The human CD1-restricted T cell repertoire is limited to cross-reactive antigens: implications for host responses against immunologically related pathogens

The repertoires of CD1- and MHC-restricted T cells are complementary, permitting the immune recognition of both lipid and peptide Ags, respectively. To compare the breadth of the CD1-restricted and MHC-restricted T cell repertoires, we evaluated T cell responses against lipid and peptide Ags of mycobacteria in leprosy, comparing tuberculoid patients, who are able to restrict the pathogen, and lepromatous patients, who have disseminated infection. The striking finding was that in lepromatous leprosy, T cells did not efficiently recognize lipid Ags from the leprosy pathogen, Mycobacterium leprae, or the related species, Mycobacterium tuberculosis, yet were able to efficiently recognize peptide Ags from M. tuberculosis, but not M. leprae. To identify a mechanism for T cell unresponsiveness against mycobacterial lipid Ags in lepromatous patients, we used T cell clones to probe the species specificity of the Ags recognized. We found that the majority of M. leprae-reactive CD1-restricted T cell clones (92%) were cross-reactive for multiple mycobacterial species, whereas the majority of M. leprae-reactive MHC-restricted T cells were species specific (66%), with a limited number of T cell clones cross-reactive (34%) with M. tuberculosis. In comparison with the MHC class II-restricted T cell repertoire, the CD1-restricted T cell repertoire is limited to recognition of cross-reactive Ags, imparting a distinct role in the host response to immunologically related pathogens.     

Design, docking, and evaluation of multiple libraries against multiple targets

We present a general approach to the design, docking, and virtual screening of multiple combinatorial libraries against a family of proteins. The method consists of three main stages: docking the scaffold, selecting the best substituents at each site of diversity, and comparing the resultant molecules within and between the libraries. The core "divide-and-conquer" algorithm for side-chain selection, developed from an earlier version (Sun et al., J Comp Aided Mol Design 1998;12:597-604), provides a way to explore large lists of substituents with linear rather than combinatorial time dependence. We have applied our method to three combinatorial libraries and three serine proteases: trypsin, chymotrypsin, and elastase. We show that the scaffold docking procedure, in conjunction with a novel vector-based orientation filter, reproduces crystallographic binding modes. In addition, the free-energy-based scoring procedure (Zou et al., J Am Chem Soc 1999;121:8033-8043) is able to reproduce experimental binding data for P1 mutants of macromolecular protease inhibitors. Finally, we show that our method discriminates between a peptide library and virtual libraries built on benzodiazepine and tetrahydroisoquinolinone scaffolds. Implications of the docking results for library design are explored.     

Selection of Peptide Mimics of HIV-1 Epitope Recognized by Neutralizing Antibody VRC01

The ability to induce anti-HIV-1 antibodies that can neutralize a broad spectrum of viral isolates from different subtypes seems to be a key requirement for development of an effective HIV-1 vaccine. The epitopes recognized by the most potent broadly neutralizing antibodies that have been characterized are largely discontinuous. Mimetics of such conformational epitopes could be potentially used as components of a synthetic immunogen that can elicit neutralizing antibodies. Here we used phage display technology to identify peptide motifs that mimic the epitope recognized by monoclonal antibody VRC01, which is able to neutralize up to 91% of circulating primary isolates. Three rounds of biopanning were performed against 2 different phage peptide libraries for this purpose. The binding specificity of selected phage clones to monoclonal antibody VRC01 was estimated using dot blot analysis. The putative peptide mimics exposed on the surface of selected phages were analyzed for conformational and linear homology to the surface of HIV-1 gp120 fragment using computational analysis. Corresponding peptides were synthesized and checked for their ability to interfere with neutralization activity of VRC01 in a competitive inhibition assay. One of the most common peptides selected from 12-mer phage library was found to partially mimic a CD4-binding loop fragment, whereas none of the circular C7C-mer peptides was able to mimic any HIV-1 domains. However, peptides identified from both the 12-mer and C7C-mer peptide libraries showed rescue of HIV-1 infectivity in the competitive inhibition assay. The identification of epitope mimics may lead to novel immunogens capable of inducing broadly reactive neutralizing antibodies.     

Gradients with depth in electrospun fibrous scaffolds for directed cell behavior

A major obstacle in creating viable tissue-engineered constructs using electrospinning is the lack of complete cellularization and vascularization due to the limited porosity in these densely packed fibrous scaffolds. One potential approach to circumvent this issue is the use of various gradients of chemical and biophysical cues to drive the infiltration of cells into these structures. Toward this goal, this study focused on creating durotactic (mechanical) and haptotactic (adhesive) gradients through the thickness of electrospun hyaluronic acid (HA) scaffolds using a unique, yet simple, modification of common electrospinning protocols. Specifically, both mechanical (via cross-linking: ranging from 27-100% modified methacrylated HA, MeHA) and adhesive (via inclusion of the adhesive peptide RGD: 0-3 mM RGD) gradients were each fabricated by mixing two solutions (one ramping up, one ramping down) prior to electrospinning and fiber collection. Gradient formation was verified by fluorescence microscopy, FTIR, atomic force microscopy, and cellular morphology assessment of scaffolds at different points of collection (i.e., with scaffold thickness). To test further the functionality of gradient scaffolds, chick aortic arch explants were cultured on adhesive gradient scaffolds for 7 days, and low RGD-high RGD gradient scaffolds showed significantly greater cell infiltration compared with high RGD-low RGD gradients and uniform high RGD or uniform low RGD control scaffolds. In addition to enhanced infiltration, this approach could be used to fabricate graded tissue structures, such as those that occur at interfaces.     

Plasma chemokine levels are associated with the presence and extent of angiographic coronary collaterals in chronic ischemic heart disease

Background

In patients with chronic ischemic heart disease (IHD), the presence and extent of spontaneously visible coronary collaterals are powerful determinants of clinical outcome. There is marked heterogeneity in the recruitment of coronary collaterals amongst patients with similar degrees of coronary artery stenoses, but the biological basis of this heterogeneity is not known. Chemokines are potent mediators of vascular remodeling in diverse biological settings. Their role in coronary collateralization has not been investigated. We sought to determine whether plasma levels of angiogenic and angiostatic chemokines are associated with of the presence and extent of coronary collaterals in patients with chronic IHD.

Methodology/Principal Findings

We measured plasma concentrations of angiogenic and angiostatic chemokine ligands in 156 consecutive subjects undergoing coronary angiography with at least one ≥90% coronary stenosis and determined the presence and extent of spontaneously visible coronary collaterals using the Rentrop scoring system. Eighty-eight subjects (56%) had evidence of coronary collaterals. In a multivariable regression model, the concentration of the angiogenic ligands CXCL5, CXCL8 and CXCL12, hyperlipidemia, and an occluded artery were associated with the presence of collaterals; conversely, the concentration of the angiostatic ligand CXCL11, interferon-γ, hypertension and diabetes were associated with the absence of collaterals (ROC area 0.91). When analyzed according to extent of collateralization, higher Rentrop scores were significantly associated with increased concentration of the angiogenic ligand CXCL1 (p<0.0001), and decreased concentrations of angiostatic ligands CXCL9 (p<0.0001), CXCL10 (p = 0.002), and CXCL11 (p = 0.0002), and interferon-γ (p = 0.0004).

Conclusions/Significance

Plasma chemokine concentrations are associated with the presence and extent of spontaneously visible coronary artery collaterals and may be mechanistically involved in their recruitment.     

Pilot analysis of the plasma metabolite profiles associated with emphysematous Chronic Obstructive Pulmonary Disease phenotype

The current pilot study examined the hypothesis that cigarette smokers who developed an emphysematous phenotype of Chronic Obstructive Pulmonary Disease (COPD) were associated with distinctive patterns in their corresponding metabolomics profile as compared to those who did not. Peripheral blood plasma samples were collected from 38 subjects with different phenotypes of COPD. They were categorized into three groups: healthy non-smokers (n = 16), smokers without emphysema (n = 8), and smokers with emphysema (n = 14). Ultra High Performance Liquid Chromatography/quadrupole-Time-of-Flight Mass Spectrometry techniques were used to identify a large number of metabolite markers (3534). Unsupervised clustering analysis accurately separated the smokers with emphysema from others without emphysema and demonstrated potentials of this metabolomics data. Subsequently predictive models were created with a supervised learning set, and these predictive models were found to be highly accurate in identifying the subjects with the emphysematous phenotype of COPD with excellent sensitivity and specificity. Our methodology provides a preliminary model that differentiates an emphysematous COPD phenotype from other COPD phenotypes on the basis of the metabolomics profiles. These results also suggest that the metabolomics profiling could potentially guide the characterization of relevant metabolites that leads to an emphysematous COPD phenotype.     

High‐throughput screening of a small molecule library for promoters and inhibitors of mesenchymal stem cell osteogenic differentiation

The use of high‐throughput screening (HTS) techniques has long been employed by the pharmaceutical industry to increase discovery rates for new drugs that could be useful for disease treatment, yet this technology has only been minimally applied in other applications such as in tissue regeneration. In this work, an assay for the osteogenic differentiation of human mesenchymal stem cells (hMSCs) was developed and used to screen a library of small molecules for their potential as either promoters or inhibitors of osteogenesis, based on levels of alkaline phosphatase activity and cellular viability. From a library of 1,040 molecules, 36 promoters, and 20 inhibitors were identified as hits based on statistical criteria. Osteopromoters from this library were further investigated using standard culture techniques and a wider range of outcomes to verify that these compounds drive cellular differentiation. Several hits led to some improvement in the expression of alkaline phosphatase, osteogenic gene expression, and matrix mineralization by hMSCs when compared to the standard dexamethasone supplemented media and one molecule was investigated in combination with a recently identified biodegradable and osteoconductive polymer. This work illustrates the ability of HTS to more rapidly identify potential molecules to control stem cell differentiation. Biotechnol. Bioeng. 2011; 108:163–174. © 2010 Wiley Periodicals, Inc.