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The intracellular protein cytohesin-1 appears to be a regulatory factor for the alphaLbeta2 integrin, which is a heterodimeric integral membrane protein that mediates between cell interaction and matrix and specific cell counterreceptors. Functional and biochemical analyses of the protein show that it is homologous to the yeast SEC7 gene product and bears a pleckstrin homology domain. Results further indicate that individual PH domains in the protein are capable of discriminating between alternative targets.

Liposomes functionalized with monoclonal antibodies or their antigen-binding fragments have attracted much attention as specific drug delivery devices for treatment of various diseases including cancer. The conjugation of antibodies to liposomes is usually achieved by covalent coupling using cross-linkers in a reaction that might adversely affect the characteristics of the final product. Here we present an alternative strategy for liposome functionalization: we created a recombinant Fab antibody fragment genetically fused on its C-terminus to the hydrophobic peptide derived from pulmonary surfactant protein D, which became inserted into the liposomal bilayer during liposomal preparation and anchored the Fab onto the liposome surface. The Fab-conjugated liposomes specifically recognized antigen-positive cells and efficiently delivered their cargo, the Alexa Fluor 647 dye, into target cells in vitro and in vivo . In conclusion, our approach offers the potential for straightforward development of nanomedicines functionalized with an antibody of choice without the need of harmful cross-linkers. [ABSTRACT FROM AUTHOR]

The skin lesion erythema migrans is a characteristic early manifestation of Lyme borreliosis in humans. However, the pathomechanisms leading to development of this erythema are not fully understood. Models that mimic human skin would enhance research in this field. Human and porcine skin structures strongly resemble each other. Therefore, we attempted to induce erythema migrans lesions in experimental Borrelia burgdorferi sensu lato infection in the skin of domestic pigs. The formation of erythema migrans-like lesions was observed after intradermal injection of these spirochetes, with the lesions forming very clearly in 2/6 animals when a strain of B. garinii was used. However, no molecular or clinical proof of systemic infection of the pigs with B. afzelii , B. burgdorferi sensu stricto, or B. garinii could be achieved. [ABSTRACT FROM AUTHOR]

Ticks are vectors for many bacterial, protozoan and viral pathogens and are potential vectors for Bartonella species. Hunters and foresters, therefore, may be regarded as high-risk groups for Bartonella infections. The aims of this study were (i) to identify Bartonella species in questing Ixodes ricinus ticks collected in all provinces of Austria, and (ii) to determine the prevalence of antibodies to Bartonella species in hunters and blood donors in eastern Austria. A total of 515 larval, nymphal and adult I. ricinus , collected throughout Austria in 2005, were selected from the tick library at the Institute for Hygiene and Applied Immunology of the Medical University of Vienna and screened in a specific real-time PCR that targeted a region of the ssrA gene of Bartonella species. The overall Bartonella infection rate was 2.1% (11/515) and the highest rate, 7.5% (4/53), was found in ticks from Vienna. This finding was confirmed by screening a further 60 I. ricinus collected from Vienna in 2013: of these, 6.7% (4/60) were positive for Bartonella spp. The rate of infection was always higher in adult ticks. Sequence analysis in the Bartonella -positive ticks identified several species, including B. henselae , B. doshiae and B. grahamii . To our knowledge this is the first time that these species have been identified in I. ricinus in Austria. Prevalence of IgG antibodies against B. henselae and B. quintana was determined in serum samples from hunters (100) and blood donors (100): in hunters 23% were positive for B. quintana and in 2 samples (2%), antibodies to both B. quintana and B. henselae were detected; in blood donors 22% were positive for B. quintana , 1% for B. henselae and 5% for both. These results indicate that exposure to ticks does not constitutes a relevant risk for Bartonella infection. [ABSTRACT FROM AUTHOR]

A total of 350 ticks collected in Austria were analyzed for the presence of DNA sequences of B. miyamotoi . Three ticks gave positive results in a B. miyamotoi -specific nested PCR. Results were confirmed by sequencing the amplified glpQ gene from the positive samples. Moreover we developed a real-time PCR which unambiguously detected B. miyamotoi in all positive samples. Further genotyping of the samples found 100% identity of the 16S–23S intergenic spacer region with Swedish B. miyamotoi sequences. This is the first detection of the relapsing fever spirochete Borrelia miyamotoi in hard ticks in Austria. The results consolidate the picture of a European-wide distribution of B. miyamotoi and again underscore the need for clinical awareness to clarify possible involvement of this species in human disease. [ABSTRACT FROM AUTHOR]

Whether infection with Borrelia burgdorferi sensu lato (sl) can cause nonspecific symptoms is a matter of controversy. We investigated whether individuals infected with B. burgdorferi sl develop unspecific symptoms more frequently than non-infected controls. Eighty-nine persons having presented with B. burgdorferi sl infection between 2015 and 2019 were asked to participate. The infection was defined as erythema migrans diagnosed either in the course of a previous study, during medical visits in the outpatient department, or as seroconversion in asymptomatic subjects. The control group consisted of 85 seronegative individuals without erythema migrans in the past. About two and a half years later, participants were asked to fill out a questionnaire with a list of nonspecific symptoms. The data of 37 persons with previous Borrelia infection and 49 uninfected controls were available for analysis. Muscle pain was significantly (P = 0.040) more frequent in the control group. Fatigue occurred more often in the infected group, but this was not statistically significant (P = 0.109). Likewise, the distribution of other symptoms did not differ considerably. The analysis revealed no difference in the frequency of symptoms of persons who had EM or asymptomatic Borrelia infection 2 years prior assessment in comparison to persons without Borrelia infection. [ABSTRACT FROM AUTHOR]

Legionella infections are among the most important waterborne infections with constantly increasing numbers of cases in industrialized countries, as a result of aging populations, rising numbers of immunocompromised individuals and increased need for conditioned water due to climate change. Surveillance of water systems is based on microbiological culture-based techniques; however, it has been shown that high percentages of the Legionella populations in water systems are not culturable. In the past two decades, the relevance of such viable but non-culturable (VBNC) legionellae has been controversially discussed, and whether VBNC legionellae can directly infect human macrophages, the primary targets of Legionella infections, remains unclear. In this study, it was demonstrated for the first time that several starved VBNC Legionella strains (four L. pneumophila serogroup 1 strains, a serogroup 6 strain and a L. micdadei strain) can directly infect different types of human macrophages and amoebae even after one year of starvation in ultrapure water. However, under these conditions, the strains caused infection with reduced efficacy, as represented by the lower percentages of infected cells, prolonged time in co-culture and higher multiplicities of infection required. Interestingly, the VBNC cells remained mostly non-culturable even after multiplication within the host cells. Amoebal infection by starved VBNC Legionella, which likely occurs in oligotrophic biofilms, would result in an increase in the bacterial concentration in drinking-water systems. If cells remain in the VBNC state, the real number of active legionellae will be underestimated by the use of culture-based standard techniques. Thus, further quantitative research is needed in order to determine, whether and how many starved VBNC Legionella cells are able to cause disease in humans. [ABSTRACT FROM AUTHOR]

The plasminogen system is essential for dissolution of fibrin clots, and in addition, it is involved in a wide variety of other physiological processes, including proteolytic activation of growth factors, cell migration, and removal of protein aggregates. On the other hand, uncontrolled plasminogen activation contributes to many pathological processes (e.g. tumor cells' invasion in cancer progression). Moreover, some virulent bacterial species (e.g. Streptococci or Borrelia) bind human plasminogen and hijack the host's plasminogen system to penetrate tissue barriers. Thus, the conversion of plasminogen to the active serine protease plasmin must be tightly regulated. Here, we show that human lactoferrin, an iron-binding milk glycoprotein, blocks plasminogen activation on the cell surface by direct binding to human plasminogen.Wemapped the mutual binding sites to the N-terminal region of lactoferrin, encompassed also in the bioactive peptide lactoferricin, and kringle 5 of plasminogen. Finally, lactoferrin blocked tumor cell invasion in vitro and also plasminogen activation driven by Borrelia. Our results explain many diverse biological properties of lactoferrin and also suggest that lactoferrin may be useful as a potential tool for therapeutic interventions to prevent both invasive malignant cells and virulent bacteria from penetrating host tissues. [ABSTRACT FROM AUTHOR]

The chronic autoimmune disorder rheumatoid arthritis (RA) affects millions of adults and children every year. Chronically activated macrophages secreting enzymes and inflammatory cytokines play a key role in RA. Distinctive marker molecules on the macrophage surface could be used to design a targeted drug delivery device for the treatment of RA without affecting healthy cells and tissues. Here, different methods for covalent attachment of antibodies (mAb) recognizing MHC class II molecules found on macrophages onto human serum albumin (HSA) nanocapsules were compared. HSA nanocapsules were prepared with a hydrodynamic diameter of 500.7±9.4nm and a narrow size distribution as indicated by a polydispersity index (PDI) of 0.255±0.024. This was achieved by using a sonochemical process avoiding toxic cross linking agents and emulsifiers. Covalent binding of mAb on the surface of HSA nanocapsules was realized using polyethyleneglycol (PEG)3000 as spacer molecule. The presence of mAb was confirmed by confocal laser scanning microscopy (CLSM) and enzyme-linked immunosorbent assay (ELISA). Specific binding of mAb-HSA nanocapsules to MHC class II molecules on antigen-presenting cells was demonstrated by flow cytometry analysis. [ABSTRACT FROM AUTHOR]

The plasminogen (Plg) activation cascade on the cell surface plays a central role in cell migration and is involved in a plethora of physiological and pathological processes. Its regulation is coordinated by many receptors, in particular the urokinase-type plasminogen activator receptor (uPAR, CD87), receptors that physically interact and functionally cooperate with uPAR, and Plg binding molecules. Here we studied the impact of one of the Plg binding molecules, the mannose 6-phosphate/insulinlike growth factor 2 receptor (M6P-IGF2R, CD222), on cellular Plg activation. By developing both in vitro and in vivo Plg activation assays on size-fractionated lysates of M6P-IGF2R-silenced cells, we identified Plg-associated complexes with M6PIGF2R as the regulatory factor. Using lipid raft preserving versus dissolving detergents, we found lipid dependence of the Plg regulatory function of these complexes. Furthermore, M6P-IGF2Rsilencing in uPAR-positive human cell lines reduced internalization of Plg, resulting in elevated Plg activation. In contrast, the expression of human M6P-IGF2R in mouse embryonic fibroblasts derived from M6P-IGF2R knock-out mice enhanced Plg internalization. Finally, peptide 18-36 derived from the Plgbinding site within M6P-IGF2R enhanced Plg uptake. Thus, by targeting Plg to endocytic pathways, M6P-IGF2R appears to control Plg activation within cells that might be important to restrict plasmin activity to specific sites and substrates. [ABSTRACT FROM AUTHOR]

The plasma membrane has been hypothesized to contain nanoscopic lipid platforms, which are discussed in the context of "lipid rafts" or "membrane rafts." Based on biochemical and cell biological studies, rafts are believed to play a crucial role in many signaling processes. However, there is currently not much information on their size, shape, stability, surface density, composition, and heterogeneity. We present here a method that allows for the first time the direct imaging of nanoscopic long-lived platforms with raft-like properties diffusing in the live cell plasma membrane. Our method senses these platforms by their property to assemble a characteristic set of fluorescent marker proteins or lipids on a time scale of seconds. A special photobleaching protocol was used to reduce the surface density of labeled mobile platforms down to the level of well isolated diffraction-limited spots without altering the single spot brightness. The statistical distribution of probe molecules per platform was determined by single molecule brightness analysis. For demonstration, we used the consensus raft marker glycosylphosphatidylinositol-anchored mono- meric GFP and the fluorescent lipid analog BODIPY-GM1, which preferentially partitions into liquid-ordered phases. For both markers, we found cholesterol-dependent homo-associa- tion in the plasma membrane of living CHO and Jurkat T cells in the resting state, thereby demonstrating the existence of small, mobile, long-lived platforms containing these probes. We further applied the technology to address structural changes in the plasma membrane during fever-type heat shock: at elevated temperatures, the glycosylphosphatidyl inositol-anchored monomeric GFP homo-association disappeared, accompanied by an increase in the expression of the small heat shock protein Hsp27. [ABSTRACT FROM AUTHOR]

Abstract: Forkhead box protein 3 (Foxp3) is indispensable for the development of CD4+CD25+ regulatory T cells (Tregs). Here we analyzed three prominent evolutionary conserved regions (ECRs) upstream of the transcription start site of the human FOXP3 gene. We show that ECR2 and ECR3 fragments derived from positions −1.3 to −2.0kb and −5.0 to −6.0kb, respectively, display basal transcriptional activity. Reporter constructs derived from ECR1, located between −0.6 and +0.23kb and thus the most proximal ECR in respect of transcription initiation, remained almost inactive. However, ECR1 was transactivated by the NF-κB subunit p65 in HEK 293 cells. In Jurkat and primary T cells, in addition to p65, a second stimulus delivered by either T-cell receptor stimulation or addition of PMA was needed. Co-expression of IκBα inhibited p65-mediated FOXP3 proximal promoter transactivation, and the NF-κB inhibitor curcumin reduced Foxp3 neoexpression in IL-2/CD3/CD28/TGF-β stimulated PBMCs. Moreover, proximal FOXP3 promoter transactivation was inhibited by Foxp3 and the SP transcription factor family member SP3. Thus, the human proximal FOXP3 promoter is controlled by activation through the TCR involving PKC and the NF-κB subunit p65 and by inhibition through a negative feedback loop and SP3. [Copyright &y& Elsevier]

Palmitoylation represents a common motif for anchorage of cytosolic proteins to the plasma membrane. Being reversible, it allows for controlled exchange between cytosolic and plasma membrane-bound subpopulations. In this study, we present a live cell single molecule approach for quantifying the exchange kinetics of plasma membrane and cytosolic populations of fluorescently labeled Lck, the key Src family kinase involved in early T cell signaling. Total internal reflection (TIR) fluorescence microscopy was employed for confining the analysis to membrane-proximal molecules. Upon photobleaching Lck-YFP in TIR configuration, fluorescence recovery proceeds first via the cytosol outside of the evanescent field, so that in the early phase fluorescence signal arises predominantly from membraneproximal cytosolic Lck. The diffusion constant of each molecule allowed us to distinguish whether the molecule has already associated with the plasma membrane or was still freely diffusing in the cytosol. From the number of molecules that inserted during the recovery time we quantified the insertion kinetics: on average, membrane-proximal molecules within the evanescent field needed ∼400 ms to be inserted. The average lifetime of Lck in the plasma membrane was estimated at 50 s; together with the mobility of 0.26 μm²/s this provides sufficient time to explore the surface of the whole T cell before dissociation into the cytosol. Experiments on palmitoylation-deficient Lck mutants yielded similar on-rates, but substantially increased off-rates. We discuss our findings based on a model for the plasma membrane association and dissociation kinetics of Lck, which accounts for reversible palmitoylation on cysteine 3 and 5. [ABSTRACT FROM AUTHOR]

Abstract: The activity of the lymphocyte-function associated antigen 1 (LFA-1; CD11a/CD18) must be tightly controlled during the onset of cellular immunity. It is well known that the sialoglycoprotein CD43 can influence LFA-1-mediated cell adhesion in an either anti- or pro-adhesive manner through mechanisms not well understood. By using a yeast-2-hybrid screen and co-immunoprecipitation we identified physical association of CD43 with two novel partners, LFA-1 itself and the Ig-family member CD147 (EMMPRIN, basigin), and characterized how these interactions are involved in LFA-1-mediated cell adhesion. Monoclonal antibodies (mAbs) to both CD43 and CD147 induced similar homotypic cell aggregation and adhesion of Jurkat T cells and U937 myeloid cells. Both CD43 and CD147 mAbs induced dynamic co-capping of LFA-1 together with the CD43 and the CD147 molecule to cell contact zones. However, in contrast to CD43, we were not able to co-immunoprecipitate LFA-1 with CD147, which indicates that CD43 interacts with CD147 and LFA-1 in two distinct but similarly reorganized complexes. Co-transfection of CD43 interfered with the CD147-induced cell adhesion and aggregation, and siRNA-mediated knock down of CD43 in human T cells resulted in enhanced LFA-1 activation induced via CD147 and also the T cell antigen receptor. These results indicate that triggering CD43 and the underlying signaling pathways enhance LFA-1 adhesiveness while CD43 also negatively regulates LFA-1 induction via other receptors by dynamic interaction with either LFA-1 or CD147. [Copyright &y& Elsevier]

Objectives: This study was designed to evaluate whether replacing CD31 (PECAM-1) signaling can restore the regulation of lymphocyte activation and improve experimental atherosclerosis. Background: Atherosclerosis, the principal cause of myocardial infarction and stroke, is due to the development of a pathogenic immune response within the vascular wall and is aggravated by the reduction of regulatory T-cells. CD31, a transmembrane adhesion molecule with inhibitory signaling functions, is physiologically expressed on blood and vascular resting cells but is lost in pathologic conditions associated with atherosclerosis. Methods: Replacement therapy with a CD31 receptor globulin (Rg) was delivered by in vivo gene transfer in 6-week-old apolipoprotein E knockout mice (n = 14 per group) every 5 weeks for 6 months. Control groups were treated with a truncated CD31Rg or with vehicle alone. At the end of the study, plaque size and morphology and blood T-cell compartment were analyzed in all mice. Results: Atherosclerotic lesions of CD31Rg-treated mice were smaller (p < 0.01) and showed less neovascularization and intraplaque hemorrhage (p < 0.05) compared with control subjects. Furthermore, circulating regulatory T-cells were increased in vivo (p < 0.01) and showed normal suppressive function on proliferation of conventional T-cells in vitro. Indeed, CD31Rg treatment led to blunted blood T-cell activation (p < 0.05) and reduced T-cell infiltration within plaques (p < 0.01). Conclusions: Our data suggest that CD31 plays a key role in the regulation of the immune response linked to atherosclerosis. CD31-targeting therapeutic approaches may therefore be envisaged for preventing and treating atherosclerotic diseases. [Copyright &y& Elsevier]

Abstract: Cell–cell interactions are critical at key points of immune responses and are mediated by a complex array of adhesion receptors. One of the most important adhesion molecules on leukocytes is intercellular adhesion molecule 1 (ICAM-1, CD54). Here we demonstrate that engagement of ICAM-1 with human major group rhinoviruses (HRV) enhances adhesiveness and homotypic aggregation of human monocytes and monocyte-derived dendritic cells (DC). Cluster formation upon engagement of ICAM-1 with HRV14 represents an active process. It is temperature and energy dependent, requires divalent cations, an intact cytoskeleton and protein de novo synthesis. Homotypic interaction between monocytes induced by HRV14 can be inhibited with blocking mAbs against LFA-1 (CD11a/CD18) and ICAM-3 (CD50) as well as with a mAb against the first immunoglobulin (Ig)-domain of PECAM-1 (CD31). Induction of enhanced cytoadhesiveness by HRV14 was not accompanied with an upregulation of LFA-1, ICAM-3 or PECAM-1 expression. Binding studies with recombinant PECAM-1 proteins indicated, however, that monocyte clustering upon engagement of ICAM-1 with HRV was accompanied with increased homophilic PECAM-1 interactions. Taken together the results of our study demonstrate that signalling via ICAM-1 induces adhesiveness of mononuclear phagocytes, which critically involves PECAM-1 and is mediated via LFA-1/ICAM-3. [Copyright &y& Elsevier]

Abstract: CD147 is a leukocyte surface molecule which belongs to the immunoglobulin superfamily. It is broadly expressed on various cell types and is a lymphocyte activation-associated molecule. In order to study the function of CD147, five CD147 monoclonal antibodies (mAbs) were generated: M6-2F9; M6-1D4; M6-1F3; M6-1B9; and M6-1E9. Biochemical characterizations and cross-blocking experiments indicated that M6-1B9 and M6-1E9 recognize the same or contiguous epitopes on CD147. By employing COS transfectants expressing CD147 membrane-distal domain (domain 1) and membrane-proximal domain (domain 2), mAbs M6-2F9, M6-1D4, M6-1B9, and M6-1E9 were shown to recognize epitopes located on domain 1 of the molecule. Functional studies indicated that engagement of CD147 by mAbs M6-1B9 and M6-1E9 strongly inhibited lymphocyte proliferation induced by a CD3 mAb. In contrast, mAbs M6-2F9, M6-1D4, and M6-1F3 induced U937 homotypic cell aggregation. The results indicate that CD147 contains at least two bioactive domains. Epitopes responsible for induction of cell aggregation are different from those regulating lymphocyte activation. [Copyright &y& Elsevier]

SummaryCellular adherens junctions are formed by cadherins linked to proteins of the catenin family. In endothelial cells, not only vascular endothelial cadherin but also platelet endothelial cell adhesion molecule-1 localizes into junctions and associates with β-catenin. To explore a putative cooperation of platelet endothelial cell adhesion molecule-1 and vascular endothelial cadherin, we analyzed transfectants expressing either platelet endothelial cell adhesion (CD31 cells) or vascular endothelial cadherin (CD144 cells) or both molecules (CD31/CD144 cells), and, for comparison, human umbilical vein endothelial cells. Basic fibroblast growth factor completely dissociated vascular endothelial cadherin/β-catenin complexes and robustly moved β-catenin into the nucleus in CD144 cells, whereas in CD31/CD144 cells as well as in human umbilical vein endothelial cells, fibroblast growth factor only partially dissociated the junctional complex followed by a significantly reduced nuclear translocation of β-catenin. In contrast, in CD31 cells, the subcellular distribution of β-catenin remained unaffected by fibroblast growth factor. As a functional consequence, fibroblast growth factor induced a complete collapse of the F-actin network in CD144 cells, a limited rearrangement of F-actin fibers in CD31/CD144 cells and no F-actin rearrangement in CD31 cells. We also analyzed the effect of fibroblast growth factor-induced rearrangement of junctions on junction permeability for leukocytes: in line with our observation that vascular endothelial cadherin was required for cells to respond to fibroblast growth factor, only in CD31/CD144 cells, but not in CD31 cells, leukocyte transmigration was significantly enhanced by fibroblast growth factor. In conclusion platelet endothelial cell adhesion molecule-1 cooperates with vascular endothelial cadherin in a mutual fashion; platelet endothelial cell adhesion molecule-1 reduces and temporarily limits fibroblast growth factor-induced dissociation of vascular endothelial cadherin/β-catenin complexes, but requires vascular endothelial cadherin to control leukocyte transmigration in dependence of fibroblast growth factor. [ABSTRACT FROM AUTHOR]