Pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) and vasoactive intestinal polypeptide are structurally and functionally closely related but show differences in migraine-inducing properties. Mechanisms responsible for the difference in migraine induction are unknown. Here, for the first time, we present a head-to-head comparison study of the immediate and long-lasting observations of the migraine-inducing, arterial, physiological and biochemical responses comparing PACAP38 and vasoactive intestinal polypeptide. In a double-blind crossover study 24 female migraine patients without aura were randomly allocated to intravenous infusion of PACAP38 (10 pmol/kg/min) or vasoactive intestinal polypeptide (8 pmol/kg/min) over 20 min. We recorded incidence of migraine during and after infusion (0-24 h). Magnetic resonance angiography of selected extra- and intracranial arteries, blood samples (plasma PACAP38 and vasoactive intestinal polypeptide and serum tryptase), and vital signs (blood pressure, heart rate, respiratory frequency, and end-tidal pressure of CO2) was recorded before and up to 5 h after infusion. Twenty-two patients [mean age 24 years (range 19-36)] completed the study on both days. Sixteen patients (73%) reported migraine-like attacks after PACAP38 and four after vasoactive intestinal polypeptide (18%) infusion (P = 0.002). Three of four patients, who reported migraine-like attacks after vasoactive intestinal polypeptide, also reported attacks after PACAP38. Both peptides induced marked dilatation of the extracranial (P < 0.05), but not intracranial arteries (P > 0.05). PACAP38-induced vasodilatation was longer lasting (>2 h), whereas vasoactive intestinal polypeptide-induced dilatation was normalized after 2 h. We recorded elevated plasma PACAP38 at 1 h after the start of PACAP38 infusion only in those patients who later reported migraine attacks. Blood levels of vasoactive intestinal polypeptide and tryptase were unchanged after PACAP38 infusion. In conclusion, PACAP38-induced migraine was associated with sustained dilatation of extracranial arteries and elevated plasma PACAP38 before onset of migraine-like attacks. PACAP38 has a much higher affinity for the PAC1 receptor and we therefore suggest that migraine induction by PACAP38 may be because of activation of the PAC1 receptor, which may be a future anti-migraine drug target.

Background: Physiological changes during pregnancy may affect laboratory parameters. Reference values based on samples from non-pregnant women are not necessarily useful for clinical decisions during pregnancy. There is a need to establish reference values during pregnancy in order to recognize pathological conditions. Methods: Eight hundred and one women with expected normal pregnancies were included in the study. Of these, 391 had no complications during pregnancy, delivery, or the early postpartum period. Blood samples were obtained at gestational weeks 13-20, 21-28, 29-34, 35-42, at labor, and 1 and 2 days postpartum. Reference intervals were calculated for 36 tests as recommended by the International Federation of Clinical Chemistry and Laboratory Medicine. Results: Many tests showed such large variations indicating that gestational age-specific reference intervals were necessary. Other tests had different but stable values when compared to non-pregnant women. A minor decrease in albumin levels was observed. This was not only due to pregnancyassociated hemodilution, since other components with the same or a larger molecular diameter did not show a similar decrease. Many tests exhibited a broad distribution around vaginal delivery and in the early postpartum period. Conclusions: Only a few parameters were unaffected during uncomplicated pregnancy, delivery, and the early postpartum period suggesting that implementation of gestational agespecific reference intervals is necessary. Clin Chem Lab Med 2010;48:237-48.

Haemostatic reference intervals are generally based on samples from non-pregnant women. Thus, they may not be relevant to pregnant women, a problem that may hinder accurate diagnosis and treatment of haemostatic disorders during pregnancy. In this study, we establish gestational age-specific reference intervals for coagulation tests during normal pregnancy. Eight hundred one women with expected normal pregnancies were included in the study. Of these women, 391 had no complications during pregnancy, vaginal delivery, or postpartum period. Plasma samples were obtained at gestational weeks 13-20, 21-28, 29-34, 35-42, at active labor, and on postpartum days 1 and 2. Reference intervals for each gestational period using only the uncomplicated pregnancies were calculated in all 391 women for activated partial thromboplastin time (aPTT), fibrinogen, fibrin D-dimer, antithrombin, free protein S, and protein C and in a subgroup of 186 women in addition for prothrombin time (PT), Owren and Quick PT, protein S activity, and total protein S and coagulation factors II, V, VII, VIII, IX, X, XI, and XII. The level of coagulation factors II, V, X, XI, XII and antithrombin, protein C, aPTT, PT remained largely unchanged during pregnancy, delivery, and postpartum and were within non-pregnant reference intervals. However, levels of fibrinogen, D-dimer, and coagulation factors VII, VIII, and IX increased markedly. Protein S activity decreased substantially, while free protein S decreased slightly and total protein S was stable. Gestational age-specific reference values are essential for the accurate interpretation of a subset of haemostatic tests during pregnancy, delivery, and puerperium.

Background Intravenous infusion of pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) provokes migraine attacks in 65-70% of migraine without aura (MO) patients. We investigated whether PACAP38 infusion causes changes in the endogenous production of PACAP38, vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), tumour necrosis factor alpha (TNFα), S100 calcium binding protein B (S100B), neuron-specific enolase and pituitary hormones in migraine patients. Methods We allocated 32 previously genotyped MO patients to receive intravenous infusion PACAP38 (10 pmol/kg/minute) for 20 minutes and recorded migraine-like attacks. Sixteen of the patients were carriers of the risk allele rs2274316 ( MEF2D), which confers increased risk of MO and may regulate PACAP38 expression, and 16 were non-carriers. We collected blood samples at baseline and 20, 30, 40, 60 and 90 minutes after the start of the infusion. A control group of six healthy volunteers received intravenous saline. Results PACAP38 infusion caused significant changes in plasma concentrations of VIP ( p = 0.026), prolactin ( p = 0.011), S100B ( p < 0.001) and thyroid-stimulating hormone (TSH; p = 0.015), but not CGRP ( p = 0.642) and TNFα ( p = 0.535). We found no difference in measured biochemical variables after PACAP38 infusion in patients who later developed migraine-like attacks compared to those who did not ( p > 0.05). There was no difference in the changes of biochemical variables between patients with and without the MEF2D-associated gene variant ( p > 0.05). Conclusion PACAP38 infusion elevated the plasma levels of VIP, prolactin, S100B and TSH, but not CGRP and TNFα. Development of delayed migraine-like attacks or the presence of the MEF2D gene variant was not associated with pre-ictal changes in plasma levels of neuropeptides, TNFα and pituitary hormones.

Background-Reduced production of the vasodilator nitric oxide (NO) in fetal vessels in pregnant smokers may lower the blood flow to the fetus and result in lower birth weight, length, and head circumference. The present study measured endothelial NO synthase (eNOS) activity in fetal umbilical and chorionic vessels from nonsmokers, smokers, and ex-smokers and related the findings to the fetal outcome. Methods and Results-Of 266 healthy, singleton pregnancies, 182 women were nonsmokers, 43 were smokers, and 41 stopped smoking early in pregnancy. eNOS activity and concentration were quantified in endothelial cells of the fetal vessels. Cotinine, lipid profiles, estradiol, L-arginine, and dimethylarginines that may affect NO production were determined in maternal and fetal blood. Serum cotinine verified self-reported smoking. Newborns of smokers had a lower weight (PՅ0.001) and a smaller head circumference (PՅ0.041) and were shorter (PՅ0.001) than newborns of nonsmokers and ex-smokers. eNOS activity in umbilical veins of smokers was 36% lower (PϽ0.001), eNOS concentration was 47% lower (PϽ0.001), and the fetal plasma level of high-density lipoprotein was 18% lower (PϽ0.001) than those of nonsmokers, whereas the same levels were found in umbilical veins from ex-smokers and nonsmokers. The same patterns in eNOS activity and concentration were found in umbilical arteries and chorionic vessels. Fetal plasma levels of estradiol, L-arginine, dimethylarginines, total cholesterol, and triglycerides were similar for nonsmokers, smokers, and ex-smokers. Conclusions-The findings suggest that maternal smoking reduces eNOS activity in the fetal vascular bed, contributing to retarded fetal growth caused by the reduction of vasodilatory capacity, and suggest that smoking cessation early in pregnancy prevents these effects in newborns. (Circulation. 2009;119:857-864.)

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The present study addressed whether chronic hypoxia is associated with reduced nitric oxide (NO) release due to decreased activation of endothelial NO synthase (eNOS). Primary cultures of endothelial cells from human umbilical veins (HUVECs) were used and exposed to different oxygen levels for 24 h, after which NO release, intracellular calcium, and eNOS activity and phosphorylation were measured after 24 h. Direct measurements using a NO microsensor showed that in contrast to 1-h exposure to 5% and 1% oxygen (acute hypoxia), histamine-evoked (10 microM) NO release from endothelial cells exposed to 5% and 1% oxygen for 24 h (chronic hypoxia) was reduced by, respectively, 58% and 40%. Furthermore, chronic hypoxia also lowered the amount and activity of eNOS enzyme. The decrease in activity could be accounted for by reduced intracellular calcium and altered eNOS phosphorylation. eNOS Ser(1177) and eNOS Thr(495) phosphorylations were reduced and increased, respectively, consistent with lowered enzyme activity. Akt kinase, which can phosphorylate eNOS Ser(1177), was also decreased by hypoxia, regarding both total protein content and the phosphorylated (active) form. Moreover, the protein content of beta- actin, which is known to influence the activity of eNOS, was almost halved by hypoxia, further supporting the fall in eNOS activity. In conclusion, chronic hypoxia in HUVECs reduces histamine-induced NO release as well as eNOS expression and activity. The decreased activity is most likely due to changed eNOS phosphorylation, which is supported by decreases in Akt expression and phosphorylation. By reducing NO, chronic hypoxia may accentuate endothelial dysfunction in cardiovascular disease.

Summary Background Anti‐tumor necrosis factor‐α (TNF‐α) is used for the treatment of severe cases of IBD, including Crohn's disease (CD) and ulcerative colitis (UC). However, one‐third of the patients do not respond to the treatment. We have previously investigated whether single nucleotide polymorphisms (SNPs) in genes involved in inflammation were associated with response to anti‐TNF therapy among patients with CD or UC. Aim A new cohort of patients was established for replication of the previous findings and to identify new SNPs associated with anti‐TNF response. Methods Fifty‐three SNPs assessed previously in cohort 1 (482 CD and 256 UC patients) were genotyped in cohort 2 (587 CD and 458 UC patients). The results were analysed using logistic regression (adjusted for age and gender). Results Ten SNPs were associated with anti‐TNF response either among patients with CD (TNFRSF1A(rs4149570) (OR: 1.92, 95% CI: 1.02‐3.60, P = 0.04), IL18(rs187238) (OR: 1.35, 95% CI: 1.00‐1.82, P = 0.05), and JAK2(rs12343867) (OR: 1.35, 95% CI: 1.02‐1.78, P = 0.03)), UC (TLR2(rs11938228) (OR: 0.55, 95% CI: 0.33‐0.92, P = 0.02), TLR4(rs5030728) (OR: 2.23, 95% CI: 1.24‐4.01, P = 0.01) and (rs1554973) (OR: 0.49, 95% CI: 0.27‐0.90, P = 0.02), NFKBIA(rs696) (OR: 1.45, 95% CI: 1.06‐2.00, P = 0.02), and NLRP3(rs4612666) (OR: 0.63, 95% CI: 0.44‐0.91, P = 0.01)) or in the combined cohort of patient with CD and UC (IBD) (TLR4(rs5030728) (OR: 1.46, 95% CI: 1.01‐2.11, P = 0.04) and (rs1554973)(OR: 0.80, 95% CI: 0.65‐0.98, P = 0.03), NFKBIA(rs696) (OR: 1.25, 95% CI: 1.01‐1.54, P = 0.04), NLRP3(rs4612666) (OR: 0.73, 95% CI: 0.57‐0.95, P = 0.02), IL1RN(rs4251961) (OR: 0.81, 95% CI: 0.66‐1.00, P = 0.05), IL18(rs1946518) (OR: 1.24, 95% CI: 1.01‐1.53, P = 0.04), and JAK2(rs12343867) (OR: 1.24, 95% CI: 1.01‐1.53, P = 0.04)). Conclusions The results support that polymorphisms in genes involved in the regulation of the NFκB pathway (TLR2, TLR4, and NFKBIA), the TNF‐α signalling pathway (TNFRSF1A), and other cytokine pathways (NLRP3, IL1RN, IL18, and JAK2) were associated with response to anti‐TNF therapy. Our multi‐SNP model predicted response rate of more than 82% (in 9% of the CD patients) and 75% (in 15% of the UC patients), compared to 71% and 64% in all CD and UC patients, respectively. More studies are warranted to predict response for use in the clinic.