Reconstruction of the medial half of the lower eyelid using a "switch" split-lid procedure

Reconstruction of the medial half of the lower eyelid has one major disadvantage: It produces a scar at right angles to the eyelid rim. In contrast, use of a "switch" split-lid procedure avoids this inconvenience. The lateral half of the lower eyelid is split in two lamellae. The inner layer is transferred medially, and the resulting defect is closed with a buccal graft. The outer layer is drawn laterally to cover the raw surface of the mucosal pedicle and graft. The surplus of skin over the lateral canthal area is removed. This procedure, which so far has been used in three patients, promises to be a useful alternative for reconstruction of the medial half, but not more, of the lower eyelid.     

Histological and Ultrastructural Studies on the Conjunctiva of the Barred Owl (Strix varia)

This report is the first characterization of the histology and ultrastructure of the barred owl conjunctiva. The inferior eyelid was dominated by a large disk-shaped plate covered by a non-keratinized stratified squamous or cuboidal epithelium of variable thickness. The apical surface of the plate epithelium varied from flat to long microvilli or even short cytoplasmic extensions similar to those seen in the third eyelid. All specimens had a few goblet cells filled with mucous secretory granules in the plate region. The underlying connective tissue was a dense fibroelastic stroma. Eosinophils were surprisingly common in the epithelial layer and underlying connective tissue in the plate and more distal orbital mucosal region. The orbital mucosa contained goblet cells with heterogeneous glycosylation patterns. The leading edge and marginal plait of the third eyelid are designed to collect fluid and particulate matter as they sweep across the surface of the eye. The palpebral conjunctival surface of the third eyelid was covered by an approximately five-cell-deep stratified squamous epithelium without goblet cells. The bulbar surface of the third eyelid was a bilayer of epithelial cells whose superficial cells have elaborate cytoplasmic tapering extensions reaching out 25 μm. Narrow cytofilia radiated outwards up to an additional 15–20 μm from the cytoplasmic extensions. Lectin labeling demonstrated heterogeneous glycosylation of the apical membrane specializations but only small amounts of glycoprotein-filled secretory granules in the third eyelid.     

Microbiota-immune system interaction: an uneasy alliance

An estimated 100 trillion microbes colonize human beings, with the majority of organisms residing in the intestines. This microbiota impacts host nutrition, protection, and gut development. Alterations in microbiota composition are associated with susceptibility to various infectious and inflammatory gut diseases. The mucosal surface is not a static barrier that simply prevents microbial invasion but a critical interface for microbiota-immune system interactions. Recent work suggests that dynamic interactions between microbes and the host immune system at the mucosal surface inform immune responses both locally and systemically. This review focuses on intestinal microbiota-immune interactions leading to intestinal homeostasis, and show that these interactions at the GI mucosal surface are critical for driving both protective and pathological immune responses systemically.     

Points of control exerted along the macrophage-endothelial cell-polymorphonuclear neutrophil axis by PECAM-1 in the innate immune response of acute colonic inflammation

PECAM-1 is expressed on endothelial cells and leukocytes. Its extracellular domain has been implicated in leukocyte diapedesis. In this study, we used PECAM-1(-/-) mice and relevant cells derived from them to assess the role of PECAM-1 in an experimental model of acute colonic inflammation with a predominant innate immune response, i.e., 2,4,6-trinitrobenzine sulfonic acid (TNBS). Using chimeric approaches, we addressed the points of control exerted by PECAM-1 along the macrophage-endothelial cell-polymorphonuclear neutrophil (PMN) axis. In vivo, TNBS-induced colitis was ameliorated in PECAM-1(-/-) mice, an event attributed to PECAM-1 on hematopoietic cells rather than to PECAM-1 on endothelial cells. The in vivo innate immune response was mimicked in vitro by using a construct of the vascular-interstitial interface, i.e., PMN transendothelial migration was induced by colonic lavage fluid (CLF) from TNBS mice or macrophages (MPhi) challenged with CLF. Using the construct, we confirmed that endothelial cell PECAM-1 does not play a role in PMN transendothelial migration. Although MPhi activation (NF-kappaB nuclear binding) and function (keratinocyte-derived chemokine production) induced by CLF was diminished in PECAM-1(-/-) MPhi, this did not affect their ability to promote PMN transendothelial migration. By contrast, PECAM-1(-/-) PMN did not adhere to or migrate across endothelial cell monolayers in response to CLF. Further, as compared with PECAM-1(+/+) PMN, PECAM-1(-/-) PMN were less effective in orientating their CXCR2 receptors (polarization) in the direction of a chemotactic gradient. Collectively, our findings indicate that PECAM-1 modulation of PMN function (at a step before diapedesis) most likely contributes to the inflammation in a colitis model with a strong innate immune component.     

Signaling pathways recruited by the cardiotrophin-like cytokine/cytokine-like factor-1 composite cytokine: specific requirement of the membrane-bound form of ciliary neurotrophic factor receptor alpha component

Ciliary neurotrophic factor (CNTF) is a cytokine supporting the differentiation and survival of a number of neural cell types. Its receptor complex consists of a ligand-binding component, CNTF receptor (CNTFR), associated with two signaling receptor components, gp130 and leukemia inhibitory factor receptor (LIFR). Striking phenotypic differences between CNTF- and CNTFR-deficient mice suggest that CNTFR serves as a receptor for a second developmentally important ligand. We recently demonstrated that cardiotrophin-like cytokine (CLC) associates with the soluble orphan receptor cytokine-like factor-1 (CLF) to form a heterodimeric cytokine that displayed activities only on cells expressing the tripartite CNTF receptor on their surface. In this present study we examined the membrane binding of the CLC/CLF composite cytokine and observed a preferential interaction of the cytokine with the CNTFR subunit. Signaling pathways recruited by the CLC/CLF complex in human neuroblastoma cell lines were also analyzed in detail. The results obtained showed an activation of Janus kinases (JAK1, JAK2, and TYK2) leading to a tyrosine phosphorylation of the gp130 and LIFR. The phosphorylated signaling receptors served in turn as docking proteins for signal transducing molecules such as STAT3 and SHP-2. In vitro analysis revealed that the gp130-LIFR pathway could also stimulate the phosphatidylinositol 3-kinase and the mitogen-activated protein kinase pathways. In contrast to that reported before for CNTF, soluble CNTFR failed to promote the action CLC/CLF, and an absolute requirement of the membrane form of CNTFR was required to generate a functional response to the composite cytokine. This study reinforces the functional similarity between CNTF and the CLC/CLF composite cytokine defining the second ligand for CNTFR.     

Injection of plasmid DNA into the gastric mucosa induces mucosal and systemic immunity

Nearly all mucosal surfaces participate in a common mucosal immune system, and application of an antigen to one mucosal surface elicits local as well as distant mucosal immune responses. However, whether the gastric mucosa is a part of this network has not been examined directly. We show here that the injection of plasmid DNA encoding beta-galactosidase into the gastric wall caused transfection of gastric mucosal epithelial cells, induced systemic and mucosal antibody responses at both local (digestive tract) and distant (genital and respiratory tracts) sites, and induced cytotoxic T lymphocyte responses in the spleen and the mesenteric and iliac lymph nodes.     

Mucosal and systemic immune responses of mice to tetanus toxoid coadministered nasally with AFCo1

Mucosal immune responses are an early and important line of defense against pathogens. The current understanding of the mucosal immune system allows us to consider the use of nasal immunization for induction of antigen-specific immune responses at the mucosal surface and the systemic compartment. Mucosal adjuvants are key for developing novel mucosal vaccines and represent 1 approach to improving mucosal and systemic immunity. However, few mucosal vaccine adjuvants are currently approved for human use. Neisseria meningitidis B proteoliposome-derived cochleate (AFCo1 - Adjuvant Finlay Cochleate 1) has been demonstrated to be a potent mucosal adjuvant. The present work demonstrates that intranasal immunization of 3 doses of tetanus toxoid (TT) coadministered with AFCo1 in mice promotes high systemic and mucosal responses. The anti-TT IgG serum titers and the mucosal anti-TT IgA in saliva and vaginal wash were significantly higher than TT alone. The analysis of antibody subclasses showed that intranasal administration of AFCo1 + TT induced not only IgG1 but also IgG2a anti-TT antibodies at levels comparable to those obtained with TT vaccine (vax-TET). These data support the fact that AFCo1 is a potent mucosal adjuvant in nasal immunization to a coadministered protein antigen.     

Two different contact sites are recruited by cardiotrophin-like cytokine (CLC) to generate the CLC/CLF and CLC/sCNTFRalpha composite cytokines

The cytokines of the interleukin-6 family are multifunctional proteins that regulate cell growth, differentiation, and other cell functions in a variety of biological systems including the immune, inflammatory, hematopoietic, and nervous systems. One member of this family, ciliary neurotrophic factor (CNTF), displays biological functions more restricted to the neuromuscular axis. We have recently identified two additional ligands for the CNTF receptor complex. Both are composite cytokines formed by cardiotrophin-like cytokine (CLC) associated to either the soluble type I cytokine receptor CLF or the soluble form of CNTF receptor alpha (CNTFRalpha). The present study was aimed at analyzing the interactions between the cytokine CLC and its different receptor chains. For this purpose, we modeled CLC/receptor interactions to define the residues potentially involved in the contact sites. We then performed site-directed mutagenesis on these residues and analyzed the biological interactions between mutants and receptor chains. Importantly, we found that CLC interacts with the soluble forms of CNTFRalpha and CLF via sites 1 and 3, respectively. For site 1, the most crucial residues involved in the interaction are Trp67, Arg170, and Asp174, which interact with CNTFRalpha. Surprisingly, the residues that are important for the interaction of CLC with CLF are part of the conserved FXXK motif of site 3 known to be the interaction site of LIFRbeta. Obtained results show that the Phe151 and Lys154 residues are effectively involved in the interaction of CLC with LIFRbeta. This study establishes the molecular details of the interaction of CLC with CLF, CNTFRalpha, and LIFRbeta and helps to define the precise role of each protein in this functional receptor complex.     

Parenteral and mucosal prime-boost immunization strategies in mice with hepatitis B surface antigen and CpG DNA

Synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG ODN) are potent adjuvants to protein antigens administered by parenteral or mucosal routes to BALB/c mice. To date, there have been no studies using combined parenteral/mucosal approaches with CpG DNA as adjuvant. In this study we evaluated different parenteral prime-mucosal boost and mucosal prime-parenteral boost strategies using hepatitis B surface antigen (HBsAg) alone or with different adjuvants: aluminum hydroxide (alum), cholera toxin (CT), CpG ODN. In addition, since CpG ODN has previously been shown to act synergistically with other adjuvants after parenteral or mucosal delivery, we also evaluated adjuvant combinations: alum+CpG ODN and CT+CpG ODN. The effects of adjuvant and administration strategy on systemic and mucosal humoral responses were measured, as well as cell-mediated immune responses (cytotoxic T lymphocyte activity). These results were compared to parenteral only or mucosal only strategies. Our findings demonstrate that parenteral immunization can prime for mucosal responses even when different lymph nodes were being targeted. HBsAg-specific immune responses (IgG in plasma, cytotoxic T lymphocytes) induced by parenteral prime could all be significantly enhanced by mucosal boosting and despite the fact that intramuscular immunization alone could not induce mucosal IgA, it could prime for a subsequent mucosal boost. In addition, the presence of adjuvant at time of boosting could influence the nature of subsequent immune responses (Th1 vs. Th2). Mice primed intranasally could have their systemic immune responses boosted with a parenteral administration and it was also possible to enhance mucosal responses induced by intranasal prime with an intramuscular boost.     

Prefabricated (expander) capsule-lined transposition and advancement flaps in reconstruction of lower eyelid and oral defects: an experimental study

In six pigs with prefabricated transposition flaps and six pigs with prefabricated advancement flaps, both flap types (lined with an expander capsule) were used to reconstruct wedge excisions of the lower eyelid or defects in the cheek/oral mucosa. The capsules replaced the conjunctiva in eyelid defects and the oral mucosa in cheek defects. Histopathologic studies were performed at 5 to 7 days, 9 to 10 days, 2 weeks, 3 to 4 weeks, and 2 and 3 months after flap reconstructions. Healing was rapid and uneventful, leading to restoration of the conjunctiva/eyelid and oral mucosa between 9 days and 2 weeks. The healing of the eyelid conjunctiva was somewhat faster than of the oral mucosa. The expander capsule acted as a conjunctival/ mucosal substitute, providing a temporary physical shield, an infectious barrier, and a matrix for epithelial regeneration. All reconstructions were successful except one oral reconstruction with early flap necrosis. Flaps lined with an expander capsule could improve and facilitate clinical reconstructions in the eyelid and oral cavity.     

A DNA vaccine prime followed by a liposome-encapsulated protein boost confers enhanced mucosal immune responses and protection

A variety of DNA vaccine prime and recombinant viral boost immunization strategies have been developed to enhance immune responses in humans, but inherent limitations to these strategies exist. There is still an overwhelming need to develop safe and effective approaches that raise broad humoral and T cell-mediated immune responses systemically and on mucosal surfaces. We have developed a novel mucosal immunization regimen that precludes the use of viral vectors yet induces potent T cell responses. Using hepatitis B surface Ag (HBsAg), we observed that vaccination of BALB/c mice with an i.m. HBsAg-DNA vaccine prime followed by an intranasal boost with HBsAg protein encapsulated in biologically inert liposomes enhanced humoral and T cell immune responses, particularly on mucosal surfaces. Intranasal live virus challenge with a recombinant vaccinia virus expressing HBsAg revealed a correlation between T cell immune responses and protection of immunized mice. A shortened immunization protocol was developed that was successful in both adult and neonatal mice. These results support the conclusion that this new approach is capable of generating a Th-type-1-biased, broad spectrum immune response, specifically at mucosal surfaces. The success of this design may provide a safe and effective vaccination alternative for human use.     

Metabolomic approach by 1H NMR spectroscopy of serum for the assessment of chronic liver failure in patients with cirrhosis

Assessment of chronic liver failure (CLF) in cirrhotic patients is needed to make therapeutic decisions. A biological score is usually performed, using the Model for End-Stage Liver Disease (MELD), to evaluate CLF. Nevertheless, MELD does not take into account metabolic perturbations produced by liver-function impairment. In contrast, metabolomics can investigate many metabolic perturbations within biological systems. The purpose of this study was to assess whether metabolomic profiles of serum, obtained by proton NMR spectroscopy from cirrhotic patients, are affected by the severity of CLF. An orthogonal projection to latent-structure analysis was performed to compare MELD scores and NMR spectra of 124 patients with cirrhosis. The statistical model obtained showed a good explained variance (R(2)X = 0.87 and R(2)Y = 0.86) and a good predictability (Q(2)Y = 0.64). Metabolomic profiles showed significant differences regarding various metabolites depending of severity of CLF: levels of high-density lipoprotein and phosphocholine resonances were significantly higher in patients with mild CLF compared to severe CLF. Other metabolites such as lactate, pyruvate, glucose, amino acids, and creatinine were significantly higher in patients with severe CLF than mild CLF. Our conclusion is that metabolomic NMR analysis provides new insights into metabolic processes related to the severity of hepatic function impairment in cirrhosis.     

Identification of a peptide enhancing mucosal and systemic immune responses against EGFP after oral administration in mice

Gangliosides are receptors for various peptides and proteins including neuropeptides, beta-amyloid proteins, and prions. Recently, the role of gangliosides in mucosal immunization has attracted attention due to the emerging interest in oral vaccination. Ganglioside GM1 exists in abundance on the surface of the M cells of Peyer's patch, a well-known mucosal immunity induction site. In the present study we identified a peptide ligand for GM1 and tested whether it played a role in immune induction. GM1-binding peptides were selected from a phage-displayed dodecapeptide library and one peptide motif, GWKERLSSWNRF, was fused to the C-terminus of enhanced green fluorescent protein (EGFP). The fusion protein, but not EGFP fused with a control peptide, was concentrated around Peyer's patch after incubation in the lumen of the intestine ex vivo. Furthermore, oral feeding of the fusion protein but not control EGFP induced mucosal and systemic immune responses against EGFP resembling Th2-type immune responses.     

靶向M细胞的抗原递送¾¾增强黏膜免疫应答的关键策略

黏膜是阻止病原入侵的第一道防线,黏膜免疫系统在抵抗感染方面起着至关重要的作用。通过黏膜途径接种疫苗可以同时诱导黏膜和全身免疫反应,因此,理论上针对黏膜的免疫策略是最合理和有效的。但黏膜免疫系统的复杂性和屏障作用造成抗原诱导的免疫应答水平低下,制约了黏膜疫苗的发展。M细胞(Microfoldcells)是黏膜免疫系统所独有的,其具有捕获腔内抗原和启动抗原特异性免疫应答的功能。M细胞摄取抗原的多少直接关系到黏膜疫苗的免疫效力,而利用M细胞配体可将抗原靶向递呈给M细胞,从而实现高效的黏膜免疫应答。靶向M细胞的抗原递送策略及其应用可以提高黏膜免疫应答水平,促进黏膜疫苗的研制。尽管如此,要成功研制安全高效的黏膜疫苗,今后依然有漫长的路要走,这可能有赖于进一步探究M细胞的特性和功能及黏膜免疫机制。     

New routes of administration: epidermal, transcutaneous mucosal ways of vaccination

A successful vaccine triggers the interaction of various cells of the immune system as does a regular immune response. It is thus necessary to introduce the vaccine antigens into an anatomic site where they will contact immune cells. The route of administration is thus critical for the outcome of vaccination. Intramuscular or subcutaneous injections are the most popular. Antigens injected intramuscularly can form persistent precipitates that are dissolved and re-absorbed relatively slowly. If injecting antigens is a quick, easy and reproducible way to vaccination, it requires trained personnel. Alternatives exist, through non-invasive formulations which allow administration by the patient or a third party with no particular expertise. The skin, especially its epidermal layer, is an accessible and competent immune environment and an attractive target for vaccine delivery, through transcutaneous delivery or immunostimulant patches. Mucosal immunization is another strategy: its major rationale is that organisms invade the body via mucosal surfaces. Therefore, local protection at mucosal surface as well as systemic defense is beneficial. Various formulations of mucosal vaccines have been developed, such as the Sabin oral polio vaccine (OPV), rotavirus vaccines, cold-adapted influenza vaccines or vaccine against typhoid fever. Thus we are entering in an era where mucosal and transcutaneous immunisation will play an important role in disease management. However, it has not been so easy to obtain regulatory approval for mucosal or transcutaneous formulations and needle-based vaccines continue to dominate the market.     

Quartz sand as an adsorbent for purification of extracellular glucose oxidase from Penicillium funiculosum 46.1

The procedure of purification of extracellular glucose oxidase (GO, EC 1.1.3.4) from culture-liquid filtrate (CLF) of the fungus Penicillum funiculosum 46.1 using alluvial quartz sand as an adsorbent has been developed. The modification of sand by changing the charge and polarity did not lead to a significant increase in its adsorption capacity towards GO. The effectiveness of sand and aluminum oxide, used as sorbents for isolation of GO from CLF, was compared. Glucose oxidase, isolated from CLF by adsorption on sand, exhibited a greater catalytic activity compared to the enzyme specimens obtained by column chromatography on CLF. Sand adsorbed GO from P. funiculosum 46.1 more effectively than aluminum oxide. It is concluded that sand may be used for fractionation of partly purified GO.     

Elastohydrodynamics of the Eyelid Wiper

This paper presents an elastohydrodynamic model of the human eyelid wiper. Standard lubrication theory is applied to the fluid layer between the eyelid wiper and ocular surface. The role of the lubrication film is to reduce the shear stresses by preventing solid to solid contact between the eyelid wiper and ocular surface. For the lubrication film to be effective, it is required that the orientation of the eyelid wiper changes between the opening and closing phases of a blink. In order to model this, the hydrodynamic model is coupled with an elastic mattress model for the soft tissue of the eyelid wiper and ocular surface. This leads to a one-dimensional non-linear partial differential equation governing the fluid pressure in the lubrication film. In order to solve the differential equation, a loading condition or constraint equation must be specified. The resulting system is then solved numerically. The model allows predictions of the tear film flux from under the upper eyelid, as well as normal and shear stresses acting on the ocular surface. These factors are important in relation to dry eye syndrome, deformation of the cornea and contact lens design. It is found that the pressure and shear stress under the eyelid act across a length of approximately 0.1 mm which is consistent with clinical observations. It order to achieve a flow of tears from under the upper eyelid during a blink, the model requires that the normal force the eyelid applies to the ocular surface during the closing phase of the blink is significantly higher than during the opening phase of the blink. Electronic Supplementary Material The online version of this article () contains supplementary material, which is available to authorized users.     

Gastrointestinal tract as a part of immune defence.

The paper is devoted to reviewing the function of gastrointestinal mucosal immune system. Gut-associated lymphoid tissue (GALT) provides the host with protective mechanisms against invasion by potential pathogens across the mucosal surface, and on the other hand, it plays a role in the development of induced tolerance against harmless products of digestion and the normal intestinal flora, all of which are potentially immunogenic. First the organization of GALT, its afferent and efferent limbs are described, then the normal B cell homeostasis and finally the induced oral tolerance are discussed. Knowledge of mechanisms of mucosal immune response is essential to complete understanding of the pathogenesis and treatment of inflammatory gastrointestinal diseases as well.     

Quartz Sand as an Adsorbent for Purification of Extracellular Glucose Oxidase from Penicillium funiculosum 46.1

A procedure for purification of extracellular glucose oxidase (GO, EC 1.1.3.4) from a filtrate of culture liquid (CLF) of the fungus Penicillium funiculosum 46.1 has been developed using alluvial quartz sand as an adsorbent. Modifying the sand by changing the charge and polarity did not lead to a significant increase in its adsorption capacity towards GO. The effectiveness of sand and aluminum oxide used as adsorbents for GO isolation from CLF has been compared. Glucose oxidase isolated from CLF by adsorption on sand exhibited a greater catalytic activity than enzyme preparations obtained by column chromatography on CLF. Glucose oxidase from P. funiculosum 46.1 was adsorbed on sand more effectively than on aluminum oxide. It is concluded that sand may be used for fractionation of partially purified GO.     

A catabolin-like factor from porcine kidney

Explants of pig kidney cortex and medulla release a catabolin-like factor (CLF). The CLF of both kidney cortex and medulla can be precipitated with ammonium sulphate, mainly in the 60-95 per cent fraction. By gel chromatography the kidney CLF showed a major active fraction at a molecular weight of around 22 500. Whole glomeruli and dissociated glomerular cells in culture also released into the medium a CLF that could be bioassayed in live cartilage but displayed no effect on dead cartilage. The possible role of such a local hormone is discussed.