Constructing an in vitro cornea from cultures of the three specific corneal cell types

Summary This paper presents a reliable method for establishing pure cultures of the three types of corneal cells. This is believed to be the first time, corneal cells have been cultured from fetal pig corneas. Cell growth studies were performed in different media. Subcultures of the three corneal cell types were passaged until the 30th generation without their showing signs of senescence. For engineering an in vitro cornea, corneal epithelial cells were cultured over corneal stromal cells in an artificial biomatrix of collagen with an underlying layer of corneal endothelial cells. The morphology, histology, and differentiation of the in vitro cornea were investigated to determine the degree of comparability to the cornea in vivo. The in vitro construct displayed signs of transition to an organotypic phenotype of which the most prominent was the formation of two basement membranes.     

Lung-specific overexpression of CC chemokine ligand (CCL) 2 enhances the host defense to Streptococcus pneumoniae infection in mice: role of the CCL2-CCR2 axis

Mononuclear phagocytes are critical components of the innate host defense of the lung to inhaled bacterial pathogens. The monocyte chemotactic protein CCL2 plays a pivotal role in inflammatory mononuclear phagocyte recruitment. In this study, we tested the hypothesis that increased CCL2-dependent mononuclear phagocyte recruitment would improve lung innate host defense to infection with Streptococcus pneumoniae. CCL2 transgenic mice that overexpress human CCL2 protein in type II alveolar epithelial cells and secrete it into the alveolar air space showed a similar proinflammatory mediator response and neutrophilic alveolitis to challenge with S. pneumoniae as wild-type mice. However, CCL2 overexpressing mice showed an improved pneumococcal clearance and survival compared with wild-type mice that was associated with substantially increased lung mononuclear phagocyte subset accumulations upon pneumococcal challenge. Surprisingly, CCL2 overexpressing mice developed bronchiolitis obliterans upon pneumococcal challenge. Application of anti-CCR2 Ab MC21 to block the CCL2-CCR2 axis in CCL2 overexpressing mice, though completely abrogating bronchiolitis obliterans, led to progressive pneumococcal pneumonia. Collectively, these findings demonstrate the importance of the CCL2-CCR2 axis in the regulation of both the resolution/repair and remodelling processes after bacterial challenge and suggest that overwhelming innate immune responses may trigger bronchiolitis obliterans formation in bacterial lung infections.     

Distinct cytokine-driven responses of activated blood gammadelta T cells: insights into unconventional T cell pleiotropy

Human Vgamma9/Vdelta2 T cells comprise a small population of peripheral blood T cells that in many infectious diseases respond to the microbial metabolite, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), expanding to up to 50% of CD3(+) cells. This "transitional response," occurring temporally between the rapid innate and slower adaptive response, is widely viewed as proinflammatory and/or cytolytic. However, increasing evidence that different cytokines drive widely different effector functions in alphabeta T cells provoked us to apply cDNA microarrays to explore the potential pleiotropy of HMB-PP-activated Vgamma9/Vdelta2 T cells. The data and accompanying validations show that the related cytokines, IL-2, IL-4, or IL-21, each drive proliferation and comparable CD69 up-regulation but induce distinct effector responses that differ from prototypic alphabeta T cell responses. For example, the Th1-like response to IL-2 also includes expression of IL-5 and IL-13 that conversely are not induced by IL-4. The data identify specific molecules that may mediate gammadelta T cell effects. Thus, IL-21 induces a lymphoid-homing phenotype and high, unexpected expression of the follicular B cell-attracting chemokine CXCL13/BCA-1, suggesting a novel follicular B-helper-like T cell that may play a hitherto underappreciated role in humoral immunity early in infection. Such broad plasticity emphasizes the capacity of gammadelta T cells to influence the nature of the immune response to different challenges and has implications for the ongoing clinical application of cytokines together with Vgamma9/Vdelta2 TCR agonists.     

FMS-like tyrosine kinase 3 ligand aggravates the lung inflammatory response to Streptococcus pneumoniae infection in mice: role of dendritic cells

Pretreatment of mice with the hemopoietic growth factor, FMS-like tyrosine kinase 3 ligand (Flt3L), has been shown to increase monocyte-derived myeloid dendritic cells (DC) in lung parenchymal tissue, with possible implications for protective immunity to lung bacterial infections. However, whether Flt3L treatment improves lung innate immunity of mice to challenge with Streptococcus pneumoniae has not been investigated previously. Mice pretreated with Flt3L exhibited a peripheral monocytosis and a strongly expanded lung myeloid DC pool, but responded with a similar proinflammatory cytokine release (TNF-alpha, IL-6, keratinocyte derived cytokine, MIP-2, CCL2) and neutrophilic alveolitis upon infection with S. pneumoniae as did control mice with a normal lung DC pool. Unexpectedly, however, Flt3L-pretreated mice, but not control mice, infected with S. pneumoniae developed vasculitis and increased lung permeability by days 2-3 postinfection, and florid pneumonia accompanied by sustained increased bacterial loads by days 3-4 postinfection. This was associated with an overall increased mortality of approximately 35% by day 4 after pneumococcal challenge. Application of anti-CCR2 Ab MC21 to block inflammatory monocyte-dependent lung mononuclear phagocyte mobilization significantly reduced the lung leakage, but not vasculitis in Flt3L-pretreated mice infected with S. pneumoniae, without affecting the intra-alveolar cytokine liberation or the concomitantly developing neutrophilic alveolitis. Together, the data demonstrate that previous Flt3L-induced lung DC accumulation is not protective in lung innate immunity to challenge with S. pneumoniae, and support the concept that CCR2-dependent mononuclear phagocyte as opposed to neutrophil recruitment contributes to increased lung leakage in Flt3L-pretreated mice challenged with S. pneumoniae.     

Toc64--a preprotein-receptor at the outer membrane with bipartide function

Protein translocation across membranes is assisted by translocation machineries present in the membrane targeted by the precursor proteins. Translocon subunits can be functionally divided into receptor proteins warranting the specificity of this machine and a translocation channel. At the outer envelope of chloroplasts two sets of receptor proteins regulate protein translocation facing the cytosol or acting in the intermembrane space. One, Toc64 is a receptor of the translocon at the outer envelope of chloroplasts (Toc complex) with dual function. Toc64 recognizes Hsp90 delivered precursor proteins via a cytosolic exposed domain containing three tetratrico-peptide repeat motifs and as demonstrated in here, Toc64 functions also as a major component of a complex facing the intermembrane space. The latter complex is composed of an Hsp70 localized in the intermembrane space, its interaction partner Toc12, a J-domain containing protein and the intermembrane space protein Tic22. We analyzed the intermembrane space domain of Toc64. This domain is involved in preprotein recognition and association with the Toc-complex independent of the cytosolic domain of the Toc64 receptor. Therefore, Toc64 is involved in preprotein translocation across the outer envelope at both sites of the membrane.     

The structures of bacteriophages K1E and K1-5 explain processive degradation of polysaccharide capsules and evolution of new host specificities

External polysaccharides of many pathogenic bacteria form capsules protecting the bacteria from the animal immune system and phage infection. However, some bacteriophages can digest these capsules using glycosidases displayed on the phage particle. We have utilized cryo-electron microscopy to determine the structures of phages K1E and K1-5 and thereby establish the mechanism by which these phages attain and switch their host specificity. Using a specific glycosidase, both phages penetrate the capsule and infect the neuroinvasive human pathogen Escherichia coli K1. In addition to the K1-specific glycosidase, each K1-5 particle carries a second enzyme that allows it to infect E. coli K5, whose capsule is chemically different from that of K1. The enzymes are organized into a multiprotein complex attached via an adapter protein to the virus portal vertex, through which the DNA is ejected during infection. The structure of the complex suggests a mechanism for the apparent processivity of degradation that occurs as the phage drills through the polysaccharide capsule. The enzymes recognize the adapter protein by a conserved N-terminal sequence, providing a mechanism for phages to acquire different enzymes and thus to evolve new host specificities.     

Early stress and chronic methylphenidate cross-sensitize dopaminergic responses in the adolescent medial prefrontal cortex and nucleus accumbens

Methylphenidate (MP) is widely used to treat attention deficit/hyperactivity disorder in children. However, basic research has been mainly focused on MP treatment in adult, behaviorally normal rodents. Here we analyzed MP-evoked changes of dopamine (DA) release in the limbic system of juvenile rodents with hyperactive and attention deficit-like symptoms. Using dual probe in vivo microdialysis, DA levels were quantified in the medial prefrontal cortex and nucleus accumbens of juvenile and adolescent degus ( Octodon degus ). Acute stress- and acute MP-evoked dopaminergic responses in normal juvenile and adolescent animals were compared with (i) animals showing symptoms of hyperactivity and attention deficits induced by early life stress, i.e. repeated parental separation during the first 3 weeks of life, and (ii) animals chronically treated with MP during pre-adolescence. Our main results revealed that (i) early life stress and (ii) chronic MP treatment during pre-adolescence cross-sensitize limbic dopaminergic functions in adolescent animals. Furthermore, we demonstrated a unique pattern of acute MP-evoked DA release in the juvenile compared with the adolescent medial prefrontal cortex and nucleus accumbens. Our findings that the functional maturation of dopaminergic limbic function is significantly altered by early life experience, i.e. repeated parental separation and chronic MP treatment, allow novel insights into the etiology of attention deficit/hyperactivity disorder and into the long-term consequences of MP treatment on brain development.