H. Zeitler

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.

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.

Reverse transcriptase—polymerase chain reaction (RT—PCR): a sensitive method to examine basic fibroblast growth factor-induced expression of the early growth response gene-1 (egr-1) in human umbilical arterial endothelial cells

Immediate-early genes are expressed upon growth and differentiation in a large variety of cells and species. In the present study we investigated the effect of basic fibroblast growth factor (bFGF) on early growth response gene-1 (egr-1)-mRNA expression in human umbilical arterial endothelial cells (HUAEC). The detection of this gene in HUAEC was performed by Northern blotting and by reverse transcriptase-polymerase chain reaction (RT-PCR). For RT-PCR specific primers for egr-1 and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) were constructed and PCR conditions were optimized. bFGF induced a time- and concentration-dependent increase of egr-1 expression. Maximal expression occurred within 30 min of stimulation with bFGF at a concentration of 50-100 ng ml-1. RT-PCR gave highly reproducible and specific results. The comparison of both methods showed comparable results but a higher sensitivity for RT-PCR in detecting the egr-1 mRNA. RT-PCR is an excellent method for detecting the expression of egr-1 mRNA in HUAEC.

Increased frequency of the CTLA-4 49 A/G polymorphism in patients with acquired haemophilia A compared to healthy controls.

Summary.  Acquired haemophilia (AH) is an autoimmune disorder characterized by autoantibodies against endogenous factor VIII (FVIII). Half of the patients present with an underlying disease known to cause the FVIII autoantibodies whereas in the other half the disease is of idiopathic nature. Recently, it has been shown that variants of the polymorphic cytotoxic T lymphocyte antigen‐4 (CTLA‐4) gene are associated with autoimmune diseases and also represent a risk factor for inhibitor formation in inherited haemophilia A. In the present study, we investigated whether CTLA‐4 variants also play a role in the pathogenesis of AH. Therefore, we analyzed three single nucleotide polymorphisms (SNPs) of the CTLA‐4 gene (‐318 C/T, +49 A/G and CT60 A/G) in 57 AH patients and 98 controls. The CTLA‐4 + 49 G allele occurred with a significantly higher frequency in patients with AH compared with controls [odds ratio (OR) = 2.17, 95% confidence interval (CI): 1.36–3.48, P = 0.001]. This effect was mainly caused by a higher frequency of the 49 G allele in female patients (OR = 5.1, 95% CI: 1.76–15.02, P = 0.002), whereas in males the frequencies were not significantly different (OR = 1.4, P = 0.5). A higher frequency of the G allele was also observed in the subcohort with AH and underlying autoimmune disease (OR = 3.1, P = 0.04). Our observations of a higher frequency of the CTLA‐4 + 49 A/G SNP in AH patients are in concordance with findings in other autoimmune disorders. In conclusion, on the background of the CTLA‐4 gene polymorphism, further genetic and/or environmental factors might contribute to and finally trigger the clinical manifestation of AH.

Genetic markers in acquired haemophilia.

Summary.  Acquired haemophilia (AH) is a rare autoimmune bleeding disorder, which arises as a result of the spontaneous production of autoantibodies against endogenous factor VIII. The breakdown in immune tolerance is thought to be a result of a combination of genetic and environmental factors. Both human leucocyte antigen (HLA) and cytotoxic T lymphocyte antigen 4 (CTLA‐4) play an important role in the maintenance of peripheral T‐cell tolerance. A higher frequency of HLA class II alleles and single nucleotide polymorphisms of the CTLA‐4 gene have been observed in some autoimmune diseases and severe haemophilia A. In 57 patients with AH, significantly higher frequencies of the HLA class II alleles DRB*16 [odds ratio (OR) 10.2] and DQB1*0502 (OR 2.5) have been detected when compared with controls. The CTLA‐4 + 49 G allele has also presented with a significantly higher frequency in the same cohort of patients with AH (OR 2.17). This observation was mainly because of a higher frequency of the CTLA‐4 + 49 G allele in female patients. These findings suggest that immune response genes may contribute to the development of anti‐factor VIII autoantibodies in AH.

HLA genotype in patients with acquired haemophilia A

Summary.  Acquired haemophilia A (AH) is a rare bleeding disorder caused by an auto‐antibody to coagulation factor VIII. It is associated with various autoimmune diseases, pregnancy, cancer or drug ingestion; however, in 50% of patients, no underlying disorder is found. In the present study, we investigated the association of HLA class I (A, B and Cw) and class II (DRB1 and DQB1) alleles with AH in a cohort of 57 patients. While no association with any class I allele was detected, a significantly higher frequency of DRB1*16 [odds ratio (OR) 10.2, 95%CI: 5.32–19.57, P < 0.0001] and DQB1*0502 (OR 2.2, 95%CI: 1.12–4.54, P < 0.05) was observed. In contrast, the frequency of DRB1*15 and DQB1*0602 alleles was found to be decreased in patients with AH corresponding to an OR of 0.4 for both HLA loci. Upon comparing the frequencies of these alleles with those of patients with congenital haemophilia A with inhibitors, the data demonstrate that the high risk alleles in patients with AH DRB1*16 and DQB1*0502 are found to be low risk alleles in patients with congenital haemophilia A with inhibitors (OR 1.1 and 1.5 respectively). Conversely, the alleles that exhibit low risk in AH DRB1*15 and DQB1*0602 are found to be high risk for haemophilia A inhibitor patients (OR 2.2 and 3.7 respectively). The pathophysiological reason for this finding remains unknown. It might be speculated that the presence or absence of the FVIII antigen and the various ability of HLA molecules to present the FVIII antigen to the T‐cell receptor contribute to these findings.

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.