Characterization of binding properties of ICAM-1 peptides to LFA-1: inhibitors of T-cell adhesion

In the present study, we characterized the binding site of two intercellular adhesion molecule-1-derived cyclic peptides, cIBC and cIBR, to the LFA-1 on the surface of T cells. These peptides had been able to inhibit LFA-1/intercellular adhesion molecule-1 signal by blocking the signal-2 of immune synapse. Both peptides prefer to bind to the closed form of LFA-1 I-domain, indicating that two peptides act as allosteric inhibitors against intercellular adhesion molecule-1. Binding site mapping using monoclonal antibodies proposes that cIBC binds to around residues 266-272 of LFA-1 I-domain where this site is adjacent to the metal ion-dependent adhesion site. On the other hand, cIBR binds to the pocket called L-site where is distant from metal ion-dependent adhesion site. Cross-inhibition mapping between two peptides show that cIBR could inhibit the binding of cIBC but not vice versa, suggesting that cIBR has some properties that allow this peptide bind to more than one site. Structural comparison between cIBC and cIBR reveals that cIBR is more flexible than cIBC, allowing this peptide bind to exposed region, such as cIBC-binding site as well as cramped pocket like L-site. Our findings are important for understanding the selectivity of cIBC and cIBR peptides; thus, they can be conjugated with drugs and transported specifically to the target.     

Blood-derived macrophages infiltrate the retina and activate Muller glial cells under experimental choroidal neovascularization

Inflammation is a major mechanism in the pathogenesis of age-related macular degeneration, the most important cause of blindness in the elderly. Previous studies have focused on the role of macrophages in regulating the growth of pathological new vessels over the retina, called choroidal neovascularization (CNV). However, no research has been done to evaluate the role of inflammation as a mechanism of vision loss and retinal degeneration in the retina underlying CNV. In other neuropathological conditions, hematogenous macrophages and/or resident microglia contribute to neurodegeneration. We have combined laser-induced CNV in mice and bone marrow transplantation with GFP-labeled bone marrow to determine the relative role of recruited blood-derived macrophages versus resident microglia in the retina associated with CNV. Using these chimeric mice, we have found that many GFP-labeled cells infiltrated the retina underlying CNV but not the retina unaffected by CNV. Immunostaining for the cell adhesion molecules VCAM 1, ICAM 1, and PECAM was strongly upregulated in retinal blood vessels under CNV. All GFP-labeled cells were immunoreactive for the macrophage marker F4/80. Most (70%) of the F4/80 immunoreactive cells were GFP-labeled under CNV. The density of resident microglia did not increase. Most GFP-labeled cells were found in close proximity to activated Muller cells. Depleting circulating macrophages with clodronic acid diminished the density of F4/80 immunoreactive cells as well as the density of pERK immunoreactive Muller cells in the retina under CNV. Thus, recruitment of blood-derived macrophages more than resident microglia seems to be associated with CNV.     

Soluble adhesion molecules in young children with signs of beta-cell autoimmunity--prospective follow-up from birth

BACKGROUND: This study aimed at evaluating the relationship between the circulating concentrations of soluble intercellular adhesion molecule-1 (sICAM-1) and sL-selectin and the appearance of beta-cell autoimmunity, and at assessing whether these molecules could assist in the identification of environmental factors implicated in the immune process damaging the pancreatic beta-cells. METHODS: Serum levels of soluble adhesion molecules were measured with enzyme-linked immunosorbent assays over the first 2 years of life in 65 children seroconverting to positivity for autoantibodies and 65 control children, all with HLA-conferred susceptibility to type 1 diabetes (T1D). RESULTS: The total integrated concentrations of soluble adhesion molecules were comparable between the two groups. The autoantibody-positive children tended to have higher sL-selectin concentrations during the 3-month seroconversion (SC) period than did the control children during the corresponding period (P = 0.07), the difference being significant (P = 0.03) after excluding subjects with signs of a concurrent enterovirus infection. Autoantibody-positive children had higher concentrations of sL-selectin in the 3-month period when an enterovirus infection was detectable than did the control children (P = 0.018). No significant difference could, however, be seen after excluding the children with concomitant seroconversion to autoantibody positivity. CONCLUSIONS: Elevated concentrations of sL-selectin are temporally associated with seroconversion to autoantibody positivity suggesting that leukocyte activation might coincide with the appearance of beta-cell autoimmunity. Early-onset progressive beta-cell autoimmunity, on the other hand, is not reflected in overall increased concentrations of soluble adhesion molecules in the peripheral circulation during the first 2 years of life in children carrying increased HLA-conferred disease susceptibility. Enterovirus infections (EVIs) are not independently associated with increased circulating sL-selectin concentrations in young children with enhanced HLA-conferred susceptibility to T1D.     

Peptide‐mediated targeted drug delivery

Targeted drug delivery to specific group of cells offers an attractive strategy to minimize the undesirable side effects and achieve the therapeutic effect with a lower dose. Both linear and cyclic peptides have been explored as trafficking moiety due to ease of synthesis, structural simplicity, and low probability of undesirable immunogenicity. Peptides derived from sequence of cell surface proteins, such as intercellular adhesion molecule‐1 (ICAM‐1), LHRH, Bombesin, and LFA‐1, have shown potent binding affinity to the target cell surface receptors. Moreover, peptides derived from ICAM‐1 receptor can be internalized by the leukemic T‐cells along with the conjugated moiety offering the promise to selectively treat cancers and autoimmune diseases. Systematic analyses have revealed that physicochemical properties of the drug–peptide conjugates and their mechanism of receptor‐mediated cellular internalization are important controlling factors for developing a successful targeting system. This review is focused on understanding the factors involved in the development of an effective drug–peptide conjugate with an emphasis on the chemistry and biology of the conjugates. Reported results on several promising drug–peptide conjugates have been critically evaluated. The approaches and results presented here will serve as a guide to systematically approach targeted delivery of cytotoxic drug molecules using peptides for treatment of several diseases. © 2010 Wiley Periodicals, Inc. Med Res Rev, 32, No. 3, 637‐658, 2012     

大鼠颅脑损伤后ICAM-1在神经细胞中表达谱及其意义

目的探讨大鼠颅脑损伤后ICAM-1的表达。方法将50只大鼠随机分为10组,采用改进的Feeney自由落体硬膜外撞击方法制造大鼠颅脑损伤模型,各型损伤分别于伤后15 min、30 min、1 h、3 h、6 h、24 h、72 h、96 h及120 h处死,免疫组化法检测ICAM1的表达,采用CMIAS多功能真彩色病理图像分析系统测量神经细胞内平均灰度值。结果 ICAM1在颅脑损伤3 h后表达增高(P<0.05),至120 h表达接近正常。结论大鼠颅脑损伤后ICAM1表达随时间变化,很可能在颅脑损伤修复中发挥重要功能,且对颅脑损伤的早期判定具有重要意义。     

葛根芩连汤对糖尿病大鼠早期视网膜病变影响的实验研究

[目的]探讨葛根芩连汤对糖尿病大鼠早期视网膜病变的治疗作用。[方法]选取45只Wistar大鼠,链脲佐菌素(STZ)腹腔注射,制作糖尿病模型,糖尿病模型建立后随机分为DM组(糖尿病组)、DM+NS组(糖尿病生理盐水注射组)和实验组(DM+葛根芩连汤组)。模型建立后,DM组不做任何处理,实验组给予葛根芩连汤8.2 g/kg,DM+NS组注射等体积的生理盐水。对糖尿病大鼠的空腹血糖、血浆胰岛素、体质量、甘油三酯(TG)和高密度脂蛋白(HDL-C)的含量进行检测,以观察葛根芩连汤降糖降脂功效;通过免疫印迹(Westem blot)法检测视网膜蛋白激酶B(PKB)以及炎性相关因子——细胞间黏附分子-1(ICAM-1)的表达情况;利用苏木-伊红(HE)染色观察各组大鼠的超微结构;采用TUNEL法检测凋亡相关蛋白C-myc的凋亡指数。[结果]与DM组和DM+NS组比较,实验组大鼠空腹血糖明显降低、体质量明显增加,差异有统计学意义(P0.05);与DM组和DM+NS组比较,实验组大鼠血胰岛素含量增加,组间比较差异有统计学意义(P0.05);与DM组和DM+NS组比较,实验组大鼠TG明显降低而HDL-C明显升高(P0.05)。通过Westem blot检测可见与DM组和DM+NS组比较,实验组大鼠PKB的表达明显增加、ICAM-1的表达明显下降,差异有统计学意义(P0.05);光镜下HE染色观察到各组超微结构:DM组可见核固缩、浓染,核膜内陷,染色质边集,凋亡小体出现,实验组变化明显减轻(P0.05);与DM组和DM+NS组比较,实验组中凋亡相关蛋白C-myc明显减少(P0.05)。[结论]葛根芩连汤能够改善大鼠空腹血糖、血浆胰岛素及体质量等的变化,具有较好的降糖降脂作用,能通过增加PKB的表达及抑制糖尿病大鼠视网膜病变的炎症反应减轻或延缓糖尿病的发生和发展。     

Modulation of ICAM‐1 tissue expression in patients with liver transplantation (LT) and acute rejection (AR) after glucocorticoid treatment

Abstract Acute rejection (AR) is a frequent complication following liver transplantation (LT). ICAM‐1 may be involved in its pathogenesis. High doses of glucocorticoids are the standard treatment in these patients. The aim of this study was to describe corticoid effects on ICAM‐1 tissue expression in liver biopsies of patients with LT and AR. The study included liver biopsies performed before and after treatment in 12 patients with LT and proven AR. In 10 patients AR was reversible and in 2, was steroid resistant. For immunohistochemistry, an indirect immunoperoxidase technique was used. Each histology section was semiquantitatively evaluated as follows: 0: <10% staining, 1: 10‐25%, 2: 25‐50%, 3: >50%. The control group comprised nine patients with LT and normal liver biopsies. In pre‐treatment liver biopsy samples, ICAM‐1 was markedly expressed on sinusoidal cells (2.41 ± 0.66), and there was also expression on periportal (0.66 ± 0.65) and perivenular hepatocytes (0.83 ± 0.57). By contrast, in the liver tissue from the control group, sinusoidal ICAM‐1 reactivity was significantly lower (0.88 ± 0.33; P < 0.05), and hepatocytes showed no reliable ICAM‐1 expression. After steroid treatment the intensity of ICAM‐1 decreased significantly in sinusoids (1.5 ± 0.67; P < 0.05) and in perivenular hepatocytes (0.25 ± 0.86; P < 0.05). Additionally, we also observed a decreased ICAM‐1 reactivity in portal hepatocytes (0.25 ± 0.62), but these differences did not reach statistical significance. Remarkably, after treatment, hepatocytes did not show ICAM‐1 reactivity in resolved AR, but in corticoid‐resistant patients AR did not change or increase. In conclusion, in patients with LT and AR, ICAM‐1 was expressed in hepatocytes and with more intensity in sinusoid cells. Additionally, a down‐regulation of the ICAM‐1 tissue expression after corticoid treatment may exist, although in corticoid‐resistant AR no modulation on ICAM‐1 tissue expression was observed.     

Abdominal peritoneum as a defense organ: Analysis of ICAM‐1 expression in the LPS‐stimulated rat

To investigate the immune environment of the peritoneal cavity, ICAM‐1 (intercellular adhesion molecule) expression on the apical surface of the hepatic peritoneum of LPS (lipopolysaccharide) stimulated rats was anlayzed ultrastructurally and chronologically with immnunoTEM&SEM. ICAM‐1 expression was restricted to the side of microvilli of the mesothelial cells. Microvilli demonstrated bulbous tips and included fuzzy coats and strands. Bulbous tips sometimes expressed the antigen, but fuzzy coats and strands did not. Intervillar cell surfaces lacked its expression. Although ICAM‐1 expression increased eightfold 24 hr after stimulation, the selective expression remained unchanged. These results suggest that microvilli are closely associated with cell migration in the peritoneal cavity through adhesion molecules that establish a road for migration. Clin. Anat. 12:20–26, 1999. © 1999 Wiley‐Liss, Inc.     

ICAM‐1 and B7‐1 provide similar but distinct costimulation for CD8+ T cells,while CD4+ T cells are poorly costimulated by ICAM‐1

The function of purified ICAM‐1 in costimulating CD4⁺ and CD8⁺ T cell responses has been directly compared to that of B7‐1 in a model system that minimizes contributions of other receptor‐ligand interactions. While B7‐1 costimulates both subsets of T cells, ICAM‐1 is much more effective in the costimulation of CD8⁺ cells. ICAM‐1 also synergizes with B7‐1 for the induction of IL‐2 production in CD8⁺ but not CD4⁺ T cells. These differences are not explained by differences in LFA‐1 receptor expression on the two subsets of T cells. The CD8⁺ T cell response to ICAM‐1 costimulation is associated with increased proliferation and IL‐2 production at levels similar to those seen with B7‐1 costimulation, but clonal expansion in response to ICAM‐1 is not as great due to decreased cell survival. ICAM‐1‐mediated costimulation is effective for both naive and memory CT8⁺ T cells, is independent of CD28 engagement, and does not appear to be due solely to effects on adhesion. These results suggest that ICAM‐1‐dependent, B7‐independent costimulation may be important in initiating a CTL response to class I antigen presented by cells that are not professional APC.