Assessment of longitudinal serum neutrophil extracellular trap-inducing activity in anti-neutrophil cytoplasmic antibody-associated vasculitis and glomerulonephritis in a prospective cohort using a novel bio-impedance technique.

Neutrophil extracellular traps (NET) have been implicated in the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Here, we developed a novel, label-free, high-throughput bio-impedance technique to effectively measure serum NET-inducing activity. Using this technique, NET-inducing activity of serum derived from patients with AAV was assessed in a prospective cohort of 62 patients presenting with active AAV with major organ involvement. Thirty-five patients presented with new and 27 patients presented with relapsing AAV, of whom 38 had kidney and/or 31 had lung involvement. NET-inducing activity was assessed at diagnosis of active AAV (time zero), during the first 6 weeks of treatment, and after 6 months of treatment. Forty-seven patients revealed elevated NET-inducing activity at time zero. After initiation of immunosuppressive treatment, NET-inducing activity was reduced at six weeks. A subsequent increase at six months could potentially identify patients with relapsing disease (hazard ratio, 11.45 [interquartile range 1.36-96.74]). NET-inducing activity at time zero correlated with kidney function and proteinuria. Importantly, in kidney tissue, NETs co-localized with lesions typical of ANCA-associated glomerulonephritis and even correlated with systemic serum NET-inducing activity. Thus, our prospective data corroborate the importance of NET formation in AAV and ANCA-associated glomerulonephritis and the potential of longitudinal evaluation, as monitored by our novel bio-impedance assay and detailed histological evaluation.

C5b9 Formation on Endothelial Cells Reflects Complement Defects among Patients with Renal Thrombotic Microangiopathy and Severe Hypertension.

Background Severe hypertension can induce thrombotic microangiopathy (TMA) in the renal vasculature, the occurrence of which has been linked to mechanical stress to the endothelium. Complement defects may be the culprit of disease in patients who present with severe renal disease and often progress to ESRD, despite BP control.Methods We studied a well defined cohort of 17 patients with hypertension-associated TMA to define the prevalence of complement defects by a specific ex vivo serum-based microvascular endothelial cell assay.Results Compared with normal human serum and samples from patients with hypertensive arterionephrosclerosis, 14 of 16 (87.5%) serum samples collected at presentation from 16 patients with hypertension-associated TMA induced abnormal C5b9 formation on microvascular endothelial cells. We detected rare variants in complement genes in eight of 17 (47%) patients. ESRD occurred in 14 of 17 (82%) patients, and recurrent TMA after transplant occurred in seven of 11 (64%) donor kidneys. Eculizumab improved the renal function in three patients and prevented TMA recurrence in an allograft recipient.Conclusions These observations point to complement defects as the key causative factor of ESRD and recurrent TMA after transplant in patients presenting with severe hypertension. Complement defects can be identified by measurements of complement activation on microvascular endothelial cells, which should substantially influence treatment and prognosis.

CD4+CD28null T Cells are related to previous cytomegalovirus infection but not to accelerated atherosclerosis in ANCA-associated vasculitis.

Previous studies have suggested an increased risk for cardiovascular events in antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). We analyzed the presence of atherosclerotic damage in patients with AAV in relation to the presence of CD4+CD28null T cells and antibodies against cytomegalovirus (CMV) and human Heat-Shock Protein 60 (hHSP60). In this cross-sectional study, patients with inactive AAV were compared with healthy controls (HC). Carotid intima-media thickness (IMT) and aortic pulse-wave velocity (PWV) were measured. In addition, CD4+CD28null T cells, anti-CMV, and anti-hHSP60 levels were determined. Forty patients with AAV were included. Patients' spouses were recruited as HC (N = 38). CD4+CD28null T cells are present in patients with AAV in a higher percentage (median 3.1, range 0.01-85) than in HC (0.28, 0-36, P < 0.0001). No significant difference in IMT (mm) between patients and controls was detected (mean 0.77 ± standard deviation 0.15 and 0.73 ± 0.11, respectively, P = 0.20). PWV standardized for MAP was increased in AAV patients (9.80 ± 2.50 m/s, compared to 8.72 ± 1.68 in HC, P = 0.04). There was a strong association between a previous CMV infection and the presence and percentage of CD4+CD28null T cells (0.33 vs 13.8, P < 0.001). There was no relationship between CD4+CD28null T cells and/or a previous CMV infection and IMT or PWV. There was no relation between anti-hHSP60 and CD4+CD28null T cells. Increased PWV values suggest atherosclerotic damage in patients with AAV. Plaque size, as determined by IMT, did not differ. CD4+CD28null T cells are increased in AAV and related to the previous CMV infection.

Phosphorylcholine antibodies are diminished in ANCA-associated vasculitis.

BACKGROUND/AIM: ANCA-associated (AAV) vasculitis is an autoimmune small-vessel vasculitis and may be associated with accelerated atherosclerosis as suggested by current literature. Antibodies against oxidized lipoproteins (OxLDL) and phosphorylcholine (Pc) protect from atherosclerosis. This study characterizes these antibodies in AAV. METHODS: Pc- and anti-OxLDL antibodies were determined in sera of 39 AAV patients and 44 healthy controls (HC). Intima-media thickness (IMT, carotids) and pulse wave velocity (PWV, A. femoralis) were measured. RESULTS: Pc-/OxLDL IgM antibodies were significantly reduced in AAV. IMT and PWV were negatively associated with anti-Pc antibodies in HC only. CONCLUSION: Atheroprotective anti-Pc/anti-OxLDL antibodies are significantly reduced in AAV possibly explaining accelerated atherosclerosis in vasculitis patients.

DNA extraction from long-term stored urine.

BACKGROUND: Traditionally, for DNA analyses, DNA is recovered from buffy coats. Since DNA in urine has been reported to deteriorate quickly, this option is often not considered. To complete our DNA database in patients with ANCA-associated vasculitis, we aimed to extract DNA from stored urine. METHODS: Urine was stored at the time of kidney biopsy from patients included in our regional kidney biopsy database, who had given informed consent for further study. Urine was subsequently filtered, dialyzed, concentrated and freeze dried and finally resolubilized and centrifuged. DNA was extracted using the high pure PCR template preparation kit (Roche Diagnostics). Next, concentration and purity were determined by Nanodrop analysis and by Quant-iT analysis. RESULTS: One hundred and eighty-one patients with ANCA-associated vasculitis were included. Of 114 patients (63%), DNA was available. From 53 of the remaining 67 patients, stored urine was available. Of the 53 samples that were processed, 46 (86.8%) yielded DNA with a mean concentration of 258.7 ng/µL (range 33.2-529) with a mean purity ratio of 1.81 (λ 260/280). CONCLUSION: DNA extraction from fresh urine has been described before, yielding DNA usable for PCR analysis in healthy subjects. Storage of fresh urine at 4 °C or lower temperatures results in significant degradation of the DNA, making recovery of DNA more difficult with longer periods of storage. In the current study, we demonstrated that DNA could be retrieved from subsequently filtered, dialyzed, concentrated and freeze dried urine that was stored at room temperature. In addition, we demonstrated that this DNA could be used for PCR analysis. This method is useful when no other material from these patients is available.

Autoantibodies against oxidized low-density lipoprotein in cerebral small vessel disease.

BACKGROUND AND PURPOSE: Oxidized low-density lipoprotein (oxLDL) induces endothelial dysfunction and antibody formation. Because endothelial dysfunction is involved in cerebral small vessel disease (CSVD) (dilated Virchow Robin spaces, lacunar infarcts, and white matter lesions), oxLDL antibodies could play a role in CSVD pathogenesis. Therefore, we studied oxLDL antibodies in patients with high prevalence of CSVD: lacunar stroke patients and essential hypertensive patients. METHODS: A total of 158 lacunar stroke patients, 158 hypertensive patients, and 43 healthy controls were included. We determined levels of IgG and IgM against hypochlorite (HOCl) and malondialdehyde (MDA) oxLDL using ELISA (values in optical density). RESULTS: Patients with CSVD had higher levels of IgG-HOCl-oxLDL (0.77 versus 0.70; P<0.01), as well as lower levels of IgM-MDA-oxLDL (0.55 versus 0.65; P<0.05) than patients without such lesions. Higher IgG-HOCl-oxLDL levels were only independently associated with higher numbers of Virchow Robin spaces at the level of the basal ganglia (ß=0.218; P<0.001). CONCLUSIONS: An autoinflammatory process with lower levels of IgM antibodies and higher levels of IgG antibodies against oxLDL may be involved in CSVD.

Adipogenesis from Bovine Precursors.

Cultured meat, or tissue engineered meat, is a promising alternative to conventional meat production. In order to realistically mimic the multiple tissue types found in beef, food-compatible methods for bovine fat tissue engineering must be developed. We present a protocol for the isolation of adipose tissue-derived preadipocytes and subsequent adipogenic differentiation through free fatty acid stimulation. Differentiating preadipocytes can be either grown in 2D culture conditions or seeded in 3D alginate scaffolds. Differentiation is visually confirmed through lipophilic staining.

Vitamin D3 supplementation and the IL-2/IL-2R pathway in multiple sclerosis: Attenuation of progressive disturbances?

Vitamin D3 upregulates IL-2 receptor alpha (IL2RA, CD25)-expression on CD4+ T cells in vitro. We investigated effects of 48-weeks vitamin D3 supplements on CD25-expression by CD4+ T cells of patients with multiple sclerosis (MS). There was no significant difference between the vitamin D3 (n=30) and placebo group (n=23) in IL2RA mRNA-expression by PBMC. Likewise, CD25 cell surface-expression by conventional or regulatory T cells (Treg) did not differ between groups, although Treg CD25-expression and circulating soluble-CD25 levels decreased significantly in the placebo but not vitamin D3-group. We speculate that vitamin D3 may promote the maintenance of CD25-related immune homeostasis in MS.

Monocytic microRNA profile associated with coronary collateral artery function in chronic total occlusion patients.

An expansive collateral artery network is correlated with improved survival in case of adverse cardiac episodes. We aimed to identify cellular microRNAs (miRNA; miR) important for collateral artery growth. Chronic total occlusion (CTO) patients (n = 26) were dichotomized using pressure-derived collateral flow index (CFIp) measurements; high collateral capacity (CFIp > 0.39; n = 14) and low collateral (CFIp < 0.39; n = 12) capacity. MiRNA profiling via next generation sequencing from various monocyte phenotypes (freshly isolated monocytes, monocytes cultured without stimulant, or stimulation with lipopolysaccharide, interleukin 4, transforming growth factor beta-1, or interferon gamma) revealed significantly different miRNA expression patterns between high versus low collateral capacity patients. Validation by real-time polymerase chain reaction demonstrated significantly decreased expression of miR339-5p in all stimulated monocyte phenotypes of low collateral capacity patients. MiR339-5p showed significant correlation with CFIp values in stimulated monocytes. Ingenuity pathway analysis of predicted gene targets of miR339-5p and differential gene expression data from high versus low CFIp patients (n = 20), revealed significant association with STAT3 pathway, and also suggested a possible regulatory role for this signaling pathway. These results identify a novel association between miR339-5p and coronary collateral function. Future work examining modulation of miR339-5p and downstream effects on the STAT3 pathway and subsequent collateral vessel growth are warranted.

Expression of NLRP3 inflammasome and T cell population markers in adipose tissue are associated with insulin resistance and impaired glucose metabolism in humans.

Recent studies in rodents indicate that the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome and a proinflammatory shift in the T cell population in adipose tissue (AT) contribute to AT inflammation and insulin resistance. We investigated: (1) the interplay between the NLRP3 inflammasome and T cell populations in abdominal subcutaneous AT in obese and lean humans in relation to AT inflammatory processes, and (2) involvement of the NLRP3 inflammasome and T cell populations in insulin resistance. Abdominal subcutaneous AT biopsies were collected in 10 obese men with impaired glucose tolerance and 9 lean normal glucose tolerant age-matched controls. AT gene expression of NLRP3 inflammasome-related genes and markers of T cell populations, chemoattraction, macrophage infiltration and other aspects of inflammation were examined. Furthermore, we examined systemic adaptive immune activation and insulin sensitivity (hyperinsulinemic-euglycemic clamp). CASPASE-1 mRNA and the proportion of T(h)1 transcripts (TBX21/CD3ɛ) were significantly higher in AT from obese compared with lean subjects. CASPASE-1 expression and a relative increase in T(h)1 transcripts in AT were strongly associated with insulin resistance and impairments in glucose homeostasis. Gene expression of NLRP3, CASPASE-1, CD3ɛ (pan T cells), TBX21 (T(h)1 cells) and RORC (T(h)17 cells) was positively, whereas GATA3 (T(h)2 cells) was inversely correlated with AT inflammation. Our data suggest that NLRP3 inflammasome activation and a T(h)1 shift in the T cell population in AT of obese subjects is related to insulin resistance and impaired glucose metabolism, which may be explained by AT inflammatory processes.

Vascular inflammation in cerebral small vessel disease.

Cerebral small vessel disease (CSVD) is considered to be caused by an increased permeability of the blood-brain barrier and results in enlargement of Virchow Robin spaces (VRs), white matter lesions, brain microbleeds, and lacunar infarcts. The increased permeability of the blood-brain barrier may relate to endothelial cell activation and activated monocytes/macrophages. Therefore, we hypothesized that plasma markers of endothelial activation (adhesion molecules) and monocyte/macrophage activation (neopterin) relate to CSVD manifestations. In 163 first-ever lacunar stroke patients and 183 essential hypertensive patients, we assessed CSVD manifestations on brain magnetic resonance imaging (MRI) and levels of C-reactive protein (CRP), neopterin, as well as circulating soluble adhesion molecules (sICAM-1, sVCAM-1, sE-selectin, sP-selectin). Neopterin, sICAM-1 and sVCAM-1 levels were higher in patients with extensive CSVD manifestations than in those without (p < 0.01). Neopterin levels independently related to higher numbers of enlarged Virchow Robin spaces (p < 0.001). An inflammatory process with activated monocytes/macrophages may play a role in the increased permeability of the blood brain barrier in patients with CSVD.

Vitamin D-related gene expression profiles in immune cells of patients with relapsing remitting multiple sclerosis.

An impaired vitamin D (vit-D) processing by immune cells of relapsing remitting multiple sclerosis (RRMS) patients may increase their vulnerability for a poor vit-D status. We assessed with qPCR the expression of vit-D related genes in PBMC and CD4+ T-cells. Gene expression profiles of vit-D receptor (VDR), CYP27B1 and CYP24A1 did not differ between RRMS patients and healthy controls. Interestingly, more VDR expression in PBMC correlated with less circulating IFN-γ+ CD4+ T-cells. Our results suggest that vit-D processing by immune cells is not impaired in RRMS, and is potentially relevant for the composition of the peripheral CD4+ T-cell compartment.

Evaluation of antibodies against human HSP60 in patients with MPO-ANCA associated glomerulonephritis: a cohort study.

BACKGROUND: Human Heat Shock Protein 60 (hHSP60) has been implicated in autoimmunity through molecular mimicry, based on the high degree of homology with HSP65 of micro-organisms leading to autoimmune recognition of the human protein. Additionally, sequence homology between hHSP60 and myeloperoxidase (MPO) has been described. MPO is a major autoantigen in vasculitis associated with antineutrophil cytoplasmic antibodies (ANCA). We hypothesized that infections may trigger the ANCA response against MPO through hHSP60. METHODS: In 86 consecutive patients with ANCA-associated vasculitis (AAV), anti-hHSP60 and anti-mycobacterial HSP65 were measured by ELISA. Patients were compared with 69 healthy controls (HC). Continuous data between groups were compared using Wilcoxon signed rank test and Kruskal-Wallis test with Dunn's post-test when appropriate. Correlations between data were derived using Spearman correlation. Odds ratios and 95% confidence intervals were obtained using Fisher's exact test. RESULTS: At diagnosis, median anti-mHSP65 level was higher in AAV (median [range]: 42.5 [0-500]), and subsequently in MPO-ANCA (44 [7-500]), compared to HC (22 [0-430]). Anti-hHSP60 levels in AAV were not higher compared to HC (18 [0-319] and 18.5 [0-98], respectively). However, in MPO-ANCA anti-hHSP60 levels were increased (32.5 [0-319]) compared to PR3-ANCA (13 [0-79]) and HC. We could not detect cross-reactivity between hHSP60 and MPO-ANCA. There was a correlation between anti-mHSP65 and anti-hHSP60 levels (r = 0.32, P = 0.003) but not between anti-hHSP60 and MPO-ANCA (r = -0.064, P = 0.69). CONCLUSION: Antibodies against mHSP65 are higher in AAV compared to HC, and anti-hHSP60 antibodies are higher in patients with MPO-ANCA than in patients with PR3-ANCA and HC. Although this finding may be indicative for cross-reactivity between MPO-ANCA and hHSP60, additional assays did not support this hypothesis.

Neutrophil myeloperoxidase activity and the influence of two single-nucleotide promoter polymorphisms.

Myeloperoxidase (MPO) catalyses the formation of hypochlorous acid and is involved in many (patho)physiological processes. The present study was designed to determine the effect of two MPO promoter polymorphisms (463G/A and 129G/A) on enzyme activity. In 243 healthy controls, genotypes were determined and MPO activity was measured on a single-cell level using a haematological analyser. The 129G/A polymorphism reduces MPO activity in neutrophils, whereas for the 463G/A polymorphism, only gender-dependent differences in MPO activity in older age groups could be found. When studying these polymorphisms in disease, therefore, age and gender should be included in the analysis.