Increased immune complexes of hypocretin autoantibodies in narcolepsy

Background

Hypocretin peptides participate in the regulation of sleep-wake cycle while deficiency in hypocretin signaling and loss of hypocretin neurons are causative for narcolepsy-cataplexy. However, the mechanism responsible for alteration of the hypocretin system in narcolepsy-cataplexy and its relevance to other central hypersomnias remain unknown. Here we studied whether central hypersomnias can be associated with autoantibodies reacting with hypocretin-1 peptide present as immune complexes.

Methodology

Serum levels of free and dissociated (total) autoantibodies reacting with hypocretin-1 peptide were measured by enzyme-linked immunosorbent assay and analyzed with regard to clinical parameters in 82 subjects with narcolepsy-cataplexy, narcolepsy without cataplexy or idiopathic hypersomnia and were compared to 25 healthy controls.

Principal Findings

Serum levels of total but not free IgG autoantibodies against hypocretin-1 were increased in narcolepsy-cataplexy. Increased levels of complexed IgG autoantibodies against hypocretin-1 were found in all patients groups with a further increase in narcolepsy-cataplexy. Levels of total IgM hypocretin-1 autoantibodies were also elevated in all groups of patients. Increased levels of anti-idiotypic IgM autoantibodies reacting with hypocretin-1 IgG autoantibodies affinity purified from sera of subjects with narcolepsy-cataplexy were found in all three groups of patients. Disease duration correlated negatively with serum levels of hypocretin-1 IgG and IgM autoantibodies and with anti-idiotypic IgM autoantibodies.

Conclusion

Central hypersomnias and particularly narcolepsy-cataplexy are characterized by higher serum levels of autoantibodies directed against hypocretin-1 which are present as immune complexes most likely with anti-idiotypic autoantibodies suggesting their relevance to the mechanism of sleep-wake cycle regulation.     

Distinct Patterns of IgG and IgA against Food and Microbial Antigens in Serum and Feces of Patients with Inflammatory Bowel Diseases

Background

Inflammatory bowel disease (IBD) is associated with a defective intestinal barrier and enhanced adaptive immune responses against commensal microbiota. Immune responses against food antigens in IBD patients remain poorly defined.

Methods

IgG and IgA specific for food and microfloral antigens (wheat and milk extracts; purified ovalbumin; Escherichia coli and Bacteroides fragilis lysates; mannan from Saccharomyces cerevisiae) were analyzed by ELISA in the serum and feces of patients with Crohn''s disease (CD; n = 52 for serum and n = 20 for feces), ulcerative colitis (UC; n = 29; n = 17), acute gastroenteritis/colitis (AGE; n = 12; n = 9) as well as non-inflammatory controls (n = 61; n = 39).

Results

Serum anti-Saccharomyces cerevisiae antibodies (ASCA) and anti-B. fragilis IgG and IgA levels were increased in CD patients whereas antibody (Ab) levels against E. coli and food antigens were not significantly different within the patient groups and controls. Subgroup analysis revealed that CD patients with severe diseases defined by stricturing and penetrating lesions have slightly higher anti-food and anti-microbial IgA levels whereas CD and UC patients with arthropathy have decreased anti-food IgG levels. Treatment with anti-TNF-α Abs in CD patients was associated with significantly decreased ASCA IgG and IgA and anti-E. coli IgG. In the feces specific IgG levels against all antigens were higher in CD and AGE patients while specific IgA levels were higher in non-IBD patients. Anti-food IgG and IgA levels did not correlate with food intolerance.

Summary

In contrast to anti-microbial Abs, we found only minor changes in serum anti-food Ab levels in specific subgroups of IBD patients. Fecal Ab levels towards microbial and food antigens show distinct patterns in controls, CD and UC patients.     

Salivary Immune Responses to the 7-Valent Pneumococcal Conjugate Vaccine in the First 2 Years of Life

Background

The CRM197-conjugated 7-valent pneumococcal vaccine (PCV7) is protective against vaccine serotype disease and nasopharyngeal carriage. Data on PCV7-induced mucosal antibodies in relation to systemic or natural anticapsular antibodies are scarce.

Methods

In a randomized controlled setting, children received PCV7 at age 2 and 4 months (2-dose group), at age 2, 4 and 11 months (2+1-dose group) or no PCV7 (control group). From 188 children paired saliva samples were collected at 12 and 24 months of age. From a subgroup of 15 immunized children also serum samples were collected. IgG and IgA antibody-levels were measured by multiplex immunoassay.

Results

At 12 months, both vaccine groups showed higher serum and saliva IgG-levels against vaccine serotypes compared with controls which sustained until 24 months for most serotypes. Salivary IgG-levels were 10–20-fold lower compared to serum IgG, however, serum and saliva IgG-levels were highly correlated. Serum and salivary IgA-levels were higher in both vaccine groups at 12 months compared with controls, except for serotype 19F. Higher salivary IgA levels remained present for most serotypes in the 2+1-dose group until 24 months, but not in the 2-dose group. Salivary IgA more than IgG, increased after documented carriage of serotypes 6B, 19F and 23F In contrast to IgG, salivary IgA-levels were comparable with serum, suggesting local IgA-production.

Conclusions

PCV7 vaccination results in significant increases in salivary IgG and IgA-levels, which are more pronounced for IgG when compared to controls. In contrast, salivary anticapsular IgA-levels seemed to respond more to natural boosting. Salivary IgG and IgA-levels correlate well with systemic antibodies, suggesting saliva might be useful as potential future surveillance tool.     

Impact of interleukin-21 in the pathogenesis of primary Sjogren's syndrome: increased serum levels of interleukin-21 and its expression in the labial salivary glands

Introduction

Interleukin (IL)-21 is a cytokine that controls the functional activity of effector T helper cells and the differentiation of Th17 cells, and promotes B-cell differentiation. To test whether IL-21 participates in the pathogenesis of primary Sjögren''s syndrome (SS), serum IL-21 level was measured and IL-21 expression in the labial salivary glands (LSG) was examined.

Methods

Serum IL-21 levels in 40 primary SS, 40 rheumatoid arthritis (RA), and 38 systemic lupus erythematosus (SLE) patients and 20 healthy controls were measured. Serum IL-21 levels of SS patients were assessed for correlations with laboratory data, including anti-nuclear antibody, anti-Ro/La antibodies, globulin, immunoglobulin (Ig) class, and IgG subclass. LSGs from 16 primary SS and 4 controls with sicca symptoms were evaluated for IL-21 and IL-21 receptor (IL-21R) expression by immunohistochemistry. Confocal microscopy was performed to further characterize the IL-21 positive cells.

Results

Primary SS patients had significantly higher serum IL-21 levels than controls, and these increments correlated positively with levels of IgG, IgG1. Serum IgG1 levels correlated with anti-Ro antibody titers. Immunohistochemical analyses showed that lymphocytic foci and the periductal area of the LSGs from SS patients expressed high levels of IL-21 and lower levels of IL-21R, whereas the control LSGs showed minimal expression of both antigens. The more the lymphocyte infiltrated, IL-21expression in LSGs showed a tendency to increase. Confocal microscopic analyses revealed that IL-21 expressing infiltrating lymphocytes in the LSGs of SS patients also expressed CXCR5.

Conclusions

Primary SS is associated with high serum IL-21 levels that correlate positively with serum IgG, especially IgG1, levels. The expression of IL-21 is increased as more lymphocytes infiltrated in LSGs. These observations suggest that IL-21 may play an important role in primary SS pathogenesis.     

Anti-dsDNA Antibody Isotypes in Systemic Lupus Erythematosus: IgA in Addition to IgG Anti-dsDNA Help to Identify Glomerulonephritis and Active Disease

Objectives

To evaluate the role of serum IgG, IgM and IgA anti-dsDNA antibody isotypes in the diagnosis of systemic lupus erythematosus (SLE), and their association with clinical features and disease activity, in a large cohort of SLE patients.

Methods

Sera of 200 SLE patients (mean age 34±10.3 years; 26 male and 174 female; median duration of disease 115 months, range 7–378), and of 206 controls, including 19 Sjögren''s syndrome, 27 rheumatoid arthritis, 26 psoriatic arthritis, 15 idiopathic inflammatory myopathies (IIM), 13 systemic sclerosis, 49 infectious diseases and 57 healthy subjects, were tested for anti-dsDNA IgG, IgM and IgA isotypes.

Results

Selecting a cutoff corresponding to 95% specificity, the sensitivity of IgG, IgM and IgA anti-dsDNA antibodies in SLE was 55%, 30% and 49%, respectively; 12.5%, 1% and 7.5% of SLE patients had positive IgG, IgM or IgA isotype alone, respectively. SLE patients with glomerulonephritis showed higher levels of IgA anti-dsDNA (p = 0.0002), anti-dsDNA IgG/IgM (p = 0.001) and IgA/IgM (p<0.0001) ratios than patients without renal disease. No significant associations have been found between anti-dsDNA isotypes and other clinical features. IgA anti-dsDNA (p = 0.01) (but not IgG or IgM) and IgG/IgM ratio (p = 0.005) were significantly higher in patients with more active disease (ECLAM score >4).

Conclusions

The detection of IgA anti-dsDNA autoantibodies seems to improve our ability to diagnose SLE and to define lupus nephritis phenotype and active disease. By contrast, IgM anti-dsDNA antibodies might be protective for renal involvement. These data support the hypothesis that anti-dsDNA antibody class clustering may help to refine SLE diagnosis and prognosis.     

Serum BAFF and APRIL levels in patients with IgG4-related disease and their clinical significance

Introduction

B cell-activating factor of the tumor necrosis factor family (BAFF) and a proliferation-inducing ligand (APRIL) play a crucial role in B cell development, survival, and antibody production. Here we analyzed the serum levels of BAFF and APRIL and their respective clinical associations in patients with an immunoglobulin (Ig) G4-related disease (IgG4-RD).

Methods

We measured serum levels of BAFF and APRIL in patients with IgG4-RD, primary Sjögren''s syndrome (pSS), and healthy individuals. Serum BAFF and APRIL levels in IgG4-RD were assessed for correlations with serological parameters, including Ig, particularly IgG4, and the number of affected organs. Serum BAFF and APRIL levels in IgG4-RD were monitored during glucocorticoid (GC) therapy.

Results

Serum BAFF and APRIL levels in patients with IgG4-RD were significantly higher (P < 0.01) than in healthy individuals. The BAFF levels of patients with IgG4-RD were comparable to those of patients with pSS. Although clinical parameters, such as serum IgG4 and the number of affected organs, were not correlated with the levels of BAFF, serum APRIL levels were inversely correlated with serum IgG4 levels (r = -0.626, P < 0.05). While serum BAFF levels decreased following GC therapy, serum APRIL levels increased during follow-up.

Conclusion

These results indicate that BAFF and APRIL might be useful markers for predicting disease activity in IgG4-RD. Further studies are needed to elucidate the role of BAFF and APRIL in the pathogenesis of IgG4-RD.     

Increased intrathecal high-avidity anti-tau antibodies in patients with multiple sclerosis

Background

Antibodies against tau protein indicate an interaction between the immune system and the neurocytoskeleton and therefore may reflect axonal injury in multiple sclerosis (MS).

Methodology/Principal Findings

The levels and avidities of anti-tau IgG antibodies were measured using ELISA in paired cerebrospinal fluid (CSF) and serum samples obtained from 49 MS patients and 47 controls. Anti-tau antibodies were significantly elevated intrathecally (p<0.0001) in the MS group. The CSF anti-tau antibody levels were lower in MS patients receiving therapy than those without treatment (p<0.05). The avidities of anti-tau antibodies were higher in the CSF than in the serum (MS group p<0.0001; controls p<0.005). Anti-tau avidities in the CSF were elevated in MS patients in comparison with controls (p<0.05), but not in serum.

Conclusions

MS patients have higher levels of intrathecal anti-tau antibodies. Anti-tau antibodies have different avidities in different compartments with the highest values in the CSF of MS patients.     

Circulating endoglin concentration is not elevated in chronic kidney disease

Background

Soluble endoglin, a TGF-β receptor, plays a key role in cardiovascular physiology. Whether circulating concentrations of soluble endoglin are elevated in CKD or underlie the high risk of cardiovascular death associated with chronic kidney disease (CKD) is unknown.

Methods

Individuals with and without CKD were recruited at a single center. Estimated glomerular filtration rate (eGFR) was estimated using the modified MDRD study equation and the serum creatinine at the time of recruitment, and patients were assigned to specific CKD stage according to usual guidelines. Serum endoglin concentration was measured by ELISA and univariate and multivariable regression was used to analyze the association between eGFR or CKD stage and the concentration of soluble endoglin.

Results

Serum endoglin was measured in 216 patients including 118 with stage 3 or higher CKD and 9 individuals with end stage renal disease (ESRD). Serum endoglin concentration did not vary significantly with CKD stage (increase of 0.16 ng/mL per 1 stage increase in CKD, P = 0.09) or eGFR (decrease -0.06 ng/mL per 10 mL/min/1.73 m² increase in GFR, P = 0.12), and was not higher in individuals with ESRD than in individuals with preserved renal function (4.2±1.1 and 4.3±1.2 ng/mL, respectively). Endoglin concentration was also not significantly associated with urinary albumin excretion.

Conclusions

Renal function is not associated with the circulating concentration of soluble endoglin. Elevations in soluble endoglin concentration are unlikely to contribute to the progression of CKD or the predisposition of individuals with CKD to develop cardiovascular disease.     

Serological Assessment for Celiac Disease in IgA Deficient Adults

Purpose

Selective immunoglobulin A deficiency is the most common primary immunodeficiency disorder that is strongly overrepresented among patients with celiac disease (CD). IgG antibodies against tissue transglutaminase (tTG) and deamidated gliadin peptides (DGP) serve as serological markers for CD in IgA deficient individuals, although the diagnostic value remains uncertain. The aim of this study was to investigate the prevalence of these markers in a large cohort of IgA deficient adults with confirmed or suspected CD and relate the findings to gluten free diet.

Methods

Sera from 488,156 individuals were screened for CD in seven Swedish clinical immunology laboratories between 1998 and 2012. In total, 356 out of 1,414 identified IgA deficient adults agreed to participate in this study and were resampled. Forty-seven IgA deficient blood donors served as controls. Analyses of IgG antibodies against tTG and DGP as well as HLA typing were performed and a questionnaire was used to investigate adherence to gluten free diet. Available biopsy results were collected.

Results

Out of the 356 IgA deficient resampled adults, 67 (18.8%) were positive for IgG anti-tTG and 79 (22.2%) for IgG anti-DGP, 54 had biopsy confirmed CD. Among the 47 IgA deficient blood donors, 4 (9%) were positive for IgG anti-tTG and 8 (17%) for anti-DGP. Four were diagnosed with biopsy verified CD, however, 2 of the patients were negative for all markers. Sixty-eight of 69 individuals with positive IgG anti-tTG were HLA-DQ2/DQ8 positive whereas 7 (18.9%) of the 37 individuals positive for IgG anti-DGP alone were not.

Conclusions

IgG anti-tTG seems to be a more reliable marker for CD in IgA deficient adults whereas the diagnostic specificity of anti-DGP appears to be lower. High levels of IgG antibodies against tTG and DGP were frequently found in IgA deficient adults despite adhering to gluten free diet.     

Clinical utility of diagnostic guidelines and putative biomarkers in lymphangioleiomyomatosis

Background

Lymphangioleiomyomatosis is a rare disease occurring almost exclusively in women. Diagnosis often requires surgical biopsy and the clinical course varies between patients with no predictors of progression. We evaluated recent diagnostic guidelines, clinical features and serum biomarkers as diagnostic and prognostic tools.

Methods

Serum vascular endothelial growth factor-D (VEGF-D), angiotensin converting enzyme (ACE), matrix metalloproteinases (MMP) -2 and -9, clinical phenotype, thoracic and abdominal computerised tomography, lung function and quality of life were examined in a cohort of 58 patients. 32 healthy female controls had serum biomarkers measured.

Results

Serum VEGF-D, ACE and total MMP-2 levels were elevated in patients. VEGF-D was the strongest discriminator between patients and controls (median = 1174 vs. 332 pg/ml p < 0.0001 with an area under the receiver operating characteristic curve of 0.967, 95% CI 0.93-1.01). Application of European Respiratory Society criteria allowed a definite diagnosis without biopsy in 69%. Adding VEGF-D measurement to ERS criteria further reduced the need for biopsy by 10%. VEGF-D was associated with lymphatic involvement (p = 0.017) but not the presence of angiomyolipomas.

Conclusions

Combining ERS criteria and serum VEGF-D reduces the need for lung biopsy in LAM. VEGF-D was associated with lymphatic disease but not lung function.     

Serum Autoantibody Measurement for the Detection of Hepatocellular Carcinoma

Background

Individuals with liver disease, and especially those with Hepatitis B or C, are at an increased risk of developing hepatocellular carcinoma (HCC) which is the third most common cause of cancer-related death worldwide. Inadequate screening tests largely account for presentation of advanced tumours and high mortality rates. Early detection of HCC amongst high-risk groups is paramount in improving prognosis. This research aimed to further characterise the previously described humoral immune response raised to tumour-associated antigens (TAAs) in the serum of patients with HCC.

Methods

Serum from 96 patients with confirmed HCC, 96 healthy controls matched for age and sex, 78 patients with confirmed liver cirrhosis and 91 patients with confirmed chronic liver disease were analysed for the presence of IgG autoantibodies raised to 41 recombinant TAAs/antigen fragments by ELISA.

Results

Varying autoantibody specificities (97–100%) and sensitivities (0–10%) were observed to individual TAAs. A 21-antigen panel achieved a specificity of 92% and sensitivity of 45% for the detection of HCC. This same panel identified 21% of 169 high-risk controls as having elevated autoantibody levels. A reproducible panel of 10 antigens achieved a specificity of 91% and sensitivity of 41% in HCC. 15% of 152 high-risk controls gave positive results with this panel.

Conclusions

This minimally invasive blood test has the potential to offer advantages over currently available tools for the identification of HCC amongst pre-disposed patients. Results are comparable to current gold standards in HCC (Ultrasonography) and to similar tests in other cancers (EarlyCDT-Lung).     

FCGR2B and FCRLB Gene Polymorphisms Associated with IgA Nephropathy

Background

IgA nephropathy (IgAN) is a complex syndrome characterized by deposition of IgA and IgA containing immune complexes (ICs) composed of IgG and complement C3 proteins in the mesangial area of glomeruli. The low-affinity receptors for the Fc region of IgG (FcγRs) are involved in autoantibody/immune complex-induced organ injury as well as ICs clearance. The aim of the study was to associate multiple polymorphisms within FCGR gene locus with IgAN in a large Chinese cohort.

Patients and Methods

60 single nucleotide polymorphisms (SNPs) spanning a 400 kb range within FCGR gene locus were analyzed in 2100 DNA samples from patients with biopsy proven IgAN and healthy age- and sex-matched controls from the same population in Chinese.

Results

Among the 60 SNPs investigated, 15 gene polymorphisms within FCGR gene locus (25%) were associated with susceptibility to IgAN. The most significantly associated SNPs within individual genes were FCGR2B rs12118043 (p = 8.74*10⁻³, OR 0.76, 95% CI 0.62–0.93), and FCRLB rs4657093 (p = 2.28*10⁻³, OR 0.77, 95% CI 0.65–0.91). Both conditional analysis and linkage disequilibrium analysis suggested they were independent signals associated with IgAN. Associations between FCGR2B rs12118043 and proteinuria (p = 3.65×10⁻²) as well as gross hematuria (p = 4.53×10⁻²), between FCRLB rs4657093 and levels of serum creatinine (p = 2.67×10⁻²) as well as eGFR (p = 5.41*10⁻³) were also observed. Electronic cis-expression quantative trait loci analysis supported their possible functional significance, with protective genotypes correlating lower gene expressions.

Conclusion

Our data from genetic associations and expression associations revealed potentially pathogenic roles of Fc receptor gene polymorphisms in IgAN.     

The Relation between Serum Phosphorus Levels and Clinical Outcomes after Acute Myocardial Infarction

Background

Elevated serum phosphorus levels have been linked with cardiovascular disease and mortality with conflicting results, especially in the presence of normal renal function.

Methods

We studied the association between serum phosphorus levels and clinical outcomes in 1663 patients with acute myocardial infarction (AMI). Patients were categorized into 4 groups based on serum phosphorus levels (<2.50, 2.51–3.5, 3.51–4.50 and >4.50 mg/dL). Cox proportional-hazards models were used to examine the association between serum phosphorus and clinical outcomes after adjustment for potential confounders.

Results

The mean follow up was 45 months. The lowest mortality occurred in patients with serum phosphorus between 2.5–3.5 mg/dL, with a multivariable-adjusted hazard ratio of 1.24 (95% CI 0.85–1.80), 1.35 (95% CI 1.05–1.74), and 1.75 (95% CI 1.27–2.40) in patients with serum phosphorus of <2.50, 3.51–4.50 and >4.50 mg/dL, respectively. Higher phosphorus levels were also associated with increased risk of heart failure, but not the risk of myocardial infarction or stroke. The effect of elevated phosphorus was more pronounced in patients with chronic kidney disease (CKD). The hazard ratio for mortality in patients with serum phosphorus >4.5 mg/dL compared to patients with serum phosphorus 2.50–3.50 mg/dL was 2.34 (95% CI 1.55–3.54) with CKD and 1.53 (95% CI 0.87–2.69) without CKD.

Conclusion

We found a graded, independent association between serum phosphorus and all-cause mortality and heart failure in patients after AMI. The risk for mortality appears to increase with serum phosphorus levels within the normal range and is more prominent in the presence of CKD.     

Hemolytic Streptococcus May Exacerbate Kidney Damage in IgA Nephropathy through CCL20 Response to the Effect of Th17 Cells

Background

The exacerbation of IgA nephropathy (IgAN) is related to respiratory tract infection with hemolytic streptococcus (HS), but the mechanism is unknown. In this study we investigated the role of chemokine ligand 20 (CCL20) in response to the effect of T helper 17 (Th17) cells in the pathogenesis of IgAN associated with HS.

Methods

Thirty mice were randomly divided into five groups: control mice (control), IgAN mice (IgAN), HS-infected IgAN mice (HS-IgAN), CCL20-treated IgAN mice (CCL20-IgAN), and CCL20-treated HS infected IgAN mice (CCL20-HS-IgAN). IgAN mice were induced with lipopolysaccharide, carbon tetrachloride and bovine serum albumin. Then the mice were sensitized with CCL20 antibody and infected with alpha-hemolytic streptococcus (α-HS) isolated from tonsils in sequence. Urine Albumin-Creatinine ratio and sediments were measured. The pathological changes in kidney and lung tissues were observed under microscopy. Th17 cells and regulatory T cells (Tregs) in kidneys were tested by flow cytometry. CCL20, IL-17A, IL-6 and IL-21 in the kidneys were detected by ELISA.

Results

The IgAN mice had albuminuria and microscopic hematuria, renal mesangial proliferation, IgA deposition, high electron dense deposition in glomerular mesangial region, decreased frequency of Tregs, increased frequency of Th17 and Th17-Treg ratio. Furthermore, Th17-related cytokines CCL20, IL-17A, IL-6 and IL-21 were all increased in the kidneys of IgAN mice. Compared with IgAN mice, the manifestations in HS-IgAN mice were more severe, but alleviated in CCL20-treated groups.

Conclusion

α-HS may exacerbate kidney damage in IgAN through CCL20 response to the effect of Th17 cells.     

Decreased circulating C3 levels and mesangial C3 deposition predict renal outcome in patients with IgA nephropathy

Background and Aims

Mesangial C3 deposition is frequently observed in patients with IgA nephropathy (IgAN). However, the role of complement in the pathogenesis or progression of IgAN is uncertain. In this observational cohort study, we aimed to identify the clinical implications of circulating C3 levels and mesangial C3 deposition and to investigate their utility as predictors of renal outcomes in patients with IgAN.

Methods

A total of 343 patients with biopsy-proven IgAN were enrolled between January 2000 and December 2008. Decreased serum C3 level (hypoC3) was defined as C3 <90 mg/dl. The study endpoint was end-stage renal disease (ESRD) and a doubling of the baseline serum creatinine (D-SCr).

Results

Of the patients, there were 66 patients (19.2%) with hypoC3. During a mean follow-up of 53.7 months, ESRD occurred in 5 patients (7.6%) with hypoC3 compared with 9 patients (3.2%) with normal C3 levels (P = 0.11). However, 12 patients (18.2%) with hypoC3 reached D-SCr compared with 17 patients (6.1%) with normal C3 levels [Hazard ratio (HR), 3.59; 95% confidence interval (CI), 1.33–10.36; P = 0.018]. In a multivariable model in which serum C3 levels were treated as a continuous variable, hypoC3 significantly predicted renal outcome of D-SCr (per 1 mg/dl increase of C3; HR, 0.95; 95% CI, 0.92–0.99; P = 0.011). The risk of reaching renal outcome was significantly higher in patients with mesangial C3 deposition 2+ to 3+ than in patients without deposition (HR 9.37; 95% CI, 1.10–80.26; P = 0.04).

Conclusions

This study showed that hypoC3 and mesangial C3 deposition were independent risk factors for progression, suggesting that complement activation may play a pathogenic role in patients with IgAN.     

HIV-1 Specific IgA Detected in Vaginal Secretions of HIV Uninfected Women Participating in a Microbicide Trial in Southern Africa Are Primarily Directed Toward gp120 and gp140 Specificities

Background

Many participants in microbicide trials remain uninfected despite ongoing exposure to HIV-1. Determining the emergence and nature of mucosal HIV-specific immune responses in such women is important, since these responses may contribute to protection and could provide insight for the rational design of HIV-1 vaccines.

Methods and Findings

We first conducted a pilot study to compare three sampling devices (Dacron swabs, flocked nylon swabs and Merocel sponges) for detection of HIV-1-specific IgG and IgA antibodies in vaginal secretions. IgG antibodies from HIV-1-positive women reacted broadly across the full panel of eight HIV-1 envelope (Env) antigens tested, whereas IgA antibodies only reacted to the gp41 subunit. No Env-reactive antibodies were detected in the HIV-negative women. The three sampling devices yielded equal HIV-1-specific antibody titers, as well as total IgG and IgA concentrations. We then tested vaginal Dacron swabs archived from 57 HIV seronegative women who participated in a microbicide efficacy trial in Southern Africa (HPTN 035). We detected vaginal IgA antibodies directed at HIV-1 Env gp120/gp140 in six of these women, and at gp41 in another three women, but did not detect Env-specific IgG antibodies in any women.

Conclusion

Vaginal secretions of HIV-1 infected women contained IgG reactivity to a broad range of Env antigens and IgA reactivity to gp41. In contrast, Env-binding antibodies in the vaginal secretions of HIV-1 uninfected women participating in the microbicide trial were restricted to the IgA subtype and were mostly directed at HIV-1 gp120/gp140.     

Significance of Urinary Full-Length Megalin in Patients with IgA Nephropathy

Background and Objectives

Megalin is highly expressed at the apical membranes of proximal tubular epithelial cells. A urinary full-length megalin (C-megalin) assay is linked to the severity of diabetic nephropathy in type 2 diabetes. This study examined the relationship between levels of urinary C-megalin and histological findings in adult patients with IgA nephropathy (IgAN).

Design, Setting, Participants, & Measurements

Urine samples voided in the morning on the day of renal biopsy were obtained from 73 patients with IgAN (29 men and 44 women; mean age, 33 years) and 5 patients with membranous nephropathy (MN). Renal pathologic variables were analyzed using the Oxford classification of IgAN, the Shigematsu classification and the Clinical Guidelines of IgAN in Japan. The levels of urinary C-megalin were measured by sandwich ELISA.

Results

Histological analysis based on the Oxford classification revealed that the levels of urinary C-megalin were correlated with mesangial hypercellularity in IgAN patients (OR = 1.76, 95% CI: 1.04–3.27, P<0.05). There was a significant correlation between the levels of urinary C-megalin and the severity of chronic extracapillary abnormalities according to the Shigematsu classification in IgAN patients (β = 0.33, P = 0.008). The levels of urinary C-megalin were significantly higher in all risk levels of IgAN patients requiring dialysis using the Clinical Guidelines of IgAN in Japan than in the control group. The levels of urinary C-megalin were significantly higher in the high risk and very high risk grades than in the low risk grade (P<0.05). The levels of urinary C-megalin were significantly higher in MN patients compared to the control group.

Conclusions

The levels of urinary C-megalin are associated with histological abnormalities in adult IgAN patients. There is a possibility that urinary C-megalin is an independent predictor of disease progression of IgAN. In addition, our results suggest that urinary C-megalin is a marker of glomerular abnormalities in various glomerular diseases as well as IgAN.     

Cartilage oligomeric matrix protein-induced complement activation in systemic sclerosis

Introduction

Complexes between cartilage oligomeric matrix protein (COMP) and the complement activation product C3b have been found in the circulation of patients with rheumatoid arthritis and systemic lupus erythematosus. In systemic sclerosis (SSc) COMP expression in the skin is upregulated both in lesional and non-lesional skin, which is also reflected in an increased amount of circulating COMP. We investigated the presence of COMP-C3b complexes in serum and skin biopsies of patients with SSc.

Methods

The presence of COMP and COMP-C3b complexes in the serum of 80 patients with limited cutaneous SSc (lcSSc, n = 40) and diffuse cutaneous SSc (dcSSc, n = 40) and 97 healthy controls was measured by ELISA and correlated to different clinical parameters. Samples were collected both at baseline and after three to five years to assess longitudinal changes in COMP-C3b complex levels. Furthermore, skin biopsies from seven patients with dcSSc and three healthy controls were analyzed for expression of COMP and deposition of C3b and IgG.

Results

Serum levels of COMP-C3b were found to be elevated in both dcSSc and lcSSc compared to healthy controls and decreased at the second measurement in patients on immunosuppressive therapy. No co-localization of COMP and C3b was found in the skin biopsies, indicating that the COMP-C3b complexes are formed upon release of COMP into the circulation.

Conclusion

COMP-C3b complexes are found in the serum of patients with SSc. The lack of co-localization between COMP and C3b in the skin suggests that COMP does not drive complement activation in the skin in SSc.     

Serum CXCL16 as a Novel Marker of Renal Injury in Type 2 Diabetes Mellitus

Background

Soluble C-X-C chemokine ligand 16 (CXCL16), a scavenger receptor for oxidized low density lipoprotein, has been shown to promote atherogenic effects in vivo and to predict long-term mortality in acute coronary syndrome. The aim of this study was to explore the association of circulating CXCL16 levels with diabetic subjects with and without renal disease.

Methodology/Principal Findings

One hundred twenty Chinese subjects, which included patients with type 2 diabetes mellitus (T2DM), diabetic nephropathy (DN), and CKD, as well as healthy controls, were enrolled in this study. Serum CXCL16 levels were examined by immunoassay and other clinical biochemical parameters were tested based on standard methods. Our results indicated that, HDL and LDL cholesterol levels are significantly different in DN but not in T2D patients in comparison with healthy subjects. On the other hand, Serum CXCL16 levels were significantly increased in DN subjects compared with age and gender matched healthy and T2DM subjects (p<0.05 respectively). However, no significant changes in serum CXCL16 levels were found between T2DM and healthy subjects. Furthermore, serum CXCL16 concentration negatively correlated with estimated glomerular filtrate rate, creatinine clearance rate and blood albumin, and positively with 24 h proteinuria, blood urea nitrogen (BUN), creatinine, and uric acid after adjusting for age, gender and BMI in subjects with DN. Multiple stepwise regression analyses indicated that serum CXCL16 levels were independently associated with serum 24 h proteinuria, and BUN (p<0.05 respectively).

Conclusion

Serum CXCL16 may be an indicator of renal injury in subjects with T2DM. Understanding the exact mechanism of elevated CXCL16 in subjects with DN requires further study.     

Enhanced neutrophil extracellular trap generation in rheumatoid arthritis: analysis of underlying signal transduction pathways and potential diagnostic utility

Introduction

Neutrophil extracellular traps (NETs) have recently been implicated in a number of autoimmune conditions, including rheumatoid arthritis (RA). We examined the underlying signaling pathways triggering enhanced NETosis in RA and ascertained whether the products of NETosis had diagnostic implications or usefulness.

Methods

Neutrophils were isolated from RA patients with active disease and from controls. Spontaneous NET formation from RA and control neutrophils was assessed in vitro with microscopy and enzyme-linked immunosorbent assay (ELISA) for NETosis-derived products. The analysis of the signal-transduction cascade included reactive oxygen species (ROS) production, myeloperoxidase (MPO), neutrophil elastase (NE), peptidyl arginine deiminase 4 (PAD4), and citrullinated histone 3 (citH3). NET formation was studied in response to serum and synovial fluid and immunoglobulin G (IgG) depleted and reconstituted serum. Serum was analyzed for NETosis-derived products, for which receiver operator characteristic (ROC) curves were calculated.

Results

Neutrophils from RA cases exhibited increased spontaneous NET formation in vitro, associated with elevated ROS production, enhanced NE and MPO expression, nuclear translocation of PAD4, PAD4-mediated citrullination of H3, and altered nuclear morphology. NET formation in both anti-citrullinated peptide antibody (ACPA)-positive and -negative RA was abolished by IgG depletion, but restored only with ACPA-positive IgG. NETosis-derived products in RA serum demonstrated diagnostic potential, the ROC area under the curve for cell-free nucleosomes being >97%, with a sensitivity of 91% and a specificity of 92%. No significant difference was observed between ACPA-positive and -negative cases.

Conclusions

Signaling elements associated with the extrusion of NETs are significantly enhanced to promote NETosis in RA compared with healthy controls. NETosis depended on the presence of ACPA in ACPA-positive RA serum. The quantitation of NETosis-derived products, such as cell-free nucleosomes in serum, may be a useful complementary tool to discriminate between healthy controls and RA cases.