Takanari Nakano

Free immunoglobulin light chain: Its biology and implications in diseases

Immunoglobulin light chain (IgLC) is a component of antibodies, but its free form is observed in the circulation, which originates from 10 to 40% excess synthesis over heavy chain in B cells. Complete antibodies function as a defined tetramer structure unit, H2L2; thus, separation of heavy and light chains results in considerable or complete loss of antigen-binding ability. Free IgLC has been considered as an inconsequential spillover during antibody assembly because, unlike heavy chain, neither effector functions such as complement activation nor specific-receptor binding has been identified in IgLCs. Free IgLC in sera and cerebrospinal fluids increases in inflammatory diseases such as autoimmune diseases and infections, presumably as a result of B-cell activation. This may be just a concomitant event during elevated disease activity, but recent findings suggest that free IgLC is involved in a wide range of immunological phenomena as a signaling effector or an anti-inflammatory molecule. These effects are likely to be intrinsic to IgLC. In this review, we attempt to give a comprehensive view about the biological roles of free IgLC together with the gene expression, secretion, antigen-binding ability, and its metabolic characteristics.

ELISAs for free light chains of human immunoglobulins using monoclonal antibodies: comparison of their specificity with available polyclonal antibodies

For the measurement of human immunoglobulin free light chains (LCs) in clinical samples, highly specific assays for free LCs are required to discriminate them from LC portions of intact immunoglobulins (bound LCs). To develop specific enzyme-linked immunosorbent assays (ELISAs) for free LCs, two anti-free LC kappa and lambda monoclonal antibodies (MAbs) were raised by a mouse/mouse hybridoma technique. We compared the specificities of these two MAbs with those of six commercially available anti-free LC antisera, which are widely used in free LC immunoassays. Comparative titrations against free LCs and intact IgGs showed the MAbs had less cross-reactivity to intact IgGs, while the four of six antisera had high reactivity to intact IgGs. Using these MAbs, we developed LC kappa and LC lambda ELISAs with ranges from 7.8 to 500 micro g/l of free LCs and less cross-reactivity to intact IgGs (less than 0.12%). On the other hand, ELISAs with anti-free LC antisera showed low specificity and/or sensitivity. Thus, the use of these MAbs may provide reliable methods for specific measurements of free LCs in clinical samples.

Immunochemical quantification of free immunoglobulin light chains from an analytical perspective

Abstract Immunoglobulin light chains are components of antibodies, but some exist in a free form in serum and urine as a result of their excess production over heavy chains. Free light chain (FLC) levels are of the order of milligram per liter in normal serum and urine, but marked increases have been observed in various disease conditions. It has now been established that the measurement of FLC levels contributes to diagnosis and clinical management in monoclonal gammopathies. Recent developments in FLC assays have been adapted to several automated platforms and they have now become available in laboratories. There have, however, been some concerns regarding the analytical aspects. The current assay specificity appears to be insufficient to prevent the influence of intact light chains of several orders of magnitude greater than FLCs in serum. Moreover, the heterogeneous nature of light chains makes accurate quantification unreliable. FLC assays have never been standardized because of the lack of an international reference calibrator. In this review, we summarize the reports on FLC measurements and examine the specificity of anti-FLC antibodies and the reliability of FLC assays. We also discuss difficulties in the standardization and setting of normal reference intervals for FLC assays.

NAD(P)H oxidase p22phox Gene C242T polymorphism and lipoprotein oxidation.

BACKGROUND Vascular NAD(P)H oxidase is a key enzyme of superoxide anion production in human vessel walls. The C242T mutation in the CYBA gene coding for p22phox, a component of the enzyme, may change the redox state. The aim of this study was to evaluate the influence of the polymorphism on serum concentrations of oxidative stress markers. METHODS Serum samples were collected from 134 Type 2 diabetic patients and analyzed for oxidized high-density lipoprotein (HDL) by in-house ELISA, and oxidized low-density lipoprotein (LDL) and thiobarbituric acid reactive substance (TBARS) by commercial kits. For genotyping, the Taqman PCR method was adapted to detect the polymorphism. RESULTS Circulating concentrations of oxidized HDL were about 1.5-fold lower in those of the CT/TT genotypes than the CC genotype [3.3 +/- 0.3 and 5.0 +/- 0.3 U/dl (mean +/- S.E.M.), respectively; multiple regression analysis, p=0.006], whereas concentrations of oxidized LDL were slightly greater (1.1-fold, p=0.01) in those with the CT/TT genotypes. However, no significant difference was observed in TBARS between the genotypes. CONCLUSIONS The effect was inconsistent among the markers, but these results suggest that the CYBA C242T polymorphism is involved in NAD(P)H oxidase activity and affects oxidation of lipoproteins by altering the redox state in the vasculature.

Subtle Immunodeficiency in Severe Asthma: IgA and IgG2 Correlate with Lung Function and Symptoms

Background: Atopy, increased serum IgE and eosinophilic airway inflammation are common in asthma and may indicate aberrant immune responses, but the cause(s) are unknown. It was hypothesized that differences in serum immunoglobulins, immunoglobulin free light chains (FLC) and secretory IgA (sIgA) would exist between subjects with asthma of varying severity and normal subjects, and the levels would correlate with lung function, symptoms and airway inflammation. Methods: Serum IgG, IgA, IgE and IgM, IgG subclasses and FLC, and bronchoalveolar lavage sIgA were evaluated from 15 normal subjects, 9 mild and 22 severe asthmatics with similar atopic status. Asthma symptoms were obtained by questionnaire, and airway inflammation was assessed by immunostaining for five inflammatory cell types. Results: Immunoglobulin levels in all groups were generally within the normal range. However, IgA and IgG were lower in severe asthmatics than normal subjects (overall p = 0.006 and 0.02, respectively). IgA, but not IgG, correlated with lung function and asthma symptoms (r-values >0.58; p-values <0.009). Although similar among the groups, higher sIgA and IgG2 also positively correlated with lung function and negatively with asthma symptoms (r-values >0.63; p-values <0.009). IgA and IgG/IgG1 positively correlated with tissue mast cells. Conclusions: Subtle alterations in IgA- and IgG2-mediated responses in asthma may be disease-related. As their levels are generally normal, it is possible that the quality/repertoire of immune protection provided by these isotypes, perhaps against carbohydrate epitopes, may be altered in asthma.

Immunochemical detection of circulating oxidized high-density lipoprotein with antioxidized apolipoprotein A-I monoclonal antibody

The oxidative susceptibility of high-density lipoprotein (HDL) may play a role in its antiatherogenic effects. In an effort to determine circulating levels of oxidized HDL in the bloodstream, we produced a monoclonal antibody (mAb), 3C11, specific to oxidized apolipoprotein A-I and developed an enzyme-linked immunosorbent assay (ELISA) for oxidized HDL that incorporates the mAb. The examination of oxidized forms of several lipoproteins showed that the ELISA had a high specificity for oxidized HDL and did not react appreciably with native, acetylated, or malondialdehyde-modified HDL or with the other lipoproteins and their oxidized forms. Using the ELISA, we detected oxidized HDL in human serum samples and determined serum levels of oxidized HDL in 40 healthy volunteers. The mean serum concentration of oxidized HDL was 4.65 +/- 2.65 U/dL (mean +/- SD; range 1.47-12.81 U/dL). Further analysis showed no correlation between serum concentrations of oxidized HDL and those of six serum markers: HDL, apolipoprotein A-I, oxidized low-density lipoprotein, C-reactive protein, thiobarbituric acid-reactive substances, and serum iron. The ELISA provides a method for measuring oxidized HDL in the circulation, and this determination may elucidate the clinical significance of HDL oxidation in human beings.

A possible role of lysophospholipids produced by calcium-independent phospholipase A2 in membrane-raft budding and fission

Phospholipase A(2) (PLA(2)) not only plays a role in the membrane vesiculation system but also mediates membrane-raft budding and fission in artificial giant liposomes. This study aimed to demonstrate the same effects in living cells. Differentiated Caco-2 cells were cultured on filter membranes. MDCK cells were challenged with Influenza virus. The MDCK cultures were harvested for virus titration with a plaque assay. Alkaline phosphatase (ALP), a membrane-raft associated glycosylphosphatidylinositol (GPI)-anchored protein, was 70% released by adding 0.2 mmol/l lysophosphatidylcholine, which was abolished by treatment with a membrane-raft disrupter, methyl-beta-cyclodextrin. Activation of calcium-independent PLA(2) (iPLA(2)) by brefeldin A increased the apical release of ALP by approximately 1.5-fold (p<0.01), which was blocked by PLA(2) inhibitor bromoenol lactone (BEL). BEL also reduced Influenza virus production into the media (<10%) in the MDCK culture. These results suggest that cells utilize inverted corn-shaped lysophospholipids generated by PLA(2) to modulate plasma membrane structure and assist the budding of raft-associated plasma membrane particles, which virus utilizes for its budding. Brush borders are enriched with membrane-rafts and undergo rapid turnover; thus, PLA(2) may be involved in the regulatory mechanism in membrane dynamism. Further, iPLA(2) may provide a therapeutic target for viral infections.

Endogenous Luminal Surface Adenosine Signaling Regulates Duodenal Bicarbonate Secretion in Rats

Luminal ATP increases duodenal bicarbonate secretion (DBS) via brush border P2Y receptors. Because ATP is sequentially dephosphorylated to adenosine (ADO) and the brush border highly expresses adenosine deaminase (ADA), we hypothesized that luminal [ADO] regulators and sensors, including P1 receptors, ADA, and nucleoside transporters (NTs) regulate DBS. We measured DBS with pH and CO2 electrodes, perfusing ADO ± adenosine receptor agonists or antagonists or the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTRinh-172 on DBS. Furthermore, we examined the effect of inhibitors of ADA or NT on DBS. Perfusion of AMP or ADO (0.1 mM) uniformly increased DBS, whereas inosine had no effect. The A1/2 receptor agonist 5′-(N-ethylcarboxamido)-adenosine (0.1 mM) increased DBS, whereas ADO-augmented DBS was inhibited by the potent A2B receptor antagonist N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]-acetamide (MRS1754) (10 μM). Other selective adenosine receptor agonists or antagonists had no effect. The A2B receptor was immunolocalized to the brush border membrane of duodenal villi, whereas the A2A receptor was immunolocalized primarily to the vascular endothelium. Furthermore, ADO-induced DBS was enhanced by 2′-deoxycoformycin (1 μM) and formycin B (0.1 mM), but not by S-(4-nitrobenzyl)-6-thioinosine (0.1 mM), and it was abolished by CFTRinh-172 pretreatment (1 mg/kg i.p). Moreover, ATP (0.1 mM)-induced DBS was partially reduced by (1R,2S,4S,5S)-4–2-iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphonooxy)bicyclo[3.1.0]hexane-1-methanol dihydrogen phosphate ester tetraammonium salt (MRS2500) or 8-[4-[4-(4-chlorophenzyl)piperazide-1-sulfonyl)phenyl]]-1-propylxanthine (PSB603) and abolished by both, suggesting that ATP is sequentially degraded to ADO. Luminal ADO stimulates DBS via A2B receptors and CFTR. ATP release, ecto-phosphohydrolases, ADA, and concentrative NT may coordinately regulate luminal surface ADO concentration to modulate ADO-P1 receptor signaling in rat duodenum.

Ratio of urinary free immunoglobulin light chain κ to λ in the diagnosis of bence jones proteinuria

Abstract The aim of this study was to evaluate the diagnostic efficacy of the ratio of urinary free light chain (FLC) kappa to lambda (κ/λ ratio) for the detection of Bence Jones protein (BJP). Urine specimens were collected from 243 patients suspected of having BJP. Immunofixation identified 59 BJP-positive specimens among them. The κ/λ ratios of all specimens were determined by FLC immunoassays and then the cutoffs for the κ/λ ratio were defined as 5.5 for BJP κ and 0.1 for BJP λ by ROC curve analyses. Using the cutoffs, we detected abnormal κ/λ ratios in 51 (86%) of the 59 BJP-positives and 11 (6%) of the 184 BJP-negatives identified by the results of immunofixation. High-resolution urinary protein electrophoresis (UPE), a sensitive method for BJP screening, showed almost equal sensitivity to the κ/λ ratio, detecting monoclonal band(s) in 52 (88%) of the 59 BJP-positives. However, in UPE analysis these positive specimens should be followed by redundant immunofixation analysis to determine the isotypes. We further evaluated the combination method of FLC assays with UPE that correctly diagnosed 82% of the specimens as positive or negative for BJP, with only two false-negative results. These results suggest that quantitative FLC immunoassays provide an alternative or complementary method for the detection of BJP.

Statins Activate Human PPARα Promoter and Increase PPARα mRNA Expression and Activation in HepG2 Cells

Statins increase peroxisome proliferator-activated receptor α (PPARα) mRNA expression, but the mechanism of this increased PPARα production remains elusive. To examine the regulation of PPARα production, we examined the effect of 7 statins (atorvastatin, cerivastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin) on human PPARα promoter activity, mRNA expression, nuclear protein levels, and transcriptional activity. The main results are as follows. (1) Majority of statins enhanced PPARα promoter activity in a dose-dependent manner in HepG2 cells transfected with the human PPARα promoter. This enhancement may be mediated by statin-induced HNF-4α. (2) PPARα mRNA expression was increased by statin treatment. (3) The PPARα levels in nuclear fractions were increased by statin treatment. (4) Simvastatin, pravastatin, and cerivastatin markedly enhanced transcriptional activity in 293T cells cotransfected with acyl-coenzyme A oxidase promoter and PPARα/RXRα expression vectors. In summary, these data demonstrate that PPARα production and activation are upregulated through the PPARα promoter activity by statin treatment.