Mary Alice Garlipp

Impaired Phagocytosis of Nontypeable Haemophilus influenzae by Human Alveolar Macrophages in Chronic Obstructive Pulmonary Disease

BACKGROUND Interactions of nontypeable Haemophilus influenzae (NTHI) with human alveolar macrophages are implicated in the persistence of NTHI in chronic obstructive pulmonary disease (COPD). However, the immunologic mechanisms that mediate NTHI-induced macrophage responses are poorly understood. We hypothesized that immunologic responses of alveolar macrophages to NTHI are impaired in COPD. METHODS Blood and alveolar macrophages--obtained from ex-smokers with COPD (n = 14), ex-smokers without COPD (n = 15), and nonsmokers (n = 9)--were incubated with 3 distinct NTHI strains obtained from patients with COPD. Phagocytosis of 3H-NTHI, expressed as a percentage of the mean total radioactivity, and of intracellular viability, assessed as a percentage of viable cell-associated NTHI, were measured. RESULTS Alveolar macrophages from donors with COPD, compared with those from donors without COPD, had impaired phagocytosis (median [interquartile range]) for each NTHI strain: 14P13H5, 0.26 (0.08-0.61) versus 1.36 (0.69-1.95); 6P5H1, 0.92 (0.32-1.82) versus 1.90 (1.32-2.68); and 14P14H1, 0.79 (0.23-1.32) versus 2.13 (1.13-2.40) (P < or = .01 for each). However, phagocytosis of all NTHI strains by blood macrophages from donors with COPD was indistinguishable from that of blood macrophages from donors without COPD and from nonsmokers. The intracellular killing of NTHI was not impaired in alveolar macrophages from donors with COPD. CONCLUSIONS These results support a paradigm of impaired phagocytosis by alveolar macrophages, but not blood macrophages, in COPD and provide an immunologic basis for persistence of NTHI in the airways of adults with COPD.

Retinol-Binding Site in Interphotoreceptor Retinoid-Binding Protein (IRBP): A Novel Hydrophobic Cavity

PURPOSE Interphotoreceptor retinoid-binding protein (IRBP) appears to target and protect retinoids during the visual cycle. X-ray crystallographic studies had noted a betabetaalpha-spiral fold shared with crotonases and C-terminal protein transferases. The shallow cleft formed by the fold was assumed to represent the retinol-binding site. However, a second hydrophobic site consisting of a highly restricted cavity was more recently appreciated during in silico ligand-docking studies. In this study, the ligand-binding environment within the second module of Xenopus IRBP (X2IRBP) is defined. METHODS Pristine recombinant polypeptide corresponding to X2IRBP was expressed in a soluble form and purified to homogeneity without its fusion tag. Phenylalanine was substituted for tryptophan at each of the putative retinol-binding domains (W450F, hydrophobic cavity; W587F, shallow cleft). Binding of 11-cis and all-trans retinol were observed in titrations monitoring retinol fluorescence enhancement, quenching of tryptophan fluorescence, and energy transfer. The effect of oleic acid on retinol binding was also examined. RESULTS A ligand-binding stoichiometry of approximately 1:1 was observed for 11-cis and all-trans with K(d) in the tens of nanomolar range. The substitution mutants showed little effect on retinol binding in titrations after fluorescence enhancement. However, the W450F and not the W587F mutant showed a markedly reduced capacity for fluorescence quenching for both 11-cis and all-trans retinol. Oleic acid inhibited the binding of 11-cis and all-trans retinol in an apparent noncompetitive manner. CONCLUSIONS The binding site for 11-cis and all-trans retinol is a novel hydrophobic cavity that is highly restrictive and probably distinct from the long chain fatty acid-binding site.

Cone outer segment extracellular matrix as binding domain for interphotoreceptor retinoid-binding protein

Cones are critically dependent on interphotoreceptor retinoid‐binding protein (IRBP) for retinoid delivery in the visual cycle. Cone‐dominant vertebrates offer an opportunity to uncover the molecular basis of IRBPs role in this process. Here, we explore the association of IRBP with the interphotoreceptor matrix (IPM) of cones vs. rods in cone dominant retinas from chicken (Gallus domesticus), turkey (Meleagris gallopavo), and pig (Sus scrofa). Retinas were detached and fixed directly or washed in saline prior to fixation. Disassociated photoreceptors with adherent matrix were also prepared. Under 2 mM CaCl2, insoluble matrix was delaminated from saline washed retinas. The distribution of IRBP, as well as glycans binding peanut agglutinin (cone matrix) and wheat germ agglutinin (rod/cone matrix), was defined by confocal microscopy. Retina flat mounts showed IRBP diffusely distributed in an interconnecting, lattice‐like pattern throughout the entire matrix. Saline wash replaced this pattern with fluorescent annuli surrounding individual cone outer segments. In isolated cones and matrix sheets, IRBP colocalized with the peanut agglutinin binding matrix glycans. Our results reveal a wash‐resistant association of IRBP with a matrix domain immediately surrounding cone outer segments. The cone matrix sheath may be responsible for IRBP‐mediated cone targeting of 11‐cis retinoids. J. Comp. Neurol., 2012.

Pericellular interphotoreceptor matrix dictates outer retina critical surface tension

Abstract Retinal detachments create two pathological surfaces, the surface of the outer neural retinal, and an apical retinal‐pigmented epithelium (RPE) surface. The physicochemical properties of these two new surfaces are poorly understood. At a molecular level little is known how detachments form, how to optimize reattachment, or prevent extension of the detachment. A major limitation is lack of information about the biophysical consequences of the retina–RPE separation. The primary challenge is determining the molecular properties of the pathological interface surfaces. Here, using detached bovine retina, we show that this hurdle can be overcome through a combination of biophysical and ultrastructural approaches. The outer surface of freshly detached bovine neural retina, and isolated molecular components of the outer retina were subjected to: 1) Contact angle goniometry to determine the critical surface tension of the outer retinal surface, isolated insoluble interphotoreceptor matrix (IPM) and purified interphotoreceptor retinoid binding protein (IRBP); 2) Multiple attenuated internal reflectance infrared (MAIR‐IR) spectroscopy was used to characterize the molecular composition of the retinal surface. MAIR‐IR depth penetration was established through ellipsometric measurement of barium‐stearate films. Light microscopy, immunohistochemistry and electron microscopy defined the structures probed spectroscopically. Furthermore, the data were correlated to IR spectra of docosahexaenoic acid, hyaluronan, chondroitin‐6‐sulfate and IRBP, and imaging by IR‐microscopy. We found that the retinal critical surface tension is 24 mN/m, similar to isolated insoluble IPM and lower than IRBP. Barium‐stearate calibration studies established that the MAIR‐IR spectroscopy penetration depth was 0.2 &mgr;m. Ultrastructural observations and MAIR‐IR studies of isolated outer retina components determined that the pericellular IPM coating the outer retinal surface is primarily responsible for these surface properties. The critical surface tension of detached bovine retina is dictated not by the outer segments, but by a pericellular IPM covering the outer segment tips. HighlightsPhysiochemical and structural properties of the detached bovine outer retinal surface were accessed.A 0.28 &mgr;m thick matrix of critical surface tension 24 mN/m, typical of low adhesive “Theta Surfaces”, coats the retina.Multiple attenuated internal reflectance infrared (MAIR‐IR) spectroscopy probes the surface to a depth of 0.2 &mgr;m.The pericellular matrix contains chondroitin‐6 sulfate, interphotoreceptor retinoid‐binding protein, but not DHA.The critical surface tension of detached retina is dictated by a pericellular matrix covering the outer segment tips.