Origination of New Immunological Functions in the Costimulatory Molecule B7-H3: The Role of Exon Duplication in Evolution of the Immune System

B7-H3, a recently identified B7 family member, has different isoforms in human and mouse. Mouse B7-H3 gene has only one isoform (2IgB7-H3) with two Ig-like domains, whereas human B7-H3 has two isoforms (2IgB7-H3 and 4IgB7-H3). In this study a systematic genomic survey across various species from teleost fishes to mammals revealed that 4IgB7-H3 isoform also appeared in pigs, guinea pigs, cows, dogs, African elephants, pandas, megabats and higher primate animals, which resulted from tandem exon duplication. Further sequence analysis indicated that this duplication generated a new conserved region in the first IgC domain, which might disable 4IgB7-H3 from releasing soluble form, while 2IgB7-H3 presented both membrane and soluble forms. Through three-dimensional (3D) structure modeling and fusion-protein binding assays, we discovered that the duplicated isoform had a different structure and might bind to another potential receptor on activated T cells. In T cell proliferation assay, human 2IgB7-H3 (h2IgB7-H3) and mouse B7-H3 (mB7-H3) both increased T cell proliferation and IL-2, IFN-γ production, whereas human 4IgB7-H3 (h4IgB7-H3) reduced cytokine production and T cell proliferation compared to control. Furthermore, both h2IgB7-H3 and mB7-H3 upregulated the function of lipopolysacharide (LPS)-activated monocyte in vitro. Taken together, our data implied that during the evolution of vertebrates, B7-H3 exon duplication contributed to the generation of a new 4IgB7-H3 isoform in many mammalian species, which have carried out distinct functions in the immune responses

Predicting olfactory receptor neuron responses from odorant structure

Background Olfactory receptors work at the interface between the chemical world of volatile molecules and the perception of scent in the brain. Their main purpose is to translate chemical space into information that can be processed by neural circuits. Assuming that these receptors have evolved to cope with this task, the analysis of their coding strategy promises to yield valuable insight in how to encode chemical information in an efficient way. Results We mimicked olfactory coding by modeling responses of primary olfactory neurons to small molecules using a large set of physicochemical molecular descriptors and artificial neural networks. We then tested these models by recording in vivo receptor neuron responses to a new set of odorants and successfully predicted the responses of five out of seven receptor neurons. Correlation coefficients ranged from 0.66 to 0.85, demonstrating the applicability of our approach for the analysis of olfactory receptor activation data. The molecular descriptors that are best-suited for response prediction vary for different receptor neurons, implying that each receptor neuron detects a different aspect of chemical space. Finally, we demonstrate that receptor responses themselves can be used as descriptors in a predictive model of neuron activation. Conclusions The chemical meaning of molecular descriptors helps understand structure-response relationships for olfactory receptors and their 'receptive fields'. Moreover, it is possible to predict receptor neuron activation from chemical structure using machine-learning techniques, although this is still complicated by a lack of training data

Intradermal Electroporation of Naked Replicon RNA Elicits Strong Immune Responses

RNA-based vaccines represent an interesting immunization modality, but suffer from poor stability and a lack of efficient and clinically feasible delivery technologies. This study evaluates the immunogenic potential of naked in vitro transcribed Semliki Forest virus replicon RNA (RREP) delivered intradermally in combination with electroporation. Replicon-immunized mice showed a strong cellular and humoral response, contrary to mice immunized with regular mRNA. RREP-elicited induction of interferon-γ secreting CD8+ T cells and antibody responses were significantly increased by electroporation. CD8+ T cell responses remained substantial five weeks post vaccination, and antigen-specific CD8+ T cells with phenotypic characteristics of both effector and central memory cells were identified. The immune response during the contraction phase was further increased by a booster immunization, and the proportion of effector memory cells increased significantly. These results demonstrate that naked RREP delivered via intradermal electroporation constitute an immunogenic, safe and attractive alternative immunization strategy to DNA-based vaccines

Investigation of the retention/pH profile of zwitterionic fluoroquinolones in reversed-phase and ion-interaction high performance liquid chromatography

The retention/pH profiles of three fluoroquinolones, ofloxacin, norfloxacin and ciprofloxacin, was investigated by means of reversed-phase high performance liquid chromatography (RP-HPLC) and reversed-phase ion-interaction chromatography (RP-IIC), using an octadecylsilane stationary phase and acetonitrile as organic modifier. Sodium hexanesulphonate and tetrabutylammonium hydroxide were used as sources of counter ions in ion-interaction chromatography. The retention/pH profiles under in RP-HPLC were compared to the corresponding lipophilicity/pH profiles. Despite the rather hydrophilic nature of the three fluoroquinolones positive retention factors were obtained while there was a shift of the retention maximum towards more acidic pH values. This behavior was attributed mainly to non-hydrophobic silanophilic interactions with the silanized silica gel material of the stationary phase. In ion-interaction chromatography the effect of counter ions over a broad pH range was found to be ruled rather by the ion pair formation in the mobile phase which led to a drastic decrease in retention as a consequence of the disruption of the zwitterionic structure and thereupon the deliberation of a net charge in the molecules. At pH values at which zwitterionic structure was not favored both the ion-exchange and ion pair formation mechanisms were assumed to contribute to the retention. © 2005 Elsevier B.V. All rights reserved

Retention of structurally diverse drugs in human serum albumin chromatography and its potential to simulate plasma protein binding

The retention behavior of 39 structurally diverse neutral, basic and acidic drugs was investigated on an HSA stationary phase using PBS buffer (pH 7.0) and acetonitrile or 2-propanol as organic modifiers. Extrapolated or directly measured logkw values as well as isocratic retention factors were correlated with plasma protein binding data taken from the literature. Retention factors determined in the presence of 10% acetonitrile led to high quality 1:1 correlation with apparent logKHSA values. The derived reference equation was successfully validated using a secondary set of 24 drugs. Further analysis of HSA retention into more fundamental properties revealed the involvement of anionic species in solute-stationary phase interactions, expressed by the negatively charged fraction, besides the partitioning mechanism which was reflected by lipophilicity. Protonation of basic drugs, although less important, may also influence retention, leading to reduced partitioning into the HSA surface as a net effect, while it seems to have no effect on HSA binding. The above results were further confirmed by linear solvation energy relationships (LSER). © 2010 Elsevier B.V

The effect of ion pairing reagents in the retention profile of zwitterionic cephalosporins

The retention of three zwitterionic cephalosporins, Cefepime, Cefpirome, and Ceftazidime, in a broad pH range was studied by means of reversed phase high performance liquid chromatography (RP-HPLC) and reversed phase ion-pair liquid chromatography (RP-IPC), using an octadecylsilane stationary phase and acetonitrile as organic modifier. Sodium hexane-, heptane-, and octane-sulphonate and tetrabutylammonium hydroxide were used as sources of counter ions in ion pair chromatography. The presence of the permanently charged quaternary nitrogen in the cephalosporin molecules leads to zwitterionic species over a broad pH range, masking the carboxylic acid function and stabilizing the retention. The effect of the counter ion depends on the ionization state of the compounds. Formation of ion pairs with the opposite charged center of the molecules led to significant increase in retention. At pH values favoring the zwitterionic species a decrease in retention was observed as a result of the disruption of the intramolecular interactions and the deliberation of the free charge. The extent of ion pair formation and the competition with the zwitterionic species, depended on the counter ion concentration in the mobile phase and was reflected in retention. The effect of the size of the counter anion alkyl chain on retention was investigated at different pH as well

Lipophilicity, biomimetic retention profile and antioxidant activity of selenium species

The lipophilicity and biomimetic retention profile of three selenium species, methyl-seleninic acid (MSA), Se-Methyl-selenocysteine (MeSeC) and dimethyl-selenourea (DMeSeU), were established. Lipophilicity was measured by the shaking flask method, while for chromatography one reversed-phase (Discovery C-18) column and three biomimetic stationary phases, namely immobilized artificial membrane (IAM), human serum albumin (HSA) and α1-acid glycoprotein (AGP) were employed, under different mobile phase conditions. These results were combined with previous data obtained under analogous conditions on eight selenium species including selenites (Se(IV)), selenates (Se(VI)), selenomethionine (SeMet), selenocystine (SeCyst), selenocystamine (SeCM), selenourea (SeU), dimethylselenide ((CH3)2Se) and dimethyldiselenide ((CH3)2Se2). The combined data were further used to establish relations between biomimetic retention factors and lipophilicity, the effect of the presence of reduced glutathione (GSH) to retention, as well as to determine the oxidation potential by means of cyclic voltammetry. According to their anode potentials in cyclic voltammetry, only the urea analogues, SeU and, especially, DMeSeU, exhibited considerable antioxidant properties. Moreover IAM retention of Se(IV), Se(VI), SeMet and MeSeC was correlated with Caco-2 apparent permeability coefficient (Papp) available in literature. IAM retention data were further converted to percentage of human oral absorption according to a relevant equation reported in the literature, while from HSA retention results the plasma protein binding was estimated. According to these values, low biopersistence in the human body may be anticipated. © 2013 Elsevier B.V

Exploring the elution mechanism of selenium species on liquid chromatography

In the present work, the chromatographic behavior of eight selenium species, namely selenites (Se(IV)), selenates (Se(VI)), seleno-DL-methionine (Se-Met), selenocystine (Se-Cyst), selenocystamine (Se-CM), selenourea (Se-U), dimethylselenide ((CH3)2Se) and dimethyldiselenide ((CH3)2Se2), was investigated under different stationary and mobile phase conditions, in an effort to unravel secondary interferences in their underlying elution mechanism. For this purpose, two end-capped and a polar-embedded reversed-phase stationary phases were employed using different mobile phase conditions. Retention factors (log kw) were compared with octanol-water distribution coefficients (log D) as well as with log kw values on two immobilized artificial membrane (IAM) columns and two immobilized artificial plasma proteins stationary phases, obtained in our previous work. The role of electrostatic interactions was confirmed by introducing the net charge of the investigated Se species as an additional term in the log kw/log D interrelation, which in most cases proved to be statistically significant. Principal component analysis of retention factors on all stationary phases and octanol-water log D values, however, showed that the elution of the investigated selenium species is mainly governed by partitioning mechanism under all different chromatographic conditions, while the pH of the mobile phase and the special column characteristics have only a minor effect. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim