Long-term suboptimal dietary trace element supply does not affect trace element homeostasis in murine cerebellum.

The ageing process is associated with alterations of systemic trace element (TE) homeostasis increasing the risk, e.g. neurodegenerative diseases. Here, the impact of long-term modulation of dietary intake of copper, iron, selenium, and zinc was investigated in murine cerebellum. Four- and 40-wk-old mice of both sexes were supplied with different amounts of those TEs for 26 wk. In an adequate supply group, TE concentrations were in accordance with recommendations for laboratory mice while suboptimally supplied animals received only limited amounts of copper, iron, selenium, and zinc. An additional age-adjusted group was fed selenium and zinc in amounts exceeding recommendations. Cerebellar TE concentrations were measured by inductively coupled plasma-tandem mass spectrometry. Furthermore, the expression of genes involved in TE transport, DNA damage response, and DNA repair as well as selected markers of genomic stability [8-oxoguanine, incision efficiency toward 8-oxoguanine, 5-hydroxyuracil, and apurinic/apyrimidinic sites and global DNA (hydroxy)methylation] were analysed. Ageing resulted in a mild increase of iron and copper concentrations in the cerebellum, which was most pronounced in the suboptimally supplied groups. Thus, TE changes in the cerebellum were predominantly driven by age and less by nutritional intervention. Interestingly, deviation from adequate TE supply resulted in higher manganese concentrations of female mice even though the manganese supply itself was not modulated. Parameters of genomic stability were neither affected by age, sex, nor diet. Overall, this study revealed that suboptimal dietary TE supply does not substantially affect TE homeostasis in the murine cerebellum.

Ageing-associated effects of a long-term dietary modulation of four trace elements in mice.

Trace elements (TEs) are essential for diverse processes maintaining body function and health status. The complex regulation of the TE homeostasis depends among others on age, sex, and nutritional status. If the TE homeostasis is disturbed, negative health consequences can result, e.g., caused by impaired redox homeostasis and genome stability maintenance. Based on age-related shifts in TEs which have been described in mice well-supplied with TEs, we aimed to understand effects of a long-term feeding with adequate or suboptimal amounts of four TEs in parallel. As an additional intervention, we studied mice which received an age-adapted diet with higher concentrations of selenium and zinc to counteract the age-related decline of both TEs. We conducted comprehensive analysis of diverse endpoints indicative for the TE and redox status, complemented by analysis of DNA (hydroxy)methylation and markers denoting genomic stability maintenance. TE concentrations showed age-specific alterations which were relatively stable and independent of their nutritional supply. In addition, hepatic DNA hydroxymethylation was significantly increased in the elderly mice and markers indicative for the redox status were modulated. The reduced nutritional supply with TEs inconsistently affected their status, with most severe effects regarding Fe deficiency. This may have contributed to the sex-specific differences observed in the alterations related to the redox status and DNA repair activity. Overall, our results highlight the complexity of factors impacting on the TE status and its physiological consequences. Alterations in TE supply, age, and sex proved to be important determinants that need to be taken into account when considering TE interventions for improving general health and supporting convalescence in the clinics.

Experiencia de padres/madres al cuidado de niños de 5 a 10 años con trastorno del espectro autista / Parent's experience caring for children 5 to 10 years old with autism spectrum disorder / Experiência dos pais a cuidar de crianças dos 5 aos10 anos com desordem do espectro do autismo

INTRODUCCIÓN: El trastorno del espectro autista contempla una serie de trastornos evolutivos comprometiendo diversos ejes. OBJETIVO: Develar experiencias vividas por los padres/madres al cuidado de niños/as de 5-10 años con Trastorno de espectro autista (TEA). METODOLOGÍA: Estudio cualitativo paradigma interpretativo diseño fenomenológico, en el que se recogieron las vivencias a través de entrevistas en profundidad a madres/padres que participan en las fundaciones de Chile, las que fueron grabadas y transcritas textualmente por las investigadoras con previo consentimiento de los participantes. Para el análisis de los discursos se cumplió con el rigor metodológico. RESULTADOS: Tras analizar las entrevistas, se develó un proceso dinámico y continuo de múltiples vivencias que permitieron a los participantes pasar desde la experiencia al cuidado de niños con TEA de alto funcionamiento, hasta las mejoras que se podrían aplicar en salud. CONCLUSIÓN: El hecho de tener un hijo/a con TEA lo convierte en un proceso complejo, el cual genera diversas emociones en los padres/madres, pero que a pesar de ello les ha permitido vivir un proceso de adaptación, mayor conocimiento teórico y práctico de la condición, permitiendo un crecimiento y desarrollo favorable para el niño/niña y las familias.

INTRODUCTION: Autism spectrum disorder contemplates a series of developmental disorderscompromising several axes. OBJECTIVE: To unveil experiences lived by parents caring forchildren aged 5-10 years with a high-functioning Autism Spectrum Disorder. METHODOLOGY: Qualitative study, interpretative paradigm,phenomenological design,the experiences collected through in-depth interviews with parents participating in foundations in Chile, which were recorded and transcribed verbatim by there searchers with the prior consent of the participants.For the analysis of the speeches, the methodological rigor meets. RESULTS: After analyzing the nterviews, a dynamic and continuous process of multiple experiences was revealed, which allowed the participants to go from the experience of caring for children with a high-functioning Autism Spectrum Disorderto the applicable improvements in health.CONCLUSION:The fact of having a child with ASD makes it a complex process, which generates diverseemotions in the parents, but despite this, it has allowed them to live a process of adaptation,greater theoretical and practical knowledge of the condition, allowing a favorable growth anddevelopmentforthechild and the families.

INTRODUÇÃO: A perturbação do espectro autista contempla uma série de perturbações dedesenvolvimento que comprometem vários eixos. OBJETIVO: Revelar experiências vividas por pais que cuidam decrianças dos5-10 anos comTranstorno do Espectro Autista(TEA). METODOLOGIA: Estudo qualitativo fenomenológico, no qualforamrecolhidas experiênciasatravés de entrevistas aprofundadas com mães e pais que participam em fundações no Chile, as quais foram gravadas e transcritas textualmente como consentimento prévio dos participantes.Para a análise dos dados,foi respeitado o rigor metodológico.RESULTADOS: Após análised as entrevistas,foi revelado um processo dinâmico e contínuo de múltiplas experiências,o que permitiua os participantes passar da experiência de cuidar de crianças comTEA de alta funcionalidade para as melhorias que poderiam ser aplicadas na saúde.CONCLUSÃO: Ofacto deter uma criança comTEA é um processo complexo,que gera diversas emoções nos pais,mas que a pesar disso puderam viver um processo de adaptação,maior conhecimento teórico e prático da condição, permitindo um crescimento e desenvolvimento favorável para a criança e para as famílias

Effects of a Cumulative, Suboptimal Supply of Multiple Trace Elements in Mice: Trace Element Status, Genomic Stability, Inflammation, and Epigenetics.

SCOPE: Trace element (TE) deficiencies often occur accumulated, as nutritional intake is inadequate for several TEs, concurrently. Therefore, the impact of a suboptimal supply of iron, zinc, copper, iodine, and selenium on the TE status, health parameters, epigenetics, and genomic stability in mice are studied. METHODS AND RESULTS: Male mice receive reduced or adequate amounts of TEs for 9 weeks. The TE status is analyzed mass-spectrometrically in serum and different tissues. Furthermore, gene and protein expression of TE biomarkers are assessed with focus on liver. Iron concentrations are most sensitive toward a reduced supply indicated by increased serum transferrin levels and altered hepatic expression of iron-related genes. Reduced TE supply results in smaller weight gain but higher spleen and heart weights. Additionally, inflammatory mediators in serum and liver are increased together with hepatic genomic instability. However, global DNA (hydroxy)methylation is unaffected by the TE modulation. CONCLUSION: Despite homeostatic regulation of most TEs in response to a low intake, this condition still has substantial effects on health parameters. It appears that the liver and immune system react particularly sensitive toward changes in TE intake. The reduced Fe status might be the primary driver for the observed effects.

Characterization of the Filovirus-Resistant Cell Line SH-SY5Y Reveals Redundant Role of Cell Surface Entry Factors.

Filoviruses infect a wide range of cell types with the exception of lymphocytes. The intracellular proteins cathepsin B and L, two-pore channel 1 and 2, and bona fide receptor Niemann⁻Pick Disease C1 (NPC1) are essential for the endosomal phase of cell entry. However, earlier steps of filoviral infection remain poorly characterized. Numerous plasma membrane proteins have been implicated in attachment but it is still unclear which ones are sufficient for productive entry. To define a minimal set of host factors required for filoviral glycoprotein-driven cell entry, we screened twelve cell lines and identified the nonlymphocytic cell line SH-SY5Y to be specifically resistant to filovirus infection. Heterokaryons of SH-SY5Y cells fused to susceptible cells were susceptible to filoviruses, indicating that SH-SY5Y cells do not express a restriction factor but lack an enabling factor critical for filovirus entry. However, all tested cell lines expressed functional intracellular factors. Global gene expression profiling of known cell surface entry factors and protein expression levels of analyzed attachment factors did not reveal any correlation between susceptibility and expression of a specific host factor. Using binding assays with recombinant filovirus glycoprotein, we identified cell attachment as the step impaired in filovirus entry in SH-SY5Y cells. Individual overexpression of attachment factors T-cell immunoglobulin and mucin domain 1 (TIM-1), Axl, Mer, or dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) rendered SH-SY5Y cells susceptible to filovirus glycoprotein-driven transduction. Our study reveals that a lack of attachment factors limits filovirus entry and provides direct experimental support for a model of filoviral cell attachment where host factor usage at the cell surface is highly promiscuous.

SEC14L2, a lipid-binding protein, regulates HCV replication in culture with inter- and intra-genotype variations.

BACKGROUND & AIMS: The lipid-binding protein, SEC14L2, is crucial for the efficient viral replication of clinical hepatitis C virus (HCV) isolates in cell culture. Given the role of SEC14L2 in HCV replication, we aimed to study a large number of HCV positive sera carrying genotypes 1-4, to identify viral factors associated with efficient replication in culture. Additionally, we investigated whether 13 single nucleotide polymorphisms (SNPs) of SEC14L2 have an impact on RNA replication of naturally occurring HCV isolates. METHODS: We generated Huh-7.5 cell lines overexpressing SEC14L2 or 13 coding SNPs and tested 73 different HCV positive sera for in vitro replication. Furthermore, we genotyped a cohort of 262 patients with chronic HCV for the common SNP (rs757660) and investigated its effect on the clinical phenotype. RESULTS: HCV isolates from genotype 1, 2, 3 and 4 replicate in Huh-7.5 cells overexpressing SEC14L2. Interestingly, only subgenomic replicons from genotypes 1 and 3 showed enhanced replication whereas genotypes 2 and 4 remained unaffected. Furthermore, replication was independent of viral load. Importantly, all tested SNPs supported HCV RNA replication in vitro, while 1 SNP was associated with decreased SEC1L2 expression and viral RNA. All SNPs exhibited comparable cellular cholesterol and vitamin E abundance in naïve Huh-7.5 cells. CONCLUSIONS: This large screen of natural HCV isolates of 4 genotypes underscores the relevance of SEC14L2 as an in vitro HCV host factor. Additionally, SEC14L2 variants appear to recapitulate the wild-type enhancement of HCV replication. Variant rs191341134 showed a decreased effect due to lowered stability, whereas variant rs757660, a high prevalence mutant, showed a similar phenotype to the wild-type. LAY SUMMARY: Until the year 2015, consistent replication of patient-derived isolates of hepatitis C virus (HCV) in an in vitro model remained a limitation in HCV research. In 2015 a group of authors identified a protein named SEC14L2 that enabled the replication of HCV isolates in cell culture. We performed a large screen encompassing 73 isolates of 4 different HCV genotypes. Additionally, we replaced the natural SEC14L2 with 13 different mutants to test if the protein variation significantly altered its HCV replication enhancing functions. We showed that different genotypes of HCV react differently to the presence of this protein and the variants of the protein mimic the behavior of the wild-type.

Multiparametric Functional MRI: Non-Invasive Imaging of Inflammation and Edema Formation after Kidney Transplantation in Mice.

BACKGROUND: Kidney transplantation (ktx) in mice is used to learn about rejection and to develop new treatment strategies. Past studies have mainly been based on histological or molecular biological methods. Imaging techniques to monitor allograft pathology have rarely been used. METHODS: Here we investigated mice after isogenic and allogenic ktx over time with functional MRI with diffusion-weighted imaging (DWI) and mapping of T2-relaxation time (T2-mapping) to assess graft inflammation and edema formation. To characterize graft pathology, we used PAS-staining, counted CD3-positive T-lymphocytes, analyzed leukocytes by means flow cytometry. RESULTS: DWI revealed progressive restriction of diffusion of water molecules in allogenic kidney grafts. This was paralleled by enhanced infiltration of the kidney by inflammatory cells. Changes in tissue diffusion were not seen following isogenic ktx. T2-times in renal cortex were increased after both isogenic and allogenic transplantation, consistent with tissue edema due to ischemic injury following prolonged cold ischemia time of 60 minutes. Lack of T2 increase in the inner stripe of the inner medulla in allogenic kidney grafts matched loss of tubular autofluorescence and may result from rejection-driven reductions in tubular water content due to tubular dysfunction and renal functional impairment. CONCLUSIONS: Functional MRI is a valuable non-invasive technique for monitoring inflammation, tissue edema and tubular function. It permits on to differentiate between acute rejection and ischemic renal injury in a mouse model of ktx.

T Cell CX3CR1 Mediates Excess Atherosclerotic Inflammation in Renal Impairment.

Reduced kidney function increases the risk for atherosclerosis and cardiovascular death. Leukocytes in the arterial wall contribute to atherosclerotic plaque formation. We investigated the role of fractalkine receptor CX3CR1 in atherosclerotic inflammation in renal impairment. Apoe(-/-) (apolipoprotein E) CX3CR1(-/-) mice with renal impairment were protected from increased aortic atherosclerotic lesion size and macrophage accumulation. Deficiency of CX3CR1 in bone marrow, only, attenuated atherosclerosis in renal impairment in an independent atherosclerosis model of LDL receptor-deficient (LDLr(-/-)) mice as well. Analysis of inflammatory leukocytes in atherosclerotic mixed bone-marrow chimeric mice (50% wild-type/50% CX3CR1(-/-) bone marrow into LDLr(-/-) mice) showed that CX3CR1 cell intrinsically promoted aortic T cell accumulation much more than CD11b(+)CD11c(+) myeloid cell accumulation and increased IL-17-producing T cell counts. In vitro, fewer TH17 cells were obtained from CX3CR1(-/-) splenocytes than from wild-type splenocytes after polarization with IL-6, IL-23, and TGFß Polarization of TH17 or TREG cells, or stimulation of splenocytes with TGFß alone, increased T cell CX3CR1 reporter gene expression. Furthermore, TGFß induced CX3CR1 mRNA expression in wild-type cells in a dose- and time-dependent manner. In atherosclerotic LDLr(-/-) mice, CX3CR1(+/-) T cells upregulated CX3CR1 and IL-17A production in renal impairment, whereas CX3CR1(-/-) T cells did not. Transfer of CX3CR1(+/-) but not Il17a(-/-) T cells into LDLr(-/-)CX3CR1(-/-) mice increased aortic lesion size and aortic CD11b(+)CD11c(+) myeloid cell accumulation in renal impairment. In summary, T cell CX3CR1 expression can be induced by TGFß and is instrumental in enhanced atherosclerosis in renal impairment.

Circulating ADAM17 Level Reflects Disease Activity in Proteinase-3 ANCA-Associated Vasculitis.

ANCA-associated vasculitides are characterized by inflammatory destruction of small vessels accompanied by enhanced cleavage of membrane-bound proteins. One of the main proteases responsible for ectodomain shedding is disintegrin and metalloproteinase domain-containing protein 17 (ADAM17). Given its potential role in aggravating vascular dysfunction, we examined the role of ADAM17 in active proteinase-3 (PR3)-positive ANCA-associated vasculitis (AAV). ADAM17 concentration was significantly increased in plasma samples from patients with active PR3-AAV compared with samples from patients in remission or from other controls with renal nonvascular diseases. Comparably, plasma levels of the ADAM17 substrate syndecan-1 were significantly enhanced in active AAV. We also observed that plasma-derived ADAM17 retained its specific proteolytic activity and was partly located on extracellular microparticles. Transcript levels of ADAM17 were increased in blood samples of patients with active AAV, but those of ADAM10 or tissue inhibitor of metalloproteinases 3, which inhibits ADAMs, were not. We also performed a microRNA (miR) screen and identified miR-634 as significantly upregulated in blood samples from patients with active AAV. In vitro, miR-634 mimics induced a proinflammatory phenotype in monocyte-derived macrophages, with enhanced expression and release of ADAM17 and IL-6. These data suggest that ADAM17 has a prominent role in AAV and might account for the vascular complications associated with this disease.

Interleukin 17 receptor A modulates monocyte subsets and macrophage generation in vivo.

Interleukin (IL)-17A signaling via Interleukin 17 receptor A (Il17ra) contributes to the inflammatory host response by inducing recruitment of innate immune cells, but also plays a role in homeostatic neutrophilic granulocyte regulation. Monocytes, the other main innate immune cell, have a longer life span and can pursue multiple differentiation pathways towards tissue macrophages. Monocytes are divided into two subpopulations by expression of the Ly6C/Gr1 surface marker in mice. We here investigated the role of Il17ra in monocyte homeostasis and macrophage generation. In Il17ra(-/-) and in mixed bone marrow chimeric wt/Il17ra(-/-) mice, the concentrations of circulating Il17ra(-/-) Gr1(low) monocytes were significantly decreased compared to wt cells. Pulmonary, splenic and resident peritoneal Il17ra(-/-) macrophages were significantly fewer than of wt origin. Bone marrow progenitor and monocyte numbers were equal, but the proportion of Il17ra(-/-) Gr1(low) monocytes was already decreased at bone marrow level. After monocyte depletion, initial Gr1(high) and Gr1(low) monocyte regeneration of Il17ra(-/-) and wt cells was very similar. However, Il17ra(-/-) Gr1(low) counts were not sustained. After labeling with either fluorescent beads or BrdU, Il17ra(-/-) Gr1(high) monocyte transition to Gr1(low) cells was not detectable unlike wt cells. Monocyte recruitment in acute peritonitis, which is known to be largely due to Gr1(high) cell migration, was unaffected in an identical environment. Unilateral ureteral obstruction induces a less acute inflammatory and fibrotic kidney injury. Compared to wt cells in the same environment, Il17ra(-/-) macrophage accumulation in the kidney was decreased. In the absence of Il17ra on all myeloid cells, renal fibrosis was significantly attenuated. Our data show that Il17ra modulates Gr1(low) monocyte counts and suggest defective Gr1(high) to Gr1(low) monocyte transition as an underlying mechanism. Lack of Il17ra altered homeostatic tissue macrophage formation and diminished renal inflammation and fibrosis. Il17ra appears to be a novel modulator of monocyte phenotype and possible therapeutic target in renal fibrosis.

Increased atherosclerotic lesion formation and vascular leukocyte accumulation in renal impairment are mediated by interleukin-17A.

RATIONALE: Atherosclerosis is a major cause of death in patients with chronic kidney disease. Chronic inflammation of the arterial wall including invasion, proliferation, and differentiation of leukocytes is important in atherosclerotic lesion development. How atherosclerotic inflammation is altered in renal impairment is incompletely understood. OBJECTIVE: This study analyzed leukocytes of the atherosclerotic aorta in mice with impaired and normal renal function and studied a mechanism for the alteration in aortic myeloid leukocytes. METHODS AND RESULTS: Unilateral nephrectomy significantly decreased glomerular filtration rate and increased atherosclerotic lesion size and aortic leukocyte numbers in 2 murine atherosclerosis models, apolipoprotein E (Apoe(-/-)) and low-density lipoprotein (LDL) receptor-deficient (LDLr(-/-)) mice. The number of aortic myeloid cells increased significantly. They took-up less oxidized LDL, whereas CD11c expression, interaction with T cells, and aortic T cell proliferation were significantly enhanced in renal impairment. In human peripheral blood mononuclear cell cultures, chronic kidney disease serum decreased lipid uptake and increased human leukocyte antigen II (HLA II) expression. Supplementation with interleukin-17A similarly increased HLA II and CD11c expression and impaired oxidized LDL uptake. Interleukin-17A expression was increased in atherosclerotic mice with renal impairment. Ablation of interleukin-17A in LDLr(-/-) mice by lethal irradiation and reconstitution with Il17a(-/-) bone marrow abolished the effect of renal impairment on aortic CD11b(+) myeloid cell accumulation, CD11c expression, and cell proliferation. Atherosclerotic lesion size was decreased to levels observed in normal kidney function. CONCLUSIONS: Kidney function modifies arterial myeloid cell accumulation and phenotype in atherosclerosis. Our results suggest a central role for interleukin-17A in aggravation of vascular inflammation and atherosclerosis in renal impairment.

Angiopoietin-2 is a potential mediator of endothelial barrier dysfunction following cardiopulmonary bypass.

INTRODUCTION: Endothelial activation leading to vascular barrier dysfunction and organ failure is a well-recognized complication of cardiovascular surgery with cardiopulmonary bypass (CPB). The endothelial-specific angiopoietin-Tie2 ligand-receptor system has been identified as a non-redundant regulator of endothelial activation. Binding of angiopoietin-2 (Ang-2) to the Tie2 receptor antagonizes Tie2 signaling and renders the endothelial barrier responsive to pro-inflammatory cytokines. We aimed to study the time course and potential triggering factors of Ang-2 release after CPB, as well as the association of Ang-2 changes with surrogates of increased vascular permeability, organ dysfunction, and outcome. METHODS: Serum levels of Ang-2 from 25 adult patients (140 screened) were measured before and at 0, 12, and 24h following CPB procedure by in-house immuno-luminometric assay (ILMA), and compared with indices of organ dysfunction, duration of mechanical ventilation (MV), length of stay (LOS) in the intensive care unit (ICU), and hospital mortality. The effect of Ang-2 was studied in vitro by incubating high Ang-2 patient serum with endothelial cells (EC). RESULTS: Ang-2 levels steadily increased from 2.6 ± 2.4 ng/mL at 0 h up to 7.3 ± 4.6 ng/mL at 24h following CPB (P<0.001). The release of Ang-2 correlated with the duration of CPB, aortic cross-clamp time, and post-CPB lactate levels. Changes in Ang-2 during follow-up correlated with partial pressure of oxygen in arterial blood (PaO(2))/fraction of inspired oxygen (FiO(2)) ratio, alveolar-arterial oxygen tension difference (AaDO(2)), hemodynamics, fluid balance, and disease severity measures. Ang-2 levels at 12h predicted the duration of MV, ICU-LOS, and hospital mortality. High Ang-2 patient sera disrupted EC architecture in vitro, an effect reversed by treatment with the competitive Tie2 ligand angiopoietin-1 (Ang-1). CONCLUSIONS: Collectively, our results suggest that Ang-2 is a putative mediator of endothelial barrier dysfunction after CPB. These findings suggest that targeting the Ang/Tie2 pathway may mitigate organ dysfunction and improve outcome in patients undergoing CPB.

Microparticle generation and leucocyte death in Shiga toxin-mediated HUS.

BACKGROUND: Shiga toxin-induced haemolytic uraemic syndrome (STEC-HUS) is an acute multisystem disorder characterized by renal failure, neurological dysfunction, haemolysis and intravascular thrombosis. Circulating microparticles originating from a number of cell types including thrombocytes and leucocytes are elevated in paediatric patients. In vitro data also suggest modification of leucocyte death by Shiga toxin. Here, we investigated microparticle generation and leucocyte cell death in vivo in adult STEC-HUS patients during acute disease and recovery. METHODS: Multi-colour flow cytometry and immunofluorescence were used to assess microparticle concentration and provenience thrombocyte microparticle seeding to leucocytes and leucocyte cell death in adult STEC-HUS patients treated at a tertiary care centre during the STEC-HUS outbreak in Germany in 2011. RESULTS: Plasma microparticle concentrations of both platelet and leucocyte origin were elevated during acute STEC-HUS. Platelet microparticles (MP) were detected on a high proportion of monocytes and granulocytes. Among therapeutic interventions, plasma exchange reduced platelet marker expression on leucocytes, inhibition of complement had only moderate impact on the number of circulating MP and did not alter platelet microparticle binding to leucocytes. Numbers of apoptotic and necrotic monocytes and granulocytes were significantly increased in patients with STEC-HUS compared to healthy controls. Complement inhibition significantly increased the number of circulating apoptotic cells. Monocyte apoptosis on admission was significantly higher in patients subsequently assigned to plasma exchange or admitted to the intensive care unit. CONCLUSIONS: In STEC-HUS, elevated numbers of circulating MP and dead leucocytes were detected. Monocyte and granulocyte deaths are novel markers of acute STEC-HUS that may actively contribute to tissue destruction by liberation of pro-inflammatory enzymes and cytokines.

Detection of circulating microparticles by flow cytometry: influence of centrifugation, filtration of buffer, and freezing.

The clinical importance of microparticles resulting from vesiculation of platelets and other blood cells is increasingly recognized, although no standardized method exists for their measurement. Only a few studies have examined the analytical and preanalytical steps and variables affecting microparticle detection. We focused our analysis on microparticle detection by flow cytometry. The goal of our study was to analyze the effects of different centrifugation protocols looking at different durations of high and low centrifugation speeds. We also analyzed the effect of filtration of buffer and long-term freezing on microparticle quantification, as well as the role of Annexin V in the detection of microparticles. Absolute and platelet-derived microparticles were 10- to 15-fold higher using initial lower centrifugation speeds at 1500 × g compared with protocols using centrifugation speeds at 5000 × g (P < 0.01). A clear separation between true events and background noise was only achieved using higher centrifugation speeds. Filtration of buffer with a 0.2 µm filter reduced a significant amount of background noise. Storing samples for microparticle detection at -80°C decreased microparticle levels at days 28, 42, and 56 (P < 0.05 for all comparisons with fresh samples). We believe that staining with Annexin V is necessary to distinguish true events from cell debris or precipitates. Buffers should be filtered and fresh samples should be analyzed, or storage periods will have to be standardized. Higher centrifugation speeds should be used to minimize contamination by smaller size platelets.

Engulfment of apoptotic cells by microvascular endothelial cells induces proinflammatory responses.

Circulating endothelial cells (CECs) have been detected in a variety of vascular disorders, but their interactions with healthy endothelium remain unknown. The aim of this study was to evaluate the response of human endothelial cells (ECs) to apoptotic or necrotic ECs in an in vitro model and to delineate pathogenetic pathways. Here we show that incubation of the human microvascular endothelial cell line (HMEC-1) with apoptotic ECs resulted in increased expression of chemokines and enhanced binding of leukocytes to HMEC-1 cells, whereas exposure of HMEC-1 cells to necrotic ECs caused no changes in leukocyte-binding affinity. Both apoptotic and necrotic cells were bound and engulfed by HMEC-1 cells and primary human umbilical vein endothelial cells (HUVECs). We therefore suggest that exposures to apoptotic and necrotic ECs induce different patterns of chemokine synthesis and leukocyte adhesion in healthy ECs. These data indicate that CECs are not only markers of vascular damage but may induce proinflammatory signals in the endothelium.

Stimulation of endothelial progenitor cells: a new putative therapeutic effect of angiotensin II receptor antagonists.

The number of circulating endothelial progenitor cells (EPCs) correlates with endothelial dysfunction and cardiovascular risk in humans. We explored whether angiotensin II receptor antagonist therapy affects the number of regenerative EPCs in patients with type 2 diabetes. In a prospective double-blind parallel group study, we randomly treated 18 type 2 diabetics with olmesartan (40 mg) or placebo for 12 weeks. We analyzed circulating CD34+ hematopoietic progenitor cells (flow cytometry) and EPCs (in vitro assay) before and after therapy. We verified the results in a second open trial treating 20 type 2 diabetics with 300 mg of irbesartan for 12 weeks. The number of EPCs was significantly lower in diabetic patients as compared with 38 age-matched healthy subjects (210+/-10 versus 258+/-18 per high-power field; P<0.05), whereas there was no significant difference with respect to hematopoietic progenitor cells. Treatment with olmesartan (n=9) significantly increased EPCs from 231+/-24 to 465+/-71 per high-power field (P<0.05), but not hematopoietic progenitor cells. In contrast, placebo treatment (n=9) did not affect EPCs and hematopoietic progenitor cells. With irbesartan therapy, EPC number increased significantly from 196+/-15 to 300+/-23 per high-power field (P<0.05) already after 4 weeks of treatment. At the end of 12-week therapy, patients had 310+/-23 EPCs per high-power field (P<0.05 versus baseline). Angiotensin II receptor antagonists increase the number of regenerative EPCs in patients with type 2 diabetes mellitus. This action may be of therapeutic relevance contributing to their beneficial cardiovascular effects.

Erythropoietin regulates endothelial progenitor cells.

Circulating bone marrow-derived endothelial progenitor cells (EPCs) promote vascular reparative processes and neoangiogenesis, and their number in peripheral blood correlates with endothelial function and cardiovascular risk. We tested the hypothesis that the cytokine erythropoietin (EPO) stimulates EPCs in humans. We studied 11 patients with renal anemia and 4 healthy subjects who received standard doses of recombinant human EPO (rhEPO). Treatment with rhEPO caused a significant mobilization of CD34(+)/CD45(+) circulating progenitor cells in peripheral blood (measured by flow cytometry), and increased the number of functionally active EPCs (measured by in vitro assay) in patients (week 2, 312% +/- 31%; week 8, 308% +/- 40%; both P <.01 versus baseline) as well as in healthy subjects (week 8, 194% +/- 15%; P <.05 versus baseline). The effect on EPCs was already observed with an rhEPO dose of about 30 IU/kg per week. Administration of rhEPO increased the number of functionally active EPCs by differentiation in vitro in a dose-dependent manner, assessed in cell culture and by tube formation assay. Furthermore, rhEPO activates the Akt protein kinase pathway in EPCs. Erythropoietin increases the number of functionally active EPCs in humans. Administration of rhEPO or EPO analogs may open new therapeutic strategies in regenerative cardiovascular medicine.