The role of variant alleles of the mannose-binding lectin in the inhibitor development in severe hemophilia A.

INTRODUCTION: The administration of FVIII leads to inhibitors in up to 30% of patients with hemophilia A (HA), the most severe treatment complication. FVIII-mannosylation fosters the presentation of FVIII to CD4+-T-lymphocytes. Mannose as primary ligand for the mannose-binding lectin (MBL) activates the lectin pathway of complement. MBL2 single nucleotide polymorphisms (SNPs) lead to low peripheral MBL concentrations that may hamper the removal of mannosylated FVIII. OBJECTIVE: Investigation of the association between the inhibitor development in hemophilia A and MBL2-SNPs. METHODS: In a case-control study the MBL2-SNPs in exon 1 at codons 52, 54 and 57 (C, B, D-Alleles respectively) were determined in 237 patients with severe hemophilia A with and without inhibitors to FVIII (119 vs 118). The association of MBL2-SNPs and the -308 G>A TNF-α-polymorphism with the presence of inhibitors were determined. RESULTS: In the inhibitor group higher frequencies of the B allele (codon 54) (OR: 1.77, P < 0.05) were present. Summarising the MBL2 SNPs (alleles B, C and D) as 0, the 0/0 type occurred only in the inhibitor group (frequencies: 0.08 vs 0, P = 0.003). Based on the genetic background a functional immune response phenotype was determined. 11.8% of patients with inhibitors were of the low MBL/high TNF-α phenotype vs 0.03% of the non-inhibitor patients (OR: 3.71). CONCLUSION: Data suggest an association of MBL2-SNPs alone or combined with the 308-TNF-α polymorphism in the inhibitor development. Investigations of components of all three complement pathways are required to comprehend their individual and overall contribution to the inhibitor development in HA.

Salicylate-based phytopharmaceuticals induce adaptive cytokine and chemokine network responses in human fibroblast cultures.

BACKGROUND: Cytokines and chemokines (CC) play a central role in immunoregulatory and inflammatory processes. Neutralising antibodies for single proinflammatory cytokines have developed into a powerful, though expensive and not always curative therapeutic strategy for severe diseases. Considering the redundancy of CC functions, network (N) rather than single target approaches are essential. Phytopharmaceuticals, common adjuvant therapies, are known modulators of a broad spectrum of CCs, but as complex mixtures with multiple targets they have not been systematically investigated. We investigated the effect of clinically established salicylate-based phytopharmaceuticals alone or in combination on CCNs under non-inflammatory and inflammatory conditions, using fibroblasts being a major source of cytokines in connective tissue diseases. METHODS: Synchronised human skin fibroblasts (HSKF) were treated for 6 h with standardised fluid plant extracts (E) of Populus tremula L. [end concentration: 0.06%, 0.1%], Solidago virgaurea L. [0.02%, 0.1%], Fraxinus excelsior L. [0.02%, 0.1%], an established combination of the three extracts-STW1 [0.05, 0.1%] and acetyl salicylic acid (ASA) [30 µg/ml], individually or in the presence of lipopolysaccharides (LPS) [10 µg/ml]. Cell lysates were profiled for 23 cytokines. Supernatants were investigated for IL-6 and IL-8 release (ELISA). Total RNA was isolated for gene-expression profiling. RESULTS: Under non-inflammatory conditions P. tremula E and ASA increased cellular proteins (P) IL-8 and IL-10; S. virgaurea E modulated IL-1α, IL-10, IL-15 and Groα (P). F. excelsior decreased IL-1α and IL-15 (P). The combination of the three extracts (STW1) modulated IL-1α, IL-3 and TNF-ß (P). LPS stimulation increased cellular IL-8, Groα, MCP-1 and RANTES (P) and increased the secretion of IL-6 and IL-8 into the medium. Under these inflammatory conditions F. excelsior reduced GMCSF, GCSF and RANTES. STW1 reduced IL-1α, IL-8, Groα, and MCP-1(P). Secretion of IL-8 and IL-6 was reduced by STW1 and ASA. Gene expression profiles supported non-additive CCN profiles. CONCLUSION: Salicylate based phytopharmaceuticals provoke cellular pro-and anti-inflammatory CCN responses under non-stress conditions, which adapt to anti-inflammatory responses after LPS-stimulation. CCN-profiles of the single extracts are not additives in combination. A simultaneous activation of cellular pro- and anti-inflammatory cytokines might heighten the immunological reactivity status of a cell.

Extracorporeal Treatment in Severe Hypertriglyceridemia-Induced Pancreatitis.

Plasmapheresis is a well-accepted treatment option in severe hypertriglyceridemia-induced pancreatitis (HTGP). The rationale behind this approach is the depletion of triglycerides and the reduction of inflammatory cytokines. The time span between onset of clinical symptoms and start of plasmapheresis might have an important impact on mortality. Hyperviscosity of patients' plasma represents another special challenge for the applied separation technology. The procedures can be performed either by centrifugal device (CFD) or membrane based (MBS) units. The present study reports the outcome of 10 patients suffering from HTG. The expected mortality of the collective was 25%. Plasmapheresis was started after an average 16.3 h (SD ± 6.7 h) after onset of symptoms. No mortality occurred. Apheresis was statistically equally effective with both devices. A median of 3 sessions reduced the TG level to normal and correlated with patients' improvement. During follow up, three patients developed a pancreatic pseudocyst requiring surgical intervention without further complication.

Immunoadsorption for pregnancy-associated severe acquired hemophilia.

Postpartum hemorrhage is a common cause of maternal mortality. Acquired hemophilia (AH) is a rare, life-threatening bleeding disorder induced by autoantibodies against coagulation factors (inhibitors). We report about eight patients with postpartum AH (out of 82). Seven AH patients with severe bleeding complications were treated by the "Modified Bonn-Malmö Protocol (MBMP)" which consists of inhibitor elimination via immunoadsorption (IA) in combination with immunosuppression and high-dose Factor VIII substitution. One patient was treated only by immunosuppression. Seven out of eight patients with severe AH and mean inhibitor titers (IT) of 118 BU/mL were referred to our center. They were severe cases with a median delay of diagnosis of 30.5 days (range 7-278 days). After a median of 3 IA sessions (range 3-5 days), no inhibitor was detectable. The factor substitution was discontinued after a median of 13 IA sessions (range 8-24 days) and IA was terminated after a median of 15 sessions (range 9-27 days). One less severe affected patient (IT: 2.1 BU/mL) received prednisolone (1.5 mg/kg BW) for 120 days. Complete remission was achieved in all patients with a median follow-up of 100 months (range 56-126 m). The delayed diagnosis of pregnancy-associated AH leads to a high bleeding risk with bleeding associated complications. Immunoadsorption offers an important treatment option in severe AH, enabling a fast reconstitution of the blood coagulation with a reduced time for the Factor VIII substitution and for immunosuppressive treatment. In cases of postpartum bleeding the diagnosis of AH should be routinely considered.

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.

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.

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.

Multiple benign symmetric lipomatosis--a differential diagnosis of obesity. Is there a rationale for fibrate treatment?

Multiple benign symmetric lipomatosis (MSL) is characterized by a rapid progression of multiple, symmetric nonencapsulated fat masses in the face, neck, and extremities. The lipomas are thought to be the result of defective brown adipose tissue (BAT). In up to 90% MSL is associated with chronic alcohol abuse. Prognosis depends on the concomitant presence of a neuropathy with a mortality of 25.8%. Therapeutic options are limited to alcohol abstinence and surgical interventions. We report here about a 53-year-old MSL patient who increased his body weight by 37 kg over 10 years. Multiple lipectomies were performed, but disease progressed. We treated him with fenofibrates (200 mg/day). Disease progression discontinued and circumferences of abdominal adipose tissue reduced. Fibrates, peroxisome proliferator-activated receptor alpha (PPAR alpha) agonists, are pleiotropic hypolipidemic drugs, and might have worked by suppression of protein expressions involved in the architecture of BAT keeping it in a quiescent state.

Aplicación de las tecnologías ómicas a la Fitoterapia / No disponible

La demostración de la eficacia de los preparados fitoterápicos y la determinación de sus mecanismos de acción son retos permanentes para la fitoterapia basada en la evidencia. La genómica, proteómica y metabolómica son tecnologías de alto rendimiento, que permiten detectar simultáneamente un gran número de proteínas/genes, así como relacionar mezclas complejas con efectos complejos en forma de perfiles de expresión génica/proteica. La descripción de perfiles de expresión específicos para un determinado preparado fitoterápico puede ser útil para su estandarización química y farmacológica, así como para la evaluación de su toxicidad. A largo plazo, pueden economizar los estudios de eficacia y de mecanismos de acción, y facilitar la investigación de extractos vegetales carentes de principio(s) activo(s) predominantes. Se ha descubierto que los perfiles de expresión génica inducidos por fármacos individuales o sus combinaciones pueden ser completamente diferentes. La aplicación de las tecnologías “ómicas” puede suponer un cambio de paradigma en cuanto a la aplicación de mezclas complejas en medicina y abrir nuevos campos como la fitogenómica, fitoproteómica y fitometabolómica (AU)

The proof of efficacy of phytopreparations and the determination of their mode of action are permanent challenges for an evidence-based phytotherapy. Genomics, proteomics and metabolomics are high-throughput technologies that allow the simultaneous detection of a high number of proteins/genes and have the potential to relate complex mixtures to complex effects in the form of gene/ protein expression profiles. The development of phytopreparation-specific expression profiles will be useful for its chemical and pharmacological standardization and its toxicological assessment. Over a long-term perspective they may economize the studies on efficacy and mode of action of phytomedicines and allow to investigate herbal extracts without prominent active principle(s). The application of genomics revealed already that the gene expression profiles induced by single drugs and their combinations can be entirely different. The application of the “-omic-” technologies may lead to a change of paradigms towards the application of complex mixtures in medicine and open the new fields of phytogenomics, -proteomics and -metabolomics (AU)

Protective effects of taurine on endothelial cells impaired by high glucose and oxidized low density lipoproteins.

BACKGROUND: Endothelial dysfunction, common to diabetes and cardiovascular diseases, is an early step in the development of atherosclerosis and diabetic angiopathies. Deficiencies of taurine have been related to diabetes and cardiovascular diseases. AIMS OF THE STUDY: We investigated whether taurine provides protective action against endothelial dysfunction induced by hyperglycemia and/or oxidized low density lipoproteins (oxLDL). METHODS: Quiescent human umbilical cord venous endothelial cells were exposed for 20 h to high glucose (35 mM) and/or oxLDL (60 microg/ml) alone and in presence of taurine (0.5-2.5 mg/ml). Apoptosis, caspase-3 activity, soluble(s) and cell surface expressions of vascular cellular (VCAM-1) and intercellular (ICAM-1) adhesion molecules were determined. Results are given as a percentage of the low glucose medium control. Apoptosis, VCAM-1 and ICAM-1 expressions were related to cell number. RESULTS: Hyperglycemia increased apoptosis to 162.5 +/- 19.2%, caspase-3 activity to 153.2 +/- 10.3%, cell-surface expression of VCAM-1 to 125.1 +/- 5.8%, the expression of ICAM-1 to 123.7 +/- 2.8% and sICAM-1 to 146.5 +/- 7.9%. Taurine (0.5-2.5 mg/ml) restored apoptosis, caspase-3 activity and expressions of VCAM-1 and ICAM-1. OxLDL (60 microg/ml) increased apoptosis to 114.8 +/- 3.1%; taurine (2.5 mg/ml) reduced this apoptosis to 40.5 +/- 4.1%. The combination of hyperglycemia and oxLDL increased apoptosis to 211.7 +/- 11.6%. This increase was normalized by taurine (2.5 mg/ml) to 97.9 +/- 12.8%. CONCLUSION: Taurine protects HUVECs from endothelial dysfunction induced by hyperglycemia through down-regulation of apoptosis and adhesion molecules. Counteracting the combination of oxLDL and hyperglycemia requires pharmacological concentrations of taurine.

Vitamin C promotes human endothelial cell growth via the ERK-signaling pathway.

BACKGROUND: Epidemiological, secondary prevention and small interventional trials suggest a preventive role of vitamin C for cardiovascular diseases (CAD), especially through improving endothelial dysfunction. Large primary prevention trials failed to confirm this. Mechanistic studies may contribute to resolve this discrepancy. AIM OF THE STUDY: We examined whether vitamin C activates mitogen-activated protein kinases (MAPK) in human umbilical cord venous endothelial cells (HUVECs) and whether reactive oxygen species (ROS) play a role in this process. METHODS: Subconfluent quiescent HUVECs were incubated with vitamin C alone or in combination with catalase (CAT) and/or hydrogenperoxide (H2O2). Intracellular MAPK were determined by Western blot, proliferation by cell count and DNA-synthesis by [3H]-thymidine-uptake. RESULTS: HUVECs were incubated with vitamin C (60 microM) for 5-60 min or for 20 min (30-90 microM). A dose-dependent phosphorylation of extracellular signal-regulated-kinases (ERKs)-1 and -2 with a maximum of phosphorylation at 15-20 min was observed and inhibitable by MEK1/2-inhibitor U0126 (5-10 microM). Vitamin C (60 microM) stimulated phosphorylation of ERK5, but not of p38 and c-Jun, demonstrating a different MAPK-activation pattern compared to H2O2. Vitamin C (60 microM) induced proliferation and a dose-dependent [3H]-thymidine-uptake (30-120 microM) within 20 h. CAT (0.3 U/ml) did neither suppress the vitamin C induced [3H]-thymidine-uptake nor ERK1/2-phosphorylation. CAT (0.3 U/ml), but not vitamin C (60 microM) abrogated the inhibitory effects of H2O2 (100 microM) on [3H]-thymidine-uptake. CONCLUSION: Physiological vitamin C-concentrations promote proliferation of subconfluent ECs by activating an ERK1/2 controlled pathway. Targeting MAPK by vitamin C may improve, besides antioxidant mechanisms, endothelial dysfunction by promoting a fast regeneration of the endothelium after tissue injury, particularly required during secondary prevention and early development.

Long-term effects of a multimodal approach including immunoadsorption for the treatment of myasthenic crisis.

A multimodal treatment protocol with immunoadsorption (IA) as the central element was used in the treatment of myasthenic crisis (MC). Fifteen patients with MC were treated in repeated, uninterrupted 7-day cycles until mobilization with: (i) large-volume IA using an antihuman-IgG adsorber, days 1-5; (ii) intravenous immunoglobulin substitution (0.3-0.5 g/kg body weight [BW]/day), days 5-7; and (iii) immunosuppression with cyclophosphamide (1-2 mg/kg BW/day) and prednisolone (0.5-1 mg/kg BW/day), until remission. Patients required a median of 8 days of mechanical ventilation, 12 days in the intensive care unit, and 35 days of hospitalization. Functional improvement compared to their precrisis condition was attained by 14 of 15 patients. MG severity score improved by a mean of 10 points, quality of life score by 9.8 points, and Karnofsky index by 29 points in 14 of 15 patients. Improvements remained stable and no further crises occurred during long-term follow-up, which averaged 4.4 years. No fatalities due to MC occurred. The results demonstrate that our protocol is a potent therapeutic approach in the treatment of MC.

Treatment of acquired hemophilia by the Bonn-Malmo Protocol: documentation of an in vivo immunomodulating concept.

Acquired hemophilia (AH) is an extremely rare condition in which autoantibodies (inhibitors) against clotting factor VIII induce acute and life-threatening hemorrhagic diathesis because of abnormal blood clotting. The mortality rate of AH is as high as 16%, and current treatment options are associated with adverse side effects. We investigated a therapeutic approach for AH called the modified Bonn-Malmo Protocol (MBMP). The aims of MBMP include suppression of bleeding, permanent elimination of inhibitors, and development of immune tolerance, thereby avoiding long-term reliance on coagulation products. The protocol included immunoadsorption for inhibitor elimination, factor VIII substitution, intravenous immunoglobulin, and immunosuppression. Thirty-five high-titer patients with critical bleeding who underwent MBMP were evaluated. Bleeding was rapidly controlled during 1 or 2 apheresis sessions, and no subsequent bleeding episodes occurred. Inhibitor levels decreased to undetectable levels within a median of 3 days (95% confidence interval [95% CI], 2-4 days), factor substitution was stopped within a median of 12 days (95% CI, 11-17 days), and treatment was completed within a median of 14 days (95% CI, 12-17 days). Long-term follow-up (7 months-7 years) showed an overall response rate of 88% for complete remission (CR). When cancer patients were excluded, the CR rate was 97%.

Risk of hepatitis C after immunoadsorption.

An episode of acute hepatitis in a patient with hemophilia during immunoadsorption therapy initially was misinterpreted as a reactivated hepatitis C virus (HCV) infection, but ultimately was shown to be an exogenous reinfection during cohort treatment with another HCV-positive patient. This incident illustrates that policies for the prevention of nosocomial transmission of blood-borne pathogens, especially in cohort treatment units, may need to be reassessed.