Total serum tryptase levels are higher in young infants.

BACKGROUND: Mast cells participate in immune defense and allergic disease. At baseline, serum tryptase levels primarily reflect mast cell burden, while mast cell degranulation leads to granule tryptase release, which may be detectable as a transitory elevation of serum tryptase levels. Thus, mast cell burden and mast cell activity are reflected by serum tryptase levels, but reports are scarce in infants under 1 yr. We aimed at defining levels of total serum tryptase levels in this population. METHODS: Total serum tryptase levels (ImmunoCAP; Phadia) were measured in 372 sera from infants younger than 1 yr. Two hundred and forty-two sera came from non-atopic, non-allergic infants in good condition, who had blood drawn for routine follow-up or diagnosis of illnesses that are not known to induce changes in serum tryptase levels. Seventy-two sera were from atopic and/or allergic infants, and 58 sera were from non-atopic, non-allergic infants requiring intensive care. RESULTS: Median serum tryptase levels were highest in infant2s under 3 months (6.12 ± 3.47 µg/l) and gradually decreased before reaching levels similar to those described in adults and older children (3.85 ± 1.8 µg/l between 9 and 12 months). Atopic/allergic status was associated with even higher tryptase levels (14.20 ± 10.22 µg/l in infants younger than 3 months). Intensive care patients had lower levels of serum tryptase (4.12 ± 3.38 µg/l in infants younger than 3 months). Longitudinal follow-up was performed in 27 patients and showed tryptase levels decrease over time in individual patients. Infants'sex was not found to interfere with serum tryptase levels. CONCLUSION: Total serum tryptase levels are significantly higher in younger infants compared with older ones. In infants of the same age, serum tryptase levels may vary according to the clinical condition and thus suggest mast cell involvement in the physiologic as well as in the allergic immune responses of young infants.

Production of an agonist-like monoclonal antibody to the human A2A receptor of adenosine for clinical use.

The second extracellular loop of the A(2A) receptor (A(2A)R) of adenosine was used to immunize mice for production of Adonis, an IgM monoclonal antibody. Adonis bound to the immunogen peptide and the native receptor in ELISA with K(D) values in 6.51-12.35 nM range. It recognized a linear epitope of 7 amino acids (LFEDVVP) at the C-terminal part of the external loop. Adonis revealed a 45-kDa band in lysate of human peripheral blood mononuclear cells in Western blotting in denaturing conditions. This served to monitor the up-regulation of the A(2A)R expression by caffeine. Adonis stimulated the cAMP production and inhibited the cell proliferation of an A(2A)R transfected stable cell line. These results confirm the immunogenicity and the functional relevance of the second extracellular loop of the A(2A)R. They suggest that Adonis may be of clinical use in various pathological situations to measure the regulation of the A(2A)R expression and to act as A(2A)R agonist drug.

Anti-thyroperoxidase antibodies from patients with Hashimoto's encephalopathy bind to cerebellar astrocytes.

A cohort of 10 Hashimoto's encephalopathy (HE) patients, 33 patients with unrelated neurological symptoms, 12 Hashimoto's thyroiditis patients and 4 healthy adult donors was studied to explore the neurological targets of anti-thyroperoxidase (TPO) autoantibodies (aAb) in HE. High levels of anti-TPO aAb were only detected in HE group's cerebrospinal fluids. In immunofluorescence assays on monkey brain cerebellum sections, both HE patients' sera and anti-TPO monoclonal antibodies (mAb) were able to bind cerebellar cells expressing glial fibrillary acid protein. Normal human astrocytes from primary cultures also reacted with anti-TPO mAb. Specific astrocyte binding of anti-TPO aAb suggests a role of these aAb in the HE pathogenesis.

Managing irritable bowel syndrome: The impact of micro-physiotherapy.

Background Irritable bowel syndrome (IBS) has a complex pathology, high prevalence and large impact on patients' quality of life. As conventional therapy may yield unsatisfactory results, a more holistic approach may be desirable. The current study assessed the effect of micro-physiotherapy on the severity of IBS symptoms. Methods In a double-blind study, 61 recurrent IBS patients were randomised to two sessions of micro-physiotherapy or sham micro-physiotherapy. Inclusion criteria were the presence of ≥1 IBS symptom from abdominal pain, constipation, diarrhoea or bloating. Exclusion criteria were previous major intestinal surgery and the presence of chronic diseases. The mean patient age was 53.5±15.3 years. Micro-physiotherapy consisted of micro-palpatory examination to identify osteopathic lesions, followed by micro-massage to stimulate self-healing. The control group underwent a sham procedure. The presence and severity of symptoms was assessed at baseline and at 1-month follow-up by the same gastroenterologist. Results Two patients did not complete the study. There was a significant difference in percentage of patients that improved after the first session, at 74 % for the micro-physiotherapy group and 38 % for the sham group, respectively (p=0.005). After the second session, the initial improvement was maintained in both groups, although with no further gains, and the differences between the study groups remained significant (p=0.007). Conclusions Micro-physiotherapy significantly improves IBS symptoms and should be explored further for use in mainstream healthcare.

Detection of thyroid peroxidase mRNA and protein in orbital tissue.

OBJECTIVE: We have previously reported that the absence of thyroid peroxidase antibodies (TPOAb) in Graves' disease (GD) was associated with an increased risk of Graves' ophthalmopathy (GO). This observation raised the possibility that TPOAb could act as a protective factor. However, the presence of thyroid peroxidase (TPO) in the orbit has not been previously reported. The aim of this study was to confirm or exclude the presence of orbital TPO. METHODS AND DESIGN: Relative TPO mRNA expression from GO (n=6) and normal (n=5) orbital fat tissue was determined using real-time PCR technique. Orbital fat in the normal group from blepharoplasty represents extraconal (anterior) fat. mRNA expression in fibroblasts grown from these tissues before and after adipocyte differentiation was also documented. Finally, Western blotting was carried out to verify translation of TPO mRNA transcripts. RESULTS AND DISCUSSION: TPO transcripts were detected in the orbital fat tissue obtained from normal and GO subjects using the real-time PCR technique. TPO expression was increased in GO compared to normal (N) tissues. However, TPO expression in cultured fibroblasts was similar in both groups and adipogenesis did not appear to alter TPO expression. Protein was detected by Western blot analysis using the TPO MAB 47 (mAb 47). The predicted 110-kDa band was detected in orbital fat as well as in orbital fibroblasts. Our results suggest the presence of TPO in GO and N orbital tissues. We hypothesise that immune responses directed against orbital TPO might play a role in modulating the clinical expression of GO.

Identification and pharmacological characterization of SRBP-2: a novel SR31747A-binding protein.

SR31747A is a sigma ligand with potent antiproliferative activity against tumor cells and for which three binding proteins have been identified to date: (a) SRBP-1 (also called sigma 1); (b) HIS; and (c) sigma 2. In this study, we characterized an additional SR31747A binding site, i.e., SRBP-2 (SR31747A-binding protein 2). Using an in silico screening approach, we identified this novel sequence, which exhibits 41% homology with HSI. The 1142-bp cDNA was found to encode a 206 amino acid protein not related to SRBP-1. Northern blot analysis of SRBP-2 mRNA expression revealed a single 1.1-kb transcript that was widely expressed in organs; the liver was particularly enriched, and the brain showed the lowest abundance. A murine homologue that exhibited a similar expression pattern was also characterized. Subcellular localization analysis using specific polyclonal antibodies revealed that SRBP-2 had the same nuclear membrane and endoplasmic reticulum localization as other members of the SR31747A-binding protein family. Considering SRBP-2-binding properties, pharmacological analysis clearly highlighted that SRBP-2 was distinct from sigma 2. Scatchard plot analysis revealed K(d) values of 10 and 3 nM for SR31747A and Tamoxifen, respectively. In contrast with HSI, the protein also did not exhibit detectable isomerase activity. When analyzing SRBP-2 expression in human breast cancer biopsies, we obtained evidence that SRBP-2 expression, together with SRBP-1 and HSI, may be of interest as a prognostic marker. These findings demonstrated that SRBP-2 represents an additional molecular target for SR31747A, which could help to understand the immunosuppressive and antiproliferative effects of the molecule.

Androgen-dependent expression of FcgammaRIIB2 by thyrocytes from patients with autoimmune Graves' disease: a possible molecular clue for sex dependence of autoimmune disease.

Thyrocyte expression of HLA class I and class II antigens and related accessory molecules would convert these epithelial cells into functional antigen-presenting cells. Here we show that whereas normal thyrocytes express FcRn, Graves' disease thyrocytes also express FcgammaRIIB2. We further find that expression of FcgammaRIIB2, but not FcRn, is repressed by dihydrotestosterone. By mediating the uptake and transport of autoantibodies, we suggest that these IgG Fc receptors contribute in various ways to the onset and/or progression of autoimmune thyroid diseases. The androgen-mediated decrease of FcgammaRIIB2 expression in Graves' disease thyrocytes also provides a rationale for the predominant susceptibility of women to develop an autoimmune thyroid disease. Our findings open up a new prospect to autoimmunity, linking the role of the target organ to the sex dependence in autoimmune disease.

Complement activation by direct C4 binding to thyroperoxidase in Hashimoto's thyroiditis.

Biosynthesis of thyroid hormones is an oxidative process that generates reactive oxygen species (ROS) and involves thyroperoxidase (TPO) that is one of the main autoantigens involved in autoimmune thyroid diseases. The ectodomain of TPO consists of a large N-terminal myeloperoxidase-like module followed by a complement control protein (CCP)-like module and an epidermal growth factor-like module. The presence of these two additional gene modules suggests that they may play some crucial, hitherto unsuspected role associated with thyroid function. Because the CCP module is a constituent of the molecules involved in the activation of C4 complement component, we investigated the possibility that C4 may bind to TPO and activate the complement pathway in autoimmune conditions. We showed that TPO via its CCP module directly activated complement without any mediation by Ig. We suggested that this additional complement pathway requires the production of ROS and specially hydroxyl radicals that aggregate TPO and oxidize methionines of C4. Moreover, we found, in patients with Hashimoto's thyroiditis, that thyrocytes overexpress C4 and all the downstream components of the complement pathway. These results indicate that TPO has some as yet unknown function, which may contribute along with other mechanisms to the massive cell destruction observed in Hashimoto's thyroiditis. Investigating this complement pathway, therefore, would provide an excellent means of reaching a better understanding of the etiology of other degenerative diseases.

Antigenicity and immunogenicity of the C-terminal peptide of human thyroglobulin.

Thyroglobulin (Tg) is cleaved into several peptides during thyroid hormone synthesis, an oxidative process. P40, an iodinated C-terminal peptide from human Tg, has a molecular weight of about 40 kDa and contains two hormonogenic sites. P40 is the smallest peptide that is still recognized by monoclonal antibodies from mice immunized with human Tg directed against its immunodominant region. Since P40 also contains several T-cell epitopes, it is a good candidate for studying the primary events involved in the process of hormone synthesis leading to thyroid autoimmunity. The present results show that P40 is recognized by Tg antibodies from patients with thyroid disorders and induces Tg antibodies in CBA mice. P40 may therefore be involved in the autoimmune process, thus providing a useful tool for diagnostic and therapeutic purposes.

Evidence that the complement control protein-epidermal growth factor-like domain of thyroid peroxidase lies on the fringe of the immunodominant region recognized by autoantibodies.

There is no consensus regarding the location of the immunodominant region (IDR) on thyroid peroxidase (TPO) recognized by the majority of autoantibodies. Strong evidence indicates that it lies upstream of amino acid 741. However, an epitope has been localized to downstream residues 742-848 encompassing a disulfide-rich complement control protein (CCP)-like and epidermal growth factor (EGF)-like domain. To determine whether these domains comprise part of the IDR, we used a recombinant CCP/EGF-like polypeptide to screen a thyroid B-cell-derived immunoglobulin gene phage display library. Two unusual TPO autoantibodies were isolated. Neither was among the 83 clones previously obtained by panning the same library on native or denatured TPO, or TPO with the IDR masked. Fab from these clones bound native TPO, one with high affinity (Kd 6 x 10(-10) M), and both recognized TPO expressed on the surface of mammalian cells. Phage-expressing multiple copies of the antibody (multivalent), but not monovalent Fab from these clones, bound to the CCP/EGF polypeptide. Most important, inhibition of TPO binding by autoantibodies to the IDR indicated that the epitopes of the two new autoantibodies overlap with this region. The value of these two rare clones lies in the insight they provide into the location of the TPO IDR. From their binding characteristics, we deduce that the CCP/EGF-like domain lies on the fringe of the TPO immunodominant region.

Dairy fat blends high in α-linolenic acid are superior to n-3 fatty-acid-enriched palm oil blends for increasing DHA levels in the brains of young rats.

Achieving an appropriate docosahexaenoic acid (DHA) status in the neonatal brain is an important goal of neonatal nutrition. We evaluated how different dietary fat matrices improved DHA content in the brains of both male and female rats. Forty rats of each gender were born from dams fed over gestation and lactation with a low α-linolenic acid (ALA) diet (0.4% of fatty acids) and subjected for 6 weeks after weaning to a palm oil blend-based diet (10% by weight) that provided either 1.5% ALA or 1.5% ALA and 0.12% DHA with 0.4% arachidonic acid or to an anhydrous dairy fat blend that provided 1.5% or 2.3% ALA. Fatty acids in the plasma, red blood cells (RBCs) and whole brain were determined by gas chromatography. The 1.5% ALA dairy fat was superior to both the 1.5% ALA palm oil blends for increasing brain DHA (14.4% increase, P<.05), and the 2.3% ALA dairy blend exhibited a further increase that could be ascribed to both an ALA increase and n-6/n-3 ratio decrease. Females had significantly higher brain DHA due to a gender-to-diet interaction, with dairy fats attenuating the gender effect. Brain DHA was predicted with a better accuracy by some plasma and RBC fatty acids when used in combination (R(2) of 0.6) than when used individually (R(2)=0.47 for RBC n-3 docosapentaenoic acid at best). In conclusion, dairy fat blends enriched with ALA appear to be an interesting strategy for achieving optimal DHA levels in the brain of postweaning rats. Human applications are worth considering.

Kinin B1 receptor enhances the oxidative stress in a rat model of insulin resistance: outcome in hypertension, allodynia and metabolic complications.

BACKGROUND: Kinin B(1) receptor (B(1)R) is induced by the oxidative stress in models of diabetes mellitus. This study aims at determining whether B(1)R activation could perpetuate the oxidative stress which leads to diabetic complications. METHODS AND FINDINGS: Young Sprague-Dawley rats were fed with 10% D-Glucose or tap water (controls) for 8-12 weeks. A selective B(1)R antagonist (SSR240612) was administered acutely (3-30 mg/kg) or daily for a period of 7 days (10 mg/kg) and the impact was measured on systolic blood pressure, allodynia, protein and/or mRNA B(1)R expression, aortic superoxide anion (O(2)(*-)) production and expression of superoxide dismutase (MnSOD) and catalase. SSR240612 reduced dose-dependently (3-30 mg/kg) high blood pressure in 12-week glucose-fed rats, but had no effect in controls. Eight-week glucose-fed rats exhibited insulin resistance (HOMA index), hypertension, tactile and cold allodynia and significant increases of plasma levels of glucose and insulin. This was associated with higher aortic levels of O(2)(*-), NADPH oxidase activity, MnSOD and catalase expression. All these abnormalities including B(1)R overexpression (spinal cord, aorta, liver and gastrocnemius muscle) were normalized by the prolonged treatment with SSR240612. The production of O(2)(*-) in the aorta of glucose-fed rats was also measured in the presence and absence of inhibitors (10-100 microM) of NADPH oxidase (apocynin), xanthine oxidase (allopurinol) or nitric oxide synthase (L-NAME) with and without Sar[D-Phe(8)]des-Arg(9)-BK (20 microM; B(1)R agonist). Data show that the greater aortic O(2)(*-) production induced by the B(1)R agonist was blocked only by apocynin. CONCLUSIONS: Activation of kinin B(1)R increased O(2)(*-) through the activation of NADPH oxidase in the vasculature. Prolonged blockade of B(1)R restored cardiovascular, sensory and metabolic abnormalities by reducing oxidative stress and B(1)R gene expression in this model.

Kinin B1 receptor deficiency leads to leptin hypersensitivity and resistance to obesity.

OBJECTIVE: Kinins mediate pathophysiological processes related to hypertension, pain, and inflammation through the activation of two G-protein-coupled receptors, named B(1) and B(2). Although these peptides have been related to glucose homeostasis, their effects on energy balance are still unknown. RESEARCH DESIGN AND METHODS: Using genetic and pharmacological strategies to abrogate the kinin B(1) receptor in different animal models of obesity, here we present evidence of a novel role for kinins in the regulation of satiety and adiposity. RESULTS: Kinin B(1) receptor deficiency in mice (B(1)(-/-)) resulted in less fat content, hypoleptinemia, increased leptin sensitivity, and robust protection against high-fat diet-induced weight gain. Under high-fat diet, B(1)(-/-) also exhibited reduced food intake, improved lipid oxidation, and increased energy expenditure. Surprisingly, B(1) receptor deficiency was not able to decrease food intake and adiposity in obese mice lacking leptin (ob/ob-B(1)(-/-)). However, ob/ob-B(1)(-/-) mice were more responsive to the effects of exogenous leptin on body weight and food intake, suggesting that B(1) receptors may be dependent on leptin to display their metabolic roles. Finally, inhibition of weight gain and food intake by B(1) receptor ablation was pharmacologically confirmed by long-term administration of the kinin B(1) receptor antagonist SSR240612 to mice under high-fat diet. CONCLUSIONS: Our data suggest that kinin B(1) receptors participate in the regulation of the energy balance via a mechanism that could involve the modulation of leptin sensitivity.

Structural and functional aspects of thyroid peroxidase.

Thyroperoxidase (TPO) is the enzyme involved in thyroid hormone synthesis. Although many studies have been carried out on TPO since it was first identified as being the thyroid microsomal antigen involved in autoimmune thyroid disease, previous authors have focused more on the immunological than on the biochemical aspects of TPO during the last few years. Here, we review the latest contributions in the field of TPO research and provide a large reference list of original publications. Given this promising background, scientists and clinicians will certainly continue in the future to investigate the mechanisms whereby TPO contributes to hormone synthesis and constitutes an important autoantigen involved in autoimmune thyroid disease, and the circumstances under which the normal physiological function of this enzyme takes on a pathological role.

An in vitro model based on cell monolayers grown on the underside of large- pore filters in bicameral chambers for studying thyrocyte-lymphocyte interactions.

In the processes underlying thyroid autoimmunity, thyrocytes probably act as antigen-presenting cells exposing T-cell epitopes to intrathyroid lymphocytes. To study the interactions between lymphocytes and thyrocytes, which are arranged in a tight, polarized monolayer, we developed a new in vitro model based on human thyrocytes grown on the underside of a filter placed in a bicameral chamber. Thyrocytes from Graves' disease glands were plated onto the upper face of a 8-mum-pore polyethylene terephthalate culture insert filter placed in the inverted position and grown for 24 h before the insert was returned to the normal position for a week in the cell culture plate wells. Thyrocytes grown in the presence of thyroid stimulating hormone, forming a homogeneous monolayer on the underside of the filter, reached confluence after 8 days in vitro. The cells developed a transepithelial electrical resistance >1,000 Omega.cm(2), and the ZO-1 tight junction protein showed a junctional pattern of distribution. Thyrocytes showed a polarized pattern of thyroperoxidase and thyroid stimulating hormone receptor expression in the apical and basolateral positions, respectively. They were also found to aberrantly express DR class II human leukocyte antigen and an Fc immunoglobulin receptor (FcgammaRIIB2) in the basolateral and apical positions, respectively. Autologous intrathyroidal T lymphocytes cocultured for 24 h across the filter with the thyrocyte monolayer proliferated and remained in the upper chamber without any leakage occurring through the epithelial barrier, which makes this model particularly suitable for studying the cell-cell interactions involved in antigen processing.