Glucagon‐like peptide‐1 (GLP‐1) and peptide YY (PYY), secreted by enteroendocrine L‐cells located most densely in the colon and rectum, are of fundamental importance in blood glucose and appetite ...regulation. In animal models, colonic administration of bile acids can stimulate GLP‐1 and PYY by TGR5 receptor activation. We evaluated the effects of taurocholic acid (TCA), administered as an enema, on plasma GLP‐1 and PYY, as well as gastrointestinal sensations in 10 healthy male subjects, and observed that rectal administration of TCA promptly stimulated secretion of both GLP‐1 and PYY, and increased fullness, in a dose‐dependent manner. These observations confirm that topical application of bile acids to the distal gut may have potential for the management of type 2 diabetes and obesity.
Antibody fragments have been shown to penetrate into the anterior chamber when applied to the cornea. The aim of this study was to investigate whether such fragments could penetrate into the vitreous ...cavity after topical administration to the ocular surface of rabbits.
An engineered single-chain variable-domain antibody fragment with specificity for an irrelevant rat determinant was applied as a 50 microl eye drop to the eyes of live rabbits at 20-min intervals over 12 h. Eye drops contained 0.8-1.1 mg/ml protein in a buffered salt solution supplemented with penetration and viscosity enhancers. Samples were collected by paracentesis from the vitreous cavity immediately postmortem. Antibody fragments in these samples were quantified by measuring the binding activity to specific antigen, using flow cytometry.
Topically applied antibody fragments were detectable in the vitreous of rabbit eyes after 4-12 h but had cleared at 12 h following the final eye drop. Concentrations of the antibody fragment in the vitreous samples were estimated to be 50-150 ng/ml at 12 h. Penetration of the parental whole antibody into the vitreous was not observed.
Antibody fragments penetrate into the vitreous chamber of the rabbit eye after topical administration to the ocular surface. Such fragments may have therapeutic potential for diseases affecting the posterior segment.
SUMMARY
Antibodies are powerful immunotherapeutic agents but their use for treating ocular disorders is limited by their poor penetration into the eye. We hypothesized that antibody fragments of ...relatively small size might penetrate the cornea more readily. Monovalent single chain variable region (scFv) antibody fragments and divalent miniantibodies were engineered from existing monoclonal antibodies, expressed in a bacterial expression system, and purified by metal ion affinity chromatography. Corneoscleral preparations from normal pig and cat eyes were mounted in a corneal perfusion chamber. Intact antibodies and antibody fragments were applied topically to the anterior corneal surface over 12‐h periods, and samples were collected from the artificial anterior chamber. Similar experiments were performed with whole enucleated pig and human eyes. Penetration of antibodies and fragments was quantified by high‐sensitivity flow cytometry on appropriate target cells. Both monovalent scFv and divalent miniantibody fragments (but not whole immunoglobulin molecules) passed through de‐epithelialized and intact corneas after topical administration, and could be detected by antigen binding. Addition of 0·5% sodium caprate facilitated penetration through intact corneas. Topically‐applied scFv was found to penetrate into the anterior chamber fluid of rabbit eyes in vivo. The engineered fragments were stable and resistant to ocular proteases. Monovalent and divalent antibody constructs of molecular weight 28 kD and 67 kD, respectively, can penetrate through intact corneas into the anterior chamber, with retention of appropriate antigen‐binding activity. Such constructs may form novel therapeutic agents for topical ophthalmic use.
Structure of a V3-Containing HIV-1 gp120 Core Huang, Chih-chin; Tang, Min; Zhang, Mei-Yun ...
Science (American Association for the Advancement of Science),
11/2005, Letnik:
310, Številka:
5750
Journal Article
Recenzirano
Odprti dostop
The third variable region (V3) of the HIV-1 gp120 envelope glycoprotein is immunodominant and contains features essential for coreceptor binding. We determined the structure of V3 in the context of ...an HIV-1 gp120 core complexed to the CD4 receptor and to the X5 antibody at 3.5 angstrom resolution. Binding of gp120 to cell-surface CD4 would position V3 so that its coreceptor-binding tip protrudes 30 angstroms from the core toward the target cell membrane. The extended nature and antibody accessibility of V3 explain its immunodominance. Together, the results provide a structural rationale for the role of V3 in HIV entry and neutralization.
Experimental melanin‐induced uveitis (EMIU) is a rodent model of acute anterior uveitis which was described in 1993. We investigated strain susceptibility, and age and gender characteristics of the ...model, undertook histological and immunohistochemical studies to investigate underlying cellular mechanisms, and examined several treatment options. Rats were immunized with bovine ocular melanin (250 μg), and disease was followed by slit lamp examination. Lewis, Fischer 344 and Porton rats were found to be susceptible to EMIU, whereas Wistar‐Furth, DA, and Hooded Wistar strains were resistant. EMIU was neither age‐ nor gender‐dependent. In Fischer 344 rats, EMIU was characterized clinically by florid anterior segment inflammation. Histopathological findings included infiltration of ciliary body and iris with mononuclear cells and neutrophils. Both CD4+ and CD8+ T lymphocytes were prominent. Rats were then treated with intraperitoneal injections of anti‐CD4, anti‐CD8 or irrelevant isotype‐matched MoAb on days −3, 0, 3, 6 and 9 with respect to melanin immunization. Incidence of uveitis was significantly reduced in rats treated with a non‐depleting cocktail of anti‐CD4 MoAbs (P = 0.007), whereas a depleting anti‐CD8 antibody had no effect on the disease. Mannose‐6‐phosphate inhibits lymphocyte migration in some models of T cell‐mediated inflammation. This simple sugar was administered to additional rats via intraperitoneal osmotic pumps for 14 days following disease induction, but did not influence the uveitis. We conclude that EMIU is controlled by CD4+ T cells, and disease may be abrogated by treatment with anti‐CD4 MoAbs.
AIMS To examine the hypothesis that apoptosis of infiltrating cells contributes to spontaneous resolution of uveitis in clinically relevant rodent models. METHODS Experimental melanin induced uveitis ...(EMIU) was induced in Fischer 344 rats by immunisation with 250 μg bovine ocular melanin. Endotoxin induced uveitis (EIU) was induced by injection of 200 μgEscherichia coli lipopolysaccharide. Formalin fixed, paraffin embedded ocular cross sections were stained by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick end labelling (TUNEL) to identify apoptotic cells. Indirect immunoperoxidase staining of paraformaldehyde lysine periodate fixed tissue cross sections was used to demonstrate expression of inducible nitric oxide synthase (iNOS). RESULTS TUNEL positive mononuclear cells were observed in the anterior uvea during both EMIU and EIU at all selected time points. However, whereas the majority of mononuclear cells appeared apoptotic from the outset of disease, neutrophils were notably TUNEL negative at all time points examined. Many infiltrating neutrophils expressed iNOS. CONCLUSION Apoptosis occurs early in the course of rat EMIU and EIU, and may contribute to resolution of these diseases. In general, infiltrating mononuclear cells die rapidly, while neutrophils survive, producing inducible nitric oxide synthase which may contribute to disease pathogenesis.
Context:
In vitro and animal studies suggest that bile acids have the capacity to reduce blood glucose by stimulating glucagon-like peptide-1 (GLP-1) and, thereby, insulin.
Objective:
This study ...evaluated the effects of intrajejunal taurocholic acid (TCA) on blood glucose, GLP-1, and insulin responses to jejunal glucose infusion in healthy men.
Participants and Design:
Ten healthy men were each studied on 2 days in a double-blind, randomized order. After the subjects fasted overnight, a jejunal catheter was positioned and a balloon inflated 30 cm beyond the pylorus with aspiration of endogenous bile. Two grams TCA in saline, or saline control, was infused beyond the balloon over 30 minutes, followed by 2 g TCA or control, together with 60 g glucose, over the next 120 minutes. Blood was sampled frequently for the measurements of blood glucose, total GLP-1, insulin, C-peptide, and glucagon.
Results:
Intrajejunal infusion of TCA alone (t = −30 to 0 minutes) had no effect on blood glucose, GLP-1, insulin, C-peptide, or glucagon concentrations. During intrajejunal glucose infusion (t = 0 to 120 minutes), blood glucose concentrations were lower (P < .001), and plasma GLP-1 (P < .001) and the C-peptide/glucose ratio (P = .008) were both greater, whereas plasma insulin, C-peptide, and glucagon levels were not significantly different after TCA than after control.
Conclusions:
In healthy humans, small intestinal infusion of TCA potently reduces the glycemic response to small intestinal glucose, associated with an increase in GLP-1 and C-peptide/glucose ratio. These observations indicate the potential for bile acid-based therapy in type 2 diabetes.
Orthotopic penetrating guinea pig to rat and chicken to rat corneal xenografts were performed to examine the nature of the host response. Guinea pig to rat xenografts failed at a median of day 3 ...after surgery. A similar but slightly accelerated pattern of failure was seen in guinea pig xenografts performed in prevascularized recipient rat corneas. Chicken to rat xenografts failed at a median of day 2 after grafting. Rat corneal isograft controls survived indefinitely. Corneal endothelial cells were visible by silver staining on the xenografts immediately after operation, which indicates that failure was not due to loss of these cells during surgery. Histopathology and immunoperoxidase staining indicated that xenograft failure in euthymic recipients was characterized by early corneal epithelial and endothelial cell damage, granulocytic infiltration, and hemorrhage from recipient corneal and iris capillaries, followed at 7-14 days by infiltration with T cells, macrophages, and eosinophils. An accelerated pattern of graft failure was also observed in guinea pig grafts into homozygous nude rat recipients, which suggests that preformed anti-donor antibody and complement were responsible for some of the early graft damage. Flow cytometry demonstrated the presence of pre-existing natural antibodies to guinea pig and chicken lymphocytes and erythrocytes, as expected from other studies. Immunohistochemistry showed the presence of rat IgG2a, IgG1, and IgM, but not IgD deposited on grafts in immunocompetent recipients on the first postoperative day. We conclude that orthotopic corneal xenografts undergo substantial accelerated damage mediated by pre-existing antibody, followed at 7-14 days by a cell-mediated response that causes further destruction.
In healthy individuals, intraduodenal whey protein load-dependently modulates gastrointestinal motor and hormonal functions and suppresses energy intake. The effect of oral whey, particularly the ...impact of load, has not been evaluated.
The purpose of this study was to quantify gastric emptying of 30 and 70 g of oral whey protein loads and their relation to gastrointestinal hormone, glycemic, and appetitive responses.
On 3 separate occasions in a randomized, double-blind order, 18 lean men mean ± SEM age: 24.8 ± 1.4 y; body mass index (in kg/m(2)): 21.6 ± 0.5 received iso-osmolar, equally palatable drinks (∼450 mL) containing 30 g pure whey protein isolate (L), 70 g pure whey protein isolate (H), or saline (control). Gastric emptying (with the use of 3-dimensional ultrasound), plasma cholecystokinin, glucagon-like peptide 1, glucose-dependent insulinotropic peptide, insulin, glucagon, total amino acids, and blood glucose were measured for 180 min after consumption of the drinks, and energy intake at a buffet-style lunch was quantified.
Gastric emptying of the L and H drinks was comparable when expressed in kilocalories per minute (L: 2.6 ± 0.2 kcal/min; H: 2.9 ± 0.3 kcal/min) and related between individuals (r = 0.54, P < 0.01). Gastrointestinal hormone, insulin, and glucagon responses to the L and H drinks were comparable until ∼45-60 min after ingestion, after which time the responses became more differentiated. Blood glucose was modestly reduced after the H drink between t = 45 and 150 min when compared with the L drink (all P < 0.05). Energy intake was suppressed by both L and H drinks compared with control (P < 0.05) (control: 1174 ± 91 kcal; L: 1027 ± 81 kcal; and H: 997 ± 71 kcal).
These findings indicate that, in healthy lean men, the rate of gastric emptying of whey protein is independent of load and determines the initial gastrointestinal hormone response. This study was registered at www.anzctr.org.au as 12611000706976.
Context:
Changes in gut motor and hormonal function contribute to the eating-inhibitory and glucose-lowering effects of protein. The effect of amino acids, the digestive products of protein, on ...gastrointestinal function, eating, and glycemia has not been investigated comprehensively.
Objective:
We tested the hypothesis that L-tryptophan (L-Trp) stimulates gastrointestinal motor and hormonal functions, inhibits eating, and modulates glycemia.
Design, Settings, Participants, and Intervention:
Ten healthy, normal-weight men were studied in randomized, double-blind fashion, each receiving a 90-minute intraduodenal infusion of L-Trp at 0.075 (total 6.75 kcal) or 0.15 (total 13.5 kcal) kcal/min or saline (control).
Main Outcome Measures:
Antropyloroduodenal motility, plasma ghrelin, cholecystokinin, glucagon-like peptide-1, peptide tyrosine tyrosine, insulin, glucagon, blood glucose, and appetite perceptions were measured. Food intake was quantified from a buffet meal after the infusion.
Results:
Intraduodenal L-Trp suppressed antral pressures (P < .05) and stimulated pyloric pressures (P < .01) and markedly increased cholecystokinin and glucagon (both P < .001). Glucagon-like peptide-1 and peptide tyrosine tyrosine increased modestly (both P < .001), but there was no effect on total ghrelin. Insulin increased slightly (P < .05) without affecting blood glucose. Plasma L-Trp increased substantially (P < .001). All effects were dose-related and associated with increased fullness and substantially decreased energy intake (P < .001). There was a strong inverse correlation between energy intake and plasma L-Trp (r = −0.70; P < .001).
Conclusions:
Low caloric intraduodenal loads of L-Trp affect gut motor and hormonal function and markedly reduce energy intake. A strong inverse correlation between energy intake and plasma L-Trp suggests that, beyond gut mechanisms, direct effects of circulating L-Trp mediate its eating-inhibitory effect.
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