The physiologic increase in expression of some type I IFN-inducible genes during pregnancy is not associated with improved disease activity in pregnant patients with rheumatoid arthritis

During pregnancy, most patients with rheumatoid arthritis (RA) experience a spontaneous improvement in their condition. Since type I interferons (IFN) have immunomodulatory properties, we investigated whether type I IFN-inducible genes are upregulated in pregnant patients with RA. Peripheral blood mononuclear cells were evaluated using quantitative real-time polymerase chain reaction for type I IFN-inducible genes (IFI 35, IFI44, IFI44L, IFIT3, OAS1, and Siglec1) in patients with RA and healthy women during and after pregnancy as well as in nonpregnant controls. IFN-alpha and IFN-beta levels in sera of patients and healthy donors were analyzed by enzyme linked immunosorbent assay. It was found that healthy women did not show a change of gene expression levels from the second trimester until postpartum, yet some type I IFN-inducible genes were significantly upregulated in pregnant and postpartum women compared with nonpregnant individuals. In patients with RA, a pronounced upregulation of IFI35 and IFI44 at the second trimester and a peak expression of Siglec1 at the third trimester were observed. Pregnancy levels of IFI35 and IFI44 in patients with RA were higher than those of nonpregnant patients with RA. No significant association of gene expression levels with disease activity was found. In the sera of patients and healthy women, IFN-beta was undetectable and IFN-alpha levels remained stable throughout pregnancy and postpartum. Thus, pregnancy can give rise to an increased expression of type I IFN-inducible genes, reflecting an upregulation of the innate immune system. However, an association of type I IFN-inducible genes with pregnancy induced disease amelioration seems unlikely.

Correlation properties of spontaneous motor activity in healthy infants: a new computer-assisted method to evaluate neurological maturation

Qualitative assessment of spontaneous motor activity in early infancy is widely used in clinical practice. It enables the description of maturational changes of motor behavior in both healthy infants and infants who are at risk for later neurological impairment. These assessments are, however, time-consuming and are dependent upon professional experience. Therefore, a simple physiological method that describes the complex behavior of spontaneous movements (SMs) in infants would be helpful. In this methodological study, we aimed to determine whether time series of motor acceleration measurements at 40-44 weeks and 50-55 weeks gestational age in healthy infants exhibit fractal-like properties and if this self-affinity of the acceleration signal is sensitive to maturation. Healthy motor state was ensured by General Movement assessment. We assessed statistical persistence in the acceleration time series by calculating the scaling exponent α via detrended fluctuation analysis of the time series. In hand trajectories of SMs in infants we found a mean α value of 1.198 (95 % CI 1.167-1.230) at 40-44 weeks. Alpha changed significantly (p = 0.001) at 50-55 weeks to a mean of 1.102 (1.055-1.149). Complementary multilevel regression analysis confirmed a decreasing trend of α with increasing age. Statistical persistence of fluctuation in hand trajectories of SMs is sensitive to neurological maturation and can be characterized by a simple parameter α in an automated and observer-independent fashion. Future studies including children at risk for neurological impairment should evaluate whether this method could be used as an early clinical screening tool for later neurological compromise.

Transient rhythmic network activity in the somatosensory cortex evoked by distributed input in vitro

The initiation and maintenance of physiological and pathophysiological oscillatory activity depends on the synaptic interactions within neuronal networks. We studied the mechanisms underlying evoked transient network oscillation in acute slices of the adolescent rat somatosensory cortex and modeled its underpinning mechanisms. Oscillations were evoked by brief spatially distributed noisy extracellular stimulation, delivered via bipolar electrodes. Evoked transient network oscillation was detected with multi-neuron patch-clamp recordings under different pharmacological conditions. The observed oscillations are in the frequency range of 2-5 Hz and consist of 4-12 mV large, 40-150 ms wide compound synaptic events with rare overlying action potentials. This evoked transient network oscillation is only weakly expressed in the somatosensory cortex and requires increased [K+]o of 6.25 mM and decreased [Ca2+]o of 1.5 mM and [Mg2+]o of 0.5 mM. A peak in the cross-correlation among membrane potential in layers II/III, IV and V neurons reflects the underlying network-driven basis of the evoked transient network oscillation. The initiation of the evoked transient network oscillation is accompanied by an increased [K+]o and can be prevented by the K+ channel blocker quinidine. In addition, a shift of the chloride reversal potential takes place during stimulation, resulting in a depolarizing type A GABA (GABAA) receptor response. Blockade of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA), N-methyl-D-aspartate (NMDA), or GABA(A) receptors as well as gap junctions prevents evoked transient network oscillation while a reduction of AMPA or GABA(A) receptor desensitization increases its duration and amplitude. The apparent reversal potential of -27 mV of the evoked transient network oscillation, its pharmacological profile, as well as the modeling results suggest a mixed contribution of glutamatergic, excitatory GABAergic, and gap junctional conductances in initiation and maintenance of this oscillatory activity. With these properties, evoked transient network oscillation resembles epileptic afterdischarges more than any other form of physiological or pathophysiological neocortical oscillatory activity.

Hypoxia-induced gene activity in disused oxidative muscle

Hypoxia is an important modulator of the skeletal muscle's oxidative phenotype. However, little is known regarding the molecular circuitry underlying the muscular hypoxia response and the interaction of hypoxia with other stimuli of muscle oxidative capacity. We hypothesized that exposure of mice to severe hypoxia would promote the expression of genes involved in capillary morphogenesis and glucose over fatty acid metabolism in active or disused soleus muscle of mice. Specifically, we tested whether the hypoxic response depends on oxygen sensing via the alpha-subunit of hypoxia-inducible factor-1 (HIF-1 alpha). Spontaneously active wildtype and HIF-1 alpha heterozygous deficient adult female C57B1/6 mice were subjected to hypoxia (PiO2 70 mmHg). In addition, animals were subjected to hypoxia after 7 days of muscle disuse provoked by hindlimb suspension. Soleus muscles were rapidly isolated and analyzed for transcript level alterations with custom-designed AtlasTM cDNA expression arrays (BD Biosciences) and cluster analysis of differentially expressed mRNAs. Multiple mRNA elevations of factors involved in dissolution and stabilization of blood vessels, glycolysis, and mitochondrial respiration were evident after 24 hours of hypoxia in soleus muscle. In parallel transcripts of fat metabolism were reduced. A comparable hypoxia-induced expression pattern involving complex alterations of the IGF-I axis was observed in reloaded muscle after disuse. This hypoxia response in spontaneously active animals was blunted in the HIF-1 alpha heterozygous deficient mice demonstrating 35% lower HIF-1 alpha mRNA levels. Our molecular observations support the concept that severe hypoxia provides HIF-1-dependent signals for remodeling of existing blood vessels, a shift towards glycolytic metabolism and altered myogenic regulation in oxidative mouse muscle and which is amplified by enhanced muscle use. These findings further imply differential mitochondrial turnover and a negative role of HIF-1 alpha for control of fatty acid oxidation in skeletal muscle exposed to one day of severe hypoxia.

The -308 tumour necrosis factor-alpha gene polymorphism predicts therapeutic response to TNFalpha-blockers in rheumatoid arthritis and spondyloarthritis patients

OBJECTIVE: To examine whether the G-to-A polymorphism at position -308 in the promoter of the tumour necrosis factor-alpha (TNFalpha) gene influences the therapeutic response to TNFalpha-blockers in patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA) and ankylosing spondylitis (AS). METHODS: A total of 54 patients with RA, 10 with PsA and 22 with AS were genotyped by polymerase chain reaction for the -308 TNFalpha promoter polymorphism. They were treated with infliximab (n = 63), adalimumab (n = 10) or etanercept (n = 13). Clinical response was assessed after 24 weeks by the Disease Activity Score in 28 joints (DAS28) for RA and PsA, and the Bath Ankylosing Spondylitis Activity Index (BASDAI) for AS patients. RESULTS: All patients with the A/A genotype (n = 3, all RA) and two patients with the A/G genotype (AS) failed to respond to anti-TNF treatment. Irrespective of the underlying disease, moderate response (n = 44) was predominantly associated with the A/G genotype (A/G 18/22, G/G 4/22), whereas good response (n = 59) was exclusively seen in patients with the G/G genotype. The average improvement in the DAS28 score was 0.83 in the A/A, 1.50 in the A/G and 2.64 in the G/G group of RA and PsA patients (P < 0.0001). The BASDAI score in AS improved on average by 1.21 in the A/G and by 3.30 in the G/G group (P < 0.005). CONCLUSIONS: The data suggest that humans with a TNFalpha -308 G/G genotype are better responders to anti-TNFalpha treatment than those with A/A or A/G genotypes independent of the treated rheumatic disease (RA, PsA or AS).

Evidence that N-acetylcysteine inhibits TNF-alpha-induced cerebrovascular endothelin-1 upregulation via inhibition of mitogen- and stress-activated protein kinase

N-acetylcysteine (NAC) is neuroprotective in animal models of acute brain injury such as caused by bacterial meningitis. However, the mechanism(s) by which NAC exerts neuroprotection is unclear. Gene expression of endothelin-1 (ET-1), which contributes to cerebral blood flow decline in acute brain injury, is partially regulated by reactive oxygen species, and thus a potential target of NAC. We therefore examined the effect of NAC on tumor necrosis factor (TNF)-alpha-induced ET-1 production in cerebrovascular endothelial cells. NAC dose dependently inhibited TNF-alpha-induced preproET-1 mRNA upregulation and ET-1 protein secretion, while upregulation of inducible nitric oxide synthase (iNOS) was unaffected. Intriguingly, NAC had no effect on the initial activation (i.e., IkappaB degradation, nuclear p65 translocation, and Ser536 phosphorylation) of NF-kappaB by TNF-alpha. However, transient inhibition of NF-kappaB DNA binding suggested that NAC may inhibit ET-1 upregulation by inhibiting (a) parallel pathway(s) necessary for full transcriptional activation of NF-kappaB-mediated ET-1 gene expression. Similar to NAC, the MEK1/2 inhibitor U0126, the p38 inhibitor SB203580, and the protein kinase inhibitor H-89 selectively inhibited ET-1 upregulation without affecting nuclear p65 translocation, suggesting that NAC inhibits ET-1 upregulation via inhibition of mitogen- and stress-activated protein kinase (MSK). Supporting this notion, cotreatment with NAC inhibited the TNF-alpha-induced rise in MSK1 and MSK2 kinase activity, while siRNA knock-down experiments showed that MSK2 is the predominant isoform involved in TNF-alpha-induced ET-1 upregulation.

Evidence for low-grade systemic proinflammatory activity in patients with posttraumatic stress disorder

Posttraumatic stress disorder (PTSD) may increase cardiovascular risk but the psychophysiological mechanisms involved are elusive. We hypothesized that proinflammatory activity is elevated in patients with PTSD as diagnosed by the Clinician Administered PTSD Scale (CAPS) interview. Plasma levels of proinflammatory C-reactive protein (CRP), interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha, and of anti-inflammatory IL-4 and IL-10 were measured in 14 otherwise healthy PTSD patients and in 14 age- and gender-matched healthy non-PTSD controls. Levels of TNF-alpha (p=0.038; effect size Cohen's d=0.58) and of IL-1beta (p=0.075, d=0.68) were higher in patients than in controls. CRP (d=0.10), IL-6 (d=0.18), IL-4 (d=0.42), and IL-10 (d=0.37) were not significantly different between groups. Controlling for traditional cardiovascular risk factors, mood, and time since trauma revealed lower IL-4 in patients than in controls (p=0.029) and rendered group differences in TNF-alpha and IL-1beta insignificant. In all subjects, TNF-alpha correlated with total (frequency and intensity) PTSD symptom cluster of re-experiencing (r=0.49, p=0.008), avoidance (r=0.37, p=0.050), and hyperarousal (r=0.42, p=0.026), and with PTSD total symptom score (r=0.37, p=0.054). Controlling for time since trauma attenuated these associations. The correlation between IL-1beta and total avoidance symptoms (r=0.42, p=0.028) became insignificant when controlling for anxiety and depression. IL-4 correlated with total hyperarousal symptoms (r=-0.38, p=0.047), and after controlling for systolic blood pressure and smoking status, with PTSD total symptom score (r=-0.41, p=0.035). PTSD patients showed a low-grade systemic proinflammatory state, which, moreover, was related to PTSD symptom levels suggesting one mechanism by which PTSD could contribute to atherosclerotic disease.

Tumor necrosis factor-alpha: alternative role as an inhibitor of osteoclast formation in vitro

TNFalpha is known to stimulate the development and activity of osteoclasts and of bone resorption. The cytokine was found to mediate bone loss in conjunction with inflammatory diseases such as rheumatoid arthritis or chronic aseptic inflammation induced by wear particles from implants and was suggested to be a prerequisite for the loss of bone mass under estrogen deficiency. In the present study, the regulation of osteoclastogenesis by TNFalpha was investigated in co-cultures of osteoblasts and bone marrow or spleen cells and in cultures of bone marrow and spleen cells grown with CSF-1 and RANKL. Low concentrations of TNFalpha (1 ng/ml) caused a >90% decrease in the number of osteoclasts in co-cultures, but did not affect the development of osteoclasts from bone marrow cells. In cultures with p55TNFR(-/-) osteoblasts and wt BMC, the inhibitory effect was abrogated and TNFalpha induced an increase in the number of osteoclasts in a dose-dependent manner. Osteoblasts were found to release the inhibitory factor(s) into the culture supernatant after simultaneous treatment with 1,25(OH)(2)D(3) and TNFalpha, this activity, but not its release, being resistant to treatment with anti-TNFalpha antibodies. Dexamethasone blocked the secretion of the TNFalpha-dependent inhibitor by osteoblasts, while stimulating the development of osteoclasts. The data suggest that the effects of TNFalpha on the differentiation of osteoclast lineage cells and on bone metabolism may be more complex than hitherto assumed and that these effects may play a role in vivo during therapies for inflammatory diseases.

Surface expression and functional characterization of alpha-granule factor V in human platelets: effects of ionophore A23187, thrombin, collagen, and convulxin

Factor V (FV) present in platelet alpha-granules has a significant but incompletely understood role in hemostasis. This report demonstrates that a fraction of platelets express very high levels of surface-bound, alpha-granule FV on simultaneous activation with 2 agonists, thrombin and convulxin, an activator of the collagen receptor glycoprotein VI. This subpopulation of activated platelets represents 30.7% +/- 4.7% of the total population and is referred to as convulxin and thrombin-induced-FV (COAT-FV) platelets. COAT-FV platelets are also observed on activation with thrombin plus collagen types I, V, or VI, but not with type III. No single agonist examined was able to produce COAT-FV platelets, although ionophore A23187 in conjunction with either thrombin or convulxin did generate this population. COAT-FV platelets bound annexin-V, indicating exposure of aminophospholipids and were enriched in young platelets as identified by the binding of thiazole orange. The functional significance of COAT-FV platelets was investigated by demonstrating that factor Xa preferentially bound to COAT-FV platelets, that COAT-FV platelets had more FV activity than either thrombin or A23187-activated platelets, and that COAT-FV platelets were capable of generating more prothrombinase activity than any other physiologic agonist examined. Microparticle production by dual stimulation with thrombin and convulxin was less than that observed with A23187, indicating that microparticles were not responsible for all the activities observed. These data demonstrate a new procoagulant component produced from dual stimulation of platelets with thrombin and collagen. COAT-FV platelets may explain the unique role of alpha-granule FV and the hemostatic effectiveness of young platelets. (Blood. 2000;95:1694-1702)

Amino acid sequence of the alpha subunit and computer modelling of the alpha and beta subunits of echicetin from the venom of Echis carinatus (saw-scaled viper)

Echicetin, a heterodimeric protein from the venom of Echis carinatus, binds to platelet glycoprotein Ib (GPIb) and so inhibits platelet aggregation or agglutination induced by various platelet agonists acting via GPIb. The amino acid sequence of the beta subunit of echicetin has been reported and found to belong to the recently identified snake venom subclass of the C-type lectin protein family. Echicetin alpha and beta subunits were purified. N-terminal sequence analysis provided direct evidence that the protein purified was echicetin. The paper presents the complete amino acid sequence of the alpha subunit and computer models of the alpha and beta subunits. The sequence of alpha echicetin is highly similar to the alpha and beta chains of various heterodimeric and homodimeric C-type lectins. Neither of the fully reduced and alkylated alpha or beta subunits of echicetin inhibited the platelet agglutination induced by von Willebrand factor-ristocetin or alpha-thrombin. Earlier reports about the inhibitory activity of reduced and alkylated echicetin beta subunit might have been due to partial reduction of the protein.

Variation in human platelet glycoprotein VI content modulates glycoprotein VI-specific prothrombinase activity

- Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen that figures prominently in signal transduction. An addition to binding to type I and III collagens, GPVI is also bound specifically by collagen-related peptide and convulxin (CVX), a snake venom protein. We developed a quantitative assay of platelet GPVI in which biotin-conjugated CVX binds selectively to GPVI in separated total platelet proteins by a ligand blot procedure. Using this approach, we have documented a 5-fold range in platelet GPVI content among 23 normal healthy subjects. In addition, we have determined that CVX-induced or collagen-related peptide-induced prothrombinase activity is directly proportional to the platelet content of GPVI. A statistically significant correlation was observed at 2 CVX concentrations: 14.7 ng/mL (R(2)=0.854 and P<0.001, n=11) and 22 ng/mL (R(2)=0.776 and P<0.001, n=12). In previous studies, we established a similar range of expression of the integrin collagen receptor alpha(2)beta(1) on platelets of normal subjects. Among 15 donors, there is a direct correlation between platelet alpha(2)beta(1) density and GPVI content (R(2)=0.475 and P=0.004). In view of the well-documented association of GPVI with platelet procoagulant activity, this study suggests that the variation in GPVI content is a potential risk factor that may predispose individuals to hemorrhagic or thromboembolic disorders.

Induction of EROD activity by 1-phenylimidazole and beta-naphthoflavone in rainbow trout cultured hepatocytes: a comparative study

The classical pathway for induction of cytochrome P4501A (CYP1A) by xenobiotics is ligand binding to the aryl hydrocarbon receptor (AhR). However, several studies with mammalian cell systems point out a range of xenobiotics including imidazole derivatives, which are able to activate CYP1A through non-classical mechanisms. The objective of the present work is to compare induction of CYP1A (determined at the catalytic level as 7-ethoxyresorufin-O-deethylase, EROD) in rainbow trout (Oncorhynchus mykiss) hepatocytes by the prototypic AhR ligand, beta-naphthoflavone (betaNF), and by the imidazole derivative, 1-phenylimidazole (PIM). PIM was able to induce EROD activity although its potency was clearly lower than that of betaNF. In order to assess the relative importance of classical AhR ligand binding and alternative signaling pathways in CYP1A induction by PIM, co-exposure experiments with the partial AhR antagonist alpha-naphthoflavone (alphaNF) or with inhibitors of protein kinase C (staurosporine) and tyrosine kinases (genistein, herbimicine) were performed. alphaNF and herbimicin provoked a decrease of EROD induction both by betaNF and PIM, whereas staurosporine and genistein remained without effect. The overall similarities in the response of betaNF and PIM to the various inhibitors suggest that both compounds, in apparent contrast to the behaviour of some other imidazole derivatives, induce CYP1A following similar mechanisms.

Insulin resistance in mice lacking neuronal nitric oxide synthase is related to an alpha-adrenergic mechanism

BACKGROUND: nitric oxide (NO) plays an important role in the regulation of cardiovascular and glucose homeostasis. Mice lacking the gene encoding the neuronal isoform of nitric oxide synthase (nNOS) are insulin-resistant, but the underlying mechanism is unknown. nNOS is expressed in skeletal muscle tissue where it may regulate glucose uptake. Alternatively, nNOS driven NO synthesis may facilitate skeletal muscle perfusion and substrate delivery. Finally, nNOS dependent NO in the central nervous system may facilitate glucose disposal by decreasing sympathetic nerve activity. METHODS: in nNOS null and control mice, we studied whole body glucose uptake and skeletal muscle blood flow during hyperinsulinaemic clamp studies in vivo and glucose uptake in skeletal muscle preparations in vitro. We also examined the effects of alpha-adrenergic blockade (phentolamine) on glucose uptake during the clamp studies. RESULTS: as expected, the glucose infusion rate during clamping was roughly 15 percent lower in nNOS null than in control mice (89 (17) vs 101 (12) [-22 to -2]). Insulin stimulation of muscle blood flow in vivo, and intrinsic muscle glucose uptake in vitro, were comparable in the two groups. Phentolamine, which had no effect in the wild-type mice, normalised the insulin sensitivity in the mice lacking the nNOS gene. CONCLUSIONS: insulin resistance in nNOS null mice was not related to defective insulin stimulation of skeletal muscle perfusion and substrate delivery or insulin signaling in the skeletal muscle cell, but to a sympathetic alpha-adrenergic mechanism.

Anti-inflammatory properties of alpha- and gamma-tocopherol

Natural vitamin E consists of four different tocopherol and four different tocotrienol homologues (alpha,beta, gamma, delta) that all have antioxidant activity. However, recent data indicate that the different vitamin E homologues also have biological activity unrelated to their antioxidant activity. In this review, we discuss the anti-inflammatory properties of the two major forms of vitamin E, alpha-tocopherol (alphaT) and gamma-tocopherol (gammaT), and discuss the potential molecular mechanisms involved in these effects. While both tocopherols exhibit anti-inflammatory activity in vitro and in vivo, supplementation with mixed (gammaT-enriched) tocopherols seems to be more potent than supplementation with alphaT alone. This may explain the mostly negative outcomes of the recent large-scale interventional chronic disease prevention trials with alphaT only and thus warrants further investigation.

Reduced hippocampal anisotropy related to anteriorization of alpha EEG in schizophrenia

Dysfunctions of the hippocampus have been suggested to be related to schizophrenia, and reduced connectivity with other brain regions may be a key for the pathophysiology. The aim of this study was to investigate the effect of white matter anomalies in the hippocampus, as a sign of altered connectivity, on the brain electrical activity. We investigated seven first episode schizophrenic patients and seven age, gender and education-matched controls with diffusion tensor imaging and resting EEG. Fractional anisotropy was computed based on diffusion tensor imaging data for the right and left hippocampus for both groups. No group differences were found in hippocampal fractional anisotropy, EEG spectral power and topography. However a significant correlation was found between more anterior alpha activity and lower fractional anisotropy of both hippocampi in schizophrenics, but not in controls. More anterior alpha activity has been described in schizophrenia. We conclude that this feature might depict a group of schizophrenic patients with reduced hippocampal connectivity.