Comparison of A68 levels in Alzheimer diseased and non-Alzheimer's diseased brain by two ALZ50 based methods

A total of 61 human brain specimens were analyzed with both ELISA and Western Blot using the ALZ50 monoclonal antibody. The brain specimens included: Alzheimer's Disease (AD, n=31), AD/Down's (n=2), Normal (n=14), Parkinson's Disease (n=7), Huntington's chorea (n=2), Wernicke-Korsakov's Encephalopathy (n=3), and Motor Neuron Disease (n=2). The non-AD cases (n=28) had no detectable A68 by ELISA, and showed no A68 bands by Western blot. The AD cases (n=33), all were positive for A68 by the ELISA, but only 31 of 33 had visible A68 band by Western blot. Additionally, a method for solubilization of A68 is reported.     

Polyamine levels in normal human serum. Comparison of analytical methods

A comparison of levels of the polyamines, putrescine, spermidine and spermine, in individual and pooled normal human serum, as determined by three independent methods, high pressure cation-exchange chromatography (HPCC), radioimmunoassay (RIA) and gas chromatography-mass spectrometry (GC-MS), has been made. Generally good correlations were found for values derived by the three different methods.     

Isolation of a Candida albicans mutant with reduced content of cell wall mannan and deficient mannan phosphorylation

A rough-colony mutant of Candida albicans was isolated after ultraviolet mutagenesis. The mutant contained approximately half the normal amount of the cell wall mannan, the acetolysis pattern of which was indistinguishable from that of the wild-type counterpart. However, the extent of phosphorylation in the mutant mannan was about 12% of the value for wild type.     

IgG autoantibody activity in normal mouse serum is controlled by IgM

In the serum of normal BALB/c mice, IgG antibody reactivity to mouse actin and tubulin, DNA, and TNP groups was very low compared to that of the IgM. This activity was considerably increased when IgG was separated, by affinity chromatography on protein A-Sepharose, whereas no difference in the IgM activity was observed. Addition of IgM to IgG isolated from the same serum resulted in the inhibition of IgG binding to these Ag. Isolation of IgG antibodies on actin, TNP, and tubulin immunoadsorbents has indicated that at least part of the IgG antibodies is polyreactive. In order to understand this inhibition better, experiments with F(ab')2 fragments of IgG were performed. IgM inhibited the binding of F(ab')2 to the antigens in a dose-dependent manner and reacted with immobilized F(ab')2. IgM isolated on F(ab')2 immunoadsorbent, as compared to the initial IgM preparation, were less active toward the Ag but more inhibitory for IgG binding to the Ag. In some pathologic situations, IgM failed to inhibit some IgG antibody activities. The anti-DNA IgG activity from (NZB x NZW)F1 mice was not affected by autologous IgM. Similarly the anti-tubulin IgG from mice infected with Trypanosoma cruzi were less inhibited by IgM from autologous serum than antitubulin IgG from normal mice. These results are compatible with the existence in normal mice of an idiotypic-like network, regulating via an IgM population in the serum, the binding of IgG autoantibodies to self Ag. Modifications of this idiotype-anti-idiotype system might lead to the expression and/or expansion of autoreactive IgG-producing clones.     

Upper limits of normal for serum alanine aminotransferase levels in chinese han population

Background and Objectives

Serum alanine aminotransferase (ALT) activity is the most common tool for the assessment of liver diseases. However, it is not clear whether the current normal ALT range really discriminate patients with or without liver diseases. The present study was to establish a new normal range of ALT and examine its ability to identify patients with hepatitis B or nonalcoholic fatty liver disease (NAFLD) in Chinese Han population.

Methods

53037 adults were included in this study from January 1st 2008 to August 31st 2010. The 95th percentile of ALT in population with relative low risk factors for liver diseases was set as the new upper limits of normal ALT in gender-specific manner.

Results

The 95^th percentile levels at low risk factors for liver diseases were achieved at 35 U/L for men and 23 U/L for women. The concordance statistics for detection were 0.873 (95%CI: 0.865–0.881) for HBV and 0.932 (95%CI: 0.927–0.937) for NAFLD in men while 0.857 (95%CI: 0.850–0.864) for HBV and 0.909 (95%CI: 0.903–0.915) for NAFLD in women. The median sensitivity of the current used ALT upper limit (40 U/L) was 6.6% for HBV and 29.7% for NAFLD and median specificity was 98.7% for men and 99.4% for women. Using our new-derived thresholds, the sensitivities ranged from 35.3% to 61.1% and the specificities were 94.8% for men and 94.6% for women.

Conclusions

Our results suggest that upper limits of ALT 35 U/L for men and 23 U/L for women in Chinese Han population. Re-consideration of normal limits of ALT should be recommended.

Trial Registration

ChiCTR.org ChiCTR-OCS-11001173     

Detection of Candida albicans mannan by immunodiffusion,counterimmunoelectrophoresis, and enzyme-linked immunoassay

Anti-mannan was produced in rabbits after peptidoglucomannan in adjuvant was injected. The antiserum was used to detect mannan by immunodiffusion and counterimmunoelectrophoresis (CIE) in gel and by sandwich enzyme-linked immunosorbent assay (ELISA). The antiserum detected lower concentrations of mannan of serotype A than of serotype B. Except in CIE, the reactions were more pronounced at 4°C than at higher temperatures. CIE detected 0.8 g/ml mannan A or 12.5 /ml mannan B. Sandwich ELISA detected 3 ng/ml mannan A or 10⁵ ng/ml mannan B. Mannan was not detected in the serum of patients or rabbits with candidiasis.Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health, Education and Welfare.     

Quantitative evaluation of serum IgG subclass levels by an immunoenzyme method

We developed a enzyme linked immunosorbent assay (ELISA) for measuring IgG subclasses concentration in serum. For this we used monoclonal antibodies. The specificity of these antibodies was evaluated with a panel of myeloma proteins belonging to the 4 IgG subclasses. The ELISA was sensitive (allowing the detection of subclasses at ng level) and accurate (inter-assay coefficient of variation of 14%). Using the WHO serum 67/97 as reference, we determined the concentration of IgG subclasses in a pool of sera. In addition concentrations were measured in 69 healthy adults to study the distribution of each IgG subclass. A good correlation (r = 0.78) was obtained between the sum of the subclasses measured by ELISA and total IgG measured by immunonephelometry.     

Thoracic aortic aneurysm patients with Chlamydophila pneumoniae infection showed a shift in trace element levels in serum and diseased aortic tissue

Few studies have been performed on trace elements in tissues and serum in cardiovascular disease and none in aortic aneurysm. In this study the concentrations of 10 trace elements were determined in serum and aneurysmatic aortic tissue from 23 patients undergoing thoracic surgery. Macroscopically, normal thoracic aortic tissue specimens from 10 forensic autopsies and serum from 23 healthy blood donors served as controls. DNA from the intracellular respiratory pathogen Chlamydophila pneumoniae (C. pneumoniae), which may be involved in the pathogenesis of atherosclerosis, was found in 26% (6/23) of the patients but in none of the controls. The serum copper/zinc ratio, a well-known marker of ongoing infection and/or inflammation, was higher (26%, p<0.001) in aneurysm patients. C. pneumoniae requires iron for its growth. In our aneurysm patients iron was higher in serum (by 54%, p<0.001) and aneurysmal tissue (by 60%, p<0.001). Although calcium was lower in patient sera (by 8%, p<0.001), it tended to be higher (by 20%, ns) in aneurysmatic tissue. In addition, mercury concentrations in serum and aneurysmatic tissue were positively correlated (r=0.51, p<0.05). Moreover, C. pneumoniae-positive aneurysmatic tissues had lower concentrations of manganese (46%, p<0.05) and zinc (26%, ns) but a higher concentration of mercury (50%, p<0.05) than C. pneumoniae-negative aneurysmatic tissues. In conclusion, aneurysm patients showed a shift in trace element levels in serum and in the diseased part of the aorta, the pattern being partly different in C. pneumoniae-positive compared with C. pneumoniae-negative patients. The results are compatible with active infection and/or inflammation, possibly initiated by C. pneumoniae.     

Differentiation of serum levels of trace elements in normal and malignant breast patients

The objective of this study was to determine which elements in serum best differentiated breast cancer in a case-control study. Concentrations of 13 elements in serum of 68 breast tumor patients (25 malignant and 43 benign) and 26 healthy controls were measured. Logistic regression with different variable-selection procedures was used to determine a possible configuration of elements. Sensitivity and specificity of the model were calculated to obtain the optimal cutoff point for discriminating malignant breast cancers vs other individuals (including benign breast disease and normal ones). A combination of Cd, Mn, and Fe was found to have a specificity and sensitivity of 100% using forward-type logistic regression, when the cutoff value of the combination score was 52.71. Using stepwise-type logistic regression, a combination of Cr and Mn had a sensitivity of 100% and a specificity of 97.1% when the combination score of 17.4 was chosen as the cutoff. Similar analysis could be implemented to compare the malignant and control groups. Specificity and sensitivity were 100% for Mn (forward and stepwise type) with a cutoff point of 6.40. For the backward regression, specificity was 84.6% and sensitivity was 100% for Zn, with a cutoff point of 869.1. In conclusion, there was a significant difference in concentrations of all 13 elements in serum between breast cancer patients and controls. A combination among Cd, Mn, Fe, Cr, and Zn might be important to determine a differentiating reference for breast cancers if a long-term followed-up study is to be conducted.     

Comparison of the proteins in pancreatic juice in normal and diseased humans. Determination of serum proteins and demonstration of a particular protein in chronic calcifying pancreatitis

For a better understanding of the molecular mechanism leading to intraductal precipitation of proteins in primary chronic calcifying pancreatitis in man, we studied the composition of normal and pathological human pancreatic juice by immunotechniques. We found an increased level of serum proteins in pathological juices: 12.47% of total proteins compared to 1.8% in normal ones; albumin is 8.16% of the total proteins, IgG 2.84%, IgA 0.83% and IgM 0.91%. Transferrin and alpha 2-macroglobulin are present, but were not estimated. The albumin/IgA and albumin/IgG ratios favour the hypothesis of a local synthesis of these immunoglobulins as was shown in normal juice. The cross adsorption of antisera against pancreatic juice showed the presence in the pathological juice of a normal molecule in much higher concentration. The role of these proteins in precipitation is discussed.     

Comparison of selenium levels in pre-eclamptic and normal pregnancies

Abnormal placentation is the likely cause of the slow fetal growth and the high levels of circulating lipid peroxides found in severe preeclampsia. These peroxides are probably responsible for the high thromboxane:prostacyclin ratio found in this disease and may participate in the endothelial cell damage which is its most notable feature. Selenium (Se), because of its role in glutathione peroxidase, is suggested to be an important component of the removal system for these damaging peroxides. Serum-Se concentrations have therefore been measured in 19 pairs of pre-eclamptic women and matched controls. Infant birth-weights were recorded. No significant difference was found in the concentrations of Se in pre-eclamptic and control groups. Serum Se was found to be low in both groups. Birthweights were significantly lower in the pre-eclamptic group. The interpretation of serum-Se measurements from the third trimester of a pre-eclamptic pregnancy is complicated by the reduced fetal growth and probable lower Se take-up by the fetus in such a pregnancy. The merits of alternative measurements, such as total intravascular Se, placental Se, or samples from an earlier stage of gestation, are discussed. The importance of factors other than Se to the activity of glutathione peroxidase, and of other antioxidants to pre-eclampsia, is stressed.     

Isoforms of troponin in normal and diseased myocardium

The regulatory protein troponin (Tn) located on actin filament consists of three subunits: TnT--binds troponin to tropomyosin, TnC--binds divalent calcium ions, and TnI--affects myosin-actin interactions. Tn subunits display several molecular and calcium binding variations. During ontogenetic development of cardiac and skeletal muscles the synthesis of multiple isoforms of Tn subunits was detected. Expression of Tn isoforms and the extent of phosphorylation of both TnT and TnI via protein kinase C or protein kinase A under different pathological situations (e.g. ischemia, congenital heart disease, heart failure) can affect the Ca2+-stimulated contraction function and the myofibrillar ATPase activity of the heart.     

Functions of autophagy in normal and diseased liver

Autophagy has emerged as a critical lysosomal pathway that maintains cell function and survival through the degradation of cellular components such as organelles and proteins. Investigations specifically employing the liver or hepatocytes as experimental models have contributed significantly to our current knowledge of autophagic regulation and function. The diverse cellular functions of autophagy, along with unique features of the liver and its principal cell type the hepatocyte, suggest that the liver is highly dependent on autophagy for both normal function and to prevent the development of disease states. However, instances have also been identified in which autophagy promotes pathological changes such as the development of hepatic fibrosis. Considerable evidence has accumulated that alterations in autophagy are an underlying mechanism of a number of common hepatic diseases including toxin-, drug- and ischemia/reperfusion-induced liver injury, fatty liver, viral hepatitis and hepatocellular carcinoma. This review summarizes recent advances in understanding the roles that autophagy plays in normal hepatic physiology and pathophysiology with the intent of furthering the development of autophagy-based therapies for human liver diseases.     

Association of serum levels of fibrosis-related biomarkers with disease activity in patients with IgG4-related disease

Background

The aim of this study was to identify fibrosis-related serological surrogate outcome measures in patients with immunoglobulin G4-related disease (IgG4-RD).

Methods

This was a clinical observational study of 72 patients with untreated IgG4-RD from four institutions in Japan. The serum concentrations of growth differentiation factor 15 (GDF-15), CCL2, hyaluronic acid (HA), amino-terminal propeptide of type III procollagen (PIIINP), and tissue inhibitor of metalloproteinases 1 (TIMP-1) were measured by enzyme-linked immunosorbent assays. The enhanced liver fibrosis (ELF) score was calculated from the TIMP-1, PIIINP, and HA values. We evaluated associations between the values of these biomarkers and laboratory data, the IgG4-RD responder index (IgG4-RD RI) score, and organ involvements.

Results

Compared with the 44 healthy controls, the patients with IgG4-RD showed significantly elevated serum concentrations of GDF-15, MCP-1, HA, PIIINP, and TIMP-1 and ELF scores. The patients’ serum concentrations of GDF-15, CCL2, HA, and TIMP-1 (but not PIIINP) were positively correlated with each other. Among them, serum GDF-15 most efficiently distinguished patients with IgG4-RD from healthy controls. Serum GDF-15 was not associated with the IgG4-RD RI score or the number of organ involvements but was independently associated with the presence of retroperitoneal fibrosis and with parotid gland involvement.

Conclusions

We observed increased serological surrogate outcome measures of fibrosis in IgG4-RD. GDF-15 may precisely reflect the fibrotic degree in patients with IgG4-RD.

     

Anti-bacteroides lipopolysaccharide IgG levels in healthy adults and sepsis patients

Abstract Members of the genus Bacteroides greatly outnumber enterobacteria in the human colon and therefore represent a vast potential pool of biologically active LPS. An enzyme-linked immunosorbent assay was developed to estimate the distribution of IgG levels to LPS from B. fragilis, B. vulgatus, B. thetaiotaomicron and to a mixture of rough LPS from three enterobacteria and Pseudomonas aeruginosa in sera from 641 adult blood donors. By inhibition ELISA some cross-reactivity was demonstrated between the different anti-bacteroides LPS IgG, but with very little between the anti-bacteroides LPS IgG and the anti-enterobacterial/ Pseudomonas LPS IgG. Serum IgG was measured daily over 5–9-day periods in 12 sepsis patients (6 survivors, 6 non-survivors) and in a healthy individual. In all patients IgG levels fluctuated to a greater extent than levels in a healthy subject. Variations all followed similar overall trends and indicated that exposure to bacteroides LPS had occurred. In 5 out of 6 survivors, IgG levels were rising at the end of the period, while 4 of the 6 non-survivors showed falls, with an exception showing increasing levels to B. fragilis LPS. In 5 out of 6 non-survivors, IgG levels against B. fragilis LPS were substantially higher than those against the other LPSs. In this small sample some trends in antibody kinetics have been recognised which suggest bacteroides LPS may be significant in sepsis, and indicate that this study should be extended.     

IL-33 priming regulates multiple steps of the neutrophil-mediated anti-Candida albicans response by modulating TLR and dectin-1 signals

IL-33 is known to play an important role in Th2 immunity. In this study, we investigated the effect of IL-33 pretreatment on anti-fungal response using an acute Candida albicans peritoneal infection model. IL-33 pretreatment induced a rapid fungal clearance and markedly reduced the C. albicans infection-associated mortality. The priming effect of IL-33 occurred during multiple steps of the neutrophil-mediated anti-fungal response. First, the anti-fungal effect occurred due to the rapid and massive recruitment of neutrophils to the site of infection as a result of the release of CXCR2 chemokines by peritoneal macrophages and by reversal of the TLR-induced reduction of CXCR2 expression in neutrophils during IL-33 priming. Second, conditioning of neutrophils by IL-33 activated the TLR and dectin-1 signaling pathways, leading to the upregulation of complement receptor 3 expression induced by C. albicans. Upregulated CR3 in turn increased the phagocytosis of opsonized C. albicans and resulted in the production of high levels of reactive oxygen species and the subsequent enhanced killing activity of neutrophils. Taken together, our results suggest that IL-33 can regulate the anti-fungal activity of neutrophils by collaborative modulation of the signaling pathways of different classes of innate immune receptors.