HLA class II alleles and measles virus-specific cytokine immune response following two doses of measles vaccine

Measles virus-specific T cells and the production of cytokines play a critical role in the immune response following measles immunization. To understand the genetic factors that influence variation in IFN- and IL-4 responses following measles immunization and to provide insight into the factors influencing both cellular and humoral immunity to measles, we assessed associations between human leukocyte antigen (HLA) class II genes and measles-specific Th1 and Th2-type cytokine responses in peripheral blood lymphocytes from 339 children previously vaccinated with two doses of measles-mumps-rubella vaccine (MMR-II). Median values for measles-specific IFN- and IL-4 secretion levels were 40.73 and 9.71 pg/ml, respectively. The global tests suggested associations between measles-specific IFN- response and alleles of the DRB1 and DQB1 loci (P=0.07 and P=0.02, respectively). Specifically, DRB1*0301, *0901, and *1501 alleles were significantly associated with IFN- secretion. The alleles that suggested evidence of an HLA association with IL-4 secretion were DRB1*0103, *0701, and *1101. Th1 cytokine responses and DQB1 allele associations revealed that the alleles with the strongest association with IFN- secretion were DQB1*0201, *0303, *0402, and *0602. Specific alleles with a suggestive association with low measles-specific Th2 cytokine responses were DQB1*0202 and *0503. In addition, DPB1*0101, *0201, and *0601 alleles provided suggestive evidence of an HLA association with measles-induced IFN- response, while DPB1*0501 was associated with an IL-4 response. These data suggest that IFN- and IL-4 cytokine responses to measles may be genetically restricted in part by HLA class II genes, which in turn can restrict the cellular immune response to measles vaccine.     

Immunologic significance of HLA class I genes in measles virus-specific IFN-γ and IL-4 cytokine immune responses

The variability of immune responses modulated by human leukocyte antigen (HLA) genes and secreted cytokines is a significant factor in the development of a protective effect of measles vaccine. We studied the association between type 1 helper T cells (Th1)- and Th2-like cytokine immune responses and HLA class I alleles among 339 schoolchildren who previously received two doses of the measles vaccine. Median values for measles-specific interferon gamma (IFN-γ) and interleukin-4 (IL-4) cytokines were 40.7 pg/ml [interquartile range (IQR) 8.1–176.7] and 9.7 pg/ml (IQR 2.8–24.3), respectively. Class I HLA-A (*0101 and *3101) and HLA-Cw (*0303 and *0501) alleles were significantly associated with measles-virus-induced IFN-γ secretion. HLA-A*3101 and Cw*0303 were associated with a higher median IFN-γ response, while A*0101 and Cw*0501 were associated with lower measles-specific IFN-γ response. We found limited associations between HLA class I gene polymorphisms and Th2-like (IL-4) immune responses after measles vaccination, indicating that HLA class I molecules may have a limited effect on measles-vaccine-induced IL-4 secretion. Understanding the genetic factors that influence variations in cytokine secretion following measles vaccination will provide insight into the factors that influence both cell-mediated and humoral immunity to measles.     

X-ray microanalysis of airway surface liquid in the mouse

The ionic composition of airway surface liquid (ASL) has been debated, and, in particular for the mouse, a wide range of values has been published. Two techniques were developed to measure the elemental composition of the ASL. X-ray microanalysis of ASL was carried out at low temperature on trachea removed from isoflurane-anesthetized animals and shock-frozen. In the second technique, dextran beads were placed on top of the epithelium of the trachea removed from pentobarbital-anesthetized animals, left to equilibrate with the ASL, dried, and subjected to X-ray microanalysis. Both techniques showed that mouse tracheal ASL has significantly lower concentrations of Na and Cl (approximately 60-80 mM) than serum. Differences between the two techniques were due to different sampling of mucus. CFTR(-/-) mice had significantly higher concentrations of Na and Cl in their ASL than age-matched controls. Pilocarpine or isoproterenol stimulation significantly reduced the ion concentrations in tracheal ASL. ASL was also collected with the dextran bead method from the nasal cavity in situ in pentobarbital-anesthetized animals. In control animals, the elemental composition of nasal fluid was similar to that of tracheal ASL. Pilocarpine stimulation caused a significant increase in Na, Cl, and K; stimulation with isoproterenol or phenylephrine caused a significant increase only in K. It is concluded that mouse ASL under unstimulated conditions is hypotonic, which may be related to the relative paucity of submucosal glands in the mouse trachea.     

Palm tree peroxidase-based biosensor with unique characteristics for hydrogen peroxide monitoring

Three amperometric enzyme electrodes have been constructed by adsorbing anionic royal palm tree peroxidase (RPTP), anionic sweet potato peroxidase (SPP), or cationic horseradish peroxidase (HRP-C) on spectroscopic graphite electrodes. The resulting H(2)O(2)-sensitive biosensors were characterized both in a flow injection system and in batch mode to evaluate their main bioelectrochemical parameters, such as pH dependency, I(max), K(M)(app), detection limit, linear range, operational and storage stability. The obtained results showed a distinctly different behavior for the plant peroxidase electrodes, demonstrating uniquely superior characteristics of the RPTP-based sensors. The broader linear range observed for the RPTP-based biosensor is explained by a high stability of this enzyme in presence of H(2)O(2). The higher storage and operational stability of RPTP-based biosensor as well as its capability to measure hydrogen peroxide under acidic conditions connect with an extremely high thermal and pH-stability of RPTP.     

Src64 is involved in fusome development and karyosome formation during Drosophila oogenesis

Src family tyrosine kinases respond to a variety of signals by regulating the organization of the actin cytoskeleton. Here, we show that during early oogenesis Src64 mutations lead to uneven accumulation of cortical actin, defects in fusome formation, mislocalization of septins, defective transport of Orb protein into the oocyte, and possible defects in cell division. Similar mutant phenotypes suggest that Src64, the Tec29 tyrosine kinase, and the actin crosslinking protein Kelch act together to regulate actin crosslinking, much as they do later during ring canal growth. Condensation of the oocyte chromatin into a compact karyosome is also defective in Src64, Tec29, and kelch mutants and in mutants for spire and chickadee (profilin), genes that regulate actin polymerization. These data, along with changes in G-actin accumulation in the oocyte nucleus, suggest that Src64 is involved in a nuclear actin function during karyosome condensation. Our results indicate that Src64 regulates actin dynamics at multiple stages of oogenesis.     

Trapping crystal nucleation of cholesterol monohydrate: relevance to pathological crystallization

Crystalline nucleation of cholesterol at the air-water interface has been studied via grazing incidence x-ray diffraction using synchrotron radiation. The various stages of cholesterol molecular assembly from monolayer to three bilayers incorporating interleaving hydrogen-bonded water layers in a monoclinic cholesterol.H(2)O phase, has been monitored and their structures characterized to near atomic resolution. Crystallographic evidence is presented that this multilayer phase is similar to that of a reported metastable cholesterol phase of undetermined structure obtained from bile before transformation to the triclinic phase of cholesterol.H(2)O, the thermodynamically stable macroscopic form. According to grazing incidence x-ray diffraction measurements and crystallographic data, a transformation from the monoclinic film structure to a multilayer of the stable monohydrate phase involves, at least initially, an intralayer cholesterol rearrangement in a single-crystal-to-single-crystal transition. The preferred nucleation of the monoclinic phase of cholesterol.H(2)O followed by transformation to the stable monohydrate phase may be associated with an energetically more stable cholesterol bilayer arrangement of the former and a more favorable hydrogen-bonding arrangement of the latter. The relevance of this nucleation process of cholesterol monohydrate to pathological crystallization of cholesterol from cell biomembranes is discussed.     

Identification of class II HLA-DRB1*03-bound measles virus peptides by 2D-liquid chromatography tandem mass spectrometry

Two-dimensional liquid chromatography (2D-LC), combined with gas phase fractionation tandem mass spectrometry, was used to identify 13 naturally processed peptides originating from measles virus that were presented by HLA-DRB1*03 class II molecules. The peptides are from three of the six measles structural proteins: phosphoprotein, nucleocapsid, and hemagglutinin. These peptides provide an important first step toward understanding the mechanism of immune response to measles virus and development of a new generation of peptide-based vaccines.     

Serine racemase modulates intracellular D-serine levels through an alpha,beta-elimination activity

Mammalian brain contains high levels of d-serine, an endogenous co-agonist of N-methyl D-aspartate type of glutamate receptors. D-Serine is synthesized by serine racemase, a brain enriched enzyme converting L- to D-serine. Degradation of D-serine is achieved by D-amino acid oxidase, but this enzyme is not present in forebrain areas that are highly enriched in D-serine. We now report that serine racemase catalyzes the degradation of cellular D-serine itself, through the alpha,beta-elimination of water. The enzyme also catalyzes water alpha,beta-elimination with L-serine and L-threonine. alpha,beta-Elimination with these substrates is observed both in vitro and in vivo. To investigate further the role of alpha,beta-elimination in regulating cellular D-serine, we generated a serine racemase mutant displaying selective impairment of alpha,beta-elimination activity (Q155D). Levels of D-serine synthesized by the Q155D mutant are several-fold higher than the wild-type both in vitro and in vivo. This suggests that the alpha,beta-elimination reaction limits the achievable D-serine concentration in vivo. Additional mutants in vicinal residues (H152S, P153S, and N154F) similarly altered the partition between the alpha,beta-elimination and racemization reactions. alpha,beta-Elimination also competes with the reverse serine racemase reaction in vivo. Although the formation of L- from D-serine is readily detected in Q155D mutant-expressing cells incubated with physiological D-serine concentrations, reversal with wild-type serine racemase-expressing cells required much higher D-serine concentration. We propose that alpha,beta-elimination provides a novel mechanism for regulating intracellular D-serine levels, especially in brain areas that do not possess D-amino acid oxidase activity. Extracellular D-serine is more stable toward alpha,beta-elimination, likely due to physical separation from serine racemase and its elimination activity.