mediated HIV-1 transmission. Although LFA-1 of this patient is able to recognize its ligand, no high avidity/ affinity binding to ICAM-1 is taking place. Since there is no strong binding to ICAM-1, signaling through LFA-1 into the T cell (outside-in signaling) is probably not tak- ing place either. In healthy individuals, signaling through LFA-1 after ICAM-1 binding leads to actin polymerization and remodeling, which is important for enhanced cell adhesion . Impaired cell adhesion in LAD-1 (and var- iant) patients can also be illustrated by the significantly smaller clusters of DC with T cells (Fig. 5). A smaller number of T cells that is tightly attached to DC will result in a decrease of the window of opportunity for HIV-1 transmission. Furthermore, it is likely that the creation of an 'infectious synapse' is disturbed in LAD-1 and LAD-1/ variant patients. Others have shown that DC-SIGN is an important component of the infectious synapse [9,35]. Our results strongly indicate that LFA-1 is also important for infectious synapse formation, possibly through cytoskeletal rearrangements that are induced by ICAM-1 binding.
depleting regimens. These long-term survivors could not be rechal- lenged with secondary grafts to test for donor-specific tolerance, because allograft function beyond the discontinuation of main- tenance therapy was unexpected, and donor tissue had not been preserved for this purpose. After transplant, MLCs performed dur- ing this period of operational tolerance were hyporesponsive but reactive relative to pretransplant proliferation (data not shown). The continued presence of donor-specific T cell reactivity implies that complete deletional tolerance was not the mechanism of graft survival under this regimen. This finding, in conjunction with the observed formation of alloantibodies in 1 of the 2 long-term survi- vors, suggested impending graft failure, not tolerance, as the pro- duction of DSAs very often precedes rejection. One might speculate that the use of sirolimus may have induced Treg-mediated survival, as mTOR inhibition has been shown to promote long-term engraft- ment in allotransplantation by various mechanisms, including preservation of Treg-dependent immunoregulatory networks (27). While we did not examine the effect of our LFA-1–based regimens on Tregs, further investigation is warranted.
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(phorbol 12-myristate 13-acetate) without an increase in the number of receptors expressed. The molecular details of the mechanism are unknown. To determine the effect of PMA activation on LFA-1 movement within the plasma mem- brane, we used the single particle tracking technique to measure the diffusion rate of LFA-1 molecules on EBV- transformed B cells before and after PMA activation. Diffu- sion of LFA-1 on unactivated cells was restricted compared to CR1 (CD35), another transmembrane protein of equiva- lent size. PMA caused a 10-fold increase in the diffusion rate of LFA-1 without any effect on CD35. The increased LFA-1 motion induced by PMA was random, not directed, indicating that it was due to a release of constraints rather than the application of forces. The diffusion rates of LFA-1 are consistent with cytoskeletal attachment before and free diffusion after PMA. Cytochalasin D led to an equivalent increase in mobility and, at low doses, stimulated adhesion. We propose that the release of LFA-1 from cytoskeletal con- straints is an important early step in activation of adhesion, implying that the nonadhesive state of LFA-1 is actively maintained by the lymphocyte cytoskeleton. ( J. Clin. Invest. 1996. 97:2139 – 2144 .) Key words: cytoskeleton • cell mem- brane • diffusion • microscopy • microspheres
untreated neutrophils exhibited on a control protein, glycophorin. Stimulation with fMLP significantly increased neutrophil attachment to purified ICAM-1, but not to the control protein. Anti-CD11b MAbs reduced this chemotactically augmented adherence to that of unstimulated neutrophils, and in combination with anti-CD11a MAbs reduced adherence to that on the control protein. The results in this report indicate that unstimulated neutrophils exhibit LFA-1-dependent attachment to ICAM-1, and chemotactic stimulation enhances the attachment of human neutrophils to ICAM-1 by a Mac-1-dependent process.
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(approximately 5% of normal) but detectable amounts of the heterodimeric LFA-1 antigen were found on mitogen and alloantigen-activated T cells. On granulocytes, Mo1 surface expression was also dependent on the state of cellular activation. The amount of surface Mo1 present on resting normal granulocytes increased by 3-10-fold following exposure to stimuli that induced degranulation, suggesting the presence of a major intracellular pool for this antigen. Analysis […]
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Our results demonstrate that the rate of extravasation of neutrophils in the first 4 h of thioglycollate-induced peritonitis is not significantly abnormal in mice totally deficient in CD11b. Further confirmation that Mac-1 (CD11b/CD18) was unneces- sary for neutrophil migration was provided by our observation that fibrinogen-coated PET disks in the peritoneal cavity had markedly fewer attached neutrophils in CD11b-deficient mice, even though there was as vigorous an influx of neutrophils as that seen in the normal animals. The neutrophils deficient in CD11b that migrated into the peritoneal cavity were defective in adhesion to fibrinogen-coated plastic disks, confirming that CD11b plays an essential role in the binding of neutrophils to fibrinogen (14, 27). Furthermore, this suggests that CD11c/ CD18 is not markedly upregulated in these mice because CD11c/CD18 has also been shown to mediate neutrophil ad- hesion to fibrinogen-coated surfaces (34, 35). In addition, neu- trophils from CD11b-deficient mice were also defective in neu- trophil degranulation and iC3b-mediated phagocytosis. Future experiments should better define the importance in vivo of these functions of CD11b by using Mac-1–deficient mice in murine models of infection, inflammation, and healing. In con- trast to the results which demonstrate that Mac-1 plays a criti- cal function in mediating binding of leukocytes to fibrinogen, neutrophil degranulation, and iC3b-mediated phagocytosis, our results are inconsistent with the conclusion that CD11b/ CD18 is necessary for effective emigration of neutrophils, an assumption based on earlier studies with anti-CD11b mono- clonal antibodies 5C6 (7) and M1/70 (8) in similar models.
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Integrin receptor engagement in T-cells activates several proteins by tyrosine phosphorylation that initiates signalling cascades and contributes to the assembly of a ‘signalosome’, a multiprotein complex including various enzymes, their substrates and scaffold/adapter proteins. The identification of proteins undergoing tyrosine phosphorylation in response to LFA-1 integrin stimulation is essential for understanding the molecular mechanisms and relevant signalling pathways that regulate T-cell migration. Rapid progress in mass spectrometric technology in the past few years has allowed analysis and identification of proteins undergoing such post- translational modifications with unprecedented levels of sensitivity. Technologies for selective enrichment of phosphoproteins and availability of protein sequence data have further improved the phosphoproteomic studies, particularly the identification of tyrosine phosphorylated proteins. In the present study, by selective immunoprecipitation of phosphotyrosine containing proteins coupled with mass-spectrometric analysis, we identified several proteins and their associated complexes involved in LFA-1 induced T-cell migration. A total of 72 proteins were identified as involved in LFA-1-triggred T-cell migration, and we propose these as components of the LFA-1 signalling pathway. We identified several proteins novel to LFA-1 signalling such as 14-3-3]. Using the bioinformatics software ‘PANTHER’, the identified proteins were categorized into six broad groups based on their molecular functions. Here, we discuss the relevance of some of the identified proteins.
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Valuable information about the functioning of the β2 integrins has come from study of the leukocyte adhe- sion deficiency (LAD)-1 syndrome. LAD-1 is an autoso- mal recessive disorder caused by mutation in the CD18 gene on chromosome 21 that leads to absent or aber- rant biosynthesis of the β2 subunit of leukocyte inte- grins (9–11). This lesion is reflected in the absence or markedly diminished expression on the leukocyte cell surface of the β2 integrins, CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), and CD11c/CD18 (p150,95). The patient phenotype is variable but reflects the level of β2 integrins expressed. Patients with <1% expression suffer from life-threatening infections and require bone marrow transplantation for long-term survival (12). In patients with 1%–10% expression, defects in leukocyte mobility, adherence, and endocytosis lead to periodic periodontitis, skin infections, and retarded wound heal- ing with dysplastic scarring. The heterozygotic relatives of the patients have ∼40%–60% normal levels of β2 inte-
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produce net IL-1 inhibitor bioactivity with the anticipated consequences of cell cycle arrest, suppressed virus-specific proliferation, and reduced expression of activation markers. These studies were undertaken to investigate effects of exposure and resultant net IL-1 inhibitor activity on the expression of the intercellular adhesion molecule-1 (ICAM-1), and its ligand the lymphocyte function-associated antigen (LFA-1). MNL collected at 1, 4, and 24 h after exposure to influenza virus (which induces net IL-1 bioactivity) showed enhanced expression of ICAM-1 and LFA-1 relative to sham-exposed MNL and exhibited cell
expression of the Mac-1, LFA-1, p150,95 glycoprotein family on the neutrophil surface since neutrophils from patients with leukocyte adhesion deficiency, lacking surface expression of the adhesive glycoproteins, exhibited markedly diminished adherence to human endothelial cells in response to stimulation with chemotactic factors compared to normal control
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Fig. 1 shows the functions of our link failure detection algorithm. As shown in the figure, the alarming signal Link Failure Alarm (LFA) is sent back towards the source till it reaches the first recipient of data and it returns the REPLY (REP) signal is returned to the sender. In response, the RFTR scheme  is activated to avoid the failed link and restore the connection through a backup path.
o A cascading effect of multiple LFA’s can also provide de facto node protection. Equation eq2 is not satisfied, but the combined activation of LFAs by some other neighbors of the failing node F provides (de facto) node protection. In other words, it puts the dataplane in a state such that packets forwarded by S ultimately reach a neighbor of F that has a node-protecting LFA. Note that in this case S cannot indicate the node-protecting behavior of the repair without running additional computations.
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In contrast to the requirement for virus-cell attachment, we demonstrated here for the first time that to promote efficient cell-to-cell spread of HIV, the presence of activated and highly avid LFA-1 on the cell surface is not sufficient; rather, properly functional LFA-1 that can regulate its adhesiveness is required. We observed that the presence of the constitutively active LFA-1 deletion mutant on target cells did not promote virus replication and spread as seen with wild-type LFA-1, even though this LFA-1 deletion mutant helped establish the initial virus infection. It should also be noted that the cells expressing the LFA-1 deletion mutant tend to grow in tight clusters with most cells in close contact with neighboring cells, but virus spread was not accelerated in these cells. Although the reason for this phenomenon is not fully understood, these findings suggest at least three possible explanations. First, LFA-1 ex- pression on HIV-infected cultures promotes syncytium forma- tion (11), and in the cells with constitutively active LFA-1, rapid syncytium formation and death of the fused cells may cause the virus production to drop off faster. Second, the high affinity between the virus and the constitutively active LFA-1 on the cells may hinder the cell-to-cell transfer of progeny virus. Previous studies by Weber et al. (21) revealed that J␤2.7/ LFA-1 ⌬ cells could not modulate cell-cell adhesiveness and failed to undergo transendothelial migration in response to chemokines. Similarly, virus particles produced in cells with the LFA-1 deletion mutant may adhere too tightly to the host cells and may not spread to other cells in the culture efficiently. In support of this idea, we observed, using the MAb-mediated virus capture assay, that virions produced in J␤2.7/LFA-1 ⌬ cells incorporated into their envelopes a high level of LFA-1, while virions from J␤2.7/LFA-1 wt cells acquired much fewer LFA-1 molecules (data not shown). Although it remains un- clear whether the LFA-1 deletion molecules acquired by the HIV virions from the J␤2.7/LFA-1 ⌬ cells retained their highly adhesive state, the presence of such highly avid LFA-1 could potentially prevent the budding and detachment of virus prog- eny from the host cells. Third, the deletion in the cytoplasmic domain of the ␣ chain of the LFA-1 deletion mutant may affect the intracellular signaling and cytoskeleton rearrangement in- volved in the HIV maturation and/or budding process (14, 16, 17, 19). As a result, fewer mature virions may be produced in LFA-1 deletion mutant-expressing cells than in the cells ex- pressing wild-type LFA-1. Overall, the data from this study clearly show that the expression of functional LFA-1 with well- regulated adhesiveness on cells targeted by HIV significantly promotes HIV infection and transmission.
lines, since activated T cells generally express much higher levels of adhesion molecules. This is especially true of PHA blasts, which are commonly used to passage primary HIV iso- lates. Differential acquisition of adhesion molecules by HIV may result in differential sensitivity to serum neutralization. Indeed, Sawyer and colleagues have reported differential neu- tralization of HIV by antibodies to gp120 and CD4, dependent on the type of cell from which the virus buds (31). We also have data showing that passage of HIV through different cell types alters sensitivity to neutralization (11a). Interestingly, it has been reported that HIV-1 primarily infects CD4 1 memory T cells, which are characterized by high expression of adhesion molecules (32). Several published studies have shown that sol- uble CD4 and sera from gp120 vaccinees were unable to neu- tralize primary isolates as efficiently as laboratory isolates (4, 11, 24, 32). This observation may be explained by the binding of adhesion molecules on HIV acquired from PHA blasts to counterreceptors on target cells and the resulting increase in the overall binding avidity of the virus to the cells. The result of this phenomenon would be decreased sensitivity to serum neutralization, especially where neutralization is primarily through blockade of CD4-gp120 interaction.
The transmembrane constituents of TJs, all four membrane-spanning molecules, are the claudin proteins (Heiskala et al., 2001) and occludin (Furuse et al., 1993). They co-polymerize to heteropolymer TJ strands forming a continuous circumferential seal around the apical part of the cell and mediate adhesiveness to the TJ strands of the adjacent cells independently of extracellular Ca 2+ . The members of the claudin family thereby appear to be the primary seal- forming proteins with the intrinsic ability to polymerize into continuous adhesive fibrils. They may mediate tissue-specific TJ properties by their respectively restricted expression and individual ability for homophilic (between identical molecules) and heterophilic (between different molecules) interactions in cis (both molecules anchored in the same cell membrane) and in trans (both molecules anchored in the membranes of separate, opposing cells) among themselves. Occludin exerts homophilic trans-interactions and can bind intracellularly actin filaments. Claudins and occludin bind each other and both can interact with the TJ proteins cingulin and, in a PDZ-dependent manner (see 1.7.5.), with the zonula occludens proteins ZO-1, ZO-2 and ZO-3. The latter are multi-domain proteins which interact with diverse cytoplasmic molecules mediating a direct link between the sealing proteins and the actin cytoskeleton (Lampugnani et al., 1997; Tsukita et al., 1999). Among them, ZO-1 may represent a cross- talking element between AJs and TJs since its membrane targeting may be regulated by binding initially to AJ components such as α- and β-catenins with subsequent segregation to TJs. Further interaction partners of ZO-1 are both ZO-2, ZO-3 and the PDZ domain-containing AF-6 (ALL-1 fusion partner from chromosome 6; S-afadin), a Ras substrate that is expressed at TJs as well as in AJs. Due to identical binding sites on AF-6, Ras can compete with ZO-1 for AF-6 binding which may provide a regulatory mechanism (Ebnet et al., 2000). A third TJ-specific transmembrane protein is JAM-1 that may not constitute TJ strands per se but laterally associates with them. The protein interactions of JAM-1 with TJ components are discussed below.
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The density of cattle per 1 hectare of agricultural land was declining, the fastest decrease was revealed in production areas (an average of 0.55 heads/100 ha per year), a slight increase was achieved only in the LFA M (about 0.16 heads/100 ha per year). Given the increase in the yield in average by 0.3% in the LFA O and M and 1% in the NON-LFA, beef production was slightly increasing. According to the analysis of Foltýn et al. (2010), all model results (without subsidies as well as with subsidies) with the current intensity of fattening cattle show a negative profitability of this sector. A prerequisite for achieving positive results in the sector would be a necessary increase of the intensity of the fattening level by at least 0.9 kg/day. In our group of farms, such performance is only the average enterprise in the NON-LFA and only in some years, e.g. in 2011 the daily performance reached 0.81 kg (in the LFA M) and 0.86 kg (in the LFA O) and 0.89 kg (in the NON-LFA).
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Based on comparable surface levels of both primary cellular receptor and coreceptor (i.e., CD4 and CXCR4) on naive and memory T lymphocytes (43), it is usually thought that entry of HIV-1 occurs at similar rates in these two distinct cell subsets. However, besides interactions between the external virus-en- coded envelope glycoprotein gp120 and CD4/CXCR4, it has been recognized that other interactions can promote the initial events in HIV-1 replication. Indeed, convincing studies have revealed that HIV-1 incorporates a plethora of host-derived cell surface molecules during the budding process, including the intercellular adhesion molecule 1 (ICAM-1) (9, 21, 57). Interestingly, the adhesion molecule ICAM-1 is efficiently ac- quired by all tested laboratory and clinical variants of HIV-1 bearing different tropisms (i.e., R5, X4, and R5X4) once am- plified either in established cell lines or in primary human cells (2, 9, 12, 14, 23, 36). Moreover, it has also been demonstrated that virus-associated ICAM-1 influences HIV-1 biology since the natural ability of ICAM-1 to associate with its natural counterligand LFA-1 is preserved and leads to a severalfold increase in virus infectivity (21, 22, 55). Such a significant enhancement of HIV-1 infectivity is due to a more efficient virus adsorption onto target cells and a preferential entry pro- cess by fusion rather than through endocytosis (16).
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infection with the MOR and ZAG vaccine strains and the Ph26 wt. This was illustrated by the shift to the right in the FACS- generated histograms shown in Fig. 1. In contrast, there was no LFA-1 induction in response to infection with the jm77, Chi1, or Pa2 wt strain and the CAM vaccine strain. A comparison of the average fold increases in MFI revealed that by 96 h p.i., the induction of LFA-1 expression in cells infected with MOR, ZAG, or Ph26 was nearly two to three times that in mock- infected cells (Fig. 2a). It should be noted, however, that the increase in LFA-1 expression that we observed with MOR differed from previous reports, which found no induction (1, 26). For all strains, infection was evaluated in immunofluores- cence assays by the percentage of cells expressing the MV nucleoprotein (N), the most abundant viral protein. These values were as follows: for strain MOR, 10 to 50%; ZAG, 50 to 100%; CAM, 10 to 50%; Ph26, 50 to 100%; jm77, 10 to 50%; Chi1, 10%; and Pa2, 1 to 10%.
Schaubild 19 zeigt die Ergebnisse der Aufschlüsselung der in den Mitgliedstaaten im Erhebungszeitraum 1999/2000 beobachteten besonderen Situation in Bezug auf die Zahl der Betriebe, den durchschnittlichen Standarddeckungsbeitrag je Betrieb, die durchschnittliche landwirtschaftlich genutzte Fläche je Betrieb und den durchschnittlichen Viehbestand je Betrieb (für alle Betriebe) nach BG-Status (d.h. abhängig davon, ob ein Betrieb in einem benachteiligten Gebiet angesiedelt ist oder nicht). Im Erhebungszeitraum 1999/2000 war Italien der Mitgliedstaat mit der größten Zahl von Betrieben in benachteiligten Gebieten (fast 1 Mio. Betriebe, das waren 46 % aller italienischen Betriebe), gefolgt von Spanien mit 0,93 Mio. BG-Betrieben (72 % aller spanischen Betriebe). Allein in diesen beiden Mitgliedstaaten war die Hälfte (52 %) aller EU-Betriebe ansässig, die sich in benachteiligten Gebieten befanden. Weitere Mitgliedstaaten mit einer großen Zahl von Betrieben in benachteiligten Gebieten waren Griechenland (fast 0,5 Mio.) sowie Frankreich, Deutschland und Portugal, die jeweils zwischen 0,2 und 0,3 Mio. derartige Betriebe aufwiesen.
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Inflammation and infection of the gut can be followed by re- active arthritis at a distant joint. Leukocyte recruitment into synovium is essential for this process, but nothing is known about the endothelial adhesion molecules in synovial mem- brane which direct the homing of activated, gut-derived leu- kocytes to joints. Here we analyzed the expression of the known endothelial adhesion molecules in inflamed syno1 1vium and their function in binding of mucosal leukocytes. Intercellular adhesion molecule-1 (ICAM-1/CD54) and vas- cular adhesion protein-1 (VAP-1) were most prominently expressed in synovial vessels. All other adhesion molecules were found at lower levels in inflamed synovia, except mu- cosal addressin which was absent. Binding of macrophages isolated from lamina propria of the gut to synovial endothe- lium was almost entirely P-selectin–dependent. In contrast, small intestinal lymphocytes and immunoblasts both relied mainly on VAP-1 in recognition of synovial vessels. Thus, endothelial P-selectin and VAP-1 mediate binding of mu- cosal effector cells to synovium in a leukocyte subtype-selec- tive manner. Antiadhesive therapy against these inducible molecules should ablate the pathogenetic cascade leading to inappropriate homing of leukocytes to joints in arthritis. ( J. Clin. Invest. 1997. 99:2165–2172). Key words: arthritis • vas- cular addressins • vascular adhesion protein-1 • recirculation •