The novel benzimidazole derivative BRP-7 inhibits leukotriene biosynthesis in vitro and in vivo by targeting 5-lipoxygenase-activating protein (FLAP).

BACKGROUND AND PURPOSE: Leukotrienes (LTs) are inflammatory mediators produced via the 5-lipoxygenase (5-LOX) pathway and are linked to diverse disorders, including asthma, allergic rhinitis and cardiovascular diseases. We recently identified the benzimidazole derivative BRP-7 as chemotype for anti-LT agents by virtual screening targeting 5-LOX-activating protein (FLAP). Here, we aimed to reveal the in vitro and in vivo pharmacology of BRP-7 as an inhibitor of LT biosynthesis. EXPERIMENTAL APPROACH: We analysed LT formation and performed mechanistic studies in human neutrophils and monocytes, in human whole blood (HWB) and in cell-free assays. The effectiveness of BRP-7 in vivo was evaluated in rat carrageenan-induced pleurisy and mouse zymosan-induced peritonitis. KEY RESULTS: BRP-7 potently suppressed LT formation in neutrophils and monocytes and this was accompanied by impaired 5-LOX co-localization with FLAP. Neither the cellular viability nor the activity of 5-LOX in cell-free assays was affected by BRP-7, indicating that a functional FLAP is needed for BRP-7 to inhibit LTs, and FLAP bound to BRP-7 linked to a solid matrix. Compared with the FLAP inhibitor MK-886, BRP-7 did not significantly inhibit COX-1 or microsomal prostaglandin E2 synthase-1, implying the selectivity of BRP-7 for FLAP. Finally, BRP-7 was effective in HWB and impaired inflammation in vivo, in rat pleurisy and mouse peritonitis, along with reducing LT levels. CONCLUSIONS AND IMPLICATIONS: BRP-7 potently suppresses LT biosynthesis by interacting with FLAP and exhibits anti-inflammatory effectiveness in vivo, with promising potential for further development.

Elucidation of the molecular mechanism and the efficacy in vivo of a novel 1,4-benzoquinone that inhibits 5-lipoxygenase.

BACKGROUND AND PURPOSE: 1,4-Benzoquinones are well-known inhibitors of 5-lipoxygenase (5-LOX, the key enzyme in leukotriene biosynthesis), but the molecular mechanisms of 5-LOX inhibition are not completely understood. Here we investigated the molecular mode of action and the pharmacological profile of the novel 1,4-benzoquinone derivative 3-((decahydronaphthalen-6-yl)methyl)-2,5-dihydroxycyclohexa-2,5-diene-1,4-dione (RF-Id) in vitro and its effectiveness in vivo. EXPERIMENTAL APPROACH: Mechanistic investigations in cell-free assays using 5-LOX and other enzymes associated with eicosanoid biosynthesis were conducted, along with cell-based studies in human leukocytes and whole blood. Molecular docking of RF-Id into the 5-LOX structure was performed to illustrate molecular interference with 5-LOX. The effectiveness of RF-Id in vivo was also evaluated in two murine models of inflammation. KEY RESULTS: RF-Id consistently suppressed 5-LOX product synthesis in human leukocytes and human whole blood. RF-Id also blocked COX-2 activity but did not significantly inhibit COX-1, microsomal PGE2 synthase-1, cytosolic PLA2 or 12- and 15-LOX. Although RF-Id lacked radical scavenging activity, reducing conditions facilitated its inhibitory effect on 5-LOX whereas cell stress impaired its efficacy. The reduced hydroquinone form of RF-Id (RED-RF-Id) was a more potent inhibitor of 5-LOX as it had more bidirectional hydrogen bonds within the 5-LOX substrate binding site. Finally, RF-Id had marked anti-inflammatory effects in mice in vivo. CONCLUSIONS AND IMPLICATIONS: RF-Id represents a novel anti-inflammatory 1,4-benzoquinone that potently suppresses LT biosynthesis by direct inhibition of 5-LOX with effectiveness in vivo. Mechanistically, RF-Id inhibits 5-LOX in a non-redox manner by forming discrete molecular interactions within the active site of 5-LOX.

High capacity for leukotriene biosynthesis in peripheral blood during pregnancy.

Pregnancy is accompanied by major immunological changes to maintain both tolerance for the fetus and immune competence. Leukotrienes are powerful 5-lipoxygenase-derived inflammatory mediators and the characteristics of leukotriene-related diseases (e.g., asthma, allergic rhinitis) change during pregnancy. Here, we show that pregnancy affects leukotriene synthesis in human blood and leukocytes. 5-Lipoxygenase product formation in stimulated blood of pregnant women was significantly higher than in non-pregnant females. Although a pregnancy-related increase in neutrophil and monocyte counts may explain these observations, granulocytes of pregnant donors have lower leukotriene-synthetic capacities. On the other hand, granulocytes from non-pregnant woman produced more leukotrienes when resuspended in plasma of pregnant women than of non-pregnant females. Together, we show that leukotriene biosynthesis in maternal blood is increased by the interrelations of higher leukocyte numbers, lower cellular capacity for leukotriene synthesis and stimulatory effects of plasma. This bias may affect leukotriene-related diseases during pregnancy and their pharmacological treatment.

The effect of increased centrifugation temperature on the quality of red-blood-cell concentrates of automated whole blood processing.

BACKGROUND AND OBJECTIVES: There are manual and automated methods to separate whole blood (WB) available. The Atreus whole blood processing system is an automated method, which combines centrifugation and expression of components into a single device. A major difference to conventional methods is that centrifugation temperature is not controlled at 22°C. The aim of this study was to examine the influence of increased centrifugation temperatures on the quality of red-blood-cell concentrates (RCC) after active cooling of WB prior to processing. MATERIALS AND METHODS: A total of 28 WB were processed: 16 at centrifugation temperatures of up to 28°C (1st protocol) and 12 at 34°C (2nd protocol). RCC quality parameters were tested weekly for 42 days. RESULTS: Red-blood-cell concentrates (RCC) quality complied with the European and German guidelines. Haemolysis was not significantly different throughout storage. Significant statistical differences were detected between both protocols in potassium concentration at the end of storage and in ATP levels at the day of processing. CONCLUSION: Centrifugation temperatures of up to 34°C are well tolerated by the red blood cells with minimal interference with the RCC quality parameters.

Detection of antibodies in eluates of immunoadsorption causing humoural rejection in patients after solid organ transplantation.

The influence of antibodies (AB) against human leukocyte antigen (HLA) on antibody mediated rejection (AMR) is still discussed controversially. Here we demonstrate to what extent post transplant detected HLA-AB and non-HLA-AB against Angiotensin II type 1 receptor (AT1 R-AB), endothelin-1 type A receptor (ETA R-AB) and glycoprotein (GP) IIb/IIIa, Ia/IIa, Ib/IX affect the graft outcome. A total of 13 transplant recipients (9 kidneys and 4 hearts) suffering from AMR were analysed. Before immunoadsorption (IA) treatment HLA-AB (CDC) in sera were detected in 27% versus 39% in eluates and 46% versus 87% by using ELISA. We could not find any AB against GP in sera. In eluates, however, we could detect AB against GP: GP IIb/IIIa in 86% of all samples with titres from 1:1 to 1:32, GP Ib/IX (up to 1:32) in 76% and GP Ia/IIa with titres from 1:1 to 1:16 in 82%. Further we detected anti-endothelial cell antibodies (AECA) against receptors AT1 and ETA in sera before IA in 22%, after IA in 10% and in eluates in 42% of all samples. The antibody titres vary from 1:1 to 1:256. Our investigation pointed out, that AMR is still possible without detectable AB in serum and consolidates the hypothesis that clinical relevant non-HLA-AB and HLA-AB are partly fixed on the graft. IA is qualified to detach these fixed AB.

Histamine modulates γδ-T lymphocyte migration and cytotoxicity, via Gi and Gs protein-coupled signalling pathways.

BACKGROUND AND PURPOSE: The biogenic amine, histamine plays a pathophysiological regulatory role in cellular processes of a variety of immune cells. This work analyses the actions of histamine on γδ-T lymphocytes, isolated from human peripheral blood, which are critically involved in immunological surveillance of tumours. EXPERIMENTAL APPROACH: We have analysed effects of histamine on the intracellular calcium, actin reorganization, migratory response and the interaction of human γδ T cells with tumour cells such as the A2058 human melanoma cell line, the human Burkitt's Non-Hodgkin lymphoma cell line Raji, the T-lymphoblastic lymphoma cell line Jurkat and the natural killer cell-sensitive erythroleukaemia cell line, K562. KEY RESULTS: γδ T lymphocytes express mRNA for different histamine receptor subtypes. In human peripheral blood γδ T cells, histamine stimulated Pertussis toxin-sensitive intracellular calcium increase, actin polymerization and chemotaxis. However, histamine inhibited the spontaneous cytolytic activity of γδ T cells towards several tumour cell lines in a cholera toxin-sensitive manner. A histamine H(4) receptor antagonist abolished the histamine induced γδ T cell migratory response. A histamine H(2) receptor agonist inhibited γδ T cell-mediated cytotoxicity. CONCLUSIONS AND IMPLICATIONS: Histamine activated signalling pathways typical of chemotaxis (G(i) protein-dependent actin reorganization, increase of intracellular calcium) and induced migratory responses in γδ T lymphocytes, via the H(4) receptor, whereas it down-regulated γδ T cell mediated cytotoxicity through H(2) receptors and G(s) protein-coupled signalling. Our data suggest that histamine activated γδ T cells could modulate immunological surveillance of tumour tissue.

Identification of the novel allele Cw*040110 in a German patient.

We report here the identification of a novel human leukocyte-Cw*040110 allele detected by polymerase chain reaction sequence-based typing in a German patient.

No association between transmembrane protein-tyrosine-phosphatase receptor type C (CD45) exon A 77C>G transversion and Hashimoto's thyroiditis in a German population.

The CD45 77C>G transversion (rs17612648) in exon A of the CD45 gene has been reported to be associated with the development of various autoimmune diseases. Because Hashimoto's thyroiditis (HT) is a typical autoimmune disease, we performed a study to determine the association of the 77C>G transversion with susceptibility to HT. We enrolled 170 patients and 230 healthy individuals in the study. The 77C>G transversion was genotyped by polymerase chain reaction-allele specific restriction enzyme analysis (PCR-ASRA). We found four patients and six control individuals who carried the 77C>G transversion in a heterozygous form. No homozygous individual was detected in patients with HT or control population. The frequency of the 77G allele in patients was 1.2%, which did not significantly differ from 1.3% in controls (p = 0.871). Our data did not reveal any association between CD45 77C>G transversion and susceptibility to HT in a German population.

Social and behavioural aspects and their consequences in obese teenagers -importance of family’s history / Aspectos sociales y conductuales y sus consecuencias en adolescentes obesos - importancia de los antecedentes familiares

Objectives: Overweight, the metabolic syndrome and accompanying diseases are dramatically increasing problems. We investigated social and behavioral variables that influence overweight in adolescents and tested their influence on plasma markers related to diabetes and endothelial dysfunction. Methods: 79 male adolescents were enrolled (age 13-17 years). Endothelial progenitor cells were counted by flow cytometry. Adiponectin and soluble E-selectin (sEselectin) were determined by ELISA. Results: Body weight differs significantly if the family's history was positive for arterial hypertension (p < 0.001), diabetes (p < 0.001), hypercholesterolemia (p<0.001), and coronary artery disease (CAD, p < 0.01). The hours of physical activity represent a predictor of BMI in linear regression analysis (p < 0.001; R2 = 0.195). Markers for endothelial damage are altered in adolescents with positive family history for hyperlipidemia and CAD. Conclusion: The family's history is an important variable influencing the body weight of teenagers. Via obesity and independently, it influences the early development of endothelial damage. It might serve to detect teenagers at risk for appropriate intervention (AU)

Objetivos: El sobrepeso, el síndrome metabólico y sus enfermedades asociadas son problemas que están aumentando de forma notable. Investigamos las variables sociales y conductuales que influyen en el sobrepeso en adolescentes y probamos su influencia sobre los marcadores plasmáticos relacionados con la diabetes y la disfunción endotelial. Métodos: Se reclutaron 79 adolescentes varones (edad 13-17 años). Se contaron las células progenitoras endoteliales con citometría de flujo. Se determinaron la adiponectina y la selectina-e soluble (selectina-s) mediante ELISA. Resultados: El peso corporal difiera significativamente si los antecedentes familiares son positivos para hipertensión arterial (p < 0,001), diabetes (p < 0,001), hipercolesterolemia (p < 0,001) y arteriopatía coronaria (APC, p < 0,01). Las horas de actividad física representan un predictor del IMC en el análisis de regresión linear (p < 0,001; r2 = 0,195). Los marcadores de lesión endotelial están alterados en los adolescentes con unos antecedentes familiares positivos para hiperlipidemia y APC. Conclusión: Los antecedentes familiares son una variable importante que influye en el peso corporal de los adolescentes. A través de la obesidad y de forma independiente, influye en el desarrollo precoz de lesión endotelial. Podría servir para detectar a los adolescentes con riesgo para realizar una intervención apropiada (AU)

Autologous blood donor screening indicated a lower prevalence of viral hepatitis in East vs West Germany: epidemiological benefit from established health resources.

Prevalence data concerning viral hepatitis and human immunodeficiency virus (HIV) in the general population are usually scarce. We aimed for a large cohort representative of the general population that required little funding. Autologous blood donors are relatively representative of the general population, and are tested for viral hepatitis and HIV in many countries. However, frequently these data are not captured for epidemiologic purposes. We analysed data from well over 35,000 autologous blood donors as recorded in 21 different transfusion centres for anti-hepatitis C virus (HCV), HBsAg and anti-HIV, as well as TPHA if available. We found a lower prevalence of hepatitis B virus and HCV in East vs West Germany, 0.2%vs 0.32% and 0.16%vs 0.32% respectively, which confirms earlier data in smaller cohorts, thus supporting the value of our approach. HIV was too rare to disclose significant differences, 0.01%vs 0.02%. TPHA was higher in East (0.34%) vs West Germany (0.29%) without significant differences. HCV was more frequent in women vs men. Transfusion institutes managing autologous blood donations should be used as a resource for epidemiological data relating to viral hepatitis and HIV, if such testing is performed routinely. This approach generates data relating to the general population with special emphasis on undiagnosed cases.

Differential number of CD34+, CD133+ and CD34+/CD133+ cells in peripheral blood of patients with congestive heart failure.

BACKGROUND: Endothelial progenitor cells (EPC) which are characterised by the simulateous expression of CD34, CD133 and vascular endothelial growth receptor 2 (VEGF 2) are involved in the pathophysiology of congestive heart failure (CHF) and their number and function is reduced in CHF. But so far our knowledge about the number of circulating hematopoietic stem/ progenitor cells (CPC) expressing the early hematopoietic marker CD133 and CD34 in CHF is spares and therefore we determined their number and correlated them with New York Heart Association (NYHA) functional class. METHODS: CD34 and CD133 surface expression was quantified by flow cytometry in the peripheral venous blood of 41 healthy adults and 101 patients with various degrees of CHF. RESULTS: CD34+, CD133+ and CD34+/CD133+ cells correlated inversely with age. Both the number of CD34+ and of CD34+/CD133+ cells inversely correlated with NYHA functional class. The number of CD133+ cells was not affected by NYHA class. Furthermore the number of CD133+ cells did not differ between control and CHF patients. CONCLUSION: In CHF the release of CD34+, CD133+ and CD34+/CD133+ cells from the bone marrow seems to be regulated differently. Modulating the releasing process in CHF may be a tool in CHF treatment.

Social and behavioural aspects and their consequences in obese teenagers: importance of family's history.

OBJECTIVES: Overweight, the metabolic syndrome and accompanying diseases are dramatically increasing problems. We investigated social and behavioral variables that influence overweight in adolescents and tested their influence on plasma markers related to diabetes and endothelial dysfunction. METHODS: 79 male adolescents were enrolled (age 13-17 years). Endothelial progenitor cells were counted by flow cytometry. Adiponectin and soluble E-selectin (sEselectin) were determined by ELISA. RESULTS: Body weight differs significantly if the family's history was positive for arterial hypertension (p < 0.001), diabetes (p < 0.001), hypercholesterolemia (p<0.001), and coronary artery disease (CAD, p < 0.01). The hours of physical activity represent a predictor of BMI in linear regression analysis (p < 0.001; R(2) = 0.195). Markers for endothelial damage are altered in adolescents with positive family history for hyperlipidemia and CAD. CONCLUSION: The family's history is an important variable influencing the body weight of teenagers. Via obesity and independently, it influences the early development of endothelial damage. It might serve to detect teenagers at risk for appropriate intervention.

No association of the CD45 77C>G transversion with inflammatory bowel disease in German patients.

A 77C>G transversion in exon A of the CD45 gene was investigated in patients with inflammatory bowel disease (IBD) and controls. The distribution of the 77G allele was not significantly different between patients and controls. We found no evidence for the contribution of the 77C>G transversion in susceptibility to IBD.

Binding studies of an arabinogalactan-protein from Echinacea purpurea to leucocytes.

Flow cytometric investigations show binding of an isolated arabinogalactan-protein (AGP) from pressed juice of the aerial parts of Echinacea purpurea to the cell surface of human leucocytes. AGP demonstrates binding to lymphocytes, monocytes and granulocytes of different donors (n=8). Competition assays with two antibodies, directed against CD4 and CD8, revealed no interaction of AGP with these receptors, leading to the conclusion that binding of AGP to leucocytes is mediated via other structures.

Neonatal alloimmune thrombocytopenia caused by human leucocyte antigen-B27 antibody.

Neonatal alloimmune thrombocytopenia (NAIT) occurs when maternal alloantibodies to antigens presented on foetal platelets cause their immune destruction. Whether human leucocyte antigen (HLA) antibodies can cause NAIT is controversial. Here, a patient was described who suffered from a NAIT caused by an HLA-B27 antibody. Sera from the mother and the newborn were tested for human platelet antigen antibodies and HLA antibodies by monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay, solid phase-linked immunosorbent assay (ELISA), lymphocytotoxicity assay (LCT) and flow cytometric analysis. No antibodies against cluster designation (CD)109 and platelet glycoproteins of the father were found in patient's and mother's serum. However, HLA ELISA was used to identify HLA antibody in both sera. The antibody was specified as HLA-B27 antibody. Typing results showed that the father descended HLA-B27 antigen on patient and his brother. The mother was HLA-B27 negative. It is most conceivable that the previous pregnancy of the mother induced the production of anti-HLA-B27 antibody, which crossed the placenta and subsequently caused an NAIT in the case presented.

Identification of a new HLA-B allele, HLA-B*4443, in a German family.

A novel human leukocyte antigen (HLA)-B allele is described. The allele was identified in a German blood donor of Caucasian origin. Because high-resolution HLA-typing using sequence-specific primers gave inconclusive results, sequence-based typing was performed. Nucleotide sequences of exons 2 and 3 most closely match with HLA-B*4417 and HLA-B*440301 (99.5% identity). The predicted protein sequence revealed a single amino acid substitution (D156L) compared with the HLA-B*4417 allele but two substitutions (Y113H, D116S) compared with the HLA-B*440301 allele. Therefore, the novel allele has been officially assigned HLA-B*4443 by the WHO Nomenclature Committee. The HLA-B*4443 allele was found with the A*2301, Cw*0401, B*4443, DRB1*0701, DRB4*0107, and DQB1*0202 haplotype.

A transmembrane protein-tyrosine phosphatase receptor type C (CD45) exon A point mutation (77 C to G) is not associated with the development of type 1 diabetes mellitus in a German population.

To investigate whether a C to G transversion at position 77 in exon A of the CD45 gene is associated with the development of diabetes mellitus type 1 (T1D), we studied 165 patients and 220 control individuals. The frequency of the 77G allele in the control group was 1.1%, which was not significantly different from the 1.2% found in the patient group (P = 0.922). The C to G transversion does not seem to be associated with susceptibility for T1D.

Immunoadsorption of sensitized kidney transplant candidates immediately prior to surgery.

Patients with anti-human leucocyte antigen (HLA) antibodies from previous transplantation, blood transfusion are highly sensitized and at risk to hyperacute renal graft loss. As these antibodies are identified to be of pathogenic importance, an effective removal may allow successful transplantation. Six 'high risk patients' [panel-reactive antibodies (PRA) >30% or retransplanted patients with an acutely rejected first graft within 6 months from surgery] were treated by protein A immunoadsorption (IA) immediately prior to transplantation. We treated the calculated plasma volume one to three times prior to surgery: mean 4600 mL (range 2100-10 200 mL). After transplantation we repeated the sessions according to antibody (Ab) recurrence, graft function and signs of rejection. The panel reactive Ab were reduced from mean 65% pre-IA (range 35-85) to lowest 15% (range 0-55). After the course they reappeared to 30% (range 0-90). Five of the six patients had no clinical signs of vascular rejection. At a follow-up of mean 54 months (+/-14) four grafts still function with a mean serum creatinine of 172 micromol/L (+/-57). Protein A IA is a safe and effective adjunct in the treatment of highly sensitized patients awaiting renal transplantation. The treatment immediately prior to operation can prevent hyperacute rejection and increases the graft survival in these patients.

Protein A immunoadsorption in a pregnant woman with habitual abortion.

The role of antibodies in the occurrence of recurrent spontaneous miscarriage is well known. However there are many controversial issues in the management of habitual abortion. This paper describes the effect of Protein A immunoadsorption on serum levels of these antibodies and its impact on a case of a successfully treated woman in a outpatient department without need for a central venous catheter. Given the favourable clinical results described in our paper we think it may be relevant for some worse cases in clinical practice and will interest your readers.

Allele frequencies of human platelet antigen 1, 2, 3, and 5 systems in patients with chronic refractory autoimmune thrombocytopenia and in normal persons.

BACKGROUND AND OBJECTIVES: Chronic refractory autoimmune thrombocytopenic purpura (AITP) is an autoimmune disorder due to autoantibodies against platelet glycoproteins (GP). Human platelet alloantigenic (HPA) systems are distributed to different platelet GPs. We carried out genotyping of diallelic HPA-1, -2, -3, and -5 systems to clarify potential associations between HPA alleles and the development of chronic refractory AITP. PATIENTS AND METHODS: DNA was isolated from 33 unrelated German patients with chronic refractory AITP and from 80 randomly selected German blood donors to determine the phenotype and allele frequencies for the HPA-1, -2, -3, and -5 systems. Fragments carrying the polymorphic sequences corresponding to those alleles were amplified by the polymerase chain reaction and further characterized by restriction analysis. RESULTS: Whereas HPA-1, -3, and -5 allele frequencies were identical in 33 patients with chronic refractory AITP and in controls, HPA-2 allele frequencies showed a statistically significant difference (p = 0.017). In our group of patients, the HPA-2a allele frequency was 100%, but HPA-2b was not seen. In contrast, the allele frequency of HPA-2a in the control group was 92% (n = 147), and in HPA-2b it was 8% (n = 13). CONCLUSION: This study suggests an association between the HPA-2a allele and chronic refractory AITP. The HPA-2a allele may be involved in the formation of an AITP-specific autoepitope.