Omega-6 polyunsaturated fatty acids prevent atherosclerosis development in LDLr-KO mice, in spite of displaying a pro-inflammatory profile similar to trans fatty acids

The development of atherosclerosis and the inflammatory response were investigated in LDLr-KO mice on three high-fat diets (40% energy as fat) for 16 weeks: trans (TRANS), saturated (SAFA) or omega-6 polyunsaturated (PUFA) fats. The following parameters were measured: plasma lipids, aortic root total cholesterol (TC), lesion area (Oil Red-O), ABCA1 content and macrophage infiltration (immunohistochemistry), collagen content (Picrosirius-red) and co-localization of ABCA1 and macrophage (confocal microscopy) besides the plasma inflammatory markers (IL-6, TNF-alpha) and the macrophage inflammatory response to lipopolysaccharide from Escherichia coli (LPS). As expected, plasma TC and TG concentrations were lower on the PUFA diet than on TRANS or SAFA diets. Aortic intima macrophage infiltration, ABCA1 content, and lesion area on PUFA group were lower compared to TRANS and SAFA groups. Macrophages and ABCA1 markers did not co-localize in the atherosclerotic plaque, suggesting that different cell types were responsible for the ABCA1 expression in plaques. Compared to PUFA, TRANS and SAFA presented higher collagen content and necrotic cores in atherosclerotic plaques. In the artery wall, TC was lower on PUFA compared to TRANS group; free cholesterol was lower on PUFA compared to TRANS and SAFA; cholesteryl ester concentration did not vary amongst the groups. Plasma TNF-alpha concentration on PUFA and TRANS-fed mice was higher compared to SAFA. No difference was observed in IL-6 concentration amongst groups. Regarding the macrophage inflammatory response to LPS, TRANS and PUFA presented higher culture medium concentrations of IL-6 and TNF-alpha as compared to SAFA. The PUFA group showed the lowest amount of the anti-inflammatory marker IL-10 compared to TRANS and SAFA groups. In conclusion, PUFA intake prevented atherogenesis, even in a pro-inflammatory condition. (c) 2012 Elsevier Ireland Ltd. All rights reserved.

Lateral septal area alpha(1)-and alpha(2)-adrenoceptors differently modulate baroreflex activity in unanaesthetized rats

The lateral septal area (LSA) is a limbic structure involved in autonomic, neuroendocrine and behavioural responses. An inhibitory influence of the LSA on baroreflex activity has been reported; however, the local neurotransmitter involved in this modulation is still unclear. In the present study, we verified the involvement of local LSA adrenoceptors in modulating cardiac baroreflex activity in unanaesthetized rats. Bilateral microinjection of the selective a1-adrenoceptor antagonist WB4101 (10 nmol in a volume of 100 nl) into the LSA decreased baroreflex bradycardia evoked by blood pressure increases, but had no effect on reflex tachycardia evoked by blood pressure decreases. Nevertheless, bilateral administration of the selective a2-adrenoceptor antagonist RX821002 (10 nmol in 100 nl) increased baroreflex tachycardia without affecting reflex bradycardia. Treatment of the LSA with a cocktail containing WB4101 and RX821002 decreased baroreflex bradycardia and increased reflex tachycardia. The non-selective beta-adrenoceptor antagonist propranolol (10 nmol in 100 nl) did not affect either reflex bradycardia or tachycardia. Microinjection of noradrenaline into the LSA increased reflex bradycardia and decreased the baroreflex tachycardic response, an opposite effect compared with those observed after double blockade of a1- and a2-adrenoceptors, and this effect of noradrenaline was blocked by local LSA pretreatment with the cocktail containing WB4101 and RX821002. The present results provide advances in our understanding of the baroreflex neural circuitry. Taken together, data suggest that local LSA a1- and a2-adrenoceptors modulate baroreflex control of heart rate differently. Data indicate that LSA a1-adrenoceptors exert a facilitatory modulation on baroreflex bradycardia, whereas local a2-adrenoceptors exert an inhibitory modulation on reflex tachycardia.

Temporal relationships of a pulse of prolactin (PRL) to a pulse of a metabolite of PGF2 alpha in mares

Hourly blood samples were collected from 10 mares during 24 h of each of the preluteolytic, luteolytic, and postluteolytic periods. The autocorrelation function of the R program was used to detect pulse rhythmicity, and the intra-assay CV was used to locate and characterize pulses of prolactin (PRL) and a metabolite of prostaglandin F2 alpha (PGFM). Rhythmicity of PRL and PGFM concentrations was detected in 67% and 89% of mares, respectively. Combined for the three periods (no difference among periods), the PRL pulses were 5.2 +/- 0.4 h (mean +/- SEM) at the base, 7.5 +/- 1.5 h between nadirs of adjacent pulses, and 12.3 +/- 1.5 h from peak to peak. The peaks of PRL pulses were greater (P < 0.05) during the luteolytic period (46 +/- 14 ng/mL) and postluteolytic period (52 15 ng/mL) than during the preluteolytic period (17 3 ng/mL). Concentrations of PRL during hours of a PGFM pulse were different (P < 0.003) within the luteolytic period and postluteolytic period and were greatest at the PGFM peak; PRL concentrations during a PGFM pulse were not different during the preluteolytic period. The frequency of the peak of PRL and PGFM pulses occurring at the same hour (synchrony) was greater for the luteolytic period (65%, P < 0.01) and postluteolytic period (50%, P < 0.001) than for the preluteolytic period (17%). This is the first report in mares on characterization and rhythmicity of PRL pulses, synchrony between PRL and PGFM pulses, and greater PRL activity during the luteolytic and postluteolytic periods than during the preluteolytic period. (C) 2012 Elsevier Inc. All rights reserved.

Ovarian and PGF2 alpha responses to stimulation of endogenous PRL pulses during the estrous cycle in mares

The effects of a PRL-stimulating substance (sulpiride) on PRL and PGF2 alpha secretion and on luteal and ovarian follicular dynamics were studied during the estrous cycle in mares. A control group (n = 9) and a sulpiride group (Sp; n = 10) were used. Sulpiride (25 mg) was given every 8 h from Day 13 postovulation to the next ovulation. Repeated sulpiride treatment did not appear to maintain PRL concentrations at 12-h intervals beyond Day 14. Therefore, the hypothesis that a long-term increase in PRL altered luteal and follicular end points was not testable. Hourly samples were collected from the hour of a treatment (Hour 0) to Hour 8 on Day 14. Concentrations of PRL increased to maximum at Hour 4 in the Sp group. The PRL pulses were more prominent (P < 0.008) in the sulpiride group (peak, 19.4 +/- 1.9 ng/mL; mean +/- SEM) than in the controls (11.5 +/- 1.8 ng/mL). Concentrations of a metabolite of PGF2a (PGFM), number, and characteristics of PGFM pulses, and concentrations of progesterone during Hours 0 to 8 were not affected by the increased PRL. A novel observation was that the peak of a PRL pulse occurred at the same hour or 1 h later than the peak of a PGFM pulse in 8 of 8 PGFM pulses in the controls and in 6 of 10 pulses in the Sp group (P < 0.04), indicating that sulpiride interfered with the synchrony between PGFM and PRL pulses. The hypothesis that sulpiride treatment during the equine estrous cycle increases concentrations of PRL and the prominence of PRL pulses was supported. (c) 2012 Elsevier Inc. All rights reserved.

Toll-like receptor 2/MyD88 signaling mediates zymosan-induced joint hypernociception in mice: Participation of TNF-alpha, IL-1 beta and CXCL1/KC

Arthritic pain is a serious health problem that affects a large number of patients. Toll-like receptors (TLRs) activation within the joints has been implicated in pathophysiology of arthritis. However, their role in the genesis of arthritic pain needs to be demonstrated. In the present study, it was addressed the participation of TLR2 and TLR4 and their adaptor molecule MyD88 in the genesis of joint hypernociception (a decrease in the nociceptive threshold) during zymosan-induced arthritis. Zymosan injected in the tibio-tarsal joint induced mechanical hypernociception in C57BL/6 wild type mice that was reduced in TLR2 and MyD88 null mice. On the other hand, zymosan-induced hypernociception was similar in C3H/HePas and C3H/Hej mice (TLR4 mutant mice). Zymosan-induced joint hypernociception was also reduced in TNFR1 null mice and in mice treated with IL-1 receptor antagonist or with an antagonist of CXCR1/2. Moreover, the joint production of TNF-alpha, IL-1 beta and CXCL1/KC by zymosan was dependent on TLR2/MyD88 signaling. Investigating the mechanisms by which TNF-alpha, IL-1 beta and CXCL1/KC mediate joint hypernociception, joint administration of these cytokines produced mechanical hypernociception, and they act in an interdependent manner. In last instance, their hypernociceptive effects were dependent on the production of hypernociceptive mediators, prostaglandins and sympathetic amines. These results indicate that in zymosan-induced experimental arthritis, TLR2/MyD88 is involved in the cascade of events of joint hypernociception through a mechanism dependent on cytokines and chemokines production. Thus, TLR2/MyD88 signaling might be a target for the development of novel drugs to control pain in arthritis. (C) 2011 Elsevier B.V. All rights reserved.

Intracellular mechanisms of hydroquinone toxicity on endotoxin-activated neutrophils

Circulating neutrophils promptly react to different substances in the blood and orchestrate the beginning of the innate inflammatory response. We have shown that in vivo exposure to hydroquinone (HQ), the most oxidative compound of cigarette smoke and a toxic benzene metabolite, affects circulating neutrophils, making them unresponsive to a subsequent bacterial infection. In order to understand the action of toxic molecular mechanisms on neutrophil functions, in vitro HQ actions on pro-inflammatory mediator secretions evoked by Escherichia coli lipopolysaccharide (LPS) were investigated. Neutrophils from male Wistar rats were cultured with vehicle or HQ (5 or 10 mu M; 2 h) and subsequently incubated with LPS (5 mu g/ml; 18 h). Hydroquinone treatment impaired LPS-induced nitric oxide (NO), tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-1 beta and IL-6 secretions by neutrophils. The toxic effect was not dependent on cell death, reduced expression of the LPS receptor or toll-like receptor-4 (TLR-4) or cell priming, as HQ did not induce reactive oxygen species generation or beta(2)integrin membrane expression. The action of toxic mechanisms on cytokine secretion was dependent on reduced gene synthesis, which may be due to decreased nuclear factor kappa B (NF-kappa B) nuclear translocation. Conversely, this intracellular pathway was not involved in impaired NO production because HQ treatments only affected inducible nitric oxide synthase protein expression and activity, suggesting posttranscriptional and/or posttranslational mechanisms of action. Altogether, our data show that HQ alters the action of different LPS-activated pathways on neutrophils, which may contribute to the impaired triggering of the host innate immune reaction detected during in vivo HQ exposure.

alpha 1-Acid Glycoprotein Decreases Neutrophil Migration and Increases Susceptibility to Sepsis in Diabetic Mice

The mechanisms underlying immune deficiency in diabetes are largely unknown. In the present study, we demonstrate that diabetic mice are highly susceptible to polymicrobial sepsis due to reduction in rolling, adhesion, and migration of leukocytes to the focus of infection. In addition, after sepsis induction, CXCR2 was strongly downregulated in neutrophils from diabetic mice compared with nondiabetic mice. Furthermore, CXCR2 downregulation was associated with increased G-protein coupled receptor kinase 2 (GRK2) expression in these cells. Different from nondiabetic mice, diabetic animals submitted to mild sepsis displayed a significant augment in alpha 1-acid glycoprotein (AGP) hepatic mRNA expression and serum protein levels. Administration of AGP in nondiabetic mice subjected to mild sepsis inhibited the neutrophil migration to the focus of infection, as well as induced t-selectin shedding and rise in CD11b of blood neutrophils. Insulin treatment of diabetic mice reduced mortality rate, prevented the failure of neutrophil migration, impaired GRK2-mediated CXCR2 downregulation, and decreased the generation of AGP. Finally, administration of AGP abolished the effect of insulin treatment in diabetic mice. Together, these data suggest that AGP may be involved in reduction of neutrophil migration and increased susceptibility to sepsis in diabetic mice. Diabetes 61:1584-1591, 2012

Both alpha(1)- and beta(1)-adrenoceptors in the bed nucleus of the stria terminalis are involved in the expression of conditioned contextual fear

BACKGROUND AND PURPOSE The bed nucleus of the stria terminalis (BNST) is a limbic structure that is involved in the expression of conditioned contextual fear. Among the numerous neural inputs to the BNST, noradrenergic synaptic terminals are prominent and some evidence suggests an activation of this noradrenergic neurotransmission in the BNST during aversive situations. Here, we have investigated the involvement of the BNST noradrenergic system in the modulation of behavioural and autonomic responses induced by conditioned contextual fear in rats. EXPERIMENTAL APPROACH Male Wistar rats with cannulae bilaterally implanted into the BNST were submitted to a 10 min conditioning session (6 footshocks, 1.5 ma/ 3 s). Twenty-four hours later freezing and autonomic responses (mean arterial pressure, heart rate and cutaneous temperature) to the conditioning box were measured for 10 min. The adrenoceptor antagonists were administered 10 min before the re-exposure to the aversive context. KEY RESULTS L-propranolol, a non-selective beta-adrenoceptor antagonist, and phentolamine, a non-selective a-adrenoceptor antagonist, reduced both freezing and autonomic responses induced by aversive context. Similar results were observed with CGP20712, a selective beta 1-adrenoceptor antagonist, and WB4101, a selective a1-antagonist, but not with ICI118,551, a selective beta 2-adrenoceptor antagonist or RX821002, a selective a2-antagonist. CONCLUSIONS AND IMPLICATIONS These findings support the idea that noradrenergic neurotransmission in the BNST via a1- and beta 1-adrenoceptors is involved in the expression of conditioned contextual fear.

Cross-taxon congruence of alpha and beta diversity among five leaf litter arthropod groups in Colombia

In this study alpha and beta diversity patterns of five leaf litter arthropod groups (ants, predatory ants, oribatid mites, spiders and other arachnids) were described and compared in 39 sampling patches of a transformed landscape in southwestern Colombia, that represented five vegetation types: secondary forest, riparian forest, giant bamboo forest, pasture and sugarcane crop. It was also assessed whether some taxa could be used as diversity surrogates. A total of 6,765 individuals grouped in 290 morphospecies were collected. Species richness in all groups was lower in highly transformed vegetation types (pasture, sugarcane crop) than in native ones (forests). In contrast, there were no clear tendencies of beta diversity among vegetation types. Considering sampling patches, 0.1-42% of the variation in alpha diversity of one taxonomic group could be explained from the alpha diversity of another, and 0.2-33% of the variation of beta diversity of a given taxon was explained by that in other groups. Contrary to recent findings, we concluded that patterns of alpha diversity are more congruent than patterns of beta diversity. This fact could be attributed to a sampling effect that promotes congruence in alpha diversity and to a lack of a clear regional ecological gradient that could promote congruent patterns of beta diversity. We did not find evidence for an ideal diversity surrogate although diversity patterns of predatory ants had the greatest congruencies. These results support earlier multi-taxon evaluations in that conservation planning should not be based on only one leaf litter arthropod group.

Associations of the TNF-alpha-308 G/A, IL6-174 G/C and AdipoQ 45 T/G polymorphisms with inflammatory and metabolic responses to lifestyle intervention in Brazilians at high cardiometabolic risk

Background: Cytokines secreted by the adipose tissue influence inflammation and insulin sensitivity, and lead to metabolic disturbances. How certain single-nucleotide polymorphisms (SNPs) interfere on lifestyle interventions is unclear. We assessed associations of selected SNPs with changes induced by a lifestyle intervention. Methods: This 9-month intervention on diet and physical activity included 180 Brazilians at high cardiometabolic risk, genotyped for the TNF-alpha -308 G/A, IL-6 -174 G/C and AdipoQ 45 T/G SNPs. Changes in metabolic and inflammatory variables were analyzed according to these SNPs. Individuals with at least one variant allele were grouped and compared with those with the reference genotype. Results: In the entire sample (66.7% women; mean age 56.5 +/- 11.6 years), intervention resulted in lower energy intake, higher physical activity, and improvement in anthropometry, plasma glucose, HOMA-IR, lipid profile and inflammatory markers, except for IL-6 concentrations. After intervention, only variant allele carriers of the TNF-alpha -308 G/A decreased plasma glucose, after adjusting for age and gender (OR 2.96, p = 0.025). Regarding the IL-6 -174 G/C SNP, carriers of the variant allele had a better response of lipid profile and adiponectin concentration, but only the reference genotype group decreased plasma glucose. In contrast to individuals with the reference genotype, carriers of variant allele of AdipoQ 45 T/G SNP did not change plasma glucose, apolipoprotein B, HDL-c and adiponectin concentrations in response to intervention. Conclusion: The TNF alpha -308 G/A SNP may predispose a better response of glucose metabolism to lifestyle intervention. The IL-6 -174 G/C SNP may confer a beneficial effect on lipid but not on glucose metabolism. Our findings reinforce unfavorable effects of the AdipoQ 45 T/G SNP in lipid profile and glucose metabolism after intervention in Brazilians at cardiometabolic risk. Further studies are needed to direct lifestyle intervention to subsets of individuals at cardiometabolic risk.

Associations of the TNF-alpha -308 G/A, IL6 -174 G/C and AdipoQ 45 T/G polymorphisms with inflammatory and metabolic responses to lifestyle intervention in Brazilians at high cardiometabolic risk

Abstract Background Cytokines secreted by the adipose tissue influence inflammation and insulin sensitivity, and lead to metabolic disturbances. How certain single-nucleotide polymorphisms (SNPs) interfere on lifestyle interventions is unclear. We assessed associations of selected SNPs with changes induced by a lifestyle intervention. Methods This 9-month intervention on diet and physical activity included 180 Brazilians at high cardiometabolic risk, genotyped for the TNF-α -308 G/A, IL-6 -174 G/C and AdipoQ 45 T/G SNPs. Changes in metabolic and inflammatory variables were analyzed according to these SNPs. Individuals with at least one variant allele were grouped and compared with those with the reference genotype. Results In the entire sample (66.7% women; mean age 56.5 ± 11.6 years), intervention resulted in lower energy intake, higher physical activity, and improvement in anthropometry, plasma glucose, HOMA-IR, lipid profile and inflammatory markers, except for IL-6 concentrations. After intervention, only variant allele carriers of the TNF-α -308 G/A decreased plasma glucose, after adjusting for age and gender (OR 2.96, p = 0.025). Regarding the IL6 -174 G/C SNP, carriers of the variant allele had a better response of lipid profile and adiponectin concentration, but only the reference genotype group decreased plasma glucose. In contrast to individuals with the reference genotype, carriers of variant allele of AdipoQ 45 T/G SNP did not change plasma glucose, apolipoprotein B, HDL-c and adiponectin concentrations in response to intervention. Conclusion The TNFα -308 G/A SNP may predispose a better response of glucose metabolism to lifestyle intervention. The IL-6 -174 G/C SNP may confer a beneficial effect on lipid but not on glucose metabolism. Our findings reinforce unfavorable effects of the AdipoQ 45 T/G SNP in lipid profile and glucose metabolism after intervention in Brazilians at cardiometabolic risk. Further studies are needed to direct lifestyle intervention to subsets of individuals at cardiometabolic risk.

Eine kontinuierliche Lyman-alpha-Quelle basierend auf Festkörperlasersystemen

In dieser Arbeit wird eine schmalbandige kontinuierliche kohärente Lyman-α-Quelle basierend auf Festkörperlasersystemen zur zukünftigen Kühlung von Antiwasserstoff vorgestellt. Die fundamentalen Festkörperlasersysteme ermöglichen es im Vier-Wellen-Misch-Prozess zur Erzeugung der Lyman-α-Strahlung nicht nur die 6^1S – 7^1S-Zwei-Photonen-Resonanz des Quecksilbers sondern erstmals auch die 6^1S – 6^3P-Ein-Photonen-Resonanz zur Erhöhung der Konversionseffizienz optimal zu nutzen. In ersten Messungen wurden 0,063nW Leistung bei Lyman-α erzeugt. Mit dieser Lyman-α-Quelle war es, durch die Nähe des ersten fundamentalen Lasers zur Ein-Photonen-Resonanz, erstmals möglich den kompletten Verlauf der Phasenanpassungskurve des Vier-Wellen- Misch-Prozesses aufzunehmen. Neben den fundamentalen Lasersystemen und der Lyman-alpha-Erzeugung selbst, wird in dieser Arbeit die Detektion der produzierten Lyman-α-Strahlung mit einem Photomultiplier vorgestellt, die soweit optimiert wurde, dass eine zuverlässige Abschätzung der erzeugten Leistung möglich ist. Für diesen Zweck wurde zudem ein Teststand aufgebaut, mit dem die Transmissivität der Optiken, welche in der Lyman-α-Apparatur verwendet werden, bei 121,56nm gemessen wurde. Des Weiteren wird hier eine vielseitige Rechnung vorgestellt, mit der die erzeugte Leistung bei Lyman-α, unter anderem in Abhängigkeit von der Temperatur, der Absorption des ersten fundamentalen Laserstrahls, dem Dichteprofil des Quecksilberdampfes und unter dem Einfluss eines Puffergases, bestimmt wird.

Funktionelle Analyse der Meprin-Metalloproteasen alpha und beta hinsichtlich ihrer physiologischen Regulation und biologischen Bedeutung

Im Rahmen dieser Arbeit ist es gelungen, ein besseres Verständnis der beiden Metalloproteasen Meprin α und β in ihrem proteolytischen Netzwerk hinsichtlich ihrer physiologischen Regulation durch endogene Inhibitoren, wie auch der biologischen Funktion von Meprin α für den Prozess der Angiogenese, zu erlangen. rnMit der Analyse des ersten identifizierten endogenen Meprin-Inhibitors Fetuin-A gelang die Bestimmung der Ki-Werte für Meprin α mit 4,2 x 10-5 M und 1,1 x 10-6 M für Meprin β. Des Weiteren konnte für Meprin β eine Schnittstelle im Fetuin-A validiert werden. Mit der Identifizierung von Cystatin C, einem Cystein-Protease-Inhibitor als endogener Inhibitor der Metalloprotease Meprin α, mit einem Ki-Wert von 8,5 x 10-6 M, wurden erstmals Proteasefamilie-übergreifende Inhibitionsmechanismen für Metalloproteasen offenbart.rnDie Analyse von drei potentiellen Meprin-Inhibitoren, identifiziert als Substrate in einem neuen Proteomics-Analyse-Verfahren terminal amine isotopic labeling of substrates (TAILS), ermöglichte die Charakterisierung von Elafin als spezifischen Meprin α-Inhibitor. Für Elafin ist es außerdem gelungen, die durch TAILS ermittelte Schnittstelle für Meprin α mittels Edman Sequenzierung zu validieren. Der secretory leukocyte peptidase inhibitor (SLPI), ein Elafin-Homolog, konnte als weiteres Meprin α-Substrat bestätigt werden. Außerdem gelang es, die Meprin α-Schnittstelle im SLPI zu validieren.rnEin weiteres Ziel dieser Arbeit war, ein besseres Verständnis der biologischen Funktion der Metalloprotease Meprin α zu erlangen. Hier konnte in vivo eine stark pro-angiogene Wirkung von Meprin α gezeigt werden und erstmals die Expression von Meprin α, jedoch nicht von Meprin β, in Endothelzellen nachgewiesen werden. Zugleich konnte mit der Analyse der durch die TAILS-Methode identifizierten pro-angiogenen Substrate vascular endothelial growth factor A (VEGF-A) und connective tissue growth factor (CTGF) der Regulationsmechanismus von Meprin α in der Angiogenese identifiziert werden. So ist Meprin α durch die Spaltung von CTGF in der Lage VEGF-A – gebunden und inhibiert im Komplex mit CTGF – durch proteolytische Spaltung von CTGF wieder freizusetzen. Somit wird die inhibierte VEGF-A-Aktivität wieder vollständig hergestellt. rnMit der Charakterisierung der ersten endogenen Meprin-Inhibitoren ist es gelungen, zu einem besseren Verständnis der endogenen Regulation der Meprine beizutragen und eine Proteasefamilie-übergreifende endogene Regulation aufzuzeigen. Mit der Entdeckung von Meprin α als pro-angiogene Protease und der Entschlüsselung des angiogenen Regulationsmechanismus konnte eine essentielle biologische Bedeutung dieser Protease beschrieben werden.rn

Serum anti-interferon alpha antibodies in chronic hepatitis C patients treated with pegylated interferon alpha containing therapy

The development of anti-IFNα antibodies is an occurrence described in chronic hepatitis C patients during treatment with Interferonα/PEG-Interferonα. However, its relevance, especially in difficult-to treat patients, has not been defined. Methods: We retrospectively measured the serum levels of anti-IFNα antibodies (baseline and week 12) and IFNα levels (week 12) by ELISA in 76 previous non-responders, and in 14 naive patients treated with Pegylated-IFNα and Ribavirin. A group of 57 healthy donors (HD) was also assessed as control. Positivity to anti-IFNα antibodies was established on the values of HD. Results: Baseline anti-IFNα antibodies were detected in 15.5% of patients and in 7% of HD, with significantly higher concentrations in patients than HD (181.5±389.9 vs 95.9±143.0 ng mL−1, p=0.0023). All positive patients were IFNα-experienced. At week 12, the prevalence of positivity increased to 22.3 and 28.5% in experienced and naïve patients, respectively, and the levels of anti-IFNα antibodies did not differ between the two groups (391±792.3 vs 384.7±662.6 ng mL−1, respectively). IFNα concentrations were significantly lower in antibody-positive patients than in antibody-negatives (988.2±1402 vs 3462±830.8 pg mL−1, p≤0.0001) and the levels of antibodies and IFNα were inversely correlated (r=-0.405, p=0.0001). The antibody-positive population clustered in null responders (67%) and 19/21 patients (90%) did not achieve SVR. Conclusions: The development of anti-IFNα antibodies is a non-negligible occurrence in patients treated with PEG-IFNα, is stable over time, and has a relevant clinical impact when associated with low levels of circulating PEG-IFNα. It should be considered in patients undergoing treatments including PEG-IFNα.

Faltung, Assemblierung und Stabilisierung des Transmembranproteins Cytochrom b 6

In dieser Arbeit wurde der Beitrag der interhelikalen Loops zur Faltung, Assemblierung und Stabilität des kofaktortragenden Transmembranproteins Cytochrom b6 in vitro untersucht. Cytochrom b6 ist aus vier Transmembranhelices aufgebaut, die über drei Loops miteinander verbunden sind. Die beiden nicht-kovalent gebundenen Kofaktoren werden spontan in der Häm-Bindespalte zwischen den zwei Cytochrom b6-Hälften gebunden. Die Ergebnisse zeigen, dass die Verlängerung oder Eliminierung des Loops, der die beiden Hälften verbindet, nicht die Faltung und Assemblierung des Proteins beeinflusst. Der Loop ist für eine räumliche Positionierung und Orientierung der Hälften während der Assemblierung nicht essentiell. Weiterhin scheint keiner der drei interhelikalen Loops für die Bindung der Kofaktoren notwendig zu sein. Die Cytochrom b6-Hälfte, bestehend aus den Helices A und B, besitzt eine Konformation, die stabil genug ist um Häm alleine zu binden. Ebenso zeigt Helix B alleine eine α-helikale Struktur und bindet ebenfalls Häm. In vivo wurden bislang keine Faktoren beschrieben, die an der Assemblierung beteiligt sind. Im Rahmen dieser Arbeit wurden strukturelle Merkmale des Häms identifiziert, welche die Spezifität der Häm-Bindung, wenigstens in vitro, ausmachen. Von großer Bedeutung ist dabei das zentrale Eisen-Ion, dessen Eliminierung oder Austausch die Häm-Bindung verhindert. Die Substituenten des Porphyrinrings scheinen hingegen für die Stabilität der Bindung notwendig zu sein.