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Background: In human serum, a portion of homocysteine (Hcy) exists as an N-linked form to the 1-amino group of protein lysine residues. N-homocysteinylated proteins differ structurally and functionally from native proteins. The present study strives to develop detection and potential semi-quantification methods for N-homocysteinylated apolipoprotein AI (N-HcyapoAI) in human serum. Methods: Serum treated with or without cysteamine was supplied to isoelectric focusing (IEF) followed by an immunoblot using an anti-apoAI antibody. Cysteamine treatment increased the isoelectric point for N-Hcy-apoAI, but not for unmodified apoAI, due to the presence of -SH group(s) derived from Hcy and the absence of a cysteine residue in the apoAI molecule. N-Hcy-apoAI was semi-quantified from the scanned immunoblot pattern via a computer. Results: After cysteamine treatment, N-Hcy-apoAI in the serum was identified by IEF at the position with a higher pI value compared with intact apoAI. The reproducibility (between assays) of the semi-quantification method was 19.1% CV (coefficientofvariation) foranaverage ratio5.9%ofN-Hcy-apoAI to thewholeapoAI inthe serum.Approximately1.0-7.4%of apoAI was N-homocysteinylated in the serum obtained from 27 healthy subjects. Neither the ratio of N-Hcy-apoAI nor its concentration, calculated by total apoAI concentration, indicated correlation with the so-called total (free and S-linked) Hcy concentration. Conclusions:We directly found that a portion of apoAI in the serum undergoes homocysteinylation in an N-linkage manner, and used this to develop a potential semi-quantification method for N-Hcy-apoAI. [ABSTRACT FROM AUTHOR]

The linkage between low-density lipoprotein receptor-related protein (LRP)1-mediated metabolism of apolipoprotein (apo) E-containing lipoproteins (apoE-LP) and the lipopolysaccharide (LPS)-induced inflammatory response contributes to the pathogenesis of sepsis; however, the underlying mechanisms are unclear. Therefore, in this study, the effects of apoE-LP and their constituents on the mRNA expression of interleukin (IL)-6 and LRP1 were evaluated using a culture system of human fibroblasts supplemented with LPS and apoE-containing emulsion particles (apoE-EP). The affinity of apoE-LP for LPS was examined using the interaction between fluorescence-labeled LPS and serum lipoprotein fractions. LPS-induced inflammation significantly upregulated the mRNA expression of IL-6 and LRP1. This upregulation was markedly suppressed by pre-incubation of LPS with apoE-EP or its constituents (apoE or EP). The suppressive effect of apoE-EP on IL-6 upregulation was attenuated in the presence of lactoferrin, an inhibitor of LRP1. The prepared apoE-EP and serum triglyceride-rich lipoproteins showed significant affinity for LPS. However, these affinities appeared to be lower than expected based on the extent to which IL-6 upregulation was suppressed by pre-incubation of LPS with apoE-EP. Overall, these results indicate that LPS-induced inflammation may be regulated by 1) the LPS-neutralizing effect of apoE-LP, 2) anti-inflammatory effect of apoE, and 3) LRP1-mediated metabolic pathways., (© 2024 Walter de Gruyter GmbH, Berlin/Boston.)

The free hormone hypothesis has triggered controversies regarding the measurement of free vitamin D metabolites, such as free 25-hydroxyvitamin D (25(OH)D), as a suitable indicator for total vitamin D for clinical use. This issue can be addressed by developing a precise and accurate method for free 25(OH)D measurement. In the present study, a novel assay method for free 25(OH)D3 based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Sample preparation first involved ultrafiltration to remove vitamin D-binding protein-bound and albumin-bound 25(OH)D, followed by extraction with a column, derivatization, evaporation, dissolution, and injection into the LC-MS/MS system. The coefficient of variation of repeatability and reproducibility obtained were 3.8-4.5% and 4.8-5.9%, respectively. Satisfactory linearity (r=0.999) was obtained up to 80 pg/ml. The lower quantification limit was 0.97 pg/ml and the S/N ratio on the peak of 1.0 pg/ml sample was 24.8 (which is more than the acceptable value of 10). The recovery rate was between 84.5 and 92.4% with a negligible matrix effect (94.5-104.9%). Levels of free 25(OH)D3, but not total 25(OH)D3, in the serum of the patients with chronic kidney disease (CKD) and hepatic cirrhosis (HC) were substantially lower than those in healthy subjects. The correlation coefficient between total and free 25(OH)D3 was 0.738 in all samples, while the linear regression equations were different between the patients with CKD and HC. In conclusion, LC-MS/MS assay for free 25(OH)D3 might be useful to evaluate high-throughput methods, including ELISA., (© 2022 The Author(s).)

Background: Allergic transfusion reactions (ATRs) manifest frequently as transfusion reactions, and their onset may be related to a patient's allergic predisposition. Moreover, although pediatric patients with hematological/oncological disease are more susceptible to ATRs, the relationship between allergic predisposition and ATRs remains to be fully clarified., Study Design and Methods: Patients who were diagnosed with pediatric hematological/oncological disease and received transfusion at the study institutions were included. We determined patient background information related to their allergy history, measured the levels of allergen-specific immunoglobulin E (IgE) using sera obtained on diagnosis, and analyzed their associations with ATR onset., Results: Of the 363 patients analyzed, 144 developed ATRs. Multivariate analysis identified cases with high basophils in the peripheral blood, and Dermatophagoides pteronyssinus- and egg white-specific IgEs were involved in the development of ATR in all age groups. Meanwhile, a history of food allergies, and positivity for Japanese cypress- and D. pteronyssinus-specific IgEs were risk factors for developing ATRs in the <5 years age group. Moreover, patients aged 5-<10 years with a history of asthma, allergic rhinitis, pollinosis, or atopic dermatitis, and those aged ≥10 years with positivity for dog dander-specific IgE were at risk for developing ATRs., Conclusion: The allergic constitution of patients plays a role in ATR onset even in pediatric hematological/oncological diseases. Therefore, advance confirmation of a patient's allergic constitution may partly predict the onset of ATRs. However, since multiple allergic predispositions within complex mechanisms may be involved in the onset of ATRs, further verification is required., (© 2022 AABB.)

Elevated serum IgG4 is a useful marker of IgG4-related disease (IgG4-RD) activity. However, there is no uniformity in the cut-off values of IgG4 among the various reagents. The aim of this study was to compare the measured and cut-off values of IgG4 assessed using three different reagents. This study enrolled 466 IgG4-RD and non-IgG4-RD patients who required measurement of serum IgG4 levels to diagnose or treat IgG4-RD. Serum IgG4 was measured using three reagents: N-assay LA IgG4 Nittobo (Nittobo), BS-NIA IgG4 (TBS), and N Latex IgG4 (Siemens). The values obtained using the three reagents were compared, and cut-off values were calculated for each. Although there was good correlation among the results with the three reagents, the measured and cut-off values were all different. The Nittobo values were 1.4 times the TBS values and the TBS values were almost half those of the Siemens values. ROC curve analysis showed cut-off values for the Nittobo, TBS, and Siemens reagents of 1.42, 1.31, and 2.38 g/L, respectively. The measured and cut-off values of serum IgG4 vary depending on the reagents used for the assay, although there is good correlation among the values measured by the three reagents.

Objective: This study aimed to examine whether oxidized low-density lipoprotein (oxLDL) facilitates platelet aggregation, which is one cause for development of cardiovascular disease., Methods: The susceptibility of platelets to aggregation was monitored by light transmittance aggregometry and a laser light scattering method using low-density lipoprotein (LDL) and oxLDL as agonists. β-thromboglobulin (β-TG) levels released from platelets were also measured after incubation with or without oxLDL., Results: Platelet aggregation was suppressed by oxLDL as estimated by maximum light transmission. Additionally, adenosine diphosphate-induced further aggregation was slightly reduced by the presence of oxLDL. Aggregation levels of a low number of platelets, which was determined by the laser light scattering method, were lower upon addition of oxLDL compared with unoxidized LDL. After a short time of incubation, oxLDL increased secreted β-TG levels in platelet-rich plasma. However, further incubation with oxLDL caused relatively lower secreted β-TG levels compared with incubation with unoxidized LDL. This fluctuation was not due to β-TG degradation by oxLDL., Conclusions: Levels of oxLDL in vitro weakly activate platelets at an early stage, but then inhibit platelet function, such as aggregation and β-TG secretion.

Background: IgG4-related disease (IgG4-RD) is a new syndrome characterized by elevated serum IgG4 concentration and tissue infiltration of IgG4-positive plasma cells. Here, we evaluated the analytical performance of a new IgG4 assay reagent featuring a wide dynamic range, highly specific monoclonal antibody, and the reversed passive latex agglutination assay and determined the IgG4 reference interval (RI) for the Japanese population., Methods: Performance evaluations were conducted on precision, linearity, sensitivity, interference, and method comparison with The Binding Site (TBS) and Siemens reagents. The RI was derived by the parametric method from 619 apparently healthy Japanese 18 to 65 years of age., Results: Between-day precisions ranged from 1.99 to 5.52 CV%. Linearity was confirmed up to 5.0 g/l. The limit of quantitation was 0.085 g/l. Interfering substances did not significantly influence values. Method comparison among the 3 reagents yielded correlation coefficients between 0.973 and 0.988. Values for the new reagent matched those of TBS reagent except at a higher concentration range, where reactivity dissociated. The RI was 0.11-1.21 g/l without distinction by sex and age., Conclusion: The novel IgG4 assay reagent demonstrated satisfactory analytical performance for clinical use. Because of matched value with TBS reagent at low concentrations, it is possible to use the IgG4-RD cut-off value determined by TBS reagent., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Epithelial-mesenchymal transition (EMT) is associated with pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF). In this study, we investigated EMT of human pulmonary epithelial-derived cells (A549). A549 cells was either cultured by itself or co-cultured with THP-1 macrophages under normoxic (21% O2) and hypoxic (2% O2) conditions. We evaluated the presence of EMT by determining the expression of EMT markers, E-cadherin, vimentin, and fibronectin. To determine the role of TGF-β1 and IL-1β in EMT of the A549 cells, we analyzed the effects of blocking their activity with TGF-β1 inhibitor or IL-1β neutralizing antibody respectively. The A549 cells presented EMT when they were co-cultured with THP-1 macrophages. The EMT of the A549 cells co-cultured with THP-1 macrophages was exacerbated under hypoxia. In addition, the EMT were prevented by the addition of TGF-β1 type I receptor kinase inhibitor. The hypoxic condition increased the mRNA levels of TGF-β1 in A549 cells and THP-1 macrophages and that of IL-1β in THP-1 macrophages when each cells were co-cultured. Anti-IL-1β neutralizing antibody attenuated TGF-β1 secretion in co-culture media under hypoxic conditions. Thus, the IL-1β from THP-1 macrophages up-regulated the TGF-β1 from A549 cells and THP-1 macrophages, and then the TGF-β1 from both cells induced and promoted the EMT of A549 cells when they were co-cultured under hypoxia. Together, these results demonstrate that the interaction between type II pneumocytes and macrophages under hypoxia is necessary for the development of pulmonary fibrosis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A high homocysteine (Hcy) level is a risk factor for atherosclerosis. Hcy can be added to proteins through a process known as N-homocysteinylation. This is thought to be a potential cause of atherosclerosis induction. We previously reported that N-homocysteinylated apolipoprotein A-I (N-Hcy-apoA-I) was identified in normal human plasma. In this study, the effect of N-homocysteinylation on the functions of apoA-I was examined. A kinetic study using dimyristoyl phosphatidylcholine (DMPC) liposomes indicated that N-Hcy-apoA-I showed increased lipid-binding activity compared to wild-type apoA-I. Two reconstituted high-density lipoprotein (rHDL) particles of different sizes (approximately 8.2 nm and 7.6 nm in diameter) were produced by mixing apoA-I and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC). However, an increased ratio of large to small particles was found in rHDL prepared with N-Hcy-apoA-I. The normal apoA-I antioxidant ability, estimated by the suppression of conjugated diene formation in low-density lipoprotein (LDL) induced by copper sulfate oxidation, was considerably impaired when using N-Hcy-apoA-I. Although N-Hcy-apoA-I functioned as an oxidant, no significant difference was observed in the cholesterol efflux capacity from THP-1 macrophages between wild-type apoA-I and N-Hcy-apoA-I. These results suggest that N-Hcy-apoA-I might be proatherogenic due to its oxidative behavior but not an attenuation of cholesterol efflux capacity.

Background: The efficacy of white blood cell (WBC) count and left shift in predicting bacterial infections has been controversial. The aim of this study was to prove that WBC count and left shift reflect a course of bacterial infection., Materials: Six patients in whom the onset of bacterial infection had been determined and successful treatment had been done were selected. Manual 100-cell differential counts were repeated at least every 24 hr., Results: WBC count and left shift divided a course of bacterial infection into five phases. In the first phase of bacterial infection (0-10 hr after the onset), WBC count decreased to fewer than reference range without left shift. In the second phase (about 10-20 hr), low WBC count continued and left shift appeared. In the third phase (one to some days), WBC count increased above reference range with left shift. In the fourth phase (some to several days), high WBC count continued without left shift. In the fifth phase, WBC count went down into reference range without left shift., Conclusions: A combination of WBC count and left shift real-timely reflected a course of bacterial infection from the onset to healing. And we could judge which bacterial infection is adequately treated or not only by the above two routine laboratory tests., (© 2013 Wiley Periodicals, Inc.)

An Ambler class A β-lactamase gene, bla(CIA-1), was cloned from the reference strain Chryseobacterium indologenes ATCC 29897 and expressed in Escherichia coli BL21. The bla(CIA-1) gene encodes a novel extended-spectrum β-lactamase (ESBL) that shared 68% and 60% identities with the CGA-1 and CME-1 β-lactamases, respectively. bla(CIA-1)-like genes were detected from clinical isolates. In addition to the metallo-β-lactamase IND of Ambler class B, C. indologenes has a class A ESBL gene, bla(CIA-1), located on the chromosome.

In atherosclerotic artery walls, mast cells, an inflammatory cell, are activated and secrete some proteases including chymase. Chymase, a chymotrypsin-like protease, cleaves the C-terminus of apolipoprotein A-I (apoA-I) at Phe225. This cleavage reduces the ability of apoA-I to promote the efflux of cellular cholesterol. The aim of this study is to detect C-terminally truncated apoA-I in normal human serum. For this purpose, we generated a monoclonal antibody that specifically recognizes C-terminally truncated apoA-I by immunizing mice with a peptide that corresponds to human apoA-I amino acid residues 216-225. The monoclonal antibody, termed 16-4 mAb, selectively reacted with recombinant C-terminally truncated apoA-I, but not recombinant full-length apoA-I. A two-dimensional electrophoresis analysis also indicated that only two out of six spots that contained apoA-I fragments and had a molecular mass of 26 kDa after chymase digestion reacted with the 16-4 mAb. We detected an extremely small amount of C-terminally truncated apoA-I in normal human serum by concentrating the serum through affinity chromatography using a 16-4 mAb-conjugated resin, and then performing Western blot analysis. The 16-4 mAb could be useful to examine whether C-terminally truncated apoA-I is associated with the progression of atherosclerosis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objective: In our previous study to evaluate the effects of soluble silicon (Si) on bone metabolism, Si and coral sand (CS) as a natural Si-containing material suppressed peroxisome proliferator-activated receptor γ (PPARγ), which regulates both glucose and bone metabolism and increases adipogenesis at the expense of osteogenesis, leading to bone loss. In this study, we investigated the anti-diabetic effects of bone-seeking elements, Si and stable strontium (Sr), and CS as a natural material containing these elements using obese diabetic KKAy mice., Methods: Weanling male mice were fed diets containing 1% Ca supplemented with CaCO(3) as the control and CS, and diets supplemented with 50 ppm Si or 750 ppm Sr to control diet for 56 d. The mRNA expressions related to energy expenditure in the pancreas and kidney were quantified by real-time polymerase chain reaction., Results: At the end of feeding, plasma glucose, insulin, leptin, and adiponectin levels decreased significantly in three test groups, while pancreatic PPARγ and adiponectin mRNA expression levels increased significantly toward the normal level, improving the glucose sensitivity of β-cells and inducing a significant decrease in insulin expression. The renal PPARγ, PPARα, and adiponectin expression levels, histologic indices of diabetic glomerulopathy, and plasma indices of renal function were also improved significantly in the test groups., Conclusion: Taken together, anti-osteoporotic trace minerals, Si and Sr, and CS containing them showed novel anti-diabetic effects of lowering blood glucose level, improving the tolerance to insulin, leptin, and adiponectin, and reducing the risk of glomerulopathy through modulation of related gene expression in the pancreas and kidney., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Silicon is rich in the normal human aorta but decreases with age and the development of atherosclerosis. We hypothesized that soluble silica (Si) and coral sand (CS), as a natural Si-containing material, would suppress high blood pressure (BP) in spontaneously hypertensive rats (SHRs), and clarify the observed antihypertensive mechanism by cell cultures by quantifying messenger RNA expressions in the aorta. In SHR fed diets containing 1% Ca supplemented with CaCO(3) as the control (CT) and CS in a Ca-deficient diet and containing 50 mg/kg Si in the CT diet for 8 weeks, systolic BP was significantly (P < .05) lowered by 18 mm Hg for the Si group and 16 mm Hg for the CS group compared with the control CT group with 207 mm Hg. Magnesium (Mg) uptake by rat aortic smooth muscle cells significantly increased (177%, P < .005) in cells cultured with a physiologic Mg level plus Si compared with those with no Si addition. Furthermore, the increase of systolic BP by the CT diet was significantly suppressed by 17 mm Hg (P < .001) in SHR fed the diet containing Mg along with Si, but not by the Mg-deficient diet with or without Si. Soluble silica and CS treatments suppressed the aortic gene expressions of angiotensinogen and growth factors related to vascular remodeling, whereas, Si stimulated the expression of peroxisome proliferator-activated receptor-γ, the activation of which has anti-inflammatory and antihypertensive effects on vascular cells. These findings suggest that Si reduces hypertension in SHR by stimulating the intracellular Mg uptake and related gene expression in the aorta., (Copyright © 2011 Elsevier Inc. All rights reserved.)