Gliadin-specific type 1 regulatory T cells from the intestinal mucosa of treated celiac patients inhibit pathogenic T cells

Celiac disease (CD) results from a permanent intolerance to dietary gluten and is due to a massive T cell-mediated immune response to gliadin, the main component of gluten. In this disease, the regulation of immune responses to dietary gliadin is altered. Herein, we investigated whether IL-10 could modulate anti-gliadin immune responses and whether gliadin-specific type 1 regulatory T (Tr1) cells could be isolated from the intestinal mucosa of CD patients in remission. Short-term T cell lines were generated from jejunal biopsies, either freshly processed or cultured ex vivo with gliadin in the presence or absence of IL-10. Ex vivo stimulation of CD biopsies with gliadin in the presence of IL-10 resulted in suppression of Ag-specific proliferation and cytokine production, indicating that pathogenic T cells are susceptible to IL-10-mediated immune regulation. T cell clones generated from intestinal T cell lines were tested for gliadin specificity by cytokine production and proliferative responses. The majority of gliadin-specific T cell clones had a Th0 cytokine production profile with secretion of IL-2, IL-4, IFN-gamma, and IL-10 and proliferated in response to gliadin. Tr1 cell clones were also isolated. These Tr1 cells were anergic, restricted by DQ2 (a CD-associated HLA), and produced IL-10 and IFN-gamma, but little or no IL-2 or IL-4 upon activation with gliadin or polyclonal stimuli. Importantly, gliadin-specific Tr1 cell clones suppressed proliferation of pathogenic Th0 cells. In conclusion, dietary Ag-specific Tr1 cells are present in the human intestinal mucosa, and strategies to boost their numbers and/or function may offer new therapeutic opportunities to restore gut homeostasis.     

Conjugated linoleic acid enhances glutathione synthesis and attenuates pathological signs in MRL/MpJ-Fas(lpr) mice

Conjugated linoleic acid (CLA), a naturally occurring peroxisome proliferator-activated receptor gamma (PPAR gamma) ligand, exhibits proapoptotic, immunomodulatory, and anticancer properties. In this study, we examined the biological effects of CLA administration in the MRL/MpJ-Fas(lpr) mouse, an animal model of systemic lupus erythematosus (SLE). We found that CLA exerted apparently opposed activities in in vitro experiments, depending on its concentration: 100 microM CLA downregulated IFN gamma synthesis and cell proliferation of splenocytes, in association with apoptosis induction and a decrease of intracellular thiols (GSH + GSSG), whereas 25 microM CLA did not significantly influence cell proliferation but enhanced the expression of gamma-glutamylcysteine ligase catalytic subunit (GCLC) and intracellular GSH concentration. Interestingly, the antiproliferative effect at 100 microM was not inhibited by the PPAR gamma antagonist GW9662. In vivo, CLA administration drastically reduced SLE signs (splenomegaly, autoantibodies, and cytokine synthesis), a condition paralleled by the enhancement of GCLC expression and intracellular GSH content. Moreover, CLA administration significantly downregulated nuclear factor kappaB activity independent of PPAR gamma activation and apoptosis induction. In conclusion, enhanced GSH content and GCLC expression in CLA-treated mice suggest a novel biochemical mechanism underlying its immunomodulatory activity and the beneficial effects on murine SLE signs.     

Combined leptin actions on adipose tissue and hypothalamus are required to deplete adipocyte fat in lean rats: implications for obesity treatment

Intense hyperleptinemia completely depletes adipocyte fat of normal rats within 14 days. To determine the mechanism, epididymal fat pads from normal wild-type (+/+) and obese (fa/fa) Zucker Diabetic Fatty (ZDF) donor rats were transplanted into normal +/+ and fa/fa ZDF recipients. Hyperleptinemia induced by adenovirus-leptin administration depleted all fat from native fat pads and from fat transplants from +/+ donors but not from transplants from ZDF(fa/fa) donors with defective leptin receptors. In both native and transplanted +/+ fat pads, large numbers of mitochondria were apparent, and genes involved in fatty acid oxidation were up-regulated. However, +/+ fat pads transplanted into fa/fa recipients did not respond to hyperleptinemia, suggesting lack of an essential leptin-stimulated cohormone(s). In +/+ but not in fa/fa rats, plasma catecholamine levels rose, and both P-STAT3 and P-CREB increased in adipose tissue, suggesting that both direct and indirect (hypothalamic) leptin receptor-mediated actions of hyperleptinemia are involved in depletion of adipocyte fat.     

The bis-histidyl complex in hemoproteins: A detailed conformational analysis of database protein structures and the case of Antarctic fish hemoglobins

The properties of hemoproteins strictly depend on the type and orientation of axial ligands. Here, the orientations of axially coordinated His in bis-His complexes and the heme geometry in protein data bank have been analyzed. The effect of the bis-histidyl formation on the heme cavity of Antarctic fish hemoglobins has been also evaluated. The results show that protein matrix exerts a major effect on the conformation of axially ligated histidines: the imidazoles in bis-His complexes occupy a preferred relative orientation in globins and in model systems, whereas they adopt a variety of relative orientations in other hemoproteins. The bis-histidyl adducts affect the heme geometry inducing larger distortions from planarity with respect to other ligands. These deviations are larger in bis-His multiheme cytochromes than in globins. In Antarctic fish hemoglobins the bis-histidyl adduct adopts preferentially a distorted coordination and the formation of the bis-His complex induces a slight but significant modification in the shape, area and volume of the heme cavity.     

Structural features for the mechanism of antitumor action of a dimeric human pancreatic ribonuclease variant

A specialized class of RNases shows a high cytotoxicity toward tumor cell lines, which is critically dependent on their ability to reach the cytosol and to evade the action of the ribonuclease inhibitor (RI). The cytotoxicity and antitumor activity of bovine seminal ribonuclease (BSRNase), which exists in the native state as an equilibrium mixture of a swapped and an unswapped dimer, are peculiar properties of the swapped form. A dimeric variant (HHP2‐RNase) of human pancreatic RNase, in which the enzyme has been engineered to reproduce the sequence of BSRNase helix‐II (Gln28→Leu, Arg31→Cys, Arg32→Cys, and Asn34→Lys) and to eliminate a negative charge on the surface (Glu111→Gly), is also extremely cytotoxic. Surprisingly, this activity is associated also to the unswapped form of the protein. The crystal structure reveals that on this molecule the hinge regions, which are highly disordered in the unswapped form of BSRNase, adopt a very well‐defined conformation in both subunits. The results suggest that the two hinge peptides and the two Leu28 side chains may provide an anchorage to a transient noncovalent dimer, which maintains Cys31 and Cys32 of the two subunits in proximity, thus stabilizing a quaternary structure, similar to that found for the noncovalent swapped dimer of BSRNase, that allows the molecule to escape RI and/or to enhance the formation of the interchain disulfides.     

Echocardiographic Abnormalities in Normotensive Obese Patients: Relationship with Visceral Fat

Objective: To evaluate the relationship of echocardiographic characteristics and visceral adipose tissue (VAT) distribution in normotensive obese patients. Research Methods and Procedures: Echocardiographic parameters were assessed in 28 normotensive obese patients [7 men, 21 women, mean age, 43.2 years; mean body mass index (BMI), 37.2 kg/m²; 10 with impaired glucose tolerance (IGT); 6 with type 2 diabetes] and 18 sex‐ and age‐matched healthy, normal‐weight controls (4 men, 14 women; mean age, 45.8 years; mean BMI, 22.4 kg/m²) by an M‐mode, color‐doppler videofluoroscope. VAT in the obese patients was assessed by computed tomography (at L4 level). Results: The obese patients had a significantly larger internal diastolic left ventricular (LV) diameter (p < 0.05), a thicker end‐diastolic septum (p < 0.001) and posterior wall (p < 0.001), a greater indexed (g/m^2.7) LV mass (p < 0.001), a higher atrial diastolic filling wave velocity (p < 0.001), a lower ratio between early and atrial diastolic filling wave velocities (p < 0.01), and a prolonged isovolumic relaxation time (p < 0.05). End‐diastolic septum and posterior wall thickness and the LV mass were significantly greater in patients with a VAT area >130 cm² than with <130 cm². In the multivariate regression analysis, only VAT (p < 0.0001), waist‐to‐hip ratio (p < 0.001), and sex (p < 0.001) were associated with the most important echocardiographic alterations. Discussion: The morphological and functional echocardiographic alterations usually found in normotensive obese patients closely correlate with the amount of intra‐abdominal fat deposition, even in the presence of diabetes or IGT.     

Crystal structure of a ternary complex of the alcohol dehydrogenase from Sulfolobus solfataricus

The crystal structure of a ternary complex of the alcohol dehydrogenase from the archaeon Sulfolobus solfataricus (SsADH) has been determined at 2.3 A. The asymmetric unit contains a dimer with a NADH and a 2-ethoxyethanol molecule bound to each subunit. The comparison with the apo structure of the enzyme reveals that this medium chain ADH undergoes a substantial conformational change in the apo-holo transition, accompanied by loop movements at the domain interface. The extent of domain closure is similar to that observed for the classical horse liver ADH, although some differences are found which can be related to the different oligomeric states of the enzymes. Compared to its apo form, the SsADH ternary complex shows a change in the ligation state of the active site zinc ion which is no longer bound to Glu69, providing additional evidence of the dynamic role played by the conserved glutamate residue in ADHs. In addition, the structure presented here allows the identification of the substrate site and hence of the residues that are important in the binding of both the substrate and the coenzyme.     

The unswapped chain of bovine seminal ribonuclease: Crystal structure of the free and liganded monomeric derivative

Bovine seminal ribonuclease, a homodimeric enzyme joined covalently by two interchain disulphide bonds, is an equilibrium mixture of two conformational isomers, MxM and M=M. The major form, MxM, whose crystal structure has been previously determined at 1.9 A resolution, presents the swapping of the N-terminal segments (residues 1-15) and composite active sites formed by residues of different chains. The three-dimensional domain swapping does not occur in the M=M form. The different fold of each N-terminal tail is directed by the hinge loop (residue 16-22) connecting the swapping domain to the body of the protein. Reduction and alkylation of interchain disulphide bridges produce a monomeric derivative and a noncovalent swapped dimer, which are both active. The free and nucleotide-bound forms of the monomer have been crystallized at an alkaline pH and refined at 1.45 and 1.65 A resolution, respectively. In both cases, the N-terminal fragment is folded on the main body of the protein to produce an intact active site and a chain architecture very similar to that of bovine pancreatic ribonuclease. In this new fold of the seminal chain, the hinge loop is disordered. Despite the difference between the tertiary structure of the monomer and that of the chains in the MxM form, the active sites of the two enzymes are virtually indistinguishable. Furthermore, the structure of the liganded enzyme represents the first example of a ribonuclease complex studied at an alkaline pH and provides new information on the binding of a nucleotide when the catalytic histidines are deprotonated.