Protein Adsorption on Mixed Self-Assembled Monolayers: Influence of Chain Length and Terminal Group.

Mixed self-assembled monolayers (SAMs) are often used as highly tunable substrates for biomedical and biosensing applications. It is well documented, however, that mixed SAMs can be highly disordered at the molecular level and do not pack as closely or homogeneously as single-component SAMs, particularly when the chain lengths and head groups of the SAM thiol components are significantly different. In this study, we explore the impact of SAM structure and mixing ratio (-OH and -CH3 termini) on the weak physisorption behavior of bovine serum albumin (BSA), which adsorbs more readily to hydrophobic, methyl-terminated SAMs. Our results suggest that once the mixture includes 50% or more of the methyl terminus, the mixing ratio alone is a relatively good predictor of adsorption, regardless of the relative chain lengths of the thiols used in the mixture. This trend persists at any mixing ratio for SAMs where methyl- and hydroxyl-terminated groups are the same length or where the hydroxyl-terminated thiol is longer. The only variance observed is at low mixing ratios (<50% methyl-terminated) for a mixed SAM where the methyl-terminated component has a longer chain length. Relative protein adsorption increases on these mixtures, perhaps due to the disordered exposure of the excess alkane backbone. Taken together, however, we do not find significant evidence that varying chain lengths for mixed SAMs prepared on polycrystalline substrates and analyzed in air have an outsized influence on nanoscopic adsorption behavior, despite molecular-level disorder in the SAM itself.

Oxidative Destruction of Multilayer Diisopropyl Methylphosphonate Films by O(3P) Atomic Oxygen.

We present work detailing the oxidative destruction of the nerve agent simulant diisopropyl methylphosphonate (DIMP) with O(3P) using time-resolved, in situ reflection absorption infrared spectroscopy (RAIRS) and X-ray photoelectron spectroscopy (XPS). Thermally annealed DIMP films deposited on Au(111) are observed to react upon exposure to a supersonic beam containing O(3P) with average translational energies of 0.12 eV. The reaction is initiated by a hydrogen abstraction from one of three possible sites on DIMP, and then progresses through various secondary reactions with resultant hydroxyl radicals, carbon-centered DIMP-derived radicals, and nondissociated O2 in the beam. These reactions are accompanied by uptake of oxygen into the film, leading to new hydrogen bonding with the DIMP phosphoryl group. The generated product also presents greater thermal stability than pristine DIMP, suggesting the formation of a distribution of oligomeric and polymeric products. As reactivity is observed to decrease upon continued O(3P) exposure, this product likely forms a protective layer at the vacuum-film interface, hindering destruction of thicker films. Importantly, the rate of reaction and general reactivity trends are the same between DIMP and the smaller simulant dimethyl methylphosphonate (DMMP). The comparable reaction rates of the two molecules coupled with oxygen's inability to erode thick films all the way down to the substrate have specific implications for the development of oxidation-based decontamination strategies for these and other organophosphates in the solid phase. The findings presented in this paper add significant new fundamental understanding of the oxidative chemistry of such species, knowledge needed in order to develop efficacious nerve agent decontamination strategies as well as the refinement of existing models for the dispersal, adsorption, persistence, and destruction of organophosphates in the environment.

Pneumococcal polysaccharide vaccination induces polysaccharide-specific B cells in adult peripheral blood expressing CD19⁺CD20⁺CD3⁻CD70⁻CD27⁺IgM⁺CD43⁺CD5⁺/⁻.

Pneumococcal polysaccharide vaccines have been used to elicit a protective anti-pneumococcal polysaccharide antibody response against Streptococcus pneumoniae in healthy individuals. Identifying human B cells which respond to T-cell independent type-2 antigens, such as pneumococcal polysaccharides, has been challenging. We employed pneumococcal polysaccharides directly conjugated to fluorophores in conjunction with flow cytometry to identify the phenotype of B cells that respond to pneumococcal polysaccharide vaccination. We have previously identified that the majority of pneumococcal polysaccharide-selected cells responding to vaccination are CD27(+)IgM(+) (IgM(+) memory) cells. In this study, we further characterized pneumococcal polysaccharide-selected cells in the peripheral blood to better identify how the various B cell phenotypes responded 7 and 30 days post-immunization. We show that 7 days post-immunization the majority of pneumococcal polysaccharide-selected IgM(+) memory cells (PPS14(+) 56.5%, PPS23F(+) 63.8%) were CD19(+)CD20(+)CD27(+)IgM(+)CD43(+)CD5(+/-)CD70(-), which was significantly increased compared to pre-immunization levels. This phenotype is in alignment with recent publications describing human B-1 cells. PPS-responsive B cells receded to pre-immunization levels by day-30. These findings suggest that this B-1 like cell population plays an important role in early responses to S. pneumoniae infection and possibly other T-cell independent type-2 antigens in humans.

The immune response to pneumococcal polysaccharides 14 and 23F among elderly individuals consists predominantly of switched memory B cells.

The phenotype of B cells that respond to vaccination with the purified pneumococcal polysaccharide (PPS) has been a topic of debate. We have recently identified the phenotype of cells from healthy young volunteers as CD27(+)IgM(+) B cells. However, the PPS-responding B-cell population has not yet been identified in high-risk populations, such as elderly individuals. Previous studies have shown that elderly individuals have a lower percentage of immunoglobulin M memory B cells than healthy young adults. In this study, we directly characterized the phenotype of PPS-specific B cells before and after vaccination with PPS vaccine (PPV) in elderly adults, using fluorescently labeled PPS14 and PPS23F. In contrast to our observations in healthy young volunteers, the PPS-responding B-cell population consisted primarily of switched memory (CD27(+)IgM(-)) B cells. In concurrence with these findings, postvaccination immunoglobulin M concentrations were not significantly increased in this population, and the opsonophagocytic response was decreased, compared with that in young adults. These findings identify a significant shift in the phenotype of the B-cell population in response to PPV among elderly individuals.

Phenotypic analysis of pneumococcal polysaccharide-specific B cells.

The phenotype of B cells responsible for the production of anti-pneumococcal polysaccharide Ab has been unclear. Although individuals that respond poorly to the 23-valent pneumococcal polysaccharide (PPS) vaccine, Pneumovax, such as children <2 y, the asplenic, and a subset of common variable immunodeficiency patients, are profoundly deficient or lack IgM memory cells (CD27(+)IgM(+)), they are also deficient in the switched memory (CD27(+)IgM(-)) compartment. Direct characterization of PPS-specific B cells has not been performed. In this study, we labeled PPS14 and PPS23F with fluorescent markers. Fluorescently labeled PPS were used in FACSAria flow cytometry to characterize the phenotype of PPS-specific B cells obtained from 18 young adults pre- and postimmunization with Pneumovax. The labeled PPS were capable of inhibiting binding of Ab to the native PPS. Similarly, the native PPS were able to inhibit binding of PPS-specific B cells in a flow cytometric assay demonstrating specificity and functionality. Phenotypic analysis of unselected B cells, pre- and postimmunization, demonstrated a predominance of naive CD27(-)IgM(+) cells accounting for 61.5% of B cells. Likewise, the PPS-specific B cells obtained preimmunization consisted primarily of naive, CD27(-) B cells, 55.4-63.8%. In contrast, the PPS-specific B cells obtained postimmunization were predominantly IgM memory cells displaying the CD27(+)IgM(+), 54.2% for PPS14 and 66% for PPS23F, significantly higher than both unselected B cells and PPS-specific B cells. There was no significant difference in switched memory B cell populations (CD27(+)IgM(-)) between groups. These results suggest a dominant role of IgM memory cells in the immune response to pneumococcal polysaccharides.