Gudrun Ulrich-Merzenich

Synergy research: approaching a new generation of phytopharmaceuticals.

The longstanding, successful use of herbal drug combinations in traditional medicine makes it necessary to find a rationale for the pharmacological and therapeutic superiority of many of them in comparison to isolated single constituents. This review describes many examples of how modern molecular-biological methods (including new genomic technologies) can enable us to understand the various synergistic mechanisms underlying these effects. Synergistic effects can be produced if the constituents of an extract affect different targets or interact with one another in order to improve the solubility and thereby enhance the bioavailability of one or several substances of an extract. A special synergy effect can occur when antibiotics are combined with an agent that antagonizes bacterial resistance mechanisms. The verification of real synergy effects can be achieved through detailed pharmacological investigations and by means of controlled clinical studies performed in comparison with synthetic reference drugs. All the new ongoing projects aim at the development of a new generation of phytopharmaceuticals which can be used alone or in combination with synthetic drugs or antibiotics. This new generation of phytopharmaceuticals could lend phytotherapy a new legitimacy and enable their use to treat diseases which have hitherto been treated using synthetic drugs alone.

Synergy research: vitamins and secondary plant components in the maintenance of the redox-homeostasis and in cell signaling.

The maintenance of the redox-homeostasis is an essential task of antioxidants. Reactive oxygen species (ROS) formed during oxidative stress can potentially damage the normal cellular functions and support pathological processes like atherosclerosis in vessels or malignant growth in other tissues, but also the aging process. However, recent findings link ROS also to cell survival and/or proliferation, which revolutionises the age-old dogmatic view of ROS being exclusively involved in cell damage and death. Low concentrations of hydrogenperoxide e.g. are involved in cell signaling and can activate mitogen-activated kinases (MAPK) to initiate cell growth. Nutritional antioxidants like vitamin C or E can promote endothelial cell growth, but can also inhibit growth of muscle cells, and influence MAPK. Thus, keeping the redox-homeostasis in a steady state especially in the context of tissue regeneration appears to be more important than previously known and seems to be a controlled synergistic action of antioxidants and ROS. The present review summarizes the properties and functions of ROS and nutritional antioxidants like the vitamins C and E, and polyphenols in redox-homeostasis. Their relevance in the treatment of various diseases is discussed in the context of a multitarget therapy with nutraceuticals and phytotherapeutic drugs.

Simultaneous isolation of endothelial and smooth muscle cells from human umbilical artery or vein and their growth response to low-density lipoproteins.

SummaryIn the pathogenesis of atherosclerosis the interplay of endothelial cells (ECs) and smooth muscle cells (SMCs) is disturbed. Oxidatively modified low-density lipoproteins (oxLDLs), important stimulators of atherosclerotic plaque formation in vessels, modify the growth response of both cell types. To compare growth responses of ECs and SMCs of the same vessel with oxLDLs, we developed a method to isolate both cell types from the vessel walls of umbilical cords by enzymatic digestion. The method further allowed the simultaneous isolation of venous and arterial cells from a single umbilical cord. In culture, venous ECs showed an elongated appearance compared with arterial ECs, whereas SMCs of artery and vein did not look different. Smooth muscle cells of both vessel types responded to oxLDLs (60 μg/ml) with an increase in their [3H]-thymidine incorporation into DNA. On the contrary, ECs of artery or vein decreased [3H]-thymidine incorporation and cell number in the presence of oxLDLs (60 μg/ml) of increasing oxidation grade. Thus, human umbilical SMCs and ECs of the same vessel show a disparate growth response toward oxLDLs. But the physiologically more relevant minimal oxLDLs did not decrease proliferation in venous ECs but only in arterial ECs. This difference in tolerance toward minimal oxLDLs should be taken into account while using venous or arterial ECs of umbilical cord for research in atherosclerosis. Further differences of venous and arterial ECs in tolerance toward minimal oxLDLs could be of clinical relevance for coronary artery bypass grafts.

The relevance of the bleeding severity in the treatment of acquired haemophilia – an update of a single-centre experience with 67 patients

Summary.  Acquired haemophilia (AH), an autoimmune disorder with clinical features ranging from harmless haematomas to life‐threatening bleedings, still has a mortality rate of up to 25%. Owing to its low frequency (1–4 × 106), standardized treatment protocols for its variable manifestations are not available. In case of prominent severe bleedings, the treatment should aim at rapid elimination of the antibody to protect patients from bleedings and on reinduction of long‐term immune tolerance. Clinical data, short‐ and long‐term treatment results of 67 patients diagnosed by our centre are presented. Patients were treated depending on their bleeding severity either by an immunosuppressive treatment alone, or in case of life‐threatening bleedings, by a combined protocol (modified Bonn–Malmö protocol, MBMP) consisting of antibody depletion through immunoadsorption, intravenous immunoglobulin treatment, immunosuppression and high‐dose factor VIII (FVIII) substitution. Mild bleedings occurred in two patients who were treated successfully alone by immunosuppression. Complete remission (CR) was achieved in 90% of the patients treated with MBMP (60). Of the six patients (10%) who achieved a partial remission (PR), four suffered from cancer. Mortality under MBMP was not seen. In contrast, five patients, in whom diagnosis of AH was delayed, experienced fatal outcome during surgical interventions before initiation of MBMP treatment. Prognosis in AH depends mainly on its prompt diagnosis. Treatment procedures should be adapted to bleeding severity and inhibitor titres. Under these conditions, AH is a potentially curable autoimmune disorder with an excellent prognosis.

Novel neurological and immunological targets for salicylate-based phytopharmaceuticals and for the anti-depressant imipramine.

Inflammatory processes are increasingly recognised to contribute to neurological and neuropsychatric disorders such as depression. Thus we investigated whether a standardized willow bark preparation (WB) which contains among other constituents salicin, the forerunner of non-steroidal antiphlogistic drugs, would have an effect in a standard model of depression, the forced swimming test (FST), compared to the antidepressant imipramine. Studies were accompanied by gene expression analyses. In order to allocate potential effects to the different constituents of WB, fractions of the extract with different compositions of salicyl alcohol derivative and polyphenols were also investigated. Male Sprague Dawley rats (n=12/group) were treated for 14 days (p.o.) with the WB preparation STW 33-I (group A) and its fractions (FR) (groups FR-B to E) in concentrations of 30 mg/kg. The FRs were characterized by a high content of flavone and chalcone glycosides (FR-B), flavonoid glycosides and salicyl alcohol derivatives (FR-C), salicin and related salicyl alcohol derivatives (FR-D) and proanthocyanidines (FR-E). The tricyclic antidepressant imipramine (20 mg/kg) (F) was used as positive control. The FST was performed on day 15. The cumulative immobility time was significantly (p<0.05) reduced in group A (36%), group FR-D (44%) and by imipramine (16%) compared to untreated controls. RNA was isolated from peripheral blood. RNA samples (group A, group FR-D, and imipramine) were further analysed by rat whole genome microarray (Agilent) in comparison to untreated controls. Quantitative PCR for selected genes was performed. Genes (>2 fold, p<0.01), affected by WB and/or FR-D and imipramine, included both inflammatory (e.g. IL-3, IL-10) and neurologically relevant targets. Common genes regulated by WB, FR-D and imipramine were GRIA 2 ↓, SRP54 ↓, CYP26B ↓, DNM1L ↑ and KITLG ↓. In addition, the hippocampus of rats treated (27 d) with WB (15-60 mg/kg WB) or imipramine (15 mg/kg bw) showed a slower serotonin turnover (5-hydroxyindol acetic acid/serotonin (p<0.05)) depending on the dosage. Thus WB (30 mg/kg), its ethanolic fraction rich in salicyl alcohol derivatives (FR-D) (30 mg/kg) and imipramine, by being effective in the FST, modulated known and new targets relevant for neuro- and immunofunctions in rats. These findings contribute to our understanding of the link between inflammation and neurological functions and may also support the scope for the development of co-medications from salicylate-containing phytopharmaceuticals as multicomponent mixtures with single component synthetic drugs.

Immunoadsorption in the treatment of Acquired Haemophilia

In acquired haemophilia (AH) healthy humans can suddenly develop severe bleeding due to autoantibodies (inhibitors) against clotting factors, especially factor VIII. The mortality rate of 21 % is considerable, and standardized treatment protocols have not been developed due to the low disease frequency (1-4 per million). Major goals of treatment are the control of bleeding events and rapid inhibitor elimination. Conventional treatment regimens induce immune tolerance via long-term immunosuppression with success rates between 52% and 82%. However, treatment related mortality can rise to 39%. Lack of complete remission, advanced age, underlying malignancies and infections related to immunosuppressive therapy are regarded as principal risk factors for death. The modified Bonn-Malmö Protocol (MBMP), an immune tolerance protocol consisting of antibody depletion through immunoadsorption, i.v. immunoglobulin treatment, immunosuppression and high dose FVIII supplementation, achieves rapid and safe control of acute bleeding. In the largest published single centre study of high risk patients with AH, we previously demonstrated that complete remission (CR) can be achieved in 88.5% of all patients (54/61) within a median time of 3.9 wks (range: 3.2-4.5 wks) and in 97% (54/56) of AH patients without cancer as an underlying condition. Those 5 patients, who suffered also from cancer, achieved partial remission (PR). Mortality or severe treatment-related side effects were not observed. This study confirmed that MBMP is a safe and effective treatment with a high curative potential for severe AH. However, the severity of bleeding, and therefore the cost-effectiveness of the approach, needs to be considered when initiating this treatment protocol.

The lectin-like oxidized low-density lipoprotein receptor-1 as therapeutic target for atherosclerosis, inflammatory conditions and longevity

Introduction: The lectin-like oxidized LDL receptor-1 (LOX-1) is a scavenger receptor and is regarded as a central element in the initiation of endothelial dysfunction and its further progression to atherosclerosis. Increasing numbers of studies suggest that therapeutic strategies to modulate LOX-1 will have a broad spectrum of applications ranging from cardiovascular diseases to longevity. Areas covered: The dual role of LOX-1 as a culprit molecule in the process of atherosclerosis and as a danger signal in various tissues is introduced. The structure of the receptor, its ligands and its modulation by known drugs, by natural products (e.g., statins, imipramine, salicylate-based drugs, procyanidins, curcumin) and by new strategies (antisenseRNA, miRNA, pyrrole-imidazol-polyamides, LOX-1 antibodies, lipid apheresis) are described. Expert opinion: Therapeutic approaches via transcript regulation, allowing a modulation of LOX-1, may be an easier and safer strategy than a blockade of the receptor. Considering the wide distribution of LOX-1 on different tissues, research on the mechanisms of LOX-1 modulation by drugs and natural products applying “omic”-technologies will not only allow a better understanding of the role of LOX-1 in the processes of atherosclerosis, inflammation and longevity but also support the development of specific LOX-1 modulators, avoiding the initiation of molecular mechanisms which lead to adverse events.

Extracorporeal Treatment for the Acute und Long-Term Outcome of Patients with Life-Threatening Acquired Hemophilia

Objectives: In acquired hemophilia (AH), autoantibodies (inhibitors) impede blood coagulation factors leading to severe bleedings. Cornerstones of a successful treatment are the control of bleeding and an eradication of autoantibodies. The present study is an update of our previous documentation of the treatment of high-titer AH patients with severe life-threatening bleeding undergoing the modified Bonn-Malmö-Protocol (MBMP). Methods: 64 AH patients were treated by a standard combination protocol (MBMP) consisting of antibody depletion through immunoadsorption, i.v. immunoglobulin, immunosuppression, and high-dose FVIII substitution. They underwent a long-term follow-up. Results: Primary study endpoints loss of detection of the activity of the inhibitor and FVIII recovery ≥ 5% were reached in a median time of 3 days (95% CI: 2.6–3.4 days), the median time of FVIII substitution was 13 days (95% CI 10.6–15.3 days), and the median time of immunoadsorption was 16 days (95% CI 13–18.9 days). In 5 patients the AH occurred as paraneoplastic syndrome, and partial remission was achieved. Relapses without bleeding event occurred only in second-line MBMP. Those responded excellently to short time treatment. Overall patients remained in remission over a median follow-up time of 8 years. Conclusion: Except for paraneoplastic AH, MBMP-treated patients have a remarkable prognosis which is confirmed by long-term follow-up with a complete response rate of 93% (53/57) in the first year post MBMP and 100% during long-term follow-up. These outcome in life-threatening AH is unique and until now not achievable via other treatment schedules. In life-threatening bleedings physicians should take into account MBMP as a first line treatment.

Treatment of Factor VIII Inhibitors with Selective IgG Immunoadsorption – a Single Center Experience in 50 Patients with Acquired Hemophilia*

Background: Acquired hemophilia (AH) is a potentially lifethreatening disease in which severe bleeding events lead to a mortality of up to 22%. In AH autoantibodies of the IgG subtype inactivate clotting factors. Although the incidence of this disease is low (1-3 per 106), the treatment cost can be immense due to long-term clotting factor substitution. The treatment should aim at a rapid and permanent elimination of autoantibodies and the induction of a new immune tolerance to prevent further bleedings. Patients and Methods: 50 high-titer (>5 Bethesda Units(BU)/ml) AH patients were treated by the following protocol: i) inhibitor elimination via IgG immunoadsorption, ii) immunosuppression, iii) i.v. immunoglobulin, and iv) high-dose factor VIII substitution. Follow-up time ranged between 12 months and 7 years. Results: A complete remission was achieved in 46 of 50 patients (92%). Neither bleeding nor therapy-associated mortality occurred after initiation of treatment. The median time to reach undetectable inhibitor levels was 3 days (95% CI 3-6 days), coagulation factors were given at a median of 15 days (95% CI 12-18 days). The median treatment duration was 17 days (95% CI, 14-20 days). Conclusions: IgG immunoadsorption allows for a fast and permanent inhibitor elimination, being the basis of the high immunomodulatory potency of our protocol which results in long lasting complete remissions in 92% of our patients.

Xyloglucan from Tropaeolum majus Seeds Induces Cellular Differentiation of Human Keratinocytes by Inhibition of EGFR Phosphorylation and Decreased Activity of Transcription Factor CREB.

Xyloglucan XG (molecular weight 462 kDa, 1,4-/1,4,6-(pGlc) linked backbone, side chains of 1-pXyl, 1,2-pXyl, 1-p-Gal) was isolated from the seeds of Tropaeolum majus. XG (100 μg/mL) induced terminal cellular differentiation of human keratinocytes, as demonstrated by immunofluorescence staining and Western blot using cytokeratin 10 and involucrin as marker proteins. Differentiation was also induced by XG-derived oligosaccharides (degree of polymerization 7-9). Quantitative real-time polymerase chain reaction (qPCR) revealed the induction of gene expression of typical differentiation markers (cytokeratin, filaggrin, involucrin, loricrin, transglutaminase) in a time-dependent manner. Whole human genome microarray indicated that most of upregulated genes were related to differentiation processes. Microarray findings on selected genes were subsequently confirmed by qPCR. For identification of the molecular target of xyloglucan PAGE of keratinocyte membrane preparations was performed, followed by blotting with fluorescein isothiocyanate-labeled XG. XG interacting proteins were characterized by MS. Peptide fragments of epidermal growth factor receptor (EGFR) and integrin β4 were identified. Subsequent phospho-kinase array indicated that phosphorylation of EGFR and transcription factor cAMP response element-binding protein (CREB) was decreased in the XG-treated cells. Thus, the XG-induced differentiation of keratinocytes is proposed to be mediated by the inhibition of the phosphorylation of EGFR, leading to a dimished CREB activation, which is essential for the activation of cellular differentiation.