Examination of pituitary adenylate cyclase-activating polypeptide in Parkinson's disease focusing on correlations with motor symptoms.

The neuroprotective effects of pituitary adenylate cyclase-activating polypeptide (PACAP) have been shown in numerous in vitro and in vivo models of Parkinson's disease (PD) supporting the theory that PACAP could have an important role in the pathomechanism of the disorder affecting mostly older patients. Earlier studies found changes in PACAP levels in neurological disorders; therefore, the aim of our study was to examine PACAP in plasma samples of PD patients. Peptide levels were measured with ELISA and correlated with clinical parameters, age, stage of the disorder based on the Hoehn and Yahr (HY) scale, subtype of the disease, treatment, and specific scores measuring motor and non-motor symptoms, such as movement disorder society-unified Parkinson's disease rating scale (MDS-UPDRS), Epworth sleepiness scale (ESS), Parkinson's disease sleep scale (PDSS-2), and Beck depression inventory (BDI). Our results showed significantly decreased PACAP levels in PD patients without deep brain stimulation (DBS) therapy and in akinetic-rigid subtype; additionally we also observed a further decrease in the HY stage 3 and 4. Elevated PACAP levels were found in patients with DBS. There were no significant correlations between PACAP level with MDS-UPDRS, type of pharmacological treatment, PDSS-2 sleepiness, or depression (BDI) scales, but we found increased PACAP level in patients with more severe sleepiness problems based on the ESS scale. Based on these results, we suggest that following the alterations of PACAP with other frequently used clinical biomarkers in PD patients might improve strategic planning of further therapeutic interventions and help to provide a clearer prognosis regarding the future perspective of the disease.

Lack of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Disturbs Callus Formation.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a naturally secreted signaling peptide and has important regulatory roles in the differentiation of the central nervous system and its absence results in disorders in femur development. PACAP has an important function in prevention of oxidative stress or mechanical stress in chondrogenesis but little is known about its function in bone regeneration. A new callus formation model was set to investigate its role in bone remodeling. Fracturing was 5 mm distal from the proximal articular surface of the tibia and the depth was 0.5 mm. Reproducibility of callus formation was investigated with CT 3, 7, and 21 days after the operation. Absence of PACAP did not alter the alkaline phosphatase (ALP) activation in PACAP KO healing process. In developing callus, the expression of collagen type I increased in wild-type (WT) and PACAP KO mice decreased to the end of healing process. Expression of the elements of BMP signaling was disturbed in the callus formation of PACAP KO mice, as bone morphogenic protein 4 (BMP4) and 6 showed an early reduction in bone regeneration. However, elevated Smad1 expression was demonstrated in PACAP KO mice. Our results indicate that PACAP KO mice show various signs of disturbed bone healing and suggest PACAP compensatory and fine tuning effects in proper bone regeneration.

Possible effects of pituitary adenylate cyclase activating polypeptide (PACAP) on early embryo implantation marker HB-EGF in mouse.

Pituitary adenylate cyclase activating polypeptide (PACAP) was originally isolated as a hypothalamic neuropeptide stimulating adenylate cyclase activity. Besides its neuroprotective effects, numerous data proved its role in reproductive processes. However, there are limited data on its role in preimplantation embryo development and implantation. Our aim was to analyse the mRNA expression of Adcyap1 (coding region of PACAP) and Hbegf [coding region of HB-EGF (Heparin-binding EGF-like growth factor)] in embryos and pregnant uterus to investigate the possible correlation between them. Eight-week-old BDF1 mice were superovulated and subsequently mated overnight or left in their cage after hCG treatment. Day4 embryos were flushed from mated females. After morphological analysis, Adcyap1 and Hbegf gene expression of embryos and uterine tissues was assessed with qPCR. Our results showed significantly higher Adcyap1 and Hbegf mRNA levels in females producing embryos compared to non-mated ones. Robust elevation of Adcyap1 and slight elevation of Hbegf were detected in females with blastocyst embryos compared with non-blastocysts. We found low rate of Hbegf mRNA expression in uncompacted embryos, whereas morulae and blastocysts expressed high amounts of Hbegf. However, we did not find detectable Adcyap1 mRNA in embryos. Strong correlation was found between uterine tissue and embryonic Hbegf levels, slight correlation between uterine Adcyap1 and Hbegf levels. Uterine tissue Adcyap1 and embryonic Hbegf showed no correlation. In summary, our present data show, for the first time, the correlation between PACAP and HB-EGF mRNA expression suggesting that PACAP might play a role during the peri-implantation period of early mouse embryo development.

Phenotypic characterization of testicular immune cells expressing immune checkpoint molecules in wild-type and pituitary adenylate cyclase-activating polypeptide-deficient mice.

PROBLEM: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide having several regulatory functions in the nervous system and in peripheral organs including those of the reproductive system. PACAP-deficient male mice have several morphological, biochemical, behavioral defects and show disturbed signaling in spermatogenesis affecting fertility in PACAP KO mice. Reproductive functions such as fertility, mating, and maternal behaviors have been widely investigated, but no immune analyses are available regarding the testicular immune-privileged environment in male PACAP-deficient mice. METHOD OF STUDY: We performed detailed immunophenotyping of testicular immune cells and investigated the expression of TIM-3 and PD-1 Immune checkpoint molecules of immune cells together with the detection of galectin-9 and perforin. We investigated the percentage of numerous immune cell populations in the testis of wild-type and PACAP-deficient mice. RESULTS: We demonstrated a significant increase in the frequency of testicular CD8+ T cells together with the decrease in Treg cell number obtained from PACAP KO mice compared with wild-type mice. Investigating Immune checkpoint receptors, only PD-1 showed a significantly decreased expression in CD8+ T cells in PACAP KO mice compared with wild-type suggesting an impaired PD-1/PD-L1 pathway. Regarding TIM-3 expression, we did not find any significant difference between the investigated groups. CONCLUSION: We hypothesize that these local changes may result in an immune activation with disturbed testicular immunoregulation in PACAP KO mice; however, determining the exact function requires further investigations. Our data further support the view that besides a systemic immune tolerance, localized active immunosuppression is involved in the regulation of testicular immune privilege.

Age-related alterations of articular cartilage in pituitary adenylate cyclase-activating polypeptide (PACAP) gene-deficient mice.

Pituitary adenylate cyclase activating polypeptide (PACAP) is an evolutionarly conserved neuropeptide which is produced by various neuronal and non-neuronal cells, including cartilage and bone cells. PACAP has trophic functions in tissue development, and it also plays a role in cellular and tissue aging. PACAP takes part in the regulation of chondrogenesis, which prevents insufficient cartilage formation caused by oxidative and mechanical stress. PACAP knockout (KO) mice have been shown to display early aging signs affecting several organs. In the present work, we investigated articular cartilage of knee joints in young and aged wild-type (WT) and PACAP KO mice. A significant increase in the thickness of articular cartilage was detected in aged PACAP gene-deficient mice. Amongst PACAP receptors, dominantly PAC1 receptor was expressed in WT knee joints and a remarkable decrease was found in aged PACAP KO mice. Expression of PKA-regulated transcription factors, Sox5, Sox9 and CREB, decreased both in young and aged gene deficient mice, while Sox6, collagen type II and aggrecan expressions were elevated in young but were reduced in aged PACAP KO animals. Increased expression of hyaluronan (HA) synthases and HA-binding proteins was detected parallel with an elevated presence of HA in aged PACAP KO mice. Expression of bone related collagens (I and X) was augmented in young and aged animals. These results suggest that loss of PACAP signaling results in dysregulation of cartilage matrix composition and may transform articular cartilage in a way that it becomes more prone to degenerate.

Risk factors for resistance development against lamivudine during long-term treatment of chronic hepatitis B virus infections.

BACKGROUND/AIM: The use of lamivudine for the treatment of chronic hepatitis B (CHB) is limited by high rates of lamivudine resistance. However, it is still in use in many regions. Factors associated with lamivudine resistance development have been studied in only a few European cohorts. The aim of our study was to assess the rate and risk factors for lamivudine resistance in a large real-life European cohort. PATIENTS AND METHODS: We retrospectively analyzed patients with CHB treated in three German University centers over up to 12 years. Lamivudine resistance was defined as virologic breakthrough and presence of genotypic lamivudine resistance. The probability of resistance was estimated by Kaplan-Meier analysis and resistance predictors by Cox regression. RESULTS: A total of 227 patients were included into the analysis (hepatitis B envelope antigen positive or negative). Rates of lamivudine resistance by years 1-7 were 7, 26, 35, 41, 46, 53, and 55%, respectively. Interestingly, two hepatitis B envelope antigen-negative patients developed resistance during the year 12 of treatment. Independent risk factors for resistance development were hepatitis B virus DNA levels of at least 10 copies/ml before and detectable hepatitis B virus DNA by month 6 of treatment. CONCLUSION: Even after long-term response to lamivudine more than 10 years, resistance may still develop. Our findings further discourage the use of lamivudine for the treatment of CHB.

VPAC1 receptors play a dominant role in PACAP-induced vasorelaxation in female mice.

BACKGROUND: PACAP and VIP are closely related neuropeptides with wide distribution and potent effect in the vasculature. We previously reported vasomotor activity in peripheral vasculature of male wild type (WT) and PACAP-deficient (KO) mice. However, female vascular responses are still unexplored. We hypothesized that PACAP-like activity is maintained in female PACAP KO mice and the mechanism through which it is regulated differs from that of male PACAP KO animals. METHODS: We investigated the vasomotor effects of VIP and PACAP isoforms and their selective blockers in WT and PACAP KO female mice in carotid and femoral arteries. The expression and level of different PACAP receptors in the vessels were measured by RT-PCR and Western blot. RESULTS: In both carotid and femoral arteries of WT mice, PACAP1-38, PACAP1-27 or VIP induced relaxation, without pronounced differences between them. Reduced relaxation was recorded only in the carotid arteries of KO mice as compared to their WT controls. The specific VPAC1R antagonist completely blocked the PACAP/VIP-induced relaxation in both arteries of all mice, while PAC1R antagonist affected relaxation only in their femoral arteries. CONCLUSION: In female WT mice, VPAC1 receptors appear to play a dominant role in PACAP-induced vasorelaxation both in carotid and in femoral arteries. In the PACAP KO group PAC1R activation exerts vasorelaxation in the femoral arteries but in carotid arteries there is no significant effect of the activation of this receptor. In the background of this regional difference, decreased PAC1R and increased VPAC1R availability in the carotid arteries was found.

Association of HLA-DPA1 and HLA-DPB1 polymorphisms with spontaneous HBsAg seroclearance in Caucasians.

BACKGROUND & AIMS: Acute hepatitis B virus (HBV) infections may clear spontaneously or become chronic and run through different phases. The single nucleotide polymorphisms (SNPs) rs3077, rs9277535 and rs9277534 within the human leucocyte antigen (HLA)-DP gene have been found to be associated with HBV susceptibility and persistence in Asians. However, evidence for the influence of these variants in Caucasians has been limited so far. The aim of our study was to investigate the impact of these polymorphisms on the outcome of HBV infections in a large Caucasian population. METHODS: In this case-control study, we retrospectively analysed 1111 Caucasian individuals, including 618 with chronic HBV infections (CHB), 239 with spontaneous HBsAg seroclearance (SC) and 254 healthy controls (HC). The rs3077, rs9277535 and rs9277534 SNPs were genotyped by a polymerase chain reaction from blood samples and melting curve analysis. RESULTS: A significant difference in the allele distributions was observed only for the rs3077 SNP between the HC and the CHB group as well as between the SC and CHB groups. The rs3077-C allele was associated with a lower probability for spontaneous HBsAg seroclearance in comparison with the rs3077-T allele (OR 0.704, 95% CI 0.509-0.974; P = 0.033). No association of the three SNPs with the stages of chronic HBV infection was found. CONCLUSION: This is the first study demonstrating an association of the rs3077-T allele with spontaneous HBsAg seroclearance in Caucasians. Further studies are needed to elucidate the role of HLA-DP variants in disease pathogenesis and their potential role for individualized disease management.

Protective Role of Endogenous PACAP in Inflammation-induced Retinal Degeneration.

PURPOSE: Pituitary adenylate Cyclase-Activating Polypeptide (PACAP) is a neuroprotective peptide that has been shown to exert protective effects in different models of neurodegenerative diseases, including retinal degenerations. Data obtained from PACAP-deficient (PACAP KO) mice provide evidence that endogenous PACAP has a neuroprotective role in different pathologies. PACAP KO mice show enhanced sensitivity to different insults, such as oxidative stress, hypoxia and inflammation. The aim of the present study was to investigate the protective effects of endogenous PACAP in retinal inflammation. METHODS: Endotoxin-induced eye inflammation was induced by intraperitoneal injection of lipopolysaccharide (LPS) in PACAP KO and wild-type (Wt) mice. After LPS treatment, retinas were processed for histological examination. To detect the alterations of different proteins and cytokines, immunohistochemical, western blot and cytokine array were used. We also performed dark-adapted electroretinography (ERG) to detect the functional differences. RESULTS: The thickness of nearly all layers was significantly less in LPS-injected PACAP KO mice compared to Wt animals. Increased expression of Glial Fibrillary Acidic Protein (GFAP) was induced in Müller glial cells after LPS treatment, which was more intense in PACAP KO mice. The levels of pAkt and pGSK were decreased in PACAP KO group during inflammation. LPS treatment significantly increased cytokines (sICAM-1, JE, TIMP-1) in both treated groups, but it was more expressed in PACAP KO animals. Furthermore, ERG responses were disturbed after LPS injection in PACAP KO mice. CONCLUSION: Our results showed that endogenous PACAP has a protective role in LPS-caused retinal inflammation.

Polymorphisms in the Toll-like receptor 3 (TLR3) gene are associated with the natural course of hepatitis B virus infection in Caucasian population.

Innate immunity can induce spontaneous hepatitis B surface antigen (HBsAg) seroclearance (SC) of hepatitis B virus (HBV) infection or transition towards an inactive carrier state. Toll-like receptor (TLR) 3 signalling has been linked to these processes. Alterations in the TLR3 gene might impair immune responses against HBV. In our study, we analysed the impact of the TLR3 polymorphisms rs3775291 and rs5743305 on the natural course of HBV infection. In this retrospective study, a Caucasian cohort of 621 patients with chronic HBV infection (CHB), 239 individuals with spontaneous HBsAg SC, and 254 healthy controls were enrolled. In the CHB group, 49% of patients were inactive carriers, and 17% were HBeAg-positive. The TLR3 rs3775291 A allele was associated with a reduced likelihood of spontaneous HBsAg SC and HBeAg SC, and an increased risk of developing chronic hepatitis B. In haplotype analysis, the haplotype including both risk variants rs3775291A and rs5743305A had the lowest likelihood of HBsAg SC. Further research in larger cohorts and functional analyses are needed to shed light on the impact of TLR3 signalling.

Signalling Alterations in Bones of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Gene Deficient Mice.

: Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with diverse developmental roles, including differentiation of skeletal elements. It is a positive regulatory factor of chondrogenesis and osteogenic differentiation in vitro, but little is known about its in vivo role in bone formation. In our experiments, diaphyses of long bones from hind limbs of PACAP gene-deficient mice showed changes in thickness and increased staining intensity. Our main goal was to perform a detailed morphological and molecular biological analysis of femurs from PACAP knockout (KO) and wild type (WT) mice. Transverse diameter and anterior cortical bone thickness of KO femurs showed significant alterations with disturbed Ca2+ accumulation and collagen type I expression. Higher expression and activity of alkaline phosphatase were also observed, accompanied by increased fragility PACAP KO femurs. Increased expression of the elements of bone morphogenic protein (BMP) and hedgehog signalling was also observed, and are possibly responsible for the compensation mechanism accounting for the slight morphological changes. In summary, our results show that lack of PACAP influences molecular and biomechanical properties of bone matrix, activating various signalling cascade changes in a compensatory fashion. The increased fragility of PACAP KO femur further supports the role of endogenous PACAP in in vivo bone formation.

Comparative analysis of decidual and peripheral immune cells and immune-checkpoint molecules during pregnancy in wild-type and PACAP-deficient mice.

PROBLEM: PACAP is a neuropeptide having a major relevance in the nervous system and in several peripheral organs including those of the reproductive system. PACAP-deficient mice have several morphological, biochemical, behavioral defects, and show reduced fertility. Female reproductive functions such as fertility, mating behavior, maternal behaviors, and implantation alterations have been widely investigated, but no comparative immune analyses are available in pregnant wild-type (WT) and PACAP knockout (KO) mice. METHODS OF STUDY: Therefore, we performed a detailed immunophenotyping of decidual and peripheral immune cells and investigated the expression of two immune-checkpoint molecules by immune cells together with immunohistochemistry detecting Galectin-9 in placental tissues. We investigated the percentage of numerous immune cell populations in the periphery and in the decidua of pregnant mice. RESULTS: We demonstrated a significant increase in the frequency of decidual Gal-9+ Th cells obtained from PACAP KO mice compared to the decidua of WT mice. We could not determine statistical differences in TIM-3 and programmed cell death-1 expression by different immune cells in the decidua and in the periphery between WT and KO mice. In conclusion, we could not find any significant alteration either in the distribution or in the cytotoxicity of the investigated decidual immune cells which could elucidate any reproductive alterations in PACAP KO mice. CONCLUSION: The only remarkable finding is the recruitment of Gal-9+ Th cells to the decidua promoting local immune homeostasis in PACAP KO mice, which nevertheless cannot explain the reduced fertility observed in these mice.

Accelerated pre-senile systemic amyloidosis in PACAP knockout mice - a protective role of PACAP in age-related degenerative processes.

Dysregulation of neuropeptides may play an important role in aging-induced impairments. Among them, pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent cytoprotective peptide that provides an endogenous control against a variety of tissue-damaging stimuli. We hypothesized that the progressive decline of PACAP throughout life and the well-known general cytoprotective effects of PACAP lead to age-related pathophysiological changes in PACAP deficiency, supported by the increased vulnerability to various stressors of animals partially or totally lacking PACAP. Using young and aging CD1 PACAP knockout (KO) and wild type (WT) mice, we demonstrated pre-senile amyloidosis in young PACAP KO animals and showed that senile amyloidosis appeared accelerated, more generalized, more severe, and affected more individuals. Histopathology showed age-related systemic amyloidosis with mainly kidney, spleen, liver, skin, thyroid, intestinal, tracheal, and esophageal involvement. Mass spectrometry-based proteomic analysis, reconfirmed with immunohistochemistry, revealed that apolipoprotein-AIV was the main amyloid protein in the deposits together with several accompanying proteins. Although the local amyloidogenic protein expression was disturbed in KO animals, no difference was found in laboratory lipid parameters, suggesting a complex pathway leading to increased age-related degeneration with amyloid deposits in the absence of PACAP. In spite of no marked inflammatory histological changes or blood test parameters, we detected a disturbed cytokine profile that possibly creates a pro-inflammatory milieu favoring amyloid deposition. In summary, here we describe accelerated systemic senile amyloidosis in PACAP gene-deficient mice, which might indicate an early aging phenomenon in this mouse strain. Thus, PACAP KO mice could serve as a model of accelerated aging with human relevance. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Pituitary adenylate cyclase-activating polypeptide ameliorates vascular dysfunction induced by hyperglycaemia.

BACKGROUND: Short-lasting hyperglycaemia occurs frequently in prediabetes and poorly controlled diabetes mellitus leading to vascular damage. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to play a protective role in vascular complications of diabetes; moreover, antioxidant effects of PACAP were also described. Therefore, we hypothesized that PACAP exerts protective effects in short-term hyperglycaemia-induced vascular dysfunctions. METHODS: After short-term hyperglycaemia, acetylcholine-induced and sodium nitroprusside-induced vascular relaxation of mouse carotid arteries were tested with a myograph with or without the presence of PACAP or superoxide dismutase. Potential direct antioxidant superoxide-scavenging action of pituitary adenylate cyclase-activating peptide was tested with pyrogallol autoxidation assay; furthermore, the effect of pituitary adenylate cyclase-activating peptide or superoxide dismutase was investigated on hyperglycaemia-associated vascular markers. RESULTS: PACAP administration resulted in reduced endothelial dysfunction after a 1-h hyperglycaemic episode. PACAP was able to restore acetylcholine-induced relaxation of the vessels and improved sodium nitroprusside-induced relaxation. This effect was comparable to the protective effect of superoxide dismutase, but PACAP was unable to directly scavenge superoxide produced by autoxidation of pyrogallol. Endothelial dysfunction was associated with elevated levels of fibroblast growth factor basic, matrix metalloproteinase 9 and nephroblastoma overexpressed gene proteins. Their release was reduced by PACAP administration. CONCLUSION: These results suggest a strong protective role of PACAP in the vascular complications of diabetes.

Aging-Induced Modulation of Pituitary Adenylate Cyclase-Activating Peptide- and Vasoactive Intestinal Peptide-Induced Vasomotor Responses in the Arteries of Mice.

Pituitary adenylate cyclase-activating peptide (PACAP; 1-38 and 1-27) and vasoactive intestinal peptide (VIP) are related neuropeptides of the secretin/glucagon family. Overlapping signaling through G-protein-coupled receptors mediates their vasomotor activity. We previously showed that PACAP deficiency (PACAP-KO) shifts the mechanisms of vascular response and maintains arterial relaxation through the VIP backup mechanism and (mainly) its VPAC1R, but their age-dependent modulation is still unknown. We hypothesized that backup mechanisms exist, which maintain the vasomotor activity of these peptides also in older age. Thus, we investigated the effects of exogenous VIP and PACAP peptides in isolated carotid arteries of 2- and 15-month-old wild-type (WT) and PACAP-KO mice. All peptides induced relaxation in the arteries of young WT mice, whereas in young PACAP-KO mice PACAP1-27 and VIP, but not PACAP1-38, induced relaxation. Unlike VIP, PACAP-induced vasomotor responses were reduced in aging WT mice. However, in the arteries of aging PACAP-KO mice, PACAP1-27- and VIP-induced responses were reduced, but PACAP1-38 showed a greater vasomotor response compared to that of young PACAP-KO animals. There were no significant differences between the vasomotor responses of aging WT and PACAP-KO mice. Our data suggest that, in the absence of PACAP both in young and old ages, the vascular response is mediated through backup mechanisms, most likely VIP, maintaining proper vascular relaxation in aging-induced PACAP insufficiency.

Intestinal Microbiota Changes in Mice Lacking Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) - Bifidobacteria Make the Difference.

Pituitary adenylate cyclase activating polypetide (PACAP) constitutes a neuropeptide that is widely distributed in the host exerting essential cytoprotective properties, whereas PACAP-/- mice display increased susceptibility to distinct immunopathological conditions. The orchestrated interplay between the gut microbiota and the host is pivotal in immune homeostasis and resistance to disease. Potential pertubations of the intestinal microbiota in PACAP-/- mice, however, have not been addressed so far. For the first time, we performed a comprehensive survey of the intestinal microbiota composition in PACAP-/- and wildtype (WT) mice starting 2 weeks postpartum until 18 months of age applying quantitative culture-independent techniques. Fecal enterobacteria and enterococci were lower in PACAP-/- than WT mice aged 1 month and ≥6 months, respectively. Whereas Mouse Intestinal Bacteroides were slightly higher in PACAP-/- versus WT mice aged 1 and 6 months, this later in life held true for Bacteroides/Prevotella spp. (≥12 months) and lactobacilli (>15 months of age). Strikingly, health-beneficial bifidobacteria were virtually absent in the intestines of PACAP-/- mice, even when still breastfed. In conclusion, PACAP deficiency is accompanied by distinct changes in fecal microbiota composition with virtually absent bifidobacteria as a major hallmark that might be linked to increased susceptibility to disease.

Backup Mechanisms Maintain PACAP/VIP-Induced Arterial Relaxations in Pituitary Adenylate Cyclase-Activating Polypeptide-Deficient Mice.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide in the VIP/secretin/glucagon peptide superfamily. Two active forms, PACAP1-38 and PACAP1-27, act through G protein-coupled receptors, the PAC1 and VPAC1/2 receptors. Effects of PACAP include potent vasomotor activity. Vasomotor activity and organ-specific vasomotor effects of PACAP-deficient mice have not yet been investigated; thus, the assessment of its physiological importance in vasomotor functions is still missing. We hypothesized that backup mechanisms exist to maintain PACAP pathway activity in PACAP knockout (KO) mice. Thus, we investigated the vasomotor effects of exogenous vasoactive intestinal peptide (VIP) and PACAP polypeptides in PACAP wild-type (WT) and PACAP-deficient (KO) male mice. METHODS: Carotid and femoral arteries were isolated from 8- to 12-week-old male WT and PACAP-KO mice. Vasomotor responses were measured with isometric myography. RESULTS: In the arteries of WT mice the peptides induced relaxations, which were significantly greater to PACAP1-38 than to PACAP1-27 and VIP. In KO mice, PACAP1-38 did not elicit relaxation, whereas PACAP1-27 and VIP elicited significantly greater relaxation in KO mice than in WT mice. The specific PAC1R and VPAC1R antagonist completely blocked the PACAP-induced relaxations. CONCLUSION: Our data suggest that in PACAP deficiency, backup mechanisms maintain arterial relaxations to polypeptides, indicating an important physiological role for the PACAP pathway in the regulation of vascular tone.

Early Neurobehavioral Development of Mice Lacking Endogenous PACAP.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide. In addition to its diverse physiological roles, PACAP has important functions in the embryonic development of various tissues, and it is also considered as a trophic factor during development and in the case of neuronal injuries. Data suggest that the development of the nervous system is severely affected by the lack of endogenous PACAP. Short-term neurofunctional outcome correlates with long-term functional deficits; however, the early neurobehavioral development of PACAP-deficient mice has not yet been evaluated. Therefore, the aim of the present study was to describe the postnatal development of physical signs and neurological reflexes in mice partially or completely lacking PACAP. We examined developmental hallmarks during the first 3 weeks of the postnatal period, during which period most neurological reflexes and motor coordination show most intensive development, and we describe the neurobehavioral development using a complex battery of tests. In the present study, we found that PACAP-deficient mice had slower weight gain throughout the observation period. Interestingly, mice partially lacking PACAP weighed significantly less than homozygous mice. There was no difference between male and female mice during the first 3 weeks. Some other signs were also more severely affected in the heterozygous mice than in the homozygous mice, such as air righting, grasp, and gait initiation reflexes. Interestingly, incisor teeth erupted earlier in mice lacking PACAP. Motor coordination, shown by the number of foot-faults on an elevated grid, was also less developed in PACAP-deficient mice. In summary, our results show that mice lacking endogenous PACAP have slower weight gain during the first weeks of development and slower neurobehavioral development regarding a few developmental hallmarks.

Improved pharmacodynamics and pharmacokinetics after i.v. application of peginterferon alfa-2a in hepatitis C null responders.

BACKGROUND & AIM: Mechanisms of non-responsiveness to peginterferon alfa-2a are not completely understood. Inadequate plasma levels may contribute to reduced response. The aim of this prospective, multicentre, crossover, Phase 1 study was to evaluate the pharmacokinetics and viral kinetics of intravenous vs. subcutaneous peginterferon alfa-2a in patients with genotype 1 chronic hepatitis C infection who showed null response to previous peginterferon/ribavirin. METHODS: Patients were randomized in four treatment arms to subcutaneous or intravenous peginterferon alfa-2a 180 µg, once or twice weekly for 2 weeks. After a washout phase of 6 weeks, patients first receiving intravenous administration switched to subcutaneous or vice versa for additional 2 weeks. RESULTS: Intravenous administration of pegylated interferon resulted in a stronger and faster decline in HCV RNA than subcutaneous administration with a maximum decline of 1.17 log10 vs. 0.41 log10 or 1.32 log10 vs. 0.54 log10 after a once or twice weekly application, respectively. Pharmacokinetic studies revealed significantly higher maximum concentration (C(max))(0-12) h and C(max 0-7) d following intravenous administration, irrespective of dosing frequency A rapid rebound in HCV RNA was observed in all treatment arms. Adverse events occurred more frequently following intravenous administration. CONCLUSION: Intravenous administration of peginterferon alfa-2a results in considerably higher plasma concentration and a stronger decline in HCV RNA and offers an interesting approach in order to overcome interferon non-responsive state in patients with full null response to previous peginterferon/ribavirin combination therapy.