Myeloid Derived Suppressor Cells Are Present at High Frequency in Neonates and Suppress In Vitro T Cell Responses

Over 4 million infants die each year from infections, many of which are vaccine-preventable. Young infants respond relatively poorly to many infections and vaccines, but the basis of reduced immunity in infants is ill defined. We sought to investigate whether myeloid-derived suppressor cells (MDSC) represent one potential impediment to protective immunity in early life, which may help inform strategies for effective vaccination prior to pathogen exposure. We enrolled healthy neonates and children in the first 2 years of life along with healthy adult controls to examine the frequency and function of MDSC, a cell population able to potently suppress T cell responses. We found that MDSC, which are rarely seen in healthy adults, are present in high numbers in neonates and their frequency rapidly decreases during the first months of life. We determined that these neonatal MDSC are of granulocytic origin (G-MDSC), and suppress both CD4+ and CD8+ T cell proliferative responses in a contact-dependent manner and gamma interferon production. Understanding the role G-MDSC play in infant immunity could improve vaccine responsiveness in newborns and reduce mortality due to early-life infections.     

Aquaporins in complex tissues. II. Subcellular distribution in respiratory and glandular tissues of rat

The molecular pathways for fluid transport in pulmonary, oral,and nasal tissues are still unresolved. Here we use immunocytochemistry and immunoelectron microscopy to define the sites of expression of fouraquaporins in the respiratory tract and glandular epithelia, where theyreside in distinct, nonoverlapping sites. Aquaporin-1 (AQP1) is presentin apical and basolateral membranes of bronchial, tracheal, andnasopharyngeal vascular endothelium and fibroblasts. AQP5 is localizedto the apical plasma membrane of type I pneumocytes and the apicalplasma membranes of secretory epithelium in upper airway and salivaryglands. In contrast, AQP3 is present in basal cells of tracheal andnasopharyngeal epithelium and is abundant in basolateral membranes ofsurface epithelial cells of nasal conchus. AQP4 resides in basolateralmembranes of columnar cells of bronchial, tracheal, and nasopharyngealepithelium; in nasal conchus AQP4 is restricted to basolateralmembranes of a subset of intra- and subepithelial glands. These sitesof expression suggest that transalveolar water movement, modulation ofairway surface liquid, air humidification, and generation ofnasopharyngeal secretions involve a coordinated network of aquaporinwater channels.

     

Death, democracy and public ethical choice

The Danish Council of Ethics...believed that the brain-death criterion should not be accepted without public education and debate. Following the introduction of a spectrum of educational and related activites, a Gallup poll found that 98% of the survey population was aware of the debate over brain-vs-heart criteria and that 80% favoured the adoption of a supplemental brain-death standard... This raises the fundamental question of decisionmaking in pluralist democratic societies, of the limits of democratic involvement in such choices, and of the role of bodies like the Danish Council of Ethics... It must be part of the mission of a governmental bioethical body to use its peculiar expertise to teach and to lead -- to build a popular consensus out of confusion. But in doing so, such a Commission will be steering a dangerous course....     

Structural requirements for the interaction between peptide antigens and I-Ed molecules

We have analyzed the structural characteristics of the interaction between I-Ed molecules and their peptide ligands. It was found that unrelated good I-Ed binders share structurally similar "core" regions that were experimentally demonstrated to be crucial for binding to I-Ed molecules. Single amino acid substitution analogues of one good I-Ed binder, hen egg lysozyme 107-116, were analyzed for their capacity to bind to I-Ed molecules and to activate two different I-Ed-restricted T cell hybridomas. The results illustrate the great permissiveness of I-Ed-peptide interaction and the great specificity of T cell recognition. It was concluded from these analyses that basic residues on the peptide molecule play a crucial role in binding to I-Ed. This contrasts with the structural requirements for binding to the other Iad isotype, I-Ad, the crucial hydrophobic residues. Thus, different class II molecules of the same MHC haplotype may have rather distinct peptide binding specificities, thereby expanding the repertoire of possible immunogenic peptides presented for T cell recognition.     

Experimental infection of cattle with a feline isolate of Tritrichomonas foetus.

Tritrichomonas foetus is the causative agent of bovine trichomoniasis, a sexually transmitted disease in cattle that can result in large profit losses for cattle producers. Increasing reports have suggested that T. foetus is also the causative agent of large-bowel diarrhea in cats. To determine if the trichomonads recovered from the reproductive tract of cattle and the large intestine of cats can thrive in the same host, 2 groups of virgin Angus heifers were inoculated with T. foetus. The first group of heifers was inoculated with a bovine T. foetus isolate cultured from a naturally infected cow, and heifers in the second group were inoculated with T. foetus organisms cultured from the feces of a naturally infected cat. Over an 11-wk period, vaginal, cervical, and uterine mucus samples were analyzed, along with a single transcervical uterine biopsy sample, to determine organism and disease presence. The mucus and biopsy samples collected from each group indicate that the disease caused by feline and bovine isolates of T. foetus are comparable, but not identical.     

Generation of a Family-specific Phage Library of Llama Single Chain Antibody Fragments That Neutralize HIV-1

Recently, we described llama antibody fragments (VHH) that can neutralize human immunodeficiency virus, type 1 (HIV-1). These VHH were obtained after selective elution of phages carrying an immune library raised against gp120 of HIV-1 subtype B/C CN54 with soluble CD4. We describe here a new, family-specific approach to obtain the largest possible diversity of related VHH that compete with soluble CD4 for binding to the HIV-1 envelope glycoprotein. The creation of this family-specific library of homologous VHH has enabled us to isolate phages carrying similar nucleotide sequences as the parental VHH. These VHH displayed varying binding affinities and neutralization phenotypes to a panel of different strains and subtypes of HIV-1. Sequence analysis of the homologs showed that the C-terminal three amino acids of the CDR3 loop were crucial in determining the specificity of these VHH for different subtype C HIV-1 strains. There was a positive correlation between affinity of VHH binding to gp120 of HIV-1 IIIB and the breadth of neutralization of diverse HIV-1 envelopes. The family-specific approach has therefore allowed us to better understand the interaction of the CD4-binding site antibodies with virus strain specificity and has potential use for the bioengineering of antibodies and HIV-1 vaccine development.     

Regulation of post-translational modifications of muskelin by protein kinase C

Muskelin is a member of the kelch-repeat superfamily of proteins, identified as an intracellular protein involved in cell spreading responses to thombospondin-1. Muskelin is expressed by many adult tissues and has an evolutionarily conserved, multidomain architecture consisting of an amino-terminal discoidin-like domain, a central alpha-helical region and six kelch-repeats that are predicted to form a beta-propeller structure. We previous demonstrated that muskelin molecules undergo head-to-tail association, however the physiological, post-translational regulation of muskelin is not well understood. Here, we have examined the expression of muskelin during mouse embryonic development and report widespread expression that includes muscle tissues, multiple epithelia and the brain. In cultured skeletal myoblasts and vascular smooth muscle cells, muskelin exists as a complex set of isoelectric variants. Five potential sites for phosphorylation by protein kinase C (PKC), are conserved between vertebrate and Drosophila muskelins, therefore we examined the hypothesis that muskelin is regulated post-translationally by PKC activity. We demonstrate that PKC activation or inhibition regulates the profile of endogenous muskelin isoelectric variants and that muskelin is a substrate for PKCalphain vitro. Wild-type GFP-muskelin and a panel of alanine point mutations were used to test the sensitivity of self-association to PKC activation. Mutation of two of the sites, S324 and T515, partially inhibited the ability of muskelin to self-associate in cells and inhibited responsiveness to activated PKC. Interestingly, both sites are predicted to lie in surface-exposed loops on the same side of the beta-propeller, implicating a common binding interface.     

Mitogen‐ and stress‐activated kinases regulate progenitor cell proliferation and neuron development in the adult dentate gyrus

The neurogenic niche within the subgranular zone (SGZ) of the dentate gyrus is a source of new neurons throughout life. Interestingly, SGZ proliferative capacity is regulated by both physiological and pathophysiological conditions. One outstanding question involves the molecular mechanisms that regulate both basal and inducible adult neurogenesis. Here, we examined the role of the MAPK‐regulated kinases, mitogen‐ and stress‐activated kinase (MSK)1 and MSK2. as regulators of dentate gyrus SGZ progenitor cell proliferation and neurogenesis. Under basal conditions, MSK1/2 null mice exhibited significantly reduced progenitor cell proliferation capacity and a corollary reduction in the number of doublecortin (DCX)‐positive immature neurons. Strikingly, seizure‐induced progenitor proliferation was totally blocked in MSK1/2 null mice. This blunting of cell proliferation in MSK1/2 null mice was partially reversed by forskolin infusion, indicating that the inducible proliferative capacity of the progenitor cell population was intact. Furthermore, in MSK1/2 null mice, DCX‐positive immature neurons exhibited reduced neurite arborization. Together, these data reveal a critical role for MSK1/2 as regulators of both basal and activity‐dependent progenitor cell proliferation and morphological maturation in the SGZ.     

HLA-B*57 Micropolymorphism shapes HLA allele-specific epitope immunogenicity, selection pressure, and HIV immune control

The genetic polymorphism that has the greatest impact on immune control of human immunodeficiency virus (HIV) infection is expression of HLA-B*57. Understanding of the mechanism for this strong effect remains incomplete. HLA-B*57 alleles and the closely related HLA-B*5801 are often grouped together because of their similar peptide-binding motifs and HIV disease outcome associations. However, we show here that the apparently small differences between HLA-B*57 alleles, termed HLA-B*57 micropolymorphisms, have a significant impact on immune control of HIV. In a study cohort of >2,000 HIV C-clade-infected subjects from southern Africa, HLA-B*5703 is associated with a lower viral-load set point than HLA-B*5702 and HLA-B*5801 (medians, 5,980, 15,190, and 19,000 HIV copies/ml plasma; P = 0.24 and P = 0.0005). In order to better understand these observed differences in HLA-B*57/5801-mediated immune control of HIV, we undertook, in a study of >1,000 C-clade-infected subjects, a comprehensive analysis of the epitopes presented by these 3 alleles and of the selection pressure imposed on HIV by each response. In contrast to previous studies, we show that each of these three HLA alleles is characterized both by unique CD8(+) T-cell specificities and by clear-cut differences in selection pressure imposed on the virus by those responses. These studies comprehensively define for the first time the CD8(+) T-cell responses and immune selection pressures for which these protective alleles are responsible. These findings are consistent with HLA class I alleles mediating effective immune control of HIV through the number of p24 Gag-specific CD8(+) T-cell responses generated that can drive significant selection pressure on the virus.