Platelets stimulate fibroblast-mediated contraction of collagen gels

BACKGROUND: Platelets are thought to play a role in a variety of inflammatory conditions in the lung, some of which may lead to fibrosis. In the current study we tested the hypothesis that whole platelets and platelet lysate can mediate remodelling of extracellular matrix in vitro by affecting fibroblast-mediated contraction of a collagen gel. We also sought to determine to what extent platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) contribute to this effect. METHODS: Washed platelets, isolated from healthy blood donors, and platelet lysate (freezing and thawing), were cast together with human lung fibroblasts in three-dimensional collagen gels. The gels were then released and cultured for four days. PDGF and TGF-β(1 )concentrations were measured in culture supernatants by ELISA. RESULTS: Both platelets and platelet lysate augmented fibroblast-mediated gel contraction in a time and concentration dependent manner (19.9% ± 0.1 (mean ± SEM) of initial area vs. 48.0% ± 0.4 at 48 hours; P < 0.001 and 41.5% ± 0.6 vs. 60.6% ± 0.3 at 48 hours; P < 0.001, respectively). Fixed platelets had no effect in the system. Both TGF-β(1 )and PDGF-AA/AB were released in co-culture. PDGF-AA/AB had a maximum release at 24 hours whereas TGF-β(1 )release increased with longer culture periods. Neutralising antibodies to these mediators partially inhibited platelet-induced gel contraction. CONCLUSION: We conclude that platelets may promote remodelling of extracellular matrix in vitro and that PDGF and TGF-β partially mediate this effect, also indicating a role for other mediators. The findings may be an important mechanism in regulating repair processes after injury

Reduced expression of gm-csf receptor on cord blood progenitor cells from neonates at risk for atopy

Recent evidence points to an association between allergic asthma and increased responsiveness of eosinophil progenitor cells to hematopoietic growth factors. However, it is not clear at what age this phenomenon first becomes manifest. Objective: Compare expression of cytokine receptors on CD34+ progenitor cells in cord blood from normal infants ('low risk') and infants with at least one atopic first degree relative ('at risk'). Methods: Cord blood was obtained from 21 neonates. Cells were stained with antibodies directed against CD45, CD34, and the a chains of the GM-CSF, IL-3 and IL-5 receptors, and analyzed by flow cytometry. Results: No differences in absolute CD34 numbers were observed between the two groups. Surprisingly, expression of GM-CSF receptor on CD34+ cells was reduced in the 'at risk' group (1.92±O.89%; ±se) compared to the 'low risk' group (5.62±1.30%;

Adhesion molecule crosslinking and cytokine exposure modulate IgE and non-IgE- dependent basophil activation

QC 20200818</p

Leukocyte proliferation and immune modulator production in patients with chronic kidney disease.

INTRODUCTION: In Chronic Kidney Disease (CKD), immune cells are affected by uremic retention toxins. Given this effect, we analyzed lymphocyte proliferative response and immune modulators production following in vitro stimulation. METHODS: Whole blood was drawn from healthy controls, patients with eGFR <20 ml/min/1.73 m(2) (Pre-dialysis, CKD stages 4 and 5) and hemodialysis patients (stage 5D). Peripheral cells were incubated for six days with pokeweed mitogen, concanavalin A, Staphylococcus enterotoxin A or influenza A vaccine. Peripheral lymphocyte proliferation was then analyzed by the "Flow-cytometric Assay of Specific Cell-mediated Immune response in Activated whole blood" (FASCIA) method, and cytokine profile in the cell supernatants was analyzed by the Milliplex multi-array method. RESULTS: The absolute number of lymphoblasts in response to mitogenic stimulation and the number of cells in each CD4+ and CD8+ subpopulation were similar comparing the three groups, except for a single decline in number of lymphoblasts after stimulation with Staphylococcus enterotoxin A, comparing dialysis patients with healthy controls. Levels of interleukin (IL)-2 (p=0.026), -10 (p=0.019) and -15 (p=0.027) in the Staphylococcus enterotoxin A-stimulated supernatant were lower in hemodialysis patients compared to healthy controls. Levels of IL-15 (p=0.017) from pre-dialysis patients and levels of IL-5 (p=0.019) from hemodialysis patients in influenza A vaccine-stimulated supernatants were also lower compared to controls. In pokeweed mitogen-stimulated supernatant, IL-2 levels (p=0.013) were lower in hemodialysis patients compared to pre-dialysis patients. TNF-α, IL-10, IL-12, IL-15, IL-8, MCP-1, IP-10, IFN-α2, IL-1α and eotaxin levels were all significantly higher in plasma obtained from CKD patients. CONCLUSION: Our results suggest that T-cells from CKD patients have similar proliferative response to stimulation compared with healthy individuals. Moreover, however the immune cells show inability to produce selected cytokines, most likely due to the uremic milieu or dialysis procedure

Adhesion molecule crosslinking and cytokine exposure modulate IgE and non-IgE- dependent basophil activation

QC 20200818</p

Monocyte and Neutrophil Chemotactic Activity at the Site of Interstitial Inflammation in Patients on High-Flux Hemodialysis or Hemodiafiltration

Background/Aims: We have observed a difference between patients on low-flux hemodialysis (HD) or peritoneal dialysis and patients on hemodiafiltration (HDF) or high-flux HD in the capacity of transmigrated leukocytes to mobilize CD11b in response to inflammatory stimuli compared with healthy subjects. This could be due to different interstitial chemokine concentrations. Methods: We measured concentrations of circulating and interstitial macrophage inflammatory protein-1 alpha (MIP-1 alpha), matrix metalloproteinase-9 (MMP-9)/neutrophil gelatinase-associated lipocalin (NGAL), monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) in 10 patients on HDF or high-flux HD and 11 healthy subjects by using immunoassay. Results: The interstitial concentrations of MIP-1 alpha, MMP-9/NGAL and IL-8 were similar in patients and healthy subjects, while the corresponding concentration of MCP-1 was significantly higher in patients on HDF or high-flux HD as compared with healthy subjects (p < 0.01). Conclusion: We suggest that an equal or higher concentration of chemokines in the interstitium in patients with HDF or high-flux HD might be one mechanism responsible for the preserved function of transmigrated leukocytes. Copyright (C) 2009 S. Karger AG, Base

B cell and monocyte phenotyping: A quick asset to investigate the immune status in patients with IgA nephropathy.

BackgroundIgA nephropathy (IgAN) advances from multiple pathogenic "hits" resulting in poorly O-galactosylated IgA1 glycoforms (Gd-IgA1), production of antibodies and glomerular deposition of immune complexes. A sequence of immune responses arising from plasma cells, T cells and antigen presenting cells (APCs), causes glomerular injury. This study was designed to phenotype subsets of B cells, monocytes and T cells in the peripheral circulation and their association with inflammatory cytokines and kidney function in patients with IgAN, healthy controls (HC) and disease controls with autosomal dominant polycystic kidney disease (ADPKD).MethodsPatients with IgAN (n = 13), median estimated glomerular filtration rate (eGFR) of 57 ml/min/1.73m2 (IQR 42-84), patients with ADPKD (n = 13) matched for kidney function, gender and age and gender and age-matched HC (n = 13) were recruited. CD3+ and CD3- peripheral blood mononuclear cells were isolated and profiled based on their specific surface markers for different subsets of monocytes, B and T cells and analyzed by flow cytometry. Cytokines were analyzed by ELISA.ResultsWe observed a significant decrease in the proportion of pre-switched B cells and plasmablasts, but an increase in long-lived plasma cells in the peripheral circulation of IgAN patients compared to HC. The proportion of non-classical monocytes was significantly higher in IgAN patients compared to both HC and ADPKD. We also report an association between sCD40L levels and the proportion of pre-switched B cells, as well as sCD40L and MCP-1 levels and albuminuria in IgAN patients.ConclusionsWe applied an easy-access method to analyze subsets of immune cells as well as relevant inflammatory mediators in IgAN patients. Our data demonstrate an altered B cell profile that indicates a pathophysiological role of the B cell lineage and an increased proportion of non-classical monocytes that suggests their role in the disease process