Competitively disrupting the neutrophil-specific receptor–autoantigen CD177:proteinase 3 membrane complex reduces anti-PR3 antibody-induced neutrophil activation

CD177 is a neutrophil-specific receptor presenting the proteinase 3 (PR3) autoantigen on the neutrophil surface. CD177 expression is restricted to a neutrophil subset, resulting in CD177^pos/mPR3^high and CD177^neg/mPR3^low populations. The CD177^pos/mPR3^high subset has implications for antineutrophil cytoplasmic autoantibody (ANCA)–associated autoimmune vasculitis, wherein patients harbor PR3-specific ANCAs that activate neutrophils for degranulation. Here, we generated high-affinity anti-CD177 monoclonal antibodies, some of which interfered with PR3 binding to CD177 (PR3 “blockers”) as determined by surface plasmon resonance spectroscopy and used them to test the effect of competing PR3 from the surface of CD177^pos neutrophils. Because intact anti-CD177 antibodies also caused neutrophil activation, we prepared nonactivating Fab fragments of a PR3 blocker and nonblocker that bound specifically to CD177^pos neutrophils. We observed that Fab blocker clone 40, but not nonblocker clone 80, dose-dependently reduced anti-PR3 antibody binding to CD177^pos neutrophils. Importantly, preincubation with clone 40 significantly reduced respiratory burst in primed neutrophils challenged with either monoclonal antibodies to PR3 or PR3–ANCA immunoglobulin G from ANCA-associated autoimmune vasculitis patients. After separating the two CD177/mPR3 neutrophil subsets from individual donors by magnetic sorting, we found that PR3–ANCAs provoked significantly more superoxide production in CD177^pos/mPR3^high than in CD177^neg/mPR3^low neutrophils, and that anti-CD177 Fab clone 40 reduced the superoxide production of CD177^pos cells to the level of the CD177^neg cells. Our data demonstrate the importance of the CD177:PR3 membrane complex in maintaining a high ANCA epitope density and thereby underscore the contribution of CD177 to the severity of PR3–ANCA diseases.     

What you should know about PR3-ANCA. Evidence for the role of T cells in the pathogenesis of systemic vasculitis

The pathogenesis of systemic vasculitis is complex and is likely to involve many mechanisms. There is a growing body of evidence that T cells may contribute to the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides. Predominantly, T cells and monocytes are found in inflammatory infiltrates in patients with Wegener's granulomatosis (WG). The production of ANCA appears to be T-cell-dependent. T lymphocytes from the peripheral blood of patients with ANCA-associated vasculitis have been shown to proliferate in response to proteinase 3 (PR3). These and other findings outlined in this review indicate T-cell involvement, although further studies are still needed to elucidate the exact contribution of T cells to the pathogenesis of systemic vasculitis.     

What you should know about PR3-ANCA: Evidence for the role of T cells in the pathogenesis of systemic vasculitis

The pathogenesis of systemic vasculitis is complex and is likely to involve many mechanisms. There is a growing body of evidence that T cells may contribute to the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides. Predominantly, T cells and monocytes are found in inflammatory infiltrates in patients with Wegener's granulomatosis (WG). The production of ANCA appears to be T-cell-dependent. T lymphocytes from the peripheral blood of patients with ANCA-associated vasculitis have been shown to proliferate in response to proteinase 3 (PR3). These and other findings outlined in this review indicate T-cell involvement, although further studies are still needed to elucidate the exact contribution of T cells to the pathogenesis of systemic vasculitis.     

Comparing the Cervista HPV HR Test and Hybrid Capture 2 Assay in a Dutch Screening Population: Improved Specificity of the Cervista HPV HR Test by Changing the Cut-Off

The diagnostic performance of the widely-used Cervista HPV HR test was compared to the Hybrid Capture 2 (HC2) test in a Dutch population-based cervical cancer screening program. In 900 scrapings of women with normal cytomorphology, specificity was 90% (95%CI: 87.84–91.87) for the Cervista HPV HR test and 96% (95%CI: 94.76–97.37) for the HC2 test with 93% agreement between both tests (κ = 0.5, p<0.001). The sensitivity for CIN2+ using 65 scrapings of women with histological-confirmed CIN2+ was 91% (95%CI: 80.97–96.51) for the Cervista HPV HR test and 92% (95%CI: 82.94–97.43) for the HC2 test with 95% agreement between both tests (κ = 0.7, p<0.001). Fifty-seven of 60 HC2 negative/Cervista positive cases tested HPV-negative with PCR-based HPV assays; of these cases 56% were defined as Cervista triple-positive with FOZ values in all 3 mixes higher than the second cut-off of 1.93 (as set by manufacturer). By setting this cut-off at 5.0, specificity improved significantly without affecting sensitivity. External validation of this new cut-off at 5.0 in triple-positive scrapings of women selected from the SHENCCASTII database revealed that 22/24 histological normal cases now tested HPV-negative in the Cervista HPV HR test, while CIN2+ lesions remained HPV-positive. The intra-laboratory reproducibility of the Cervista HPV HR test (n = 510) showed a concordance of 92% and 93% for cut-off 1.93 and 5.0 (κ = 0.83 and κ = 0.84, p<0.001) and inter-laboratory agreement of the Cervista HPV HR test was 90% and 93% for cut-off 1.93 and 5.0 (κ = 0.80 and κ = 0.85, p<0.001). In conclusion, the specificity of the Cervista HPV HR test could be improved significantly by increasing the second cut-off from 1.93 to 5.0, without affecting the sensitivity of the test in a population-based screening setting.     

1,25-dihydroxyvitamin D3 Protects against Macrophage-Induced Activation of NFκB and MAPK Signalling and Chemokine Release in Human Adipocytes

Increased accumulation of macrophages in adipose tissue in obesity is linked to low-grade chronic inflammation, and associated with features of metabolic syndrome. Vitamin D₃ may have immunoregulatory effects and reduce adipose tissue inflammation, although the molecular mechanisms remain to be established. This study investigated the effects of vitamin D₃ on macrophage-elicited inflammatory responses in cultured human adipocytes, particularly the signalling pathways involved. Macrophage-conditioned (MC) medium (25% with adipocyte maintenance media) markedly inhibited protein expression of the nuclear factor-κB (NFκB) subunit inhibitor κBα (IκBα) (71%, P<0.001) and increased NFκB p65 (1.5-fold, P = 0.026) compared with controls. Treatment with 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) abolished macrophage-induced activation of NFκB signalling by increasing IκBα expression (2.7-fold, P = 0.005) and reducing NFκB p65 phosphorylation (68%; P<0.001). The mitogen-activated protein kinase (MAPK) signalling was activated by MC medium, which was also blunted by 1,25(OH)₂D₃ with a downregulation of phosphorylated p38 MAPK (32%, P = 0.005) and phosphorylated Erk1/2 (49%, P = 0.001). Furthermore, MC medium (12.5% or 25%) dose-dependently upregulated secretion of key proinflammatory chemokines/cytokines (22-368-fold; all P<0.001) and this was significantly decreased by 1,25(OH)₂D₃: IL-8 (61% and 31%, P<0.001), MCP-1 (37%, P<0.001 and 36%, P = 0.002), RANTES (78% and 62%, P<0.001) and IL-6 (29%, P<0.001 and 34%, P = 0.019). Monocyte migration-elicited by adipocytes treated with 1,25(OH)₂D₃ was also reduced (up to 25%, P<0.001). In conclusion, vitamin D₃ could be anti-inflammatory in adipose tissue, decreasing macrophage-induced release of chemokines and cytokines by adipocytes and the chemotaxis of monocytes. Our data suggests these effects are mediated by inhibition of the NFκB and MAPK signalling pathways.     

Negative Regulation of NF-κB by the ING4 Tumor Suppressor in Breast Cancer

Nuclear Factor kappa B (NF-κB) is a key mediator of normal immune response but contributes to aggressive cancer cell phenotypes when aberrantly activated. Here we present evidence that the Inhibitor of Growth 4 (ING4) tumor suppressor negatively regulates NF-κB in breast cancer. We surveyed primary breast tumor samples for ING4 protein expression using tissue microarrays and a newly generated antibody. We found that 34% of tumors expressed undetectable to low levels of the ING4 protein (n = 227). Tumors with low ING4 expression were frequently large in size, high grade, and lymph node positive, suggesting that down-regulation of ING4 may contribute to breast cancer progression. In the same tumor set, we found that low ING4 expression correlated with high levels of nuclear phosphorylated p65/RelA (p-p65), an activated form of NF-κB (p = 0.018). Fifty seven percent of ING4-low/p-p65-high tumors were lymph node-positive, indicating a high metastatic tendency of these tumors. Conversely, ectopic expression of ING4 inhibited p65/RelA phosphorylation in T47D and MCF7 breast cancer cells. In addition, ING4 suppressed PMA-induced cell invasion and NF-κB-target gene expression in T47D cells, indicating that ING4 inhibited NF-κB activity in breast cancer cells. Supportive of the ING4 function in the regulation of NF-κB-target gene expression, we found that ING4 expression levels inversely correlated with the expression of NF-κB-target genes in primary breast tumors by analyzing public gene expression datasets. Moreover, low ING4 expression or high expression of the gene signature composed of a subset of ING4-repressed NF-κB-target genes was associated with reduced disease-free survival in breast cancer patients. Taken together, we conclude that ING4 negatively regulates NF-κB in breast cancer. Consequently, down-regulation of ING4 leads to activation of NF-κB, contributing to tumor progression and reduced disease-free patient survival in breast cancer.     

Antineutrophil cytoplasmic autoantibody in renal disease

ANCA are an important marker for identifying pauci-immune necrotizing and crescentic glomerulonephritis and systemic vasculitis. Patients with ANCA-associated renal disease have a spectrum of illness ranging from renal-limited disease to widespread vasculitis, including the clinicopathologic syndromes of polyarteritis nodosa and Wegener's granulomatosis. ANCA may be directly involved in the pathogenesis of pauci-immune glomerulonephritis and necrotizing systemic vasculitis.     

Immune Responses to ESAT-6 and CFP-10 by FASCIA and Multiplex Technology for Diagnosis of M. tuberculosis Infection; IP-10 Is a Promising Marker

Background

There is a need for reliable markers to diagnose active and latent tuberculosis (TB). The interferon gamma release assays (IGRAs) are compared to the tuberculin skin test (TST) more specific, but cannot discriminate between recent or remote TB infection. Here the Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA), which quantifies expanded T-lymphoblasts by flow-cytometric analysis after long-term antigen stimulation of whole blood, is combined with cytokine/chemokine analysis in the supernatant by multiplex technology for diagnosis of Mycobacterium tuberculosis (Mtb) infection.

Methods and Findings

Consecutive patients with suspected TB (n = 85), with microbiologically verified active pulmonary TB (n = 33), extra pulmonary TB (n = 21), clinical TB (n = 11), presumed latent TB infection (LTBI) (n = 23), patients negative for TB (n = 8) and 21 healthy controls were studied. Blood samples were analyzed with FASCIA and multiplex technology to determine and correlate proliferative responses and the value of 14 cytokines for diagnosis of Mtb infection: IFN- γ, IL-2, TNF-α, IP-10, IL-12, IL-6, IL-4, IL-5, IL-13, IL-17, MIP-1β, GM-CSF, IFN-α2 and IL-10. Cytokine levels for IFN-γ, IP-10, MIP-1β, IL-2, TNF-α, IL-6, IL-10, IL-13 and GM-CSF were significantly higher after stimulation with the Mtb specific antigens ESAT-6 and CFP-10 in patients with active TB compared to healthy controls (p<0.05) and correlated with proliferative responses. IP-10 was positive in all patients with verified TB, if using a combination of ESAT-6 and CFP-10 and was the only marker significantly more sensitive in detecting active TB then IFN-γ (p = 0.012). Cytokine responses in patients with active TB were more frequent and detected at higher levels than in patients with LTBI.

Conclusions

IP-10 seems to be an important marker for diagnosis of active and latent TB. Patients with active TB and LTBI responded with similar cytokine profiles against TB antigens but proliferative and cytokine responses were generally higher in patients with active TB.     

Systemic Simvastatin Rescues Retinal Ganglion Cells from Optic Nerve Injury Possibly through Suppression of Astroglial NF-κB Activation

Neuroinflammation is involved in the death of retinal ganglion cells (RGCs) after optic nerve injury. The purpose of this study was to determine whether systemic simvastatin can suppress neuroinflammation in the optic nerve and rescue RGCs after the optic nerve is crushed. Simvastatin or its vehicle was given through an osmotic minipump beginning one week prior to the crushing. Immunohistochemistry and real-time PCR were used to determine the degree of neuroinflammation on day 3 after the crushing. The density of RGCs was determined in Tuj-1 stained retinal flat mounts on day 7. The effect of simvastain on the TNF-α-induced NF-κB activation was determined in cultured optic nerve astrocytes. On day 3, CD68-positive cells, most likely microglia/macrophages, were accumulated at the crushed site. Phosphorylated NF-κB was detected in some astrocytes at the border of the lesion where the immunoreactivity to MCP-1 was intensified. There was an increase in the mRNA levels of the CD68 (11.4-fold), MCP-1 (22.6-fold), ET-1 (2.3-fold), GFAP (1.6-fold), TNF-α (7.0-fold), and iNOS (14.8-fold) genes on day 3. Systemic simvastatin significantly reduced these changes. The mean ± SD number of RGCs was 1816.3±232.6/mm² (n = 6) in the sham controls which was significantly reduced to 831.4±202.5/mm² (n = 9) on day 7 after the optic nerve was crushed. This reduction was significantly suppressed to 1169.2±201.3/mm² (P = 0.01, Scheffe; n = 9) after systemic simvastatin. Simvastatin (1.0 µM) significantly reduced the TNF-α-induced NF-κB activation in cultured optic nerve astrocytes. We conclude that systemic simvastatin can reduce the death of RGCs induced by crushing the optic nerve possibly by suppressing astroglial NF-κB activation.     

Selective κ Opioid Antagonists nor-BNI,GNTI and JDTic Have Low Affinities for Non-Opioid Receptors and Transporters

Background

Nor-BNI, GNTI and JDTic induce selective κ opioid antagonism that is delayed and extremely prolonged, but some other effects are of rapid onset and brief duration. The transient effects of these compounds differ, suggesting that some of them may be mediated by other targets.

Results

In binding assays, the three antagonists showed no detectable affinity (K _i≥10 µM) for most non-opioid receptors and transporters (26 of 43 tested). There was no non-opioid target for which all three compounds shared detectable affinity, or for which any two shared sub-micromolar affinity. All three compounds showed low nanomolar affinity for κ opioid receptors, with moderate selectivity over μ and δ (3 to 44-fold). Nor-BNI bound weakly to the α_2C-adrenoceptor (K _i = 630 nM). GNTI enhanced calcium mobilization by noradrenaline at the α_1A-adrenoceptor (EC₅₀ = 41 nM), but did not activate the receptor, displace radioligands, or enhance PI hydrolysis. This suggests that it is a functionally-selective allosteric enhancer. GNTI was also a weak M₁ receptor antagonist (K _B = 3.7 µM). JDTic bound to the noradrenaline transporter (K _i = 54 nM), but only weakly inhibited transport (IC₅₀ = 1.1 µM). JDTic also bound to the opioid-like receptor NOP (K _i = 12 nM), but gave little antagonism even at 30 µM. All three compounds exhibited rapid permeation and active efflux across Caco-2 cell monolayers.

Conclusions

Across 43 non-opioid CNS targets, only GNTI exhibited a potent functional effect (allosteric enhancement of α_1A-adrenoceptors). This may contribute to GNTI''s severe transient effects. Plasma concentrations of nor-BNI and GNTI may be high enough to affect some peripheral non-opioid targets. Nonetheless, κ opioid antagonism persists for weeks or months after these transient effects dissipate. With an adequate pre-administration interval, our results therefore strengthen the evidence that nor-BNI, GNTI and JDTic are highly selective κ opioid antagonists.     

Ataxia-Telangiectasia Group D Complementing Gene (ATDC) Promotes Lung Cancer Cell Proliferation by Activating NF-κB Pathway

Previous studies suggested Ataxia-telangiectasia group D complementing gene (ATDC) as an oncogene in many types of cancer. However, its expression and biological functions in non-small cell lung cancer (NSCLC) remain unclear. Herein, we investigated its expression pattern in 109 cases of human NSCLC samples by immunohistochemistry and found that ATDC was overexpressed in 62 of 109 NSCLC samples (56.88%). ATDC overexpression correlated with histological type (p<0.0001), tumor status (p = 0.0227) and histological differentiation (p = 0.0002). Next, we overexpressed ATDC in normal human bronchial epithelial cell line HBE and depleted its expression in NSCLC cell lines A549 and H1299. MTT and colony formation assay showed that ATDC overexpression promoted cell proliferation while its depletion inhibited cell growth. Furthermore, cell cycle analysis showed that ATDC overexpression decreased the percentage of cells in G1 phase and increased the percentage of cells in S phase, while ATDC siRNA treatment increased the G1 phase percentage and decreased the S phase percentage. Further study revealed that ATDC overexpression could up-regulate cyclin D1 and c-Myc expression in HBE cells while its depletion down-regulated cyclin D1 and c-Myc expression in A549 and H1299 cells. In addition, ATDC overexpression was also associated with an increased proliferation index, cyclin D1 and c-Myc expression in human NSCLC samples. Further experiments demonstrated that ATDC up-regulated cyclin D1 and c-Myc expression independent of wnt/β-catenin or p53 signaling pathway. Interestingly, ATDC overexpression increased NF-κB reporter luciferase activity and p-IκB protein level. Correspondingly, NF-κB inhibitor blocked the effect of ATDC on up-regulation of cyclin D1 and c-Myc. In conclusion, we demonstrated that ATDC could promote lung cancer proliferation through NF-κB induced up-regulation of cyclin D1 and c-Myc.     

Necrosis-Induced Sterile Inflammation Mediated by Interleukin-1α in Retinal Pigment Epithelial Cells

Endogenous danger signals released from necrotic cells contribute to retinal inflammation. We have now investigated the effects of necrotic cell extracts prepared from ARPE-19 human retinal pigment epithelial cells (ANCE) on the release of proinflammatory cytokines and chemokines by healthy ARPE-19 cells. ANCE were prepared by subjection of ARPE-19 cells to freeze-thaw cycles. The release of various cytokines and chemokines from ARPE-19 cells was measured with a multiplex assay system or enzyme-linked immunosorbent assays. The expression of interleukin (IL)–1α and the phosphorylation and degradation of the endogenous nuclear factor–κB (NF-κB) inhibitor IκB-α were examined by immunoblot analysis. Among the various cytokines and chemokines examined, we found that ANCE markedly stimulated the release of the proinflammatory cytokine IL-6 and the chemokines IL-8 and monocyte chemoattractant protein (MCP)–1 by ARPE-19 cells. ANCE-induced IL-6, IL-8, and MCP-1 release was inhibited by IL-1 receptor antagonist and by an IKK2 inhibitor (a blocker of NF-κB signaling) in a concentration-dependent manner, but was not affected by a pan-caspase inhibitor (Z-VAD-FMK). Recombinant IL-1α also induced the secretion of IL-6, IL-8, and MCP-1 from ARPE-19 cells, and IL-1α was detected in ANCE. Furthermore, ANCE induced the phosphorylation and degradation of IκB-α in ARPE-19 cells. Our findings thus suggest that IL-1α is an important danger signal that is released from necrotic retinal pigment epithelial cells and triggers proinflammatory cytokine and chemokine secretion from intact cells in a manner dependent on NF-κB signaling. IL-1α is therefore a potential therapeutic target for amelioration of sterile inflammation in the retina.     

Meta-Analysis of Apolipoprotein E Gene Polymorphism and Susceptibility of Myocardial Infarction

A number of case-control studies have been conducted to clarify the association between ApoE polymorphisms and myocardial infarction (MI); however, the results are inconsistent. This meta-analysis was performed to clarify this issue using all the available evidence. Searching in PubMed retrieved all eligible articles. A total of 33 studies were included in this meta-analysis, including 18752 MI cases and 18963 controls. The pooled analysis based on all included studies showed that the MI patients had a decreased frequency of the ε2 allele (OR = 0.78, 95% CI = 0.70–0.87) and an increased frequency of the ε4 allele (OR = 1.15, 95% CI = 1.10–1.20); The results also showed a decreased susceptibility of MI in the ε2ε3 vs. ε3ε3 analysis (OR = 0.79, 95% CI = 0.68–0.90) and in the ε2 vs. ε3 analysis (OR = 0.78, 95% CI = 0.69–0.89), an increased susceptibility of MI in the ε3ε4 vs. ε3ε3 analysis (OR = 1.26, 95% CI = 1.12–1.41), in the ε4 vs. ε3 analysis (OR = 1.22, 95% CI = 1.12–1.32) and in the ε4ε4 vs. ε3ε3 analysis (OR = 1.59, 95% CI = 1.15–2.19). However, there were no significant associations among polymorphisms and MI for the following genetic models: frequency of the ε3 allele (OR = 0.99, 95% CI = 0.96–1.02); ε2ε2 vs. ε3ε3 analysis (OR = 0.73, 95% CI = 0.40–1.32); or ε2ε4 vs. ε3ε3 analysis (OR = 1.10, 95% CI = 0.99–1.21). Our results suggested that the ε4 allele of ApoE is a risk factor for the development of MI and the ε2 allele of ApoE is a protective factor in the development of MI.     

Fc Gamma Receptor IIIB (FcγRIIIB) Polymorphisms Are Associated with Clinical Malaria in Ghanaian Children

Plasmodium falciparum malaria kills nearly a million people annually. Over 90% of these deaths occur in children under five years of age in sub-Saharan Africa. A neutrophil mediated mechanism, the antibody dependent respiratory burst (ADRB), was recently shown to correlate with protection from clinical malaria. Human neutrophils constitutively express Fc gamma receptor-FcγRIIA and FcγRIIIB by which they interact with immunoglobulin (Ig) G (IgG)-subclass antibodies. Polymorphisms in exon 4 of FCGR2A and exon 3 of FCGR3B genes encoding FcγRIIA and FcγRIIIB respectively have been described to alter the affinities of both receptors for IgG. Here, associations between specific polymorphisms, encoding FcγRIIA p.H166R and FcγRIIIB-NA1/NA2/SH variants with clinical malaria were investigated in a longitudinal malaria cohort study. FcγRIIA-p.166H/R was genotyped by gene specific polymerase chain reaction followed by allele specific restriction enzyme digestion. FCGR3B-exon 3 was sequenced in 585 children, aged 1 to 12 years living in a malaria endemic region of Ghana. Multivariate logistic regression analysis found no association between FcγRIIA-166H/R polymorphism and clinical malaria. The A-allele of FCGR3B-c.233C>A (rs5030738) was significantly associated with protection from clinical malaria under two out of three genetic models (additive: p = 0.0061; recessive: p = 0.097; dominant: p = 0.0076) of inheritance. The FcγRIIIB-SH allotype (CTGAAA) containing the 233A-allele (in bold) was associated with protection from malaria (p = 0.049). The FcγRIIIB-NA2*03 allotype (CTGCGA), a variant of the classical FcγRIIIB-NA2 (CTGCAA) was associated with susceptibility to clinical malaria (p = 0.0092). The present study is the first to report an association between a variant of FcγRIIIB-NA2 and susceptibility to clinical malaria and provides justification for further functional characterization of variants of the classical FcγRIIIB allotypes. This would be crucial to the improvement of neutrophil mediated functional assays such as the ADRB assay aimed at assessing the functionality of antibodies induced by candidate malaria vaccines.     

Intense immunostaining of heat shock protein 70 within renal interstitium associates with long-term renal survival in an ANCA-associated vasculitis cohort

In anti-neutrophilic cytoplasmic antibody (ANCA)-associated vasculitis (AAV) genetic predisposition, ANCA autoantibodies, neutrophil extracellular traps (NETs), complement activation, and toll-like receptor signaling are implicated in AAV pathogenesis. Heat shock proteins (HSPs), a highly conserved group of small-sized molecular chaperones, take part in protein folding during cellular stress. Although HSPs were initially observed intracellularly, it has been shown that they can be secreted in the extracellular space and modulate the immune response in various autoimmune diseases including AAV. The scope of the present study is to investigate the role of heat shock protein 60 (HSP60) and 70 (HSP70) in the long renal effects in an ANCA vasculitis cohort. In this cohort of ANCA-associated vasculitis, 29 patients were followed up over 20 years. At diagnosis, immunohistochemistry was performed for HSP60 and HSP70 within the various nephron compartments. Higher renal HSP60 expression was associated with increased interstitial inflammatory infiltrates at diagnosis, while HSP70 expression was associated with a greater extent of interstitial fibrosis at diagnosis. Notably, intense tissue expression of HSP70 at the time of biopsy was associated with a worsened kidney survival. Renal HSP70 expression was associated with poor renal outcomes during long-term follow-up. This finding may indicate a role of HSPs in renal disease progression in ANCA vasculitis. Further validating studies are needed to verify a causative association between HSP70 expression and renal outcomes in ANCA-associated vasculitis.     

Detection of Anti-Pentraxin-3 Autoantibodies in ANCA-Associated Vasculitis

Objectives

Pentraxin 3 (PTX3), in common with myeloperoxidase and proteinase 3, is stored in human neutrophil granules and is expressed on apoptotic neutrophil surface. We therefore investigated the presence of anti-PTX3 autoantibodies (aAbs) in the sera of antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) patients.

Methods

Presence of anti-PTX3 autoantibodies was analysed by a specific enzyme-linked immunosorbent assay in sera from 150 patients with microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA), and eosinophilic granulomatosis with polyangiitis (EGPA), and in sera of 227 healthy subjects (HS), 40 systemic sclerosis (SSc) patients, and 25 giant cell arteritis patients (GCA). Using indirect immunofluorescence on fixed human neutrophils, we also analyzed the staining pattern associated with the presence of anti-PTX3 aAbs.

Results

Anti-PTX3 aAbs were detected in 56 of 150 (37.3%) of the AAV patients (versus 12 of 227 (5.3%) of HS, p<0.001) and, interestingly, in 7 of 14 MPO and PR3 ANCA negative AAV patients. Moreover, by indirect immunofluorescence on fixed neutrophils, anti-PTX3 aAbs gave rise to a specific cytoplasmic fluorescence pattern distinct from the classical cytoplasmic (c-ANCA), perinuclear (p-ANCA), and atypical (a-ANCA) pattern. Anti-PTX3 aAbs levels were higher in patients with active AAV as compared to patients with inactive disease.

Conclusion

Our work suggests that PTX3 is as a novel ANCA antigen. Anti-PTX3 aAbs appear thus as a promising novel biomarker in the diagnosis of AAV, including in patients without detectable MPO and PR3 ANCA.     

Association of TNF-α, TNFRSF1A and TNFRSF1B Gene Polymorphisms with the Risk of Sporadic Breast Cancer in Northeast Chinese Han Women

Background

The interaction of tumor necrosis factor-α (TNF-α) with its receptors: TNFRSF1A and TNFRSF1B is critical for the promotion of tumor growth, invasion and metastasis. To better understand the roles of single nucleotide polymorphisms (SNPs) in the TNF-α, TNFRSF1A and TNFRSF1B genes in the development of breast cancer, we explored the associations between SNPs in these three genes and breast cancer susceptibility in northeast Chinese Han women.

Methodology/Principal Findings

This case-control study was conducted among 1016 breast cancer patients and 806 age-matched healthy controls. Seven SNPs in the TNF-α (rs1800629, rs361525), TNFRSF1A (rs767455, rs4149577 and rs1800693) and TNFRSF1B (rs1061622 and rs1061624) genes were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. In TNFRSF1B, the rs1061622 GT genotype and the G allele conferred a reduced susceptibility to breast cancer (P = 0.000662, OR = 0.706, 95% CI: 0.578–0.863; P = 0.002, OR = 0.769, 95% CI; 0.654–0.905, respectively). Moreover, the AG genotype, the AA genotype and the A allele in rs1061624 conferred an increased risk of breast cancer (P = 0.007, OR = 1.470, 95% CI:1.112–1.943; P = 0.00109, OR = 1.405 95% CI:1.145–1.724; P = 0.001, OR = 1.248 95% CI:1.092–1.426, respectively). These two SNPs also had associations with breast cancer risk under the dominant model. In haplotype analysis, the CTA (rs767455 C-rs4149577 T-rs1800693 A) haplotype in TNFRSF1A and the TA (rs1061622 T-rs1061624 A) haplotype in TNFRSF1B had higher frequencies in breast cancer patients (P = 0.00324; P = 0.000370, respectively), but the frequency of GG (rs1061622 G-rs1061624 G) haplotype in TNFRSF1B was lower in breast cancer patients (P = 0.000251). The associations of the three haplotypes remained significant after correcting for multiple testing. In addition, significant associations were also observed between TNFRSF1A polymorphisms and lymph node metastasis, P53, estrogen receptor (ER) and progesterone receptor (PR) statuses.

Conclusions

Our results suggest that rs1061622 and rs1061624 in TNFRSF1B may affect breast cancer risk, and SNPs in TNFRSF1A are associated with the clinical features of breast cancer.