Identification and characterization of epitopes recognized by T lymphocytes and autoantibodies from patients with herpes gestationis

Autoantibodies associated with herpes gestationis (HG), a pregnancy-associated autoimmune skin disease, target the hemidesmosomal protein BP180. It was shown that the major noncollagenous stretch of the BP180 ectodomain (NC16A) harbors epitopes recognized by HG sera. Furthermore, Abs reactive with the homologous domain of murine BP180 are known to trigger a cutaneous blistering disease in mice by passive transfer experiments. The present study was aimed at characterizing the T cell responses and specificities of autoantibodies from two HG patients. Using immunoblotting and T cell proliferation assays, we have identified a 14-amino-acid stretch of the BP180 ectodomain (MCW-1; aa 507-520) that is recognized by both T cells and autoantibodies produced by the HG patients. The neonate born to one of these HG patients showed no signs of skin disease and had no detectable T cell response to the BP180 Ag, but did have a low titer of circulating anti-BP180 autoantibodies, presumably of maternal origin. BP180-specific T cell lines and clones developed from an HG patient specifically reacted with the MCW-1 epitope. The proliferative responses of these clones were restricted to HLA-DR, but not -DQ or -DP. These Ag-specific T cells expressed alpha/beta TCRs and a CD4 memory T cell phenotype and secreted IFN-gamma and IL-2, but not IL-4 or IL-6, suggesting that they are Th1-type lymphocytes. Further characterization of these Ag-specific T cells and autoantibodies will aid in elucidating the autoimmune mechanism(s) leading to the development of HG.     

An in vitro airway wall model of remodeling

Recent studies have shown that mechanical forces on airway epithelial cells can induce upregulation of genes involved in airway remodeling in diseases such as asthma. However, the relevance of these responses to airway wall remodeling is still unclear since 1). mechanotransduction is highly dependent on environment (e.g., matrix and other cell types) and 2). inflammatory mediators, which strongly affect remodeling, are also present in asthma. To assess the effects of mechanical forces on the airway wall in a relevant three-dimensional inflammatory context, we have established a tissue culture model of the human airway wall that can be induced to undergo matrix remodeling. Our model contains differentiated human bronchial epithelial cells characterized by tight junctions, cilia formation, and mucus secretion atop a collagen gel embedded with human lung fibroblasts. We found that addition of activated eosinophils and the application of 50% strain to the same system increased the epithelial thickness compared with either condition alone, suggesting that mechanical strain affects airway wall remodeling synergistically with inflammation. This integrated model more closely mimics airway wall remodeling than single-cell, conditioned media, or even two-dimensional coculture systems and is relevant for examining the importance of mechanical strain on airway wall remodeling in an inflammatory environment, which may be crucial for understanding and treating pathologies such as asthma.     

Vibration spectroscopy reveals light-induced chromophore and protein structural changes in the LOV2 domain of the plant blue-light receptor phototropin 1

Phototropins (phot1 and phot2), the plant blue-light receptors for phototropism, chloroplast movement, and stomatal opening, are flavoproteins that contain two approximately 12 kDa FMN-binding domains, LOV1 and LOV2, at their N-terminus, and a serine/threonine protein kinase domain at their C-terminus. The light-activated LOV2 domain forms a metastable intermediate which has been shown to be a protein-chromophore cysteinyl adduct (Cys39) at C(4a) of FMN. This species thermally relaxes back to the ground state in the dark. We measured the light-minus-dark FTIR difference spectra for the LOV2 domain of oat phot1. These spectra show the disappearance of bands at 1580, 1550, and 1350 cm(-1) that originate from, or are strongly coupled to, the N5=C(4a) stretching vibrations, consistent with the perturbations expected upon C(4a) adduct formation. Assignment of these negative difference FTIR bands to native chromophore vibrations is based on the alignment with resonance Raman bands of FMN. Prominent positive bands include a doublet at 1516 and 1536 cm(-1) and one at 1375 and 1298 cm(-1). Normal-mode vibrational-frequency calculations for both lumiflavin and lumiflavin with a sulfur attached at the C(4a) position agree with many of the positive and negative bands observed in the difference spectra. Both calculated and experimental difference FTIR spectra for deuterium isotope substitutions at exchangeable positions in the flavin chromophore are consistent with the assignment of the above positive bands to vibrational modes involving both the newly formed tetrahedral geometry of C(4a) and the N5-H bond in the long-lived LOV2(S)(390) cysteinyl species.     

Insight into the stability of the hydrophobic binding proteins of Escherichia coli: assessing the proteins for use as biosensors

Spectroscopic methods were used to monitor the unfolding of the leucine specific (LS) and the leucine-isoleucine-valine (LIV) binding proteins. Our studies indicate that ligand-free protein undergoes a simple two-state unfolding, whereas the protein-ligand complex undergoes a three-state unfolding model. Ligand binding causes significant stabilization of both proteins. There is correlation between ligand hydrophobicity and protein stabilization: the most hydrophobic ligand, isoleucine, causes the most significant stabilization of LIV protein. A disulfide bond present in N-domain of both proteins makes a large contribution to the protein stability of these periplasmic binding receptors.     

Critical oxygen tension in rat brain: a combined (31)P-NMR and EPR oximetry study

The relationship between cerebral interstitial oxygen tension (Pt(O(2))) and cellular energetics was investigated in mechanically ventilated, anesthetized rats during progressive acute hypoxia to determine whether there is a "critical" brain Pt(O(2)) for maintaining steady-state aerobic metabolism. Cerebral Pt(O(2)), measured by electron paramagnetic resonance oximetry, decreased proportionately to inspired oxygen fraction. (31)P-nuclear magnetic resonance measurements revealed no changes in P(i), phosphocreatine (PCr)/P(i) ratio, or intracellular pH when arterial blood oxygen tension (Pa(O(2))) was reduced from 145.1 +/- 11.7 to 56.5 +/- 4.4 mmHg (means +/- SE). Intracellular acidosis, a sharp rise in P(i), and a decline in the PCr/P(i) ratio developed when Pa(O(2)) was reduced further to 40.7 +/- 2.3 mmHg. The corresponding Pt(O(2)) values were 15.1 +/- 1.8, 8.8 +/- 0.4, and 6.8 +/- 0.3 mmHg. We conclude that over a range of decreasing oxygen tensions, cerebral oxidative metabolism is not sensitive to oxygen concentration. Oxygen becomes a regulatory substrate, however, when Pt(O(2)) is decreased to a critical level.