.015]; complex II: 3.1 [2.3 to 3.8] in controls versus 3.7 [3.3 to 4.6] in norepinephrine-treated animals [P = 0.09]). No differences were observed in citrate synthase activity. CONCLUSION: Norepinephrine treatment during endotoxaemia does not increase hepatosplanchnic flow, oxygen delivery or consumption, and does not improve the hepatic lactate extraction ratio. However, norepinephrine increases the liver mitochondria complex I-dependent and II-dependent respiratory control ratios. This effect was probably mediated by a direct effect of norepinephrine on liver cells.

Intrarenal resistance index for the assessment of early renal function impairment in patients with liver cirrhosis

BACKGROUND: Renovascular vasoconstriction in patients with hepatorenal syndrome can be quantified by the renal arterial resistance index (RI). We investigated the value of RI measurement in detection of renal function impairment in patients with different stages of chronic liver disease. METHODS: Subjects were divided into 4 groups containing 21 patients with liver cirrhosis and ascites, 25 patients with liver cirrhosis without ascites, 35 patients with fatty liver disease and 78 control subjects. All patients underwent abdominal ultrasound examination with renal RI measurement and correlation with laboratory results for renal function. RESULTS: RI was significantly higher in ascitic patients compared to non-ascitic patients (0.74 vs. 0.67, p<0.01) and in non-ascitic patients with liver cirrhosis than in control subjects (0.67 vs. 0.62, p<0.01). 48% (19/40) of patients with liver cirrhosis and normal serum creatinine concentration showed elevated RI levels. There were no significant differences in RI levels between patients with fatty liver disease and controls (0.63 vs. 0.62). CONCLUSIONS: Intrarenal RI measurement is a predictor of renal vasoconstriction and serves to detect early renal function impairment in cirrhotic patients. The diagnosis of elevated RI may be taken into account in the clinical management of these patients.

In human basophils, IL-3 selectively induces RANKL expression that is modulated by IgER-dependent and IgER-independent stimuli

BACKGROUND Receptor activator of NF-κB ligand (RANKL) is expressed as either surface (hRANKL1, hRANKL2) or soluble (hRANKL3) form. RANKL is involved in multifaceted processes of immunoregulation and bone resorption such as they occur in rheumatoid arthritis (RA). Interestingly, activated basophils, which are effector cells in allergic inflammation, contribute to the progress of collagen-induced arthritis (CIA), a mouse model for RA. Here, we investigate under which conditions human basophils express RANKL. METHODS Among other stimuli, basophils were cultured with IL-3 alone. Alternatively, as a secondary stimulus, IgER-dependent or IgER-independent agents were added simultaneously either with IL-3 or after prolonged IL-3 culturing. Expression of RANKL protein and mRNA was analyzed by flow cytometry, ELISA, and real-time PCR. A coculture system was applied to investigate biological activity of basophil-derived RANKL. RESULTS We show that in human basophils, IL-3 but no other stimulus induces de novo expression of soluble and surface RANKL, of which the latter enhances survival of MoDC. Upon simultaneous stimulation, IgER cross-linking reduces surface RANKL expression, while IgER-independent stimuli have no effect. This is in contrast to consecutive stimulation, as triggering with both IgER-dependent and IgER-independent stimuli enhances RANKL expression, particularly in its soluble form. Real-time PCR analysis shows that RANKL expression is mainly regulated at the mRNA level. CONCLUSION This study identifies IL-3 as a potent inducer of RANKL expression in human basophils, suggesting them to interact with bone physiology and activation of immune cells. IgER-dependent and IgER-independent stimuli modulate the IL-3-mediated RANKL expression in a time- and stimulus-dependent fashion.

Identification of serotonin 5-HT1A receptor partial agonists in ginger

Animal studies suggest that ginger (Zingiber officinale Roscoe) reduces anxiety. In this study, bioactivity-guided fractionation of a ginger extract identified nine compounds that interact with the human serotonin 5-HT(1A) receptor with significant to moderate binding affinities (K(i)=3-20 microM). [(35)S]-GTP gamma S assays indicated that 10-shogaol, 1-dehydro-6-gingerdione, and particularly the whole lipophilic ginger extract (K(i)=11.6 microg/ml) partially activate the 5-HT(1A) receptor (20-60% of maximal activation). In addition, the intestinal absorption of gingerols and shogaols was simulated and their interactions with P-glycoprotein were measured, suggesting a favourable pharmacokinetic profile for the 5-HT(1A) active compounds.

Five-year results of a randomized controlled clinical trial comparing zirconia and titanium abutments supporting single-implant crowns in canine and posterior regions

OBJECTIVES: To test the survival rates, and the technical and biological complication rates of customized zirconia and titanium abutments 5 years after crown insertion. MATERIAL AND METHODS: Twenty-two patients with 40 single implants in maxillary and mandibular canine and posterior regions were included. The implant sites were randomly assigned to zirconia abutments supporting all-ceramic crowns or titanium abutments supporting metal-ceramic crowns. Clinical examinations were performed at baseline, and at 6, 12, 36 and 60 months of follow-up. The abutments and reconstructions were examined for technical and/or biological complications. Probing pocket depth (PPD), plaque control record (PCR) and Bleeding on Probing (BOP) were assessed at abutments (test) and analogous contralateral teeth (control). Radiographs of the implants revealed the bone level (BL) on mesial (mBL) and distal sides (dBL). Data were statistically analyzed with nonparametric mixed models provided by Brunner and Langer and STATA (P < 0.05). RESULTS: Eighteen patients with 18 zirconia and 10 titanium abutments were available at a mean follow-up of 5.6 years (range 4.5-6.3 years). No abutment fracture or loss of a reconstruction occurred. Hence, the survival rate was 100% for both. Survival of implants supporting zirconia abutments was 88.9% and 90% for implants supporting titanium abutments. Chipping of the veneering ceramic occurred at three metal-ceramic crowns supported by titanium abutments. No significant differences were found at the zirconia and titanium abutments for PPD (meanPPD(ZrO2) 3.3 ± 0.6 mm, mPPD(T) (i) 3.6 ± 1.1 mm), PCR (mPCR(Z) (rO) (2) 0.1 ± 0.3, mPCR(T) (i) 0.3 ± 0.2) and BOP (mBOP(Z) (rO) (2) 0.5 ± 0.3, mBOP(T) (i) 0.6 ± 0.3). Moreover, the BL was similar at implants supporting zirconia and titanium abutments (mBL(Z) (rO) (2) 1.8 ± 0.5, dBL(Z) (rO) (2) 2.0 ± 0.8; mBL(T) (i) 2.0 ± 0.8, dBL(T) (i) 1.9 ± 0.8). CONCLUSIONS: There were no statistically or clinically relevant differences between the 5-year survival rates, and the technical and biological complication rates of zirconia and titanium abutments in posterior regions.

Missing C-terminal filaggrin expression, NFkappaB activation and hyperproliferation identify the dog as a putative model to study epidermal dysfunction in atopic dermatitis

Filaggrin loss-of-function mutations resulting in C-terminal protein truncations are strong predisposing factors in human atopic dermatitis (AD). To assess the possibility of similar truncations in canine AD, an exclusion strategy was designed on 16 control and 18 AD dogs of various breeds. Comparative immunofluorescence microscopy was performed with an antibody raised against the canine filaggrin C-terminus and a commercial N-terminal antibody. Concurrent with human AD-like features such as generalized NFKB activation and hyperproliferation, four distinctive filaggrin expression patterns were identified in non-lesional skin. It was found that 10/18 AD dogs exhibited an identical pattern for both antibodies with comparable (category I, 3/18) or reduced (category II, 7/18) expression to that of controls. In contrast, 4/18 dogs displayed aberrant large vesicles revealed by the C-terminal but not the N-terminal antibody (category III), while 4/18 showed a control-like N-terminal expression but lacked the C-terminal protein (category IV). The missing C-terminal filaggrin in category IV strongly points towards loss-of function mutations in 4/18 (22%) of all AD dogs analysed.

CD4-guided scheduled treatment interruptions compared with continuous therapy for patients infected with HIV-1: results of the Staccato randomised trial

BACKGROUND: Stopping antiretroviral therapy in patients with HIV-1 infection can reduce costs and side-effects, but carries the risk of increased immune suppression and emergence of resistance. METHODS: 430 patients with CD4-positive T-lymphocyte (CD4) counts greater than 350 cells per muL, and viral load less than 50 copies per mL were randomised to continued therapy (n=146) or scheduled treatment interruptions (n=284). Median time on randomised treatment was 21.9 months (range 16.4-25.3). Primary endpoints were proportion of patients with viral load less than 50 copies per mL at the end of the trial, and amount of drugs used. Analysis was intention-to-treat. This study is registered at ClinicalTrials.gov with the identifier NCT00113126. FINDINGS: Drug savings in the scheduled treatment interruption group, compared with continuous treatment, amounted to 61.5%. 257 of 284 (90.5%) patients in the scheduled treatment interruption group reached a viral load less than 50 copies per mL, compared with 134 of 146 (91.8%) in the continued treatment group (difference 1.3%, 95% CI-4.3 to 6.9, p=0.90). No AIDS-defining events occurred. Diarrhoea and neuropathy were more frequent with continuous treatment; candidiasis was more frequent with scheduled treatment interruption. Ten patients (2.3%) had resistance mutations, with no significant differences between groups. INTERPRETATION: Drug savings with scheduled treatment interruption were substantial, and no evidence of increased treatment resistance emerged. Treatment-related adverse events were more frequent with continuous treatment, but low CD4 counts and minor manifestations of HIV infection were more frequent with scheduled treatment interruption.

Low-dose cyclosporine therapy in triple-drug immunosuppression for heart transplant recipients

The toxicity of long-term immunosuppressive therapy has become a major concern in long-term follow-up of heart transplant recipients. In this respect the quality of renal function is undoubtedly linked to cyclosporin A (CsA) drug levels. In cardiac transplantation, specific CsA trough levels have historically been maintained between 250 and 350 micrograms/L in many centers without direct evidence for the necessity of such high levels while using triple-drug immunosuppression. This retrospective analysis compares the incidence of acute and chronic graft rejection as well as overall mortality between groups of patients with high (250 to 350 micrograms/L) and low (150 to 250 micrograms/L) specific CsA trough levels. A total of 332 patients who underwent heart transplantation between October 1985 and October 1992 with a minimum follow-up of 30 days were included in this study (46 women and 276 men; aged, 44 +/- 12 years; mean follow-up, 1,122 +/- 777 days). Standard triple-drug immunosuppression included first-year specific CsA target trough levels of 250 to 300 micrograms/L. Patients were grouped according to their average creatinine level in the first postoperative year (group I, < 130 mumol/L, n = 234; group II, > or = 130 mumol/L, n = 98). The overall 5-year survival excluding the early 30-day mortality was 92% (group I, 216/232) and 91% (group II, 89/98) with 75% of the mortality due to chronic rejection. The rate of rejection for the entire follow-up period was similar in both groups (first year: group I, 3.2 +/- 2.6 rejection/patient/year; group II, 3.6 +/- 2.7 rejection/patient/year; p = not significant).(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of the bicyclo-[4.3.0]thymidyl nucleoside via Pd(II)-mediated ring expansion chemistry

A variety of modified nucleosides to improve antisense oligodeoxynucleotide properties such as target affinity, nuclease resistance, and pharmacokinetics were developed in the last two decades. In the context of conformational restriction we present here the synthesis of the [4.3.0]-bicyclo-DNA thymine monomer via Pd(II)-mediated ring expansion of an intermediate of the tricyclo-DNA synthesis.

Conversion of CD95 (Fas) Type II into Type I signaling by sub-lethal doses of cycloheximide

CD95 (Fas/Apo-1)-mediated apoptosis was shown to occur through two distinct pathways. One involves a direct activation of caspase-3 by large amounts of caspase-8 generated at the DISC (Type I cells). The other is related to the cleavage of Bid by low concentration of caspase-8, leading to the release of cytochrome c from mitochondria and the activation of caspase-3 by the cytochrome c/APAF-1/caspase-9 apoptosome (Type II cells). It is also known that the protein synthesis inhibitor cycloheximide (CHX) sensitizes Type I cells to CD95-mediated apoptosis, but it remains contradictory whether this effect also occurs in Type II cells. Here, we show that sub-lethal doses of CHX render both Type I and Type II cells sensitive to the apoptogenic effect of anti-CD95 antibodies but not to chemotherapeutic drugs. Moreover, Bcl-2-positive Type II cells become strongly sensitive to CD95-mediated apoptosis by the addition of CHX to the cell culture. This is not the result of a restraint of the anti-apoptotic effect of Bcl-2 at the mitochondrial level since CHX-treated Type II cells still retain their resistance to chemotherapeutic drugs. Therefore, CHX treatment is granting the CD95-mediated pathway the ability to bypass the mitochondria requirement to apoptosis, much alike to what is observed in Type I cells.