Serum IgE reactivity profiling in an asthma affected cohort.

BACKGROUND: Epidemiological evidence indicates that atopic asthma correlates with high serum IgE levels though the contribution of allergen specific IgE to the pathogenesis and the severity of the disease is still unclear. METHODS: We developed a microarray immunoassay containing 103 allergens to study the IgE reactivity profiles of 485 asthmatic and 342 non-asthmatic individuals belonging to families whose members have a documented history of asthma and atopy. We employed k-means clustering, to investigate whether a particular IgE reactivity profile correlated with asthma and other atopic conditions such as rhinitis, conjunctivitis and eczema. RESULTS: Both case-control and parent-to-siblings analyses demonstrated that while the presence of specific IgE against individual allergens correlated poorly with pathological conditions, particular reactivity profiles were significantly associated with asthma (p<10E-09). An artificial neural network (ANN)-based algorithm, calibrated with the profile reactivity data, correctly classified as asthmatic or non-asthmatic 78% of the individual examined. Multivariate statistical analysis demonstrated that the familiar relationships of the study population did not affect the observed correlations. CONCLUSIONS: These findings indicate that asthma is a higher-order phenomenon related to patterns of IgE reactivity rather than to single antibody reactions. This notion sheds new light on the pathogenesis of the disease and can be readily employed to distinguish asthmatic and non-asthmatic individuals on the basis of their serum reactivity profile.

An antigen microarray immunoassay for multiplex screening of mouse monoclonal antibodies.

The mouse monoclonal antibody (mAb) technology still represents a key source of reagents for research and clinical diagnosis, although it is relatively inefficient and expensive and therefore unsuitable for high-throughput production against a vast repertoire of antigens. In this article, we describe a protocol that combines the immunization of individual mice with complex mixtures of influenza virus strains and a microarray-based immunoassay procedure to perform a parallel screening against the viral antigens. The protocol involves testing the supernatants of somatic cell hybrids against a capture substratum containing an array of different antigens. For each fusion experiment, we carried out more than 25,000 antigen-antibody reactivity tests in less than a week, a throughput that is two orders of magnitude higher than that of traditional antibody detection assays such as enzyme-linked immunosorbent assays and immunofluorescence. Using a limited number of mice, we can develop a vast repertoire of mAbs directed against nuclear and surface proteins of several human and avian influenza virus strains.

Alteration of glyoxalase genes expression in response to testosterone in LNCaP and PC3 human prostate cancer cells.

Glyoxalase system, a ubiquitous detoxification pathway protecting against cellular damage caused by potent cytotoxic metabolites, is involved in the regulation of cellular growth. Aberrations in the expression of glyoxalase genes in several human cancers have been reported. Recently, we described a possible regulatory effect by estrogens on glyoxalase genes in human breast cancer cell lines. This result, along with those ones regarding changes in glyoxalases activity and expression in other human hormone-regulated cancers, such as prostate cancer, has prompted us to investigate whether also androgens, whose functional role in prostate cancer pathogenesis is well known, could modulate glyoxalases gene expression. Therefore, we treated LNCaP androgen-responsive and PC3 androgen-independent human prostate cancer cell lines with testosterone at the concentrations of 1 nM and 100 nM. After a two days treatment, glyoxalases mRNA levels as well as cell proliferation were evaluated by real-time RT-PCR analysis and [3H]thymidine incorporation, respectively. Results pointed out that testosterone affects the expression of glyoxalase system genes and cell proliferation in a different manner in the two cell lines. The possibility that modulation of glyoxalase genes expression by testosterone is due to glyoxalases-mediated intracellular response mechanisms to the androgen-induced oxidative stress or to the presence of androgen response elements (ARE) in glyoxalase promoters are discussed. Knowledge regarding the regulation of glyoxalases by testosterone may provide insights into the importance of these enzymes in human prostate carcinomas in vivo.

Overexpression of glyoxalase system enzymes in human kidney tumor.

PURPOSE: The purpose of this study was to investigate the messenger RNA expression and activity of glyoxalase I and glyoxalase II enzymes in a human renal carcinoma (clear cell adenocarcinoma) and in pair-matched normal tissue. PATIENTS AND METHODS: Tumor and nontumor pair-matched specimens from the same organ were collected during radical nephrectomy from a group of 12 patients of both sexes. The mean age of the patients was 52.3 years (range, 50-60 years), and none of them had previously undergone neoadjuvant therapy. Gene expression and activity were measured by ribonuclease protection assay and current spectrophotometric methods, respectively. Intracellular levels of methylglyoxal were detected by high performance liquid chromatography. RESULTS: A significant increase in the transcription levels of both glyoxalase I (about ninefold) and glyoxalase II (about threefold) was observed, compared with the pair-matched noncancerous tissues. Glyoxalase I activity was also higher in the pathological samples (about 2.5-fold) compared with the control samples and correlated with a significant decrease (about twofold) in methylglyoxal concentrations. At variance, glyoxalase II activity was significantly lower in pathological tissues than in the normal ones. DISCUSSION: Our findings suggest a possible role of the glyoxalase system enzymes in the chemoresistance displayed by the kidney tumor. In fact, such a refractory behavior involves a decrease in the methylglyoxal level, a potent apoptosis activator. In addition, glyoxalase II activity decrease in the adenocarcinoma tissue suggests a likely role of the intermediate S-D-lactoylglutathione by supplying energy in actively proliferating cells. Finally, we point out a possible use of glyoxalase I inhibitors as anticancer drugs.

A possible regulatory role of 17beta-estradiol and tamoxifen on glyoxalase I and glyoxalase II genes expression in MCF7 and BT20 human breast cancer cells.

The glutathione-dependent glyoxalases system, composed of glyoxalase I (GloI) and glyoxalase II (GloII) enzymes, is involved in the detoxification of methylglyoxal, a by-product of cell metabolism. Aberrations in the expression of glyoxalase genes in several human cancers have been reported. Sometimes, these aberrations seem to differ depending on the organs and on the sensitivity of the tumours to estrogens, as we previously detected in the hormone-responsive breast cancer compared to the hormone-independent bladder cancer. To investigate a possible regulatory role of estrogens, as well as antiestrogens, on glyoxalases system, estrogen receptor (ER)-positive MCF7 and ER-negative BT20 human breast cancer cells were cultured in the presence of 17beta-estradiol (E2) and tamoxifen (TAM) performing two independent experiments. After a 24 h or 4 days treatment, we evaluated GloI and GloII mRNA levels, by Ribonuclease Protection Assay (RPA), enzymatic activities spectrophotometrically and cell proliferation by [3H]thymidine incorporation. We found that both steroid molecules affected glyoxalases gene expression and proliferation in a different manner in the cell lines. The modifications in mRNA levels were accompanied by parallel changes in the enzymatic activities. The possibility that modulation of glyoxalase genes by E2 and TAM are due to the presence of estrogen response elements (ERE) or cross-talk mechanisms by proteins of the estrogen signal transduction pathways are discussed. Knowledge regarding the regulation of glyoxalases by E2 and TAM may provide insights into the importance of this enzymes in human breast carcinomas in vivo.

Increased acetylcholinesterase activities in specimens of Sparus auratus exposed to sublethal copper concentrations.

The present study looks at possible changes in the activity of acetylcholinesterase (AChE) in tissues (brain and white muscle) of the Mediterranean bony fish Sparus auratus after a 20 days exposure to sublethal concentrations (0.1 or 0.5 ppm) of copper in the marine water and on control untreated animals. The trials also included measurements of Cu concentration in the tissues to evaluate possible metal accumulation. Moreover, sedimentation analysis as well as V(max) and K(m) determination were carried out in tissue extracts of Cu-exposed or control animals. V(max) and K(m) were also determined with or without addition of Cu(2+) in the assay. No Cu accumulation occurred in brain and muscle after Cu exposure. AChE showed in both tissues a molecular polymorphism with putative globular (G) and asymmetric (A) forms. Cu exposition led to an increased specific activity and improved catalytic efficiency of AChE in brain and muscle, seemingly regarding G forms. The increase in catalytic efficiency also resulted from the in vitro assay with tissue extracts and Cu(2+) addition. The higher AChE activity and catalytic efficiency in both tissues after Cu exposition and without metal accumulation, suggests an increase of free Cu aliquot into the cells, likely due to mechanisms of metal homeostasis.

Different activity of glyoxalase system enzymes in specimens of Sparus auratus exposed to sublethal copper concentrations.

The present study regards possible changes in the activity of glyoxalase system enzymes (glyoxalase I, GI, and glyoxalase II, GII) in tissues (brain, liver and white muscle) of the mediterranean bony fish Sparus auratus after a 20 days exposure to sublethal concentrations (0.1 or 0.5 ppm) of Cu in the marine water and on control untreated animals. The experiments also included measurements of copper concentration in the tissues, as well as of lactate dehydrogenase (LDH) activity, to evaluate possible Cu accumulation and changes in glycolytic activity respectively. Cu accumulation only occurs in the liver. GI, GII and LDH activities kept unchanged in the brain after copper exposure. GI activity in liver and muscle of copper-exposed animals decreases probably for a slackening in the glycolytic rate, as suggested by the lowering of LDH activity. GII activity remains unchanged or increases (liver extract, 0.5 ppm of Cu), maybe to safeguard enough cellular levels of GSH.