HIV-Infected Patients: Cross Site-Specific Hydrolysis of H2a and H2b Histones and Myelin Basic Protein with Antibodies against These Three Proteins.

Anti-DNA antibodies are usually produced against histone-DNA complexes appearing during cell apoptosis, while histones are known as damage-associated molecules. A myelin sheath of axons contains myelin basic protein (MBP) playing an important role in the pathogenesis of autoimmune diseases. Antibodies with enzymatic activities (abzymes) are distinctive features of some autoimmune and viral diseases. Abzymes against different proteins can usually only hydrolyze these specific proteins. Using sequential chromatographies of homogeneous IgG preparations from sera of HIV-infected patients on columns with immobilized MBP, H2a, and H2b histones, the anti-MBP, anti-H2a, and anti-H2b antibodies were obtained. It was first shown that IgGs against H2a and H2b effectively hydrolyze these histones and MBP, while anti-MBP split MBP, H2a, and H2b, but no other control proteins. Using the MALDI mass spectrometry, the cleavage sites of H2a, H2b, and MBP by abzymes against these three proteins were found. Among 14 sites of hydrolysis of H2a by IgGs against H2a and 10 sites by anti-MBP IgGs, only one site of hydrolysis was the same for these abzymes. Eleven cleavage sites of H2b with IgGs against H2b and 10 sites of its hydrolysis with antibodies against MBP were different. Anti-H2a, anti-H2b, and anti-MBP abzymes are unpredictable examples of IgGs possessing not only cross-complexation but also catalytic cross-reactivity, which may be a common phenomenon for such abzymes in patients with different autoimmune diseases. The existence of cross-reactivity of abzymes against H2a and H2b histones and MBP represent a great danger to humans since, in contrast with MBP, histones due to cell apoptosis constantly occur in human blood. Anti-H2a, anti-H2b, and anti-MBP can attack and hydrolyze myelin basic protein of the myelin sheath of axons and plays a negative role in the pathogenesis of several pathologies.

Patterns of C1-Inhibitor/Plasma Serine Protease Complexes in Healthy Humans and in Hereditary Angioedema Patients.

C1-inhibitor (C1-INH) is an important regulator of the complement, coagulation, fibrinolytic and contact systems. The quantity of protease/C1-INH complexes in the blood is proportional to the level of the in vivo activation of these four cascade-like plasma enzyme systems. Parallel determination of C1-INH-containing activation complexes could be important to understand the regulatory role of C1-INH in diseases such as hereditary angioedema (HAE) due to C1-INH deficiency (C1-INH-HAE). We developed in-house ELISAs to measure the concentration of complexes of C1-INH formed with active proteases: C1r, C1s, MASP-1, MASP-2, plasma kallikrein, factor XIIa, factor XIa, and thrombin, as well as to determine total and functionally active C1-INH. We measured the concentration of the complexes in EDTA plasma from 6 healthy controls, from 5 with type I and 5 with type II C1-INH-HAE patients during symptom-free periods and from five patients during HAE attacks. We also assessed the concentration of these complexes in blood samples taken from one C1-INH-HAE patient during the kinetic follow-up of a HAE attack. The overall pattern of complexed C1-INH was similar in controls and C1-INH-HAE patients. C1-INH formed the highest concentration complexes with C1r and C1s. We observed higher plasma kallikrein/C1-INH complex concentration in both type I and type II C1-INH-HAE, and higher concentration of MASP-1/C1-INH, and MASP-2/C1-INH complexes in type II C1-INH-HAE patients compared to healthy controls and type I patients. Interestingly, none of the C1-INH complex concentrations changed significantly during HAE attacks. During the kinetic follow-up of an HAE attack, the concentration of plasma kallikrein/C1-INH complex was elevated at the onset of the attack. In parallel, C1r, FXIIa and FXIa complexes of C1-INH also tended to be elevated, and the changes in the concentrations of the complexes followed rather rapid kinetics. Our results suggest that the complement classical pathway plays a critical role in the metabolism of C1-INH, however, in C1-INH-HAE, contact system activation is the most significant in this respect. Due to the fast changes in the concentration of complexes, high resolution kinetic follow-up studies are needed to clarify the precise molecular background of C1-INH-HAE pathogenesis.

Serum Soluble CD89-IgA Complexes Are Elevated in IgA Nephropathy without Immunosuppressant History.

PURPOSE: CD89 (FcαRI), the receptor of IgA, can shed from cells to form complexes with IgA in serum and is supposed to participate in the pathogenesis of IgA nephropathy (IgAN). There are contradictory results on their utility in clinical practice. This study is aimed at investigating whether sCD89-IgA complexes can help in the diagnosis or evaluation of the disease. METHODS: A sandwich ELISA was established using anti-CD89 as a capture antibody and HRP-conjugated anti-IgA as a detection antibody. This method was used to measure serum levels of sCD89-IgA complexes in IgAN patients without immunosuppressant history and healthy subjects. Correlations between serum levels of sCD89-IgA complexes and disease severity were analyzed. RESULTS: Serum sCD89-IgA complexes increased with age (P < 0.001). IgAN patients had higher sCD89-IgA complex levels compared with age- and gender-matched normal healthy individuals (P < 0.001). IgAN patients had higher sCD89-IgA complex levels compared with age- and gender-matched normal healthy individuals (P < 0.001). IgAN patients had higher sCD89-IgA complex levels compared with age- and gender-matched normal healthy individuals (. CONCLUSIONS: Serum sCD89-IgA complexes can guide diagnosis of IgAN in patients without immunosuppressant history, but provide limited help in clinicopathologic prediction.

Serum complexes between C1INH and C1INH autoantibodies for the diagnosis of acquired angioedema.

Acquired angioedema due to C1-inhibitor (C1INH) deficiency (AAE) is caused by secondary C1INH deficiency leading to bradykinin-mediated angioedema episodes. AAE typically presents in adulthood and is associated with B cell lymphoproliferation. Anti-C1INH autoantibodies (antiC1INHAbs) are detectable in a subset of AAE cases and considered a hallmark of the disease. When free antiC1INHAbs and malignant tumors are not detectable, diagnosis relies on the finding of low C1INH levels and/or function, lack of family history and SERPING1 mutations, age at onset and low or undetectable C1q levels, none of which is specific for AAE. We tested the diagnostic value of a novel enzyme-linked immunosorbent assay (ELISA) for the detection of circulating complexes between C1INH and antiC1INHAbs (C1INH-antiC1INHAb) in the serum of 20 European AAE patients characterized on the basis of their complement levels and function. Free antiC1INHAbs were detected in nine of 20 patients [six of immunoglobulin (Ig)G class, two of IgM class and one simultaneously presenting IgG and IgM classes], whereas C1INH-antiC1INHAb complexes were found in 18 of 20 of the AAE cases, regardless of the presence or absence of detectable free anti-C1INHAbs. Of note, nine of 20 patients showed negative free antiC1INHabs, but positive C1INH-antiC1INHAb complexes in their first measurement. In the cohort presented, IgM-class C1INH-antiC1INHAb are specifically and strongly associated with low C1q serum levels. Detection of C1INH-antiC1-INHAbs provides an added value for AAE diagnosis, especially in those cases in whom no free anti-C1INH antibodies are detected. The link between IgM-class C1INH-antiC1INHAb complexes and C1q consumption could have further implications for the development of autoimmune manifestations in AAE.

Correlates of T50 and relationships with bone mineral density in community-living older men: the osteoporotic fractures in men (MrOS) study.

PURPOSE: T50 is a novel serum-based marker that assesses the propensity of calcification in serum. Shorter T50 indicates greater propensity to calcify and it has been associated to cardiovascular disease (CVD) and mortality among patients with kidney disease. In the general population, neither the correlates of T50 nor the relationships of T50 with bone mineral density (BMD) are known. METHODS: We performed a nested cross-sectional study selecting 150 individuals at random among participants from the Osteoporotic Fractures in Men (MrOS) Study, a study of community-living older men. We categorized individuals into tertiles of T50 and compared demographics and disease indicators across tertiles. We utilized linear regression to evaluate the cross-sectional association between T50 and hip and spine BMD in multivariable models. RESULTS: Older age was associated with shorter T50. Kidney function tended to be lower in those with shorter T50 and the prevalence of CVD and peripheral arterial disease in those with shorter T50, albeit these findings did not achieve statistical significance. We found no statistically significant associations between T50 and total hip or total spine BMD in either unadjusted or multivariable adjusted models. CONCLUSIONS: T50, a novel indicator of serum calcification propensity, is not associated with BMD in community-living older men. Future larger studies should determine if T50 may give insights to CVD in the general population above and beyond traditional risk factors.

Elevation of serum oxLDL/ß2-GPI complexes was correlated with diabetic microvascular complications in Type 2 diabetes mellitus patients.

BACKGROUND: High levels of oxLDL/ß2-GPI complexes might be a consequence of LDL atherogenic modification mediated by oxidative stress. We aimed to determine whether the levels of serum oxLDL/ß2-GPI complexes were correlated with diabetic microvascular complications in type 2 diabetes mellitus (T2DM) patients. METHODS: Levels of oxLDL/ß2-GPI complexes, oxLDL, routine lipid/lipoprotein parameters were measured in 100 healthy controls, 128 T2DM patients without any microvascular complications, and 172 T2DM patients with microvascular complications. Spearman's correlation, multivariable linear regression logistic regression analysis, and receiver operating characteristic (ROC) curve were performed. RESULTS: Levels of serum oxLDL/ß2-GPI complexes and oxLDL were significantly higher in T2DM patients with microvascular complications (oxLDL/ß2-GPI complexes: 1.10 ± 0.18 U/mL; oxLDL: 48.12 ± 7.24 mmol/L) than those in T2DM patients without microvascular complications (oxLDL/ß2-GPI complexes: 0.98 ± 0.16 U/mL; oxLDL: 41.45 ± 6.81 mmol/L) and controls (oxLDL/ß2-GPI complexes: 0.79 ± 0.15 U/mL; oxLDL: 27.85 ± 5.32 mmol/L). Variables that remained significantly associated with oxLDL/ß2-GPI complexes were oxLDL (ß = 0.568, P < 0.001), TC (ß = 0.312, P = 0.013) and microvascular complications (ß = 0.205, P = 0.027), which accounted for 58.3% of the variation of the level of oxLDL/ß2-GPI complexes in T2DM patients (R2 = 0.583). Logistic regression analysis demonstrated that elevation of oxLDL/ß2-GPI complexes (OR = 3.14, 95% CI: 1.04-9.46, P = 0.042) and oxLDL levels (OR = 3.02, 95% CI: 1.16-7.83, P = 0.023) were independently associated with occurrence of microvascular complications. Cutoff value of oxLDL/ß2-GPI for the presence of microvascular complications was 1.05 U/mL, and AUC area of ROC curve was 0.783 (95%CI: 0.713-0.853), yielding a sensitivity of 86.8% and specificity of 64.9%. CONCLUSIONS: Elevation of serum oxLDL/ß2-GPI complexes was associated with microvascular complications in T2DM patients.

Increased Levels of Serum Protein Complexes Are Associated with Type 2 Diabetes.

Objective: To screen novel biomarkers in the levels of protein complexes for type 2 diabetes mellitus (T2DM). Methods: Serum immunoinflammation-related protein complexes (IIRPCs) and diabetes-related protein complexes (DRPCs) in 1537 serum samples including 504 healthy controls, 320 patients with prediabetes, and 713 patients with T2DM were analyzed using an optimized native polyacrylamide gel electrophoresis (native-PAGE). Results: Seven patterns of serum IIRPCs and four patterns of serum DRPCs were observed in the study population, respectively. Significant increase in the levels of serum IIRPCs in T2DM was detected relative to healthy controls. Change trends of serum DRPCs are as below: patients with T2DM>patients with prediabetes> healthy controls. Conclusion: Our findings suggest that increased levels of serum IIRPCs and DRPCs were associated with T2DM.

Cystatin C-Adiponectin Complex in Plasma Associates with Coronary Plaque Instability.

AIM: Adiponectin (APN) is an adipocyte-derived bioactive molecule with antiatherogenic properties. We previously reported that cystatin C (CysC) abolished the anti-atherogenic effects of APN. We aimed to elucidate the clinical significance of CysC-APN complex in patients with coronary artery disease (CAD). METHODS: We enrolled 43 stable CAD male patients to examine the relationship between CysC-APN complex and coronary plaque characteristics. Serum was immunoprecipitated by the anti-APN antibody and immunoblotted by the anti-CysC antibody to demonstrate the presence of CysC-APN complexes in vivo. To confirm their binding in vitro, HEK293T cell lysates overexpressing myc-APN and FLAG-CysC were immunoprecipitated with an anti-myc or anti-FLAG antibody, followed by immunoblotting with an anti-APN or anti-CysC antibody. RESULTS: CysC was identified as a specific co-immunoprecipitant with APN by the anti-APN antibody in human serum. In vitro, FLAG-CysC was co-immunoprecipitated with myc-APN by the anti-myc antibody and myc-APN was co-immunoprecipitated with FLAG-CysC by the anti-FLAG antibody. Among CAD patients, serum CysC-APN complex levels negatively correlated with fibrotic components of coronary plaques and positively correlated with either necrotic or lipidic plus necrotic components. Plaque burden negatively correlated with serum APN levels but not serum CysC-APN complex levels. Serum CysC levels had no association with plaque characteristics. In multivariate analysis, CysC-APN complex levels were identified as the strongest negative factor for fibrotic components and the strongest positive factor for both necrotic and lipidic plus necrotic components. CONCLUSION: Measuring serum CysC-APN complex levels is helpful for evaluating coronary plaque instability in CAD patients.

Concomitant elevations of MMP-9, NGAL, proMMP-9/NGAL and neutrophil elastase in serum of smokers with chronic obstructive pulmonary disease.

A growing body of evidence points towards smoking-related phenotypic differences in chronic obstructive pulmonary disease (COPD). As COPD is associated with systemic inflammation, we determined whether smoking status is related to serum levels of matrix metalloproteinase-9 (pro- and active MMP-9), neutrophil gelatinase-associated lipocalin (NGAL) and the proMMP-9/NGAL complex in patients with COPD. Serum samples were collected in 100 stable-phase COPD patients (82 smokers, 18 never-smokers) and 28 healthy adults (21 smokers, 7 never-smokers). Serum levels of studied factors were measured in ELISA. Our data provide the first evidence of simultaneously elevated serum levels of MMP-9, NGAL and proMMP-9/NGAL in COPD smokers. While the triad discriminated between smokers and non-smokers in the COPD group, MMP-9 and proMMP-9/NGAL (but not NGAL) discriminated between smokers with and without COPD. Adjustment for age and smoking pack-years did not alter the findings. Serum MMP-9, NGAL and proMMP-9/NGAL levels were not correlated with the GOLD stage or FEV1 decline. Furthermore, serum levels of neutrophil elastase (NE) and MMP-3 (but not of IL-6 and MMP-12) were also higher in COPD smokers than in healthy smokers before and after adjustment for age and pack-years. Among COPD smokers, levels of MMP-9, NGAL and proMMP-9/NGAL were positively correlated with NE (P < 0.0001) but not with the remaining factors. Gelatin zymography detected proMMP-9 in serum samples of healthy and COPD smoking groups. Our results suggest that associated serum levels of proMMP-9, NGAL, proMMP-9/NGAL and NE may reflect the state of systemic inflammation in COPD related to cigarette smoking.

Diagnostic Significance of Phosphoinositide 3-Kinase and Mammalian Target of Rapamycin Complex 1 in Preeclampsia.

OBJECTIVE: To examine the association of phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin complex 1 (m-TORC1) with preeclampsia (PE) and to explore their diagnostic significance in PE. METHODS: A total of 153 singleton pregnant women were enrolled into our study, among which there were 97 patients with PE (mild PE [MPE]: n = 51; severe PE [SPE]: n = 46) and 56 healthy pregnant women (normal controls, NCs). Enzyme-linked immunosorbent assay (ELISA) and Western blot were used in this study. Moreover, a receiver-operating characteristic (ROC) curve was used to estimate the diagnostic significance. RESULTS: After adjustment for confounding factors, at 24 to 28 gestational weeks, the serum levels of PI3K and m-TORC1 were both higher in the MPE and the SPE groups compared to those in the NC group (all P < .001). The serum levels of PI3K were positively correlated with the serum levels of m-TORC1 in both the NC and the PE groups at both 15 to 21 and 24 to 28 gestational weeks (both P < .001). Multivariable linear regression indicated that both PI3K and m-TORC1 were positively correlated with the systolic pressure (both P < .001). At 24 to 28 gestational weeks, there remained relatively high sensitivity and specificity when the serum levels of PI3K and m-TORC1 were used to diagnose PE (both P < .001). A Western blot assay found that there were significant differences in the PI3K and m-TORC1 protein expression among the 3 groups (all P < .001). CONCLUSION: The serum levels of PI3K and m-TORC1 might have the potential to diagnose PE, while PI3K and m-TORC1 fail to predict PE during early pregnancy.

Dietary glycemic factors, insulin resistance, and adiponectin levels in acne vulgaris.

BACKGROUND: There is increasing evidence to support the relationship between acne vulgaris and diet. OBJECTIVE: The aim of this study was to investigate possible associations among dietary glycemic index, glycemic load, milk consumption, insulin resistance, and adiponectin levels in the pathogenesis of acne vulgaris. METHODS: The dietary glycemic index, glycemic load, milk consumption, fasting glucose, insulin, insulin-like growth factor)-1, insulin-like growth factor binding protein-3, adiponectin, and homeostasis model assessment of insulin resistance values of 50 patients with acne vulgaris and 36 healthy control subjects were measured. RESULTS: Glycemic index and glycemic load levels were significantly higher (P = .022 and P = .001, respectively) and serum adiponectin levels were significantly lower (P = .015) in patients with acne than in the control subjects. There was an inverse correlation between serum adiponectin concentration and glycemic index (P = .049, r = -0.212). LIMITATIONS: This study used a cross-sectional design and the study population was limited to young, nonobese adults. CONCLUSION: A high-glycemic-index/-load diet was positively associated with acne vulgaris. Adiponectin may be a pathogenetic cofactor contributing to the development of the disease. Further research on adiponectin levels in patients with acne in terms of development of insulin resistance might be important in this possible relationship.

Soluble B-Cell Maturation Antigen Mediates Tumor-Induced Immune Deficiency in Multiple Myeloma.

PURPOSE: Reduced uninvolved immunoglobulin (Ig) levels are a hallmark of multiple myeloma. We previously showed that B-cell maturation antigen (BCMA) is solubilized and at high levels in multiple myeloma patient serum. We hypothesize that soluble BCMA binds B-cell-activating factor (BAFF) preventing its function to stimulate late B cells, and would result in lower polyclonal antibody levels in these patients. EXPERIMENTAL DESIGN: Mice were dosed with recombinant human BCMA (rhBCMA) and BCMA-BAFF complexes were analyzed in plasma, and its effects on antibody and Ig heavy chain mRNA levels determined. Using flow cytometry, BAFF binding to B cells was examined in the presence of rhBCMA and sera from multiple myeloma patients. In multiple myeloma sera, BCMA-BAFF complex formation and BCMA, IgA, IgG levels, and heavy-light chain isoform pair levels were determined. RESULTS: rhBCMA-BAFF complexes formed in immune-competent and deficient mice. Mice with human multiple myeloma xenografts, which contain plasma hBCMA and hBCMA-BAFF complexes, showed reduced plasma-free BAFF levels. rhBCMA administered to immune competent mice markedly reduced plasma IgA, IgG, and IgM levels and splenic Ig heavy chain mRNA levels. In serum from multiple myeloma patients, BCMA-BAFF complexes were detected and BAFF levels were reduced. Multiple myeloma patient sera containing BCMA prevented binding of BAFF to B cells. There is an inverse correlation between serum BCMA and uninvolved polyclonal Ig level in multiple myeloma patients. CONCLUSIONS: Our results show that soluble BCMA sequesters circulating BAFF, thereby preventing it from performing its signaling to stimulate normal B-cell and plasma cell development, resulting in reduced polyclonal antibody levels in multiple myeloma patients. Clin Cancer Res; 22(13); 3383-97. ©2016 AACR.

α-Defensins Induce a Post-translational Modification of Low Density Lipoprotein (LDL) That Promotes Atherosclerosis at Normal Levels of Plasma Cholesterol.

Approximately one-half of the patients who develop clinical atherosclerosis have normal or only modest elevations in plasma lipids, indicating that additional mechanisms contribute to pathogenesis. In view of increasing evidence that inflammation contributes to atherogenesis, we studied the effect of human neutrophil α-defensins on low density lipoprotein (LDL) trafficking, metabolism, vascular deposition, and atherogenesis using transgenic mice expressing human α-defensins in their polymorphonuclear leukocytes (Def(+/+)). Accelerated Def(+/+) mice developed α-defensin·LDL complexes that accelerate the clearance of LDL from the circulation accompanied by enhanced vascular deposition and retention of LDL, induction of endothelial cathepsins, increased endothelial permeability to LDL, and the development of lipid streaks in the aortic roots when fed a regular diet and at normal plasma levels of LDL. Transplantation of bone marrow from Def(+/+) to WT mice increased LDL clearance, increased vascular permeability, and increased vascular deposition of LDL, whereas transplantation of WT bone marrow to Def(+/+) mice prevented these outcomes. The same outcome was obtained by treating Def(+/+) mice with colchicine to inhibit the release of α-defensins. These studies identify a potential new link between inflammation and the development of atherosclerosis.

The Levels of the Lectin Pathway Serine Protease MASP-1 and Its Complex Formation with C1 Inhibitor Are Linked to the Severity of Hereditary Angioedema.

C1 inhibitor (C1-INH) is known to form complexes with the lectin complement pathway serine proteases MASP-1 and MASP-2. Deficiency of C1-INH is associated with hereditary angioedema (HAE), an autosomal inherited disease characterized by swelling attacks caused by elevated levels of bradykinin. MASP-1 was shown to cleave high m.w. kininogen into bradykinin; therefore, we hypothesized that MASP-1 levels and the quantity of MASP-1/C1-INH complexes might be associated with different paraclinical and clinical outcomes of HAE. We measured MASP-1 serum concentrations and endogenous MASP-1/C1-INH complex levels in 128 HAE patients and 100 controls. Relatively high levels of pre-existing MASP-1/C1-INH complexes were observed in normal serum, and we found that both the serum levels of MASP-1 and the complex formation between MASP-1 and C1-INH were significantly reduced in HAE patients compared with matched controls (p < 0.0001). The level of MASP-1 and MASP-1/C1-INH complexes in HE patients correlated with the level of C1-INH (p = 0.0009 and p = 0.0047, respectively), the level of C4 (p = 0.0084 and p < 0.0001, respectively), and the number of attacks in the year of blood sampling (p = 0.0075 and p = 0.0058, respectively). In conclusion, we show that MASP-1/C1-INH complexes circulate in normal human blood. The levels of MASP-1 and MASP-1/C1-INH complexes are reduced in HAE patients compared with controls. Both MASP-1 and MASP-1/C1-INH complexes are related to the degree of complement C4 consumption, as well as the severity of disease. These results suggest that MASP-1 may exert a previously unrecognized role in the pathophysiology of HAE.

Elevated serum immunoinflammation-related protein complexes are associated with psychosis.

Emerging evidence suggests an underlying immune and inflammatory response for a variety of psychiatric disorders. Herein, we employed an optimized native-polyacrylamide gel electrophoresis to isolate psychosis-related serum immunoinflammation-related protein complexes (IIRPCs) from 147 patients with schizophrenia (SCH), 158 patients with bipolar disorder (BPD), 132 patients with other psychosis, and 145 normal controls. All participants could be classified into four categories based on serum IIRPCs, which correspond to 290, 215, 70, and 7 serum samples, correspondingly. For each category, significantly enhanced levels of serum IIRPCs in patients with SCH, BPD, and other psychosis groups were observed compared with normal controls. Receiver operating characteristic analysis indicated that serum IIRPCs have excellent diagnostic performance to differentiate SCH, BPD, and other psychosis groups from normal controls, with high sensitivities and specificities of >85%. Total serum amounts of IgG, IgA, and IgM in all patients were significantly decreased compared with normal controls.

Instability of IGF-IGFBP complex as a cause of the different performance of serum and EDTA-plasma after storage: EDTA-plasma is preferable for evaluating bioactive IGF especially in the mouse.

OBJECTIVE: The insulin-like growth factor (IGF) signaling pathway is recognized as a potential target for treating several cancers, and strategies targeting the IGF type 1 receptor (IGF-1R) have been evaluated in many clinical trials. These suggested that the pretreatment level of circulating free IGF gives an estimate of IGF bioactivity and might be a predictive biomarker of the response to anti-IGF-1R antibodies. However, there is no defined protocol for measuring free and bioactive IGF concentrations, partly because the measurement procedures, including sample collection and handling, have not been standardized. We investigated the effects of sample collection methods and storage conditions on bioactive IGF measurement using a modified kinase receptor activation (KIRA) assay in human and mouse samples. DESIGN: Blood samples were obtained from healthy men and women, and from healthy male and female wild-type BALB/c mice. Serum and ethylenediaminetetraacetic acid (EDTA)-plasma samples were collected and used immediately or stored in small quantities at 4 °C or -80 °C for 3, 7, or 14 days. A bioassay directed against the phosphorylated IGF-1R using western blot analysis was developed as a modification of the KIRA assay, in which the level of phosphorylation of IGF-1R represented the IGF bioactivity in blood samples. RESULTS: The levels of bioactive IGFs in mouse serum stored at 4 °C increased markedly in a time-dependent manner; the increase was slightly reduced in samples stored at -80 °C. Analysis of mouse EDTA-plasma stored at 4 °C showed a similar pattern, but the time-dependent increase was less than in the serum samples. By contrast, the levels of bioactive IGFs in EDTA-plasma stored at -80 °C were stable over 14 days. The levels of human bioactive IGFs in both serum and EDTA-plasma stored at 4 °C increased slightly with time, but the increases were much smaller than in mouse samples. The levels of human bioactive IGF in both serum and EDTA-plasma stored at -80 °C were stable over 14 days. CONCLUSIONS: The use of EDTA-plasma avoids the problems with long-term storage. Therefore, EDTA-plasma should be used when measuring circulating IGF bioactivity, especially in mouse samples. All samples should be stored at -80 °C when long-term storage is unavoidable. Because of the large difference in the stability of the IGF-IGF-binding protein complex between the human and mouse in vitro, all samples should be handled carefully to ensure the accurate evaluation of IGF bioactivity, especially in mouse samples.

Proteomic Investigation on Grp94-IgG Complexes Circulating in Plasma of Type 1 Diabetic Subjects.

The glucose-regulated protein94 (Grp94) has been found in complexes with IgG in plasma of Type 1 (T1) diabetic subjects; however, the pathogenetic meaning of Grp94-IgG complexes has not yet been elucidated. To shed light on the nature and structure of these complexes in vivo, we conducted a proteomic analysis on plasma of both T1 diabetic subjects and healthy control subjects. IgG purified from plasma was submitted to 2D PAGE followed by Western blotting and mass analysis. Grp94 was detected in plasma of all diabetic but not control subjects and found linked with its N-terminus to the IgG heavy chain. Mass analysis of heavy chain of IgG that binds Grp94 also in vitro, forming stable complexes with characteristics similar to those of native ones, permitted identifying CH2 and CH3 regions as those involved in binding Grp94. At the electron microscopy, IgG from diabetic plasma appeared as fibrils of various lengthes and dimensions, suggestive of elevated aggregating tendency conferred to IgG by Grp94. The nonimmune nature of complexes turned out to be responsible for the particular stability and structure adopted by complexes in plasma of diabetic subjects. Results are of relevance to understanding the pathogenetic mechanisms underlying diabetes and its complications.

Complexes between insulin-like growth factor binding proteins and alpha-2-macroglobulin in patients with tumor.

The intention of this work was to systematically study the presence of insulin-like growth factor-binding protein 1 and 2 (IGFBP-1 and IGFBP-2) complexes with alpha-2-macroglobulin (α2M) in the circulation of patients with various types of cancer. Serum samples were collected from patients diagnosed with cancer and divided into eight groups according to the tumor type: liver, pancreas, colon, lung, prostate, breast, cervix and ovary. Results obtained for each cancer group were compared with results obtained for the age and gender-matched controls. Electrophoretic separation and densitometric evaluation of immunoreactive protein bands were performed. According to our results, IGFBP-1/α2M and IGFBP-2/α2M complexes did not exhibit the same presence in different tumors and were also not uniformly related to the amount of monomer forms. Variations in relative quantities of IGFBP-1 monomers and complexes in different tumor types were much more pronounced than those of IGFBP-2. The amount of IGFBP-2 monomer increased in sera from all studied patients compared to controls, whereas the amount of IGFBP-2/α2M complexes most often decreased, being significantly reduced in patients with pancreas, colon, breast or ovary tumor. Although there is still no confirmation of the physiological role of IGFBP-2/α2M complexes, their decreased concentration in the circulation provides greater amount of free IGFBP-2.

Red blood cell storage affects the stability of cytosolic native protein complexes.

BACKGROUND: Refrigerated storage of red blood cell (RBC) units promotes the progressive accumulation of the so-called storage lesions, a widespread series of alterations to morphology, metabolism, and proteome integrity of stored RBCs. However, while storage lesions targeting the RBC membrane fraction have been widely documented, the cytosolic fraction is as yet an underinvestigated cause of the technical inconveniences related to the high abundance of hemoglobin. STUDY DESIGN AND METHODS: By exploiting a recently ideated preparative two-dimensional clear native electrophoresis, followed by mass spectrometry analysis, we could monitor the changes of soluble multiprotein complexes (MPCs) in RBCs after 0, 21, and 35 days of storage under standard blood banking conditions. RESULTS: Data indicate a substantial storage-dependent alteration of RBC MPCs, particularly of those involved in energy and redox metabolism, confirming previous evidence about the progressive dysregulation of these pathways in long-stored units. CONCLUSION: The use of native gel-based proteomics to investigate MPCs present in the RBC cytosolic fraction proved to be a powerful tool. Results collected represent a preliminary advance in the knowledge of the key role of native cytosolic MPCs in context of RBC storage lesion. Multiprotein organization and interacting partners of some key enzymes have been found to change during storage duration, suggesting that future studies will be needed to assess whether such alterations could influence their activity and efficiency.