The myokine irisin: localization and effects in swine late medium and large antral ovarian follicle.

Irisin is mainly synthesized by skeletal muscle tissue, where it is believed to be responsible for the benefits of exercise on metabolism and cardiovascular system. In adipose tissue, its best-known effect is the browning of white adipocytes, resulting in the increase of thermogenesis and energy expenditure. As it has been largely documented that metabolic dysfunctions can frequently be associated with reductions in fertility, the possible involvement of this molecule in the regulation of reproductive processes represents an issue to be addressed. On this basis, the first aim of this work was the evaluation of the presence of irisin in the swine ovary; then, we investigated the expression of the associated molecules FNDC5, PGC-1α, and PPAR-γ. To verify a potential modulatory role both on ovarian function and on redox status, cell growth, steroidogenesis, production of superoxide anion and nitric oxide, the nonenzymatic antioxidant scavengers, were assessed in vitro on granulosa cells treated with increasing concentrations of irisin (50, 100, and 150 ng/mL). The data collected demonstrate the presence of irisin in swine ovarian follicle. Moreover, the highest concentrations tested stimulated metabolic activity and inhibited cell proliferation (P < 0.05); the peptide exerted a biphasic effect on progesterone (P < 0.01) production and, at the highest concentrations, inhibited nitric oxide while stimulated the nonenzymatic antioxidant power (P < 0.05). Superoxide anion and estradiol 17ß were unaffected. The demonstration of the local presence of irisin at the ovarian level and the highlighted effects allow us to qualify this molecule as a potential physiological regulator of follicular function.

The T cell response to persistent herpes virus infections in common variable immunodeficiency.

We show that at least half of patients with common variable immunodeficiency (CVID) have circulating CD8(+) T cells specific for epitopes derived from cytomegalovirus (CMV) and/or the Epstein-Barr virus (EBV). Compared to healthy age-matched subjects, more CD8(+) T cells in CVID patients were committed to CMV. Despite previous reports of defects in antigen presentation and cellular immunity in CVID, specific CD4(+) and CD8(+) T cells produced interferon (IFN)-gamma after stimulation with CMV peptides, and peripheral blood mononuclear cells secreted perforin in response to these antigens. In CVID patients we found an association between a high percentage of circulating CD8(+) CD57(+) T cells containing perforin, CMV infection and a low CD4/CD8 ratio, suggesting that CMV may have a major role in the T cell abnormalities described previously in this disease. We also show preliminary evidence that CMV contributes to the previously unexplained severe enteropathy that occurs in about 5% of patients.

Determination of ethylenediaminetetraacetic acid at very low concentrations by high-performance liquid chromatography coupled with electrospray mass spectrometry.

Ethylenediaminetetraacetic acid (EDTA) is a metal complexing agent which is commonly used for removing metallic surface contaminations. But its presence in aqueous industrial effluents or in wastes, by example those which are generated from nuclear power facilities, is forbidden or strictly regulated by the legislation. The implementation of high-performance liquid chromatography coupled with mass spectrometry (MS) via an electrospray interface has exhibited a powerful capacity in the measurement of this complexing agent, in the form of its iron complex. The complex is eluted through a reversed stationary phase under slightly acidic conditions and the detection is performed by mass spectrometry in the single ion monitoring mode. This technique has allowed us to reach a detection limit of about 1 microg/l in EDTA for only 20 microl injected without any previous preconcentration, and to solve some analytical interferences observed in the case of industrial effluents with other analytical techniques such as ion chromatography.

Improved determination of tributyl phosphate degradation products (mono- and dibutyl phosphates) by ion chromatography.

Tributyl phosphate (TBP) is a very important compound in the nuclear industry, particularly in the area of nuclear fuel reprocessing. This compound is used in the PUREX (plutonium and uranium refining extraction) process which consists of the extraction of uranium and plutonium from an aqueous nitric acid phase, for the purpose of recycling. But TBP may be degraded to dibutyl phosphate (DBP) and monobutyl phosphate (MBP) by dealkylation of one or two butoxy groups, respectively. We have compared and evaluated the capacity of two resins manufactured by Dionex (AS11 and AS5A) in the separation and measurement of these two degradation products. AS11 generates two interferences: nitrite/DBP and carbonate/MBP. The first one is the most serious. So, we have developed a method for oxidising nitrite ions to nitrate ions which have no trouble over the measurement. The second resin tested, AS5A, allows a very efficient separation between DBP and NO2- ions and a good separation between MBP and CO3(2-) in comparison with the AS11. The detection limits for the AS5A column are 0.13 microM for MBP and 0.71 microM for DBP (injection loop=50 microl).

Cytotoxic T lymphocyte responses and CTL epitope escape mutation in HBsAg, anti-HBe positive individuals.

BACKGROUND/AIMS: Clearance of hepatitis B virus (HBV) is characterised by a strong cytotoxic T cell response. Persistence of HBV in chronic hepatitis B carriers may be related to failure of this response. The aim of this study was to determine whether HLA class I restricted cytotoxic T lymphocyte (CTL) responses persist in anti-hepatitis B e (HBe) positive / HBV DNA negative individuals, and to correlate the presence of viral CTL epitope mutation with clinical outcome. METHODS: An HLA/HBV dual transfectant model was used to demonstrate these CTL responses in individuals chronically infected with HBV. Subsequently, a known hepatitis B core (HBc) CTL epitope was sequenced in a family of five chronically infected individuals all sharing a HLA allele (HLA-A68.1). RESULTS: Low level HLA class I restricted cytotoxic T cell responses were detected in the peripheral blood of five of eight anti-HBe positive individuals. In the family of HLA-A68.1 positive chronically infected individuals, mutation of the HLA-A68.1 restricted hepatitis B core antigen (HBcAg) CTL epitope STLPETTVVRR was found in all four anti-HBe positive individuals but not in the sole hepatitis B e antigen (HBeAg) positive patient. CONCLUSION: These data are consistent with a continued immune selection pressure on HBV in anti-HBe positive chronically infected individuals with low replicating HBV infection and suggest that mutation of a CTL epitope may be a consequence of the immune response, as opposed to the cause of viral persistence.

Identification of three HLA-A*0201-restricted cytotoxic T cell epitopes in the cytomegalovirus protein pp65 that are conserved between eight strains of the virus.

The Ag specificity of the CTL response against CMV is directed almost entirely to a single CMV tegument protein, the phosphoprotein pp65. We report the identification of three peptides derived from the protein pp65 that displayed a high or intermediate binding to HLA-A*0201 molecules, which were also able to induce an in vitro CTL response in peripheral blood lymphocytes from CMV seropositive individuals. The peptide-specific CTLs generated were capable of recognizing the naturally processed pp65 either presented by CMV-infected cells or by cells infected with an adenovirus construct expressing pp65 in an HLA-A*0201-restricted manner. Thus, we were able to demonstrate responses to subdominant CTL epitopes in CMV-pp65 that were not detected in polyclonal cultures obtained by conventional stimulations. We also found that the amino acid sequences of the three peptides identified as HLA-A*0201-restricted CTL epitopes were conserved among different wild-type strains of CMV obtained from renal transplant patients, an AIDS patient, and a congenitally infected infant, as well as three laboratory strains of the virus (AD169, Towne and Davis). These observations suggest that these pp65 CTL peptide epitopes could potentially be used as synthetic peptide vaccines or for other therapeutic strategies aimed at HLA-A*0201-positive individuals, who represent approximately 40% of the European Caucasoid population. However, strain variation must be taken in consideration when the search for CTL epitopes is extended to other HLA class I alleles, because these mutations may span potential CTL epitopes for other HLA molecules, as it is described in this study.

Cells and cytokines in immunotherapy and gene therapy of cancer.

An enormous effort using a great variety of approaches has been undertaken in the last decade to translate basic and clinical research into successful cancer therapies. This review summarizes recent results of experiments and trials using cellular and cytokine therapy, as well as gene therapy, to exploit immune responses to tumors.

Synthesis of [3',5'-3H2]-alpha-fluoromethyl-tyrosine as a radioactive specific label of rat brain tyrosine hydroxylase.

The [3',5']-ditritio-alpha-fluoromethyl-tyrosine 4 (specific activity 15.0 Ci/mmol) has been synthesized and used as a radioactive probe for rat neuronal tyrosine hydroxylase (TH). The route of synthesis for the preparation of 3 and 4 allowed us to not only introduce a fluorine atom into 3/4 using an inorganic source of fluorine (CsF), but also to take advantage of the high-yielding cyclization of (alpha,beta)-acetamido alcohols mediated by diethylaminosulfur trifluoride (DAST) to give the corresponding oxazolines. The distribution and metabolism of 4 have been studied in control conditions within the rat locus caeruleus (LC). Intracisternal injection of 20 microCi of 4 was followed by a rapid disappearance of 4 (t1/2 = 1.5 h) and by a specific accumulation of radioactivity into the LC anatomical limits. This was investigated each 140 microns along the caudo-rostral axis of the noradrenergic nucleus. In each anatomical interval, its distribution correlated nicely with already described caudo-rostral distribution of TH in noradrenergic cells. Thus, 4 may provide a reliable measure of TH activity in such catecholamine structures.

The invariant chain inhibits presentation of endogenous antigens by a human fibroblast cell line.

The human fibroblast cell line, M1, expressing the products of transfected DRA and DRB1*0101 genes (M1-DR1) was unable to present intact influenza antigens to a series of DR1-restricted human T cell lines and clones, but was fully able to present synthetic peptides for T cell recognition. In contrast, M1-DR1 cells infected with live influenza virus were recognized by two polyclonal hemagglutinin- or whole virus-specific T cell lines and one of four T cell clones. This difference could not be accounted for simply by the ability of infectious virus to overcome a defect in antigen uptake by the M1-DR1 cells, in that direct studies of endocytosis showed that the M1 cells were more efficient than human B cells in the internalization of exogenous protein. These data suggested that the M1 cells were unable to present exogenous antigens but were capable of loading major histocompatibility complex (MHC) class II molecules with peptides derived from endogenous antigens. To investigate this further, the M1-DR1 cells were super-transfected with a cDNA encoding the p33 and p35 forms of the human invariant chain (Ii). Expression of the Ii chain was detected by intracytoplasmic staining of transfectants, and by metabolic labeling. Equimolar amounts of the p33 and p35 forms were detected, and the high level of p35 Ii was reflected by extensive retention of Ii protein in the endoplasmic reticulum. Addition of the Ii chain led to no recovery of presentation of intact antigens with DR1, but inhibited the presentation of live virus. These data indicate that MHC class II molecules in the M1-DR1 cells can be loaded with peptides derived from endogenous proteins, possibly in the biosynthetic pathway, and that the Ii chain has a role in limiting this route of class II antigen presentation.

Bone-marrow transplantation for severe aplastic anaemia using histocompatible unrelated volunteer donors.

Two patients with severe aplastic anaemia received bone-marrow transplants from unrelated donors selected for HLA compatibility. Graft-versus-host disease occurred in both patients but responded to treatment. Both patients had stormy courses after grafting, but subsequently their conditions improved, and one was not receiving any treatment at follow-up after day 330 while the other had mild chronic graft-versus-host disease at day 150. These results show that unrelated, histocompatible volunteers may successfully donate marrow for the treatment of severe aplastic anaemia, though many problems remain to be solved.