Immunity and tolerance to Aspergillus involve functionally distinct regulatory T cells and tryptophan catabolism

The inherent resistance to diseases caused by Aspergillus fumigatus suggests the occurrence of regulatory mechanisms that provide the host with adequate defense without necessarily eliminating the fungus or causing unacceptable levels of host damage. In this study, we show that a division of labor occurs between functionally distinct regulatory T cells (Treg) that are coordinately activated by a CD28/B-7-dependent costimulatory pathway after exposure of mice to Aspergillus conidia. Early in infection, inflammation is controlled by the expansion, activation and local recruitment of CD4+CD25+ Treg capable of suppressing neutrophils through the combined actions of IL-10 and CTLA-4 on indoleamine 2,3-dioxygenase. The levels of IFN-gamma produced in this early phase set the subsequent adaptive stage by conditioning the indoleamine 2,3-dioxygenase-dependent tolerogenic program of dendritic cells and the subsequent activation and expansion of tolerogenic Treg, which produce IL-10 and TGF-beta, inhibit Th2 cells, and prevent allergy to the fungus. The coordinate activation of Treg may, however, be subverted by the fungus, as germinating conidia are capable of interfering with anti-inflammatory and tolerogenic Treg programs. Thus, regulation is an essential component of the host response in infection and allergy to the fungus, and its manipulation may allow the pathogen to overcome host resistance and promote disease.     

Placenta-derived soluble MHC class I chain-related molecules down-regulate NKG2D receptor on peripheral blood mononuclear cells during human pregnancy: a possible novel immune escape mechanism for fetal survival

Mammalian pregnancy is an intriguing immunological phenomenon where the semiallogeneic fetus is not rejected. Tolerance toward the fetus involves a number of mechanisms associated with modifications of the immune status of the mother. In this study, we strongly suggest a novel mechanism for fetal evasion of maternal immune attack, based on the engagement and down-regulation of the activating NK cell receptor NKG2D on PBMC by soluble MHC class I chain-related proteins A and B (collectively termed MIC). A similar immune escape pathway was previously described in tumors. We found that MIC mRNA was constitutively expressed by human placenta and could be up-regulated upon heat shock treatment. Our immunomorphologic studies showed that the MIC expression in placenta was restricted to the syncytiotrophoblast. Immunoelectron microscopy revealed a dual MIC expression in the syncytiotrophoblast: on the apical and basal cell membrane and in cytoplasmic vacuoles as MIC-loaded microvesicles/exosomes. Soluble MIC molecules were present at elevated levels in maternal blood throughout normal pregnancy and were released by placental explants in vitro. Simultaneously, the cell surface NKG2D expression on maternal PBMC was down-regulated compared with nonpregnant controls. The soluble MIC molecules in pregnancy serum were able to interact with NKG2D and down-regulate the receptor on PBMC from healthy donors, with the consequent inhibition of the NKG2D-dependent cytotoxic response. These findings suggest a new physiological mechanism of silencing the maternal immune system that promotes fetal allograft immune escape and supports the view of the placenta as an immunoregulatory organ.     

Determinants of conformational dimerization of Mad2 and its inhibition by p31comet

The spindle assembly checkpoint (SAC) monitors chromosome attachment to spindle microtubules. SAC proteins operate at kinetochores, scaffolds mediating chromosome-microtubule attachment. The ubiquitous SAC constituents Mad1 and Mad2 are recruited to kinetochores in prometaphase. Mad2 sequesters Cdc20 to prevent its ability to mediate anaphase onset. Its function is counteracted by p31comet (formerly CMT2). Upon binding Cdc20, Mad2 changes its conformation from O-Mad2 (Open) to C-Mad2 (Closed). A Mad1-bound C-Mad2 template, to which O-Mad2 binds prior to being converted into Cdc20-bound C-Mad2, assists this process. A molecular understanding of this prion-like property of Mad2 is missing. We characterized the molecular determinants of the O-Mad2:C-Mad2 conformational dimer and derived a rationalization of the binding interface in terms of symmetric and asymmetric components. Mutation of individual interface residues abrogates the SAC in Saccharomyces cerevisiae. NMR chemical shift perturbations indicate that O-Mad2 undergoes a major conformational rearrangement upon binding C-Mad2, suggesting that dimerization facilitates the structural conversion of O-Mad2 required to bind Cdc20. We also show that the negative effects of p31comet on the SAC are based on its competition with O-Mad2 for C-Mad2 binding.     

Effect of anti-NGF on ovarian expression of alpha1- and beta2-adrenoceptors, TrkA, p75NTR, and tyrosine hydroxylase in rats with steroid-induced polycystic ovaries

Estradiol valerate (EV)-induced polycystic ovaries (PCO) in rats are associated with higher ovarian release and content of norepinephrine, decreased beta2-adrenoceptors (ARs), and dysregulated expression of alpha1-AR subtypes, all preceded by an increase in the production of ovarian NGF. The aim of this study was to further elucidate the role of NGF in the ovaries by blocking the action of NGF during development of EV-induced PCO in rats. Control and EV-injected rats were treated with intraperitoneal injections of IgG (control and PCO groups) or with anti-NGF antibodies (anti-NGF and PCO anti-NGF groups) every third day for 5 wk starting from the day of PCO induction. Rat weight, estrous cyclicity, ovarian morphology, ovarian mRNA, and protein expression of alpha1-AR subtypes, beta2-AR, the NGF receptor tyrosine kinase A (TrkA), p75 neurotrophin receptor (p75NTR), and tyrosine hydroxylase (TH) were analyzed. Ovaries in both PCO and PCO anti-NGF groups decreased in size as well as in number and size of corpora lutea. mRNA expression of alpha1a-AR and TrkA in the ovaries was lower, whereas expression of alpha1b- and alpha1d-AR and TH was higher, in the PCO group than in controls. Protein quantities of alpha1-ARs, TrkA, p75NTR, and TH were higher in the PCO group compared with controls, whereas the protein content of beta2-AR was lower. Anti-NGF treatment in the PCO group restored all changes in mRNA and protein content, except that of alpha1b-AR and TrkA mRNAs, to control levels. The results indicate that the NGF/NGF receptor system plays a role in the pathogenesis of EV-induced PCO in rats.     

Chaperone holdase activity of human papillomavirus E7 oncoprotein

E7 oncoprotein is the major transforming activity in human papillomavirus and shares sequence and functional properties with adenovirus E1A and SV40 T-antigen, in particular by targeting the pRb tumor suppressor. HPV 16 E7 forms spherical oligomers that display chaperone activity in thermal denaturation and chemical refolding assays of two model polypeptide substrates, citrate synthase and luciferase, and it does so at substoichiometric concentrations. We show that the E7 chaperone can stably bind model polypeptides and hold them in a state with significant tertiary structure, but does not bind the fully native proteins. The E7 oligomers bind native in vitro translated pRb without the requirement of it being unfolded, since the N-terminal domain of E7 containing the LXCXE binding motif is exposed. The N-terminal domain of E7 can interfere with pRb binding but not with the chaperone activity, which requires the C-terminal domain, as in most reported E7 activities. The ability to bind up to approximately 72 molecules of pRb by the oligomeric E7 form could be important either for sequestering pRb from Rb-E2F complexes or for targeting it for proteasome degradation. Thus, both the dimeric and oligomeric chaperone forms of E7 can bind Rb and various potential targets. We do not know at present if the chaperone activity of E7 plays an essential role in the viral life cycle; however, a chaperone activity may explain the large number of cellular targets reported for this oncoprotein.     

Binding, internalization and transport of apolipoprotein A-I by vascular endothelial cells

High density lipoproteins (HDL) and their main protein constituent, apolipoprotein A-I (apoA-I), exert potentially anti-atherogenic properties within the arterial wall. However, it is unknown how they are transported from the blood stream into the vascular wall. Here we investigated the interaction of apoA-I with endothelial cells. At 4 degrees C endothelial cells bound 125I-apoA-I with high affinity, Kd = 2.1 microg/ml and in a saturable manner (Bmax of 35 ng/mg cell protein). At 37 degrees C, the cell association of apoA-I revealed similar affinity as at 4 degrees C (Kd = 2.2 microg/ml) but the maximum specific cell association was much enhanced (Bmax = 360 ng/mg cell protein). Binding and cell association was competed by excess unlabeled apoA-I and HDL but not by albumin. Biotinylation experiments and electron microscopy studies showed that endothelial cells internalize labeled apoA-I. Only minor amounts of the internalized apoA-I were degraded. Cultivated in a Transwell system, the cells transported a fraction of 125I-apoA-I from the apical to the basolateral compartment in a competable and temperature-sensitive manner. Furthermore, after specific transport the originally prebeta-mobile and lipid-free apoA-I was recovered as particles which have electrophoretic alpha-mobility. We conclude that endothelial cells transcytose and lipidate lipid-free apoA-I.     

Transition metal derivatives of 1,10-phenanthroline-5,6-dione: Controlled growth of coordination polynuclear derivatives

The synthesis and the characterization of several mono- and polymetallic derivatives of 1,10-phenanthroline-5,6,-dione (1) are presented.The reaction of 1 with M(CO)₆ (M = Cr, Mo) gives compounds of general formula M(O,O′-C₁₂H₆N₂O₂)₃, M = Cr (2), Mo (3).Compound 3 is also obtained starting from Mo(η⁶-CH₃C₆H₅)₂, whereas the reaction of Cr(η⁶-CH₃C₆H₅)₂ with 1 affords the ionic derivative [Cr(η⁶-CH₃C₆H₅)₂][C₁₂H₆N₂O₂] (4), which has been studied by EPR spectroscopy and DFT calculations.FeCl₂(N,N′-C₁₂H₆N₂O₂)₂ (6), is obtained by thermal decomposition of [Fe(N,N′-C₁₂H₆N₂O₂)₃]Cl₂ (5).Polymetallic compounds of general formula Cr[O,O′-C₁₂H₆N₂O₂-N,N′-MCl₄]₃,containing chromium and a Group 4 element M = Ti (7), Zr (8), Hf (9), are prepared from Cr(O,O′-C₁₂H₆N₂O₂)₃ and the corresponding MCl₄ or MCl₄DME. Polynuclear derivatives of iron and chromium of formula [Fe(N,N′-C₁₂H₆N₂O₂-O,O′-CrCl₂(THF)₂)₃][PF₆]₂ (10), and Cr[O,O′-C₁₂H₆N₂O₂-N,N′-FeCl₂(THF)]₃ (11), are obtained by the reaction of [Fe(N,N′-C₁₂H₆N₂O₂)₃][PF₆]₂ with three equivalents of CrCl₂(THF)₂ and from Cr(O,O′-C₁₂H₆N₂O₂)₃ and FeCl₂(THF)_1.5, respectively. Compound 11 reacts with 1 (3 equivalents in sym-C₂H₂Cl₄ or 6 equivalents in ethanol) to give Cr[O,O′-C₁₂H₆N₂O₂-N,N′-FeCl₂(N,N′-C₁₂H₆N₂O₂)]₃ (12), and [Cr(O,O′-C₁₂H₆N₂O₂-N,N′-Fe(N,N′-C₁₂H₆N₂O₂)₂)₃]Cl₆ (13), respectively.     

A derivative of the glycopeptide A40926 produced by inactivation of the beta-hydroxylase gene in Nonomuraea sp. ATCC39727

The actinomycete Nonomuraea sp. ATCC39727 produces the glycopeptide A40926. In the corresponding dbv cluster, ORF28 encodes a putative hydroxylase. A gene replacement mutant of ORF28 in Nonomuraea produces a small amount of an A40926-related metabolite, 16 amu smaller than the parent compound, which was identified as the desoxyderivative of A40926 lacking the beta-hydroxyl group on the tyrosine moiety. This result demonstrates that ORF28 is actually involved in the formation of the beta-hydroxytyrosine residue present in A40926. The formation of an altered glycopeptide and the inability to rescue A40926 production upon feeding free beta-hydroxytyrosine are consistent with the possibility that, in contrast to balhimycin formation, hydroxylation occurs after tyrosine activation by the nonribosomal peptide synthetase.     

In vitro penetration of fresh and vitrified swine oocytes by homologous spermatozoa using different incubation systems

The present study consisted of two experiments. In the first one, ejaculates from four boars were used to compare in vitro penetration (IVP) rates of fresh and vitrified swine oocytes by homologous spermatozoa in four treatments: fresh oocytes in conventional incubation (CO2 incubator) (FC), vitrified oocytes in conventional incubation (VC), fresh oocytes in submarine (bag) incubation (FS) and vitrified oocytes in submarine incubation (VS). The IVP rates for FC, VC, FS and VS were 46.5, 44.3, 36.9 and 33.1%, respectively. Analysis through Chi-square tests identified no differences in IVP rates between FC and VC and between FS and VS (P > 0.05), but IVP rate for FC was greater (P < 0.05) than those for both FS and VS. Besides IVP rate for VC did not differ (P > 0.05) from those for FC and FS, but it was greater than that for VS (P < 0.05). Logistic regression analysis identified differential effects of treatments dependant on individual boars. The second experiment evaluated the influence of semen storage period on the semen quality of the two boars associated with greater IVP rate in the first experiment. Semen quality was estimated by IVP rate using the VC treatment and by the following methods: sperm motility, sperm morphology, hypoosmotic swelling test (HOST) and thermal stress test (TST). According to analysis using Chi-square tests, IVP rate did not differ (P > 0.05), for the first boar, between 0 (100.0%) and 24 h of semen storage (98.1%) nor after 48 and 72 h (66.0 and 59.3%, respectively), but IVP rates were greater during the 0-24 h period compared with the 48-72 h period (P < 0.05). For the second boar, IVP rate at 0 h (50.6%) was greater (P < 0.05) than at 24, 48 and 72 h of semen storage (34.3, 28.3 and 24.0%, respectively), with no further differences observed after 24 h (P > 0.05). Logistic regression analysis identified that the effect of storage on IVP rate was influenced by the effect of individual boars. No differences in semen quality during the storage period were identified by conventional methods of semen evaluation, for either boar (P > 0.05) using analysis of variance with repeated measures. These results indicate that IVP test can be used to estimate boar fertility, even when vitrified oocytes are used (if using conventional CO2 incubators) or using an alternative submarine incubation system (if using fresh oocytes). The IVP test was the only method of semen evaluation that identified the reduction in semen quality up to 72 h of storage.