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Nucleoli develop when preribosomes are synthesized at the chromosomal nucleolar organizer regions. Typically they consist of at least three nucleolar subcompartments, the fibrillar center (FC), the dense fibrillar component (DF), and the granular component (GC). The understanding of the functional arrangements of these subcompartments relates to aspects in cell biology, pathology, and virus research. In the present review morphological studies are discussed in the light of molecular findings. The available data confirm the hypothesis that rDNA transcription is connected with the DF but not necessarily with the presence of an FC. Within the DF, rDNA transcription is restricted to foci, possibly representing single transcribing genes. FCs may serve to store inactive transcription factors, to initiate rDNA transcription, and may provide structural support for transcription. The GC can be interpreted as a collection of maturing preribosomes. More recently the nucleolar subcompartments were focused on in the context of virus research and tumor biology. Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Human exposure to radiofrequency electromagnetic fields (RF-EMF) is restricted to prevent thermal effects in the tissue. However, at very low intensity exposure "non-thermal" biological effects, like oxidative stress, DNA or chromosomal aberrations, etc. collectively termed genomic-instability can occur after few hours. Little is known about chronic (years long) exposure with non-thermal RF-EMF. We identified two neighboring housing estates in a rural region with residents exposed to either relatively low (control-group) or relatively high (exposed-group) RF-EMF emitted from nearby mobile phone base stations (MPBS). 24 healthy adults that lived in their homes at least for 5 years volunteered. The homes were surveyed for common types of EMF, blood samples were tested for oxidative status, transient DNA alterations, permanent chromosomal damage, and specific cancer related genetic markers, like MLL gene rearrangements. We documented possible confounders, like age, sex, nutrition, life-exposure to ionizing radiation (X-rays), occupational exposures, etc. The groups matched well, age, sex, lifestyle and occupational risk factors were similar. The years long exposure had no measurable effect on MLL gene rearrangements and c-Abl-gene transcription modification. Associated with higher exposure, we found higher levels of lipid oxidation and oxidative DNA-lesions, though not statistically significant. DNA double strand breaks, micronuclei, ring chromosomes, and acentric chromosomes were not significantly different between the groups. Chromosomal aberrations like dicentric chromosomes (p=0.007), chromatid gaps (p=0.019), chromosomal fragments (p<0.001) and the total of chromosomal aberrations (p<0.001) were significantly higher in the exposed group. No potential confounder interfered with these findings. Increased rates of chromosomal aberrations as linked to excess exposure with ionizing radiation may also occur with non-ionizing radiation exposure. Biological endpoints can be informative for designing exposure limitation strategies. Further research is warranted to investigate the dose-effect-relationship between both, exposure intensity and exposure time, to account for endpoint accumulations after years of exposure. As established for ionizing radiation, chromosomal aberrations could contribute to the definition of protection thresholds, as their rate reflects exposure intensity and exposure time., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In clinical practice, the treatment of patients with irritable bowel syndrome (IBS) can be very challenging. The aims of the present non-interventional study (NIS) were to investigate the tolerability and efficacy of PMA-zeolite under everyday conditions in patients with diarrheic IBS type (IBS-D) or constipated type (IBS-C) or mixed type (IBS-M)., Methods: To document prospective data on tolerability and symptom frequency in the frame of a nationwide NIS, we recruited 204 IBS patients. The study focused on the IBS-related quality of life (measured by the SF-36 questionnaire) and improvements of IBS-related symptoms according to specific ROM-III criteria and stool consistency (Bristol stool scale). The participants documented their abdominal pain, bloating, number of bowel movements, and stool consistency through a web-based internet platform (initial and exit questionnaires) and daily diary entries over the period of intake (8 weeks)., Results: A total of 82.2% of the recruited patients had filled in the questionnaires before and after the 8-week treatment with PMA-zeolite. Seven of the eight subscales of the SF-36 improved significantly (p<0,001); the reduction in abdominal pain was especially significant (p<0,001). The diary entries confirmed the reduction in abdominal pain and revealed a significant reduction in days with bloating (p<0,001). The Bristol-stool-scale analysis showed improvements; particularly, patients with IBS-D benefited from the treatment (p<0,001)., Conclusion: The treatment duration of 8 weeks was well tolerated by most patients. Under everyday life conditions, PMA-zeolite alleviated the global IBS-related symptoms and raised the quality of life (QOL). The PMA-zeolite, thus, may represent a good adjuvant therapeutic option for patients with irritable bowel syndrome., Competing Interests: We declare in the manuscript, about on coauthor: SE is employed by Panaceo International GmbH, the company who sponsored the study substance. The sponsor had no influence on the project roll out, data analyses, or presentation. HV received honoraria for lectures from Panaceo. CM, WM, and RJ have no conflict of interest to declare., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial-License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Objectives: Preclinical and clinical data suggest, that the microporous mineral with large inner surface and ion exchanger capability PMA-(Panaceo-micro-activation)-zeolite can bind irritating and inflammation associated chime-constituents. We hypothesised whether or not it can ameliorate subclinical inflammation, and investigated the potential in the management of patients with Irritable Bowel syndrome (IBS)., Methods: The trial design was prospective, randomized, controlled, double-blinded, pilot study with 41 patients. They received orally 3 g of the medicinal product PMA-zeolite or microcrystalline cellulose (control) twice a day. At baseline and after three months the symptom load, blood and stool parameters, like high sensitivity C-reactive protein (hsCRP), zonulin, α1-antitrypsin, interleukin IL-10, and changes in the gut microbiome were determined by means of ANOVA, ANCOVA and non parametric statistical analyses., Results: The IBS-associated symptom scores decreased significantly in both groups (p=0,001) indicating a strong placebo effect. In the verum but not in the placebo group various inflammation related laboratory parameters decreased. The gross statistical comparison revealed a reduction of α1-antitrypsin significant (p=0,037), a lowered inflammation marker hsCRP, paralleled specific microbiome changes (Lactobacillus, Bifidobacteria, and Firmicutes)., Conclusions: The decrease of blood hsCRP and decreased stool α1-antitrypsin suggest that PMA-zeolite possibly can lower inflammation in the gut of IBS patients. The corresponding increase of the immune modulating species Bifidobacteria and Lactobacillus and the reduction of Firmicutes also point at an inflammation ameliorating effect and a possible mucous layer strengthening effect. The protocol of this explorative pilot study is feasible for a sufficiently powered trial on inflammation amelioration in IBS patients.

Objectives: Pregnancy is a period in life with a high demand of micronutrients. A prophylactic supplementation of folic acid to reduce the risk of neurological malformations in the newborn is common practice. The array of essential micronutrients during pregnancy includes neurotropic vitamins (Vitamin B6, B12 and folic acid), minerals like iron, and trace elements like zinc. As the serum level of most micronutritients is actively regulated by the organism, a prophylactic broad supplementation with a mild, but effective supplementation typically does not pose any risk for exaggerated serum levels, therefore prophylactic intake may be prefered to blood screening and specific interventions., Methods: To investigate the ingredients' bioavailability of the complex vitamin-mineral-trace element composition LaVita® we recruited healthy volunteers for six months and observed the changes of pregnancy relevant parameters by means of laboratory measures. The study design was prospective, double blind, placebo controlled, and included a "male group control". We determined baseline parameters of folate, vitamin B6 and vitamin B12, iron, zinc, homocysteine and Hb-alpha-1c. After three and six months of daily intake of the study substance the blood tests were repeated and compared to the baseline levels., Results: The regular intake resulted in an increase of the supplemented substances' serum levels. The metabolic parameter homocysteine decreased significantly, Hb-alpha-1c was slightly lowered., Conclusion: The regular intake filled up the respective storage compartments and reservoirs in the tissues, and improved the metabolic status. Female participants tended to benefit more than male. We conclude that the composition is safe, and warrants optimized micronutrient supply during pregnancy or postnatal breastfeeding.

Hintergrund: Bindungsstörungen können das Auftreten von Angst und Depression begünstigen, lassen sich aber durch korrigierende emotionale Erfahrungen verändern. Eine Massage - insbesondere wenn beide, Masseur (m/w) und Klient, sich in einem Zustand der Achtsamkeit befinden - kann eine solche korrigierende Erfahrung vermitteln. In der vorliegenden Interventionsstudie wurden die Auswirkungen von Insightouch® - einer Kombination von achtsamkeitsbasierter Massage mit Achtsamkeitsschulung - untersucht. Teilnehmer und Methoden: Wir rekrutierten 36 Personen mit psychischen Beschwerden, geringer Bindungsfähigkeit und geringer Achtsamkeit. In Intervallen von 8 Wochen wurden mittels validierter Fragebögen Parameter wie psychosomatische Symptome (Brief Symptom Inventory), Bindungsqualitäten (Adult Attachment Scale) und Achtsamkeit (Freiburger Achtsamkeitsfragebogen) erhoben. Die Hälfte der Teilnehmer (Gruppe A) startete mit der 8-wöchigen Behandlungsphase; nach weiteren 8 Wochen ohne Behandlung wurde die Nachhaltigkeit der Behandlungswirkung dokumentiert. Die Teilnehmer der Gruppe B starteten mit einer 8-wöchigen Wartephase (Kontrollphase ohne Behandlung), gefolgt von der Behandlungsphase. Ergebnisse: Während sich nach der Kontrollphase keine signifikanten Änderungen zeigten, bewirkte die aktive Behandlung statistisch signifikante Besserungen der 1) Symptomatik, 2) Bindungsdefizite und 3) Achtsamkeit. Nach der 8-wöchigen Nachbeobachtungsphase waren die psychischen und die bindungsbezogenen Symptome weiter verbessert; die behandlungsbedingt erhöhte Fähigkeit zur Achtsamkeit blieb hoch. Schlussfolgerung: Insightouch als primär nonverbale Intervention erhöht die Bindungsfähigkeit, verbessert psychosomatische Symptome und erhöht nachhaltig die Fähigkeit zur Achtsamkeit., (© 2018 S. Karger AG, Basel.)

Objective: Papaya and oats are natural food and used in traditional medicine in many parts of the world. Papaya has a high content of enzymes supporting digestive function. Oats are a source of minerals, beta-glucan fibres, immunmodulatory and antiinflammatory probiotic substances. Caricol®-Gastro combines both constituents, it was designed as vegan organic preparation for intestinal inflammatory diseases. We performed a randomized, double blind placebo controlled clinical trial to investigate the potential of Caricol®-Gastro as add on therapy in patients with diagnosed chronic gastritis., Methods: 60 Patients with endoscopically confirmed mild chronic disease were recruited. A structured interview documented the baseline data. Then the patients were allocated to the verum or placebo group by handing out a numbered study package with the study substance for the daily intake at home. A single dose was 20 g, taken twice per day. After 30 days the participants were interviewed again., Results: After the intake phase the disease related symptoms were found improved in both groups, indicating a strong placebo effect. However, the pain load reduction in the Caricol®-Gastro group was significantly larger (p=0.048)., Discussion: Due to the inherent biological activities of ingredients of papaya and of oats and their known effects (anti-inflammatory, epithelial integrity), the observed beneficial effects may be owed to the constituents synergisms to reduce chronic inflammation. We conclude, that the regular intake is a safe add on therapeutic option for patients with chronic gastritis to support standard medical care.

Typically, before and after surgical correction faces are assessed on still images by surgeons, orthodontists, the patients, and family members. We hypothesized that judgment of faces in motion and by naïve raters may closer reflect the impact on patients' real life, and the treatment impact on e.g. career chances. Therefore we assessed faces from dysgnathic patients (Class II, III and Laterognathia) on video clips. Class I faces served as anchor and controls. Each patient's face was assessed twice before and after treatment in changing sequence, by 155 naïve raters with similar age to the patients. The raters provided independent estimates on aesthetic trait pairs like ugly /beautiful, and personality trait pairs like dominant /flexible. Furthermore the perception of attractiveness, intelligence, health, the persons' erotic aura, faithfulness, and five additional items were rated. We estimated the significance of the perceived treatment related differences and the respective effect size by general linear models for repeated measures. The obtained results were comparable to our previous rating on still images. There was an overall trend, that faces in video clips are rated along common stereotypes to a lesser extent than photographs. We observed significant class differences and treatment related changes of most aesthetic traits (e.g. beauty, attractiveness), these were comparable to intelligence, erotic aura and to some extend healthy appearance. While some personality traits (e.g. faithfulness) did not differ between the classes and between baseline and after treatment, we found that the intervention significantly and effectively altered the perception of the personality trait self-confidence. The effect size was highest in Class III patients, smallest in Class II patients, and in between for patients with Laterognathia. All dysgnathic patients benefitted from orthognathic surgery. We conclude that motion can mitigate marked stereotypes but does not entirely offset the mostly negative perception of dysgnathic faces.

Background: A common pathomechanism involved in many degenerative manifestations of non-communicable diseases is nitrosative stress, giving rise to a chronic insidious inflammation causing silent inflammation at a cellular level. The release of nitric oxide inhibits multiple enzyme reactions with reduced oxidative phosphorylation and mitochondrial ATP depletion., Material and Methods: We hypothesized that enzyme-inhibition can be alleviated by micronutrient supply, and studied laboratory parameters associated with nitrosative stress (nitrotyrosine, mitochondrial activity) after a micronutrient supplementation (a multivitamin mineral and trace element formulation as verum: LaVita®) and a placebo in healthy volunteers (n=150) for six months., Results: Mean nitrotyrosine levels dropped significantly after 3 month in the verum and placebo group, whereas mitochondrial activity increased after three month in the verum group (p=0,087), but not in the placebo group (p=0,990). Ubiquinone - an essential ingredient for mitochondrial function- increased after six months in the verum group, but not after placebo consumption (p=0,001). Serum zinc and cellular zinc increased steadily after 3 and 6 month verum intake (p<0,001). As the enzyme superoxide dismutase (SOD) is mainly involved in the formation of nitrosative stress (peroxides) we measured the activity, and found significant differences in the placebo and verum group after 3 and 6 month (p=0,050 and p=0,003 respectively)., Conclusion: We conclude that a balanced combination of vital nutrients may reduce nitrosative stress and silent inflammation, and consequently the risk for various forms of degenerative diseases.

Objectives: Neurotransmitters regulate mood, attention vigilance and other clinical symptoms linked with depression. Various medications ameliorate symptoms of depression and mood disorders by interference with the serotonic metabolism. Serotonin metabolism depends on nutritional cofactors such as pyridoxin together with essential mineral and trace elements. Both, inflammation and metabolic conditions seem to affect the bioavailability of serotonin crucially. We hypothesized that serotonin supply depends on relevant gastrointestinal precursor absorption and on the availability of nutritive antiinflammatory cofactors., Methods: We performed a randomized placebo controlled clinical trial in healthy participants to study the bioavailabilty of ingredients of the multivitamin and trace element LaVita' in a prospective randomized placebo controlled double blind trial to establish the mood ameliorating potential. Serotonin and its precursor tryptophan were measured in dry blood samples. Serum parameters like chromium and zinc, as well as vitamin D, vitamin B3 and B6 were determined before intake, and after 3 months and 6 months consumption of the test substances., Results: After 3 months a slight increase of tryptophan (p=0.059) and a significant increase of serum serotonin (p < 0.013) was observed in the verum group. After 6 months the verum group showed a highly significant mean increase in niacin (p < 0.001) and the cofactors of serotonin metabolism pyridoxin (p=0.03), chromium p < 0.01), and zinc (p < 0.001). Serotonin levels droped after 6 months indicating a low risk for overdosing., Conclusion: We conclude that a continuous supply with ingredients from the natural source compound LaVita' may contribute to mood improving neurotransmitter activity.

Objectives: Neurotoxic metabolites and oxidative and nitrosative stress reactions play a crucial role in the pathways leading to neuronal cell death and neurodegeneration. The bioavailability of the many antioxidant ingredients a vitamin and trace element composition was investigated, to reveal the neuroprotective (preventive) potential of the composition., Methods: We recruited 159 healthy volunteers, assigned them randomly and double blind to a placebo and verum group. Physicians excluded volunteers with severe chronic diseases or interfeering medications. 142 participants finished the six month trial. Laboratory parameters were determined 1) before participation, and 2) after three and 3) six months. We confirmed the bioavailability of ingredients, and determined metabolic parameters associated with the integrity of the blood brain barrier, mitochondrial deficiency (Q 10), neurodegeneration (homocystein), and antioxidative capacity (e.g. lipidperoxidation), and superoxiddismutase activity., Results: Starting from baseleine, after three months neuroprotective ingredients increased within their physiological borders, folic acid (p<0.003), pyridoxin (p<0.001), cobalamin (p=0.001), and the fat soluble vitamin tocopherol (p<0.001). In parallel, homocytein decreased after 3 and 6 months (p<0.001, and p<0.025, respectively). Other paramters like zinc reacted slower, significant changes were observed only after 6 months., Conclusion: The observed metabolic changes and alteration of the oxidative status after 3 and six month of regular intake underlines the compositions' potential to ameliorate neurodegenerative processes. We conclude that the subsitution of vitamins and trace-elements with natural source in a proper manner may be effective for neuroprotection in healthy population.

Background: Many Vitamins and minerals for dietary supplements lack a standard scientific and regulatory definition that accurately reflects the bioavailabilities in humans. Especially the bioavailability of natural compounds in complex mixtures, where the different ingredients may interfere with each other, is unknown., Methods: To learn more about the bioavailability of the ingredients in the complex compound LaVita® we examined blood levels of subjects, who ingested the multivitamin and trace element composition for 6 month continuously. Blood samples for the analysis of the ingredients were taken before, during, and after administration., Results: Our data indicated a significant increase of most ingredients after 3 month, and additional three months, except for Vitamin (B9 Folic acid). The semivitamins Q10 and carnitine increased in the first 3 month (both p<0.001). While carnitine dropped during the second term, Q10 levels increased further slowly. After three months a significant increase was observed for iron (serum p=0.039; blood cells p=0.025), Selenium (serum p=0.048; cells p=0.006), and chromium (serum p=0.029). Zinc - known to interfere with the iron resorption - increased slowly in the first term of 3 months, but was raised significantly after 6 months (serum and blood cells, each p<0.001). The Copper/Zink ratio dropped accordingly (p<0.001)., Conclusion: We conclude that resorption interference between specific ingredients, and after resorption redistribution of specific ingredients to various tissue compartments precludes a linear increase of the respective serum parameters. We observed no deleterious resorption competitions for individual compounds. No parameter reached critical levels. We conclude that the test substance (LaVita®) is a sufficiently safe composite for long term consumption.

Objective: Papaya (Carica papaya L.) is used as a natural remedy in abnormal digestion in tropical and industrialized countries. Besides this wide distribution little evidence has been produced with reference to its physiological effect in humans and the proof of efficacy. Former clinical observations had revealed positive effects for patients with constipation, heartburn, and symptoms of irritable bowel syndrome (IBS) after eating papaya preparations. In line with these former positive clinical observations, we studied the clinical effects of the papaya preparation Caricol® in a double blind placebo controlled study design., Methods: In this study the participants were volunteers, with chronic (prevailing) indigestions and dysfunctions in the gastrointestinal tract. During the trial the intake of the substance of intent and placebo was 20 ml daily for 40 days. The endpoints were the frequency of 22 symptoms recorded before and after the documented intake recorded by questionnaire., Results: The symptoms "Constipation", "Bloating", and "Heartburn" were defined as primary and frequency of "painful (straining) bowel movements" as secondary endpoint. The participation ended after the intake period within two days ("early returnees"). Wash out effects were observed in "late returnees", who returned with a delay of 8.6 (±5.95 days). In the verum group early returnees revealed statistically significant improvements of the symptoms "constipation" and "bloating". The analysis of "heartburn" felt short of significant improvement because of the small number of included cases with this criteria (N=13, p=0.114). None of the significant benefits were observed after the washout phase., Conclusion: We conclude from these results, that the papaya preparation (Caricol®) contributes to the maintenance of digestive tract physiology. It ameliorates various functional disturbances, like symptoms of IBS. The mechanism of this digestive tract physiology support is discussed.

Vasoactive intestinal peptide (VIP) conveys various physiological effects in the digestive tract, nervous and cardiovascular system, airways, reproductive system, endocrine system, and more. A family of specific membrane bound receptors, termed VPAC1, VPAC2, and PAC1, bind VIP and trigger the effects. Many of them are of clinical interest. To date more than two thousand publications suggest the use of VIP in diseases like asthma, erectile dysfunction, blood pressure regulation, inflammation, endocrinology, tumours, etc. Despite this considerable potential, the peptide is not regularly used in clinical settings. A key problem is the short half life of inhaled or systemically administered VIP due to rapid enzymatic degradation. This shortcomings could be overcome with stable derivates or improved pharmacokinetics. A promising strategy is to use biocompatible and degradable depots, to protect the peptide from early degradation and allow for controlled release. This review focuses on aspects of clinical applications of VIP and the idea to use formulations based on biodegradable particles, to constitute a dispersible VIP-depot. Smart particle systems protect the peptide from early degradation, and assist the sustainable cell targeting with VIP for therapeutic or imaging purposes.

Nanomedicine is an emerging field with great opportunities to improve the treatment of diseases which are currently not curable. Pulmonary arterial hypertension (PAH) is one of these diseases treatable by inhalation of medicines that provide novel depots for drugs with short pharmacological half-lives to improve the quality of life for patients. In this context, nanostructured drug delivery systems such as liposomes and polymeric nanoparticles are depot forms that can also act as penetration enhancers and solubilizers of drugs. The pulmonary use of these drug carriers will improve the therapeutic effect of potent drugs that are currently not fully applicable. This review focuses on the design and characterization of drug delivery systems with the potential to improve the treatment options for hypertonic conditions (like PAH). Liposomes as well as polymeric nanoparticles based on lactic acid, proticles and nanocrystalline drugs have good potential to be developed toward clinical use. Preparation methods and characterization techniques of nanoparticles such as light scattering or microscopy are provided., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Purpose: To investigate whether or not low intensity radio frequency electromagnetic field exposure (RF-EME) associated with mobile phone use can affect human cells, we used a sensitive proteome analysis method to study changes in protein synthesis in cultured human cells., Methods: Four different cell kinds were exposed to 2 W/kg specific absorption rate in medium containing 35S-methionine/cysteine, and autoradiography of 2D gel spots was used to measure the increased synthesis of individual proteins., Results: While short-term RF-EME did not significantly alter the proteome, an 8-h exposure caused a significant increase in protein synthesis in Jurkat T-cells and human fibroblasts, and to a lesser extent in activated primary human mononuclear cells. Quiescent (metabolically inactive) mononuclear cells, did not detectably respond to RF-EME. Since RF exposure induced a temperature increase of less than 0.15 degrees C, we suggest that the observed cellular response is a so called "athermal" effect of RF-EME., Conclusion: Our finding of an association between metabolic activity and the observed cellular reaction to low intensity RF-EME may reconcile conflicting results of previous studies. We further postulate that the observed increased protein synthesis reflects an increased rate of protein turnover stemming from protein folding problems caused by the interference of radio-frequency electromagnetic fields with hydrogen bonds. Our observations do not directly imply a health risk. However, vis-a-vis a synopsis of reports on cells stress and DNA breaks, after short and longer exposure, on active and inactive cells, our findings may contribute to the re-evaluation of previous reports.

The receptors for vasoactive intestinal peptide (VIP), VPAC1-, VPAC2-, and PAC1-receptor are overexpressed by various tumor cells. VIP can target these receptors and transport conjugates into the cell. However, the use of VIP for tumor cell targeting is hampered by the peptides short half-lives due to enzymatic degradation. Because protamine-based nanoparticles (proticles) protect the peptide and serve as peptide depot, we explored the potential of proticles as carrier for VIP-conjugated molecules. The VIP-loaded proticles were stable as shown by Fluorescence Correlation Spectroscopy. With Confocal Laser Scanning Microscopy, we observed VIP-loaded proticles to specifically target the tumor cells. The cell binding triggered the substance release and conjugate internalization of VIP-Cy3 in vitro and ex vivo by human tumors. We observed VIP releasing proticle depots distributed in rat tissue and human tumors. Our findings warrant further studies to explore the proticles potential to enable peptide-mediated targeting for in vivo and clinical applications.

Vasoactive intestinal peptide (VIP) is one of the most abundant molecules found in the respiratory tract. Due to its anti-inflammatory and bronchodilatatory properties, it has been proposed as a novel treatment for chronic obstructive pulmonary disease (COPD). The actions of VIP are mediated via three different G-protein-coupled receptors (VPAC1, VPAC2 and PAC1) which are expressed in the respiratory tract and on immunocompetent cells including macrophages. Alveolar macrophages (AM) are key players in the pathogenesis of COPD and contribute to the severity and progression of the disease. While VPAC1 has been reported to be elevated in subepithelial cells in smokers with chronic bronchitis, little is known about VPAC expression of AM in COPD patients. AM from COPD patients show a strong VPAC1 expression which exceeds VPAC2. A similar receptor expression pattern was also observed in lipopolysaccharide (LPS)-activated monocyte-derived macrophages (MDM) from healthy volunteers and COPD patients. VIP has been shown to down-regulate interleukin 8 (IL-8) secretion significantly in MDM after LPS stimulation. The response to VIP was similar in MDM from COPD patients and healthy volunteers. Our results indicate that VPAC1 up-regulation in macrophages is a common mechanism in response to acute and chronic pro-inflammatory stimuli. Although VPAC1 up-regulation is dominant, both receptor subtypes are necessary for optimal anti-inflammatory signaling. The high VPAC1 expression in AM may reflect the chronic pro-inflammatory environment found in the lung of COPD patients. Treatment with VIP may help to decrease the chronic inflammation in the lung of COPD patients., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Many traditional healing plants successfully passed several hundred years of empirical testing against specific diseases and thereby demonstrating that they are well tolerated in humans. Although quite a few ethno-pharmacological plants are applied against a variety of conditions there are still numerous plants that have not been cross-tested in diseases apart from the traditional applications. Herein we demonstrate the anti-neoplastic potential of two healing plants used by the Maya of the Guatemala/Belize area against severe inflammatory conditions such as neuritis, rheumatism, arthritis, coughs, bruises and tumours. Phlebodium decumanum and Pluchea odorata were collected, dried and freeze dried, and extracted with five solvents of increasing polarity. We tested HL-60 and MCF-7 cells, the inhibition of proliferation and the induction of cell death were investigated as hallmark endpoints to measure the efficiency of anti-cancer drugs. Western blot and FACS analyses elucidated the underlying mechanisms. While extracts of P. decumanum showed only moderate anti-cancer activity and were therefore not further analysed, particularly the dichloromethane extract of P. odorata inhibited the cell cycle in G2-M which correlated with the activation of checkpoint kinase 2, and down-regulation of Cdc25A and cyclin D1 as well as inactivation of Erk1/2. In HL-60 and MCF-7 cells this extract was a very strong inducer of cell death activating caspase-3 followed by PARP signature type cleavage. The initiating death trigger was likely the stabilization of microtubules monitored by the rapid acetylation of alpha-tubulin, which was even more pronounced than that triggered by taxol. The dichloromethane extract of P. odorata contains apolar constituents which inhibit inflammatory responses and exhibit anti-cancer activity. The strong proapoptotic potential warrants further bioassay-guided fractionation to discover and test the active principle(s).

Drug delivery of protein and peptide-based drugs, which represent a growing and important therapeutic class, is hampered by these drugs' very short half-lives. High susceptibility towards enzymatic degradation necessitates frequent drug administration followed by poor adherence to therapy. Among these drugs is vasoactive intestinal peptide (VIP), a potent systemic and pulmonary vasodilator, which is a promising drug for the treatment of idiopathic pulmonary arterial hypertension (IPAH). Encapsulation of VIP into the nanoparticle matrix of biodegradable protamine-oligonucleotide nanoparticles (proticles) protects the peptide against rapid enzymatic degradation. Additionally, the nanoparticle matrix will be able to sustain drug release. Proticles consist of 18mer non-sense oligonucleotides and protamine, a polycationic arginine-rich peptide. VIP encapsulation occurs during self-assembly of the components. Within the present study, we evaluate nanoparticle size (hydrodynamic diameter) and zeta potential of VIP-loaded proticles as well as encapsulation efficiency and VIP release. Further, the pharmacological VIP response of "encapsulated VIP" is investigated using an ex vivo lung arterial model system. We found satisfying encapsulation efficiency (up to 80%), VIP release (77-87%), and an appropriate nanoparticle size (177-251 nm). Investigations on rat pulmonary arteries showed a modified VIP response of proticle-associated VIP. We noted differences in the profile of artery relaxation where VIP proticles lead to a 20-30% lower relaxation maximum than aqueous VIP solutions followed by prolonged vasodilatation. Our data indicate that proticles could be a feasible drug delivery system for a pulmonary VIP depot formulation.

Inhalation of vasoactive intestinal peptide (VIP) was suggested as promising treatment option of various lung diseases like asthma and pulmonary hypertension. However, the medical use of peptides is limited by their short half-life due to rapid enzymatic degradation in the airways. For that reason, we recently developed unilamellar nano-sized VIP-loaded liposomes (VLL). Now we investigated their applicability for inhalation purposes. After nebulisation by a mouthpiece ventilation inhaler we found the particle size almost unaffected, being in a size range appropriate for bronchiolar deposition; we observed no peptide release due to nebulisation. The VIP release kinetics from VLL were tested by an ex vivo vasorelaxation model. Exposure to target organs revealed an immediate response, which was significantly retarded for VLL as compared to free VIP (p=0.001). Using vasorelaxation as endpoint, we observed a sustained release and an extended pharmacological effect compared to equimolar free VIP. The liposomes have the potential to improve VIP inhalation therapy by providing a "dispersible peptide depot" in the bronchi. Thereby, the release of VIP from liposomes may be triggered by exposure to cells, i.e. directly by ligand-receptor interactions.

The endothelium and its interaction with smooth muscle play a central role in the local control of the pulmonary vasculature, and endothelial dysfunction is thought to contribute to pulmonary hypertension and chronic obstructive pulmonary disease. Vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide, relaxes the rat pulmonary artery, but there is controversy as to whether or not this action of VIP depends on the endothelium. The aim of this study, therefore, was to investigate the role of the endothelium and nitric oxide (NO), the major endothelium-derived relaxing factor, in the dilator action of VIP on the rat isolated pulmonary artery. Pulmonary artery preparations pre-contracted by the alpha(1)-adrenoceptor agonist L-phenylephrine were relaxed by VIP (0.003-1 microM) and acetylcholine (0.003-10 microM) in a concentration-dependent manner. Mechanical removal of the endothelium reduced the maximal response to VIP by about 50% and practically abolished the response to acetylcholine. Inhibition of NO synthesis by N(omega)-nitro-L-arginine methyl ester (0.5 mM) had a similar effect, abolishing the vasorelaxation caused by acetylcholine and attenuating the vasorelaxation caused by VIP by about 50%. From these data it is concluded that the relaxant action of VIP on the rat isolated pulmonary artery depends in part on the presence of the endothelium and that this part is mediated by endothelial NO.

A polymer-grafted liposomal formulation that has the potential to be developed for aerosolic pulmonary delivery of vasoactive intestinal peptide (VIP), a potent vasodilatory neuropeptide, is described. As VIP is prone to rapid proteolytic degradation in the microenvironment of the lung a proper delivery system is required to increase the half-life and bioavailability of the peptide. Here we investigate structural parameters of unilamellar liposomes composed of palmitoyl-oleoyl-phosphatidylcholine, lyso-stearyl-phosphatidylglycerol and distearyl-phosphatidyl-ethanolamine covalently linked to polyethylene glycol 2000, and report on VIP-lipid interaction mechanisms. We found that the cationic VIP is efficiently entrapped by the negatively charged spherical liposomes and becomes converted to an amphipathic alpha-helix. By fluorescence spectroscopy using single Trp-modified VIP we could show that VIP is closely associated to the membrane. Our data suggest that the N-terminal random-coiled domain is embedded in the interfacial headgroup region of the phospholipid bilayer. By doing so, neither the bilayer thickness of the lipid membrane nor the mobility of the phospholipid acyl chains are affected as shown by small angle X-ray scattering and electron spin resonance spectroscopy. Finally, in an ex vivo lung arterial model system we found that liposomal-associated VIP is recognized by its receptors to induce vasodilatory effects with comparable high relaxation efficiency as free VIP but with a significantly retarded dilatation kinetics. In conclusion, we have designed and characterized a liposomal formulation that is qualified to entrap biologically active VIP and displays structural features to be considered for delivery of VIP to the lung.

Pulmonary arterial hypertension is a progressive disease, characterised by increased proliferation of pulmonary artery smooth muscle cells, vasoconstriction and remodelling of the vascular wall leading to right heart failure and death. The idiopathic form is rare (idiopathic arterial primary hypertension (IPAH); formerly PPH, MIM# 178600). Our group correlated a deficiency in vasoactive intestinal peptide (VIP; MIM# 192320) levels in serum and lung tissue with the pathogenesis of IPAH. The aim of this study was to investigate the relevance of genetic alterations in VIP to the development of IPAH. We screened 10 patients (age 4-66 years) for alterations in the coding, the noncoding regions and the enhancer region of the VIP gene by direct sequencing. In eight of 10 patients, we found alterations compared to the wild-type sequence. We detected nine alterations. In the noncoding regions, eight alterations were in the introns 1, 2, 3 and 4 (g.448G>A g.501C>T g.764T>C g.2267A>T g.2390C>T g.3144T>C g.3912A>G g.4857A>G). In the coding regions, a single alteration in the 3' untranslated region in exon 7 (g.8129T>C) was observed in five patients. It appeared in 46% of the control group. The frequency of this alteration in the coding region of the VIP gene could therefore not be correlated with the appearance of IPAH. Apart from the importance of VIP signalling, genetic and/or environmental modifiers might therefore contribute to the development and perpetuation of the disease.

The replacement of endothelium by endothelial progenitor cells (EPCs) for therapeutic use in order to ameliorate the vascular status of ischemic organs is now in the focus of vascular research. The aim of our studies was to investigate whether EPCs derived from peripheral blood mononuclear cells (PBMNCs-derived EPCs) or EPCs propagated from CD34(+) hematopoietic stem cells (HSCs-derived EPCs), both isolated from human cord blood, are able to differentiate into early mature endothelial cells (ECs) under certain in vitro conditions. We characterized both cell populations by flow cytometry, phase contrast microscopy, fluorescence microscopy and confocal laser scanning microscopy as well as ultrastructurally using transmission and scanning electron microscopy. While PBMNCs gave rise to clusters of spindle-like EPCs after few days but did not further mature under in vitro conditions, mature ECs could only be successfully propagated from a starting population of isolated HSCs. Both, PBMNCs- and HSCs-derived EPCs, took up Dil-labeled acetylated low density lipoprotein (Dil-Ac-LDL) and could be positively stained for CD31, CD105, the vascular endothelial growth factor receptor 2 (VEGFR-2, KDR) and ulex europaeus agglutinin 1 (UEA-1) at the cell surface. EPC showed surface expression of CD54 and CD106. However, only a small portion of HSCs-derived EPCs was positive for CD54 but negative for CD106. Intracellular staining for von Willebrand factor (vWF) provided a homogenous stain in PBMNC-derived EPCs while in HSCs-derived EPCs, during cultivation for 2-3 weeks, more and more a typical punctuated staining pattern related to Weibel-Palade bodies (WPBs) was visible. By phase contrast and scanning electron microscopy, an arrangement of PBMNCs-derived EPCs in cord-like structures could be demonstrated. In these formations, cells showed parallel alignment but exhibited only few cell contacts. Well-developed WPBs could never be found in PBMNCs-derived EPCs. In contrast, differentiating HSCs-derived EPCs developed adherence junctions, interdigitating junctions as well as syndesmos. During maturation, spindle-like cell types appeared with abundant WPBs as well as cobblestone-like cell types with a fewer content of these organelles. WPBs, in the spindle-like cell types displayed conspicuous shapes and were concentrated in close proximity to mitochondria-rich areas. HSCs-derived EPCs exhibited signs of high synthetic activity such as a well-developed rough endoplasmic reticulum (RER) and multiple Golgi complexes. In the trans-Golgi network (TGN), close to the Golgi complex, a new formation of WPBs could be observed. These morphological features correlated well with a high growing capacity. Although it was not possible to demonstrate the complete differentiation line from HSCs to early matured ECs by immunologic markers because of the limited number of cells available for such investigations, distinct morphologic maturation stages could be shown at light and electron microscopical levels. In conclusion, the study presented here characterizes not only the different cell populations involved in the differentiation of early EPCs into mature ECs but also the transition stage where the maturation step takes place by demonstration of the new formation of WPBs. In this respect, these investigations provide new insights into the in vitro differentiation which could have some in vivo correlation.

Vasoactive intestinal peptide (VIP) is a potent vasorelaxing peptide that plays a role in lung physiology and possibly in pulmonary hypertension. We investigated the turnover of the VIP receptors on rat pulmonary arteries ex vivo. There was evidence for a fast receptor turnover in pulmonary arteries, which underlines the important role of VIP for the regulation of pulmonary circulation and pulmonary pathology.

We describe fluorescent-labeled peptide (FLP) studies on living cells. The new technique is nonradioactive and it allows monitoring of the binding and internalization of Vasoactive Intestinal Peptide (VIP) in VIP receptor-expressing cells. The technique is easy to perform and the observed reaction is peptide sequence specific.

There is only limited morphologic information on long-term alterations and neurotransmitter changes after perinatal asphyxia, and no long-term study showing neurodegeneration has been reported so far. We used an animal model for perinatal asphyxia well documented in the rat to investigate the guinea pig as a species highly mature at birth. Cesarean section was performed on full-term pregnant guinea pigs, and pups, still in membranes, were placed into a water bath at 37 degrees C for asphyxia periods from 2 to 4 min. Thereafter pups were given to surrogate mothers and examined at 3 mo of age. We studied brain areas reported to be hypoxia-sensitive. Neurodegeneration was evaluated by fluoro-jade, neuronal loss by Nissl, reactive gliosis by glial fibrillary acidic protein staining, and differentiation by neuroendocrine-specific protein C immunoreactivity. We tested tyrosine hydroxylase, the vesicular monoamine transporter, and dopamine beta-hydroxylase, representing the monoaminergic system; the vesicular acetylcholine transporter; and the excitatory amino acid carrier 1. Neurodegeneration was evident in cerebellum, hippocampal area CA1, and hypothalamus, and neuronal loss could be observed in cerebellum and hypothalamus; gliosis was observed in cerebellum, hippocampus, hypothalamus, and parietal cortex; dedifferentiation was found in hypothalamus and striatum; and monoaminergic, cholinergic, and amino acidergic deficits were shown in several brain regions. The major finding of the present study was that neurodegeneration and dedifferentiation evolved in the guinea pig, a species highly mature at birth. The relevance of this contribution is that a simple animal model of perinatal asphyxia resembling the clinical situation of intrauterine hypoxia-ischemia and presenting with neurodegeneration was characterized.