Protein-Peptide Arrays for Detection of Specific Anti-Hepatitis D Virus (HDV) Genotype 1, 6, and 8 Antibodies among HDV-Infected Patients by Surface Plasmon Resonance Imaging

Liver diseases linked to hepatitis B-hepatitis D virus co- or superinfections are more severe than those during hepatitis B virus (HBV) monoinfection. The diagnosis of hepatitis D virus (HDV) infection therefore remains crucial in monitoring patients but is often overlooked. To integrate HDV markers into high-throughput viral hepatitis diagnostics, we studied the binding of anti-HDV antibodies (Abs) using surface plasmon resonance imaging (SPRi). We focused on the ubiquitous HDV genotype 1 (HDV1) and the more uncommon African-HDV6 and HDV8 genotypes to define an array with recombinant proteins or peptides. Full-length and truncated small hepatitis D antigen (S-HDAg) recombinant proteins of HDV genotype 1 (HDV1) and 11 HDV peptides of HDV1, 6, and 8, representing various portions of the delta antigen were grafted onto biochips, allowing SPRi measurements to be made. Sixteen to 17 serum samples from patients infected with different HDV genotypes were injected onto protein and peptide chips. In all, Abs against HDV proteins and/or peptides were detected in 16 out of 17 infected patients (94.12%), although the amplitude of the SPR signal varied. The amino-terminal part of the protein was poorly immunogenic, while epitope 65-80, exposed on the viral ribonucleoprotein, may be immunodominant, as 9 patient samples led to a specific SPR signal on peptide 65 type 1 (65#1), independently of the infecting genotype. In this pilot study, we confirmed that HDV infection screening based on the reactivity of patient Abs against carefully chosen HDV peptides and/or proteins can be included in a syndrome-based viral hepatitis diagnostic assay. The preliminary results indicated that SPRi studying direct physical HDAg–anti-HDV Ab interactions was more convenient using linear peptide epitopes than full-length S-HDAg proteins, due to the regeneration process, and may represent an innovative approach for a hepatitis syndrome–viral etiology-exploring array.     

Interaction of different Leishmania mexicana morphotypes with plasminogen

The interaction of plasminogen with Leishmania mexicana promastigotes was found, using immunoperoxidase assays, to occur with a specific morphotype. In in vitro cultured promastigotes, the morphotype that possessed the plasminogen binding capacity had round to ovoid cell bodies. In contrast, neither slender nor metacyclic promastigotes showed this property. In vivo plasminogen immunofluorescence assays showed deposits of plasminogen exclusively on the cell surface of promastigotes. This was observed as intense patches spread around the cell, with higher intensities towards the flagellar pocket. Plasminogen binding capacity detected by plasmin activity increased with the age of the promastigote culture, at pH 7.2 and pH 5.5, as well as after heat shock. The total amastigote population, freshly isolated from a hamster lesion, also bound plasminogen in a lysine-dependent manner as detected by immunoperoxidase studies.     

Modulation of Growth Factor Action by the Extracellular Matrix

Growth factors were historically defined as molecules produced by the body to regulate cell growth and proliferation. After the identification of their receptors and the intracellular signaling machinery they activate, it is now clear that they are involved in the regulation of multiple processes essential for development and normal tissue function. The present review gives a brief overview of growth factor action. Receptor activation and signaling are discussed, highlighting the role of extracellular matrix interactions.     

Interaction of Leishmania donovani Promastigotes with Human Phagocytes

Leishmania donovani is an important intracellular protozoal pathogen of humans, which resides solely within mononuclear phagocytes. Phase-contrast microscopy and cinemicroscopy were used to examine the interaction of L. donovani promastigotes with human phagocytes to characterize and quantitate the sequence of events that results in leishmanial infection.     

Microstructural Characteristics of Extracellular Matrix Produced by Stromal Fibroblasts

The overall objective of this investigation was to characterize the extracellular matrix deposited by the stromal fibroblasts as a function of time in culture and matrix microstructure. Stromal fibroblasts were seeded onto collagen matrices and cultured for up to 5 weeks. The collagen matrices contained collagen fibrils with an average diameter of 215 ± 20 nm. When cultured on a collagen film, an average fibril diameter of 62 ± 39 nm was observed for single layer films with only slight variations with time in culture, and after 1 week of culture between two film layers 67 ± 47 nm fibrils were observed after 1 week. When the film surface was molded into 1 and 2 μm microgrooves, the initial average fibril diameter of the extracellular matrix was 73 ± 21 and 73 ± 31 nm respectively. When cultured on a collagen sponge, an average fibril diameter of 107 ± 20 nm was initially observed and decreased to 47.5 ± 17 nm after 1 week in culture. For cells cultured on a collagen sponge, Western blotting showed an increase in myofibroblast phenotype expression with time in culture. Shifts in phenotype were less distinct for cells cultured on collagen films. The microstructure, rather than geometry, of the matrix substrate appeared to influence the newly synthesized extracellular matrix and cell phenotype.     

Interaction of Leishmania mexicana promastigotes with the plasminogen-plasmin system

The binding of human plasminogen and plasmin to the promastigote form of Leishmania mexicana was investigated. L. mexicana was capable to bind both molecules, the binding being inhibited by epsilon-aminocaproic acid. Scatchard plot analysis revealed a dissociation constant (Kd) value of 2.4+/-0.8 microM and 0.9+/-0.1 x 10(4) binding sites per cell for plasminogen and a Kd value of 1.2+/-0.4 microM and 1.6+/-0.2 x 10(5) binding sites per cell for plasmin. C-terminal lysine residues are involved in plasminogen binding to cells, since carboxypeptidase B treatment reduced this binding by 34%. Ligand blotting analysis showed a group of proteins, with molecular masses between 105 and 115 kDa, capable to interact with plasminogen. Zymogram analysis showed that the protease activity acquired by L. mexicana, due to the interaction with either plasminogen or plasmin, comprises an important fraction of the total protease activity at pH 7.7. Plasminogen activation by tissue-type plasminogen activator (t-PA) was enhanced by the presence of L. mexicana promastigotes. These results raise the question whether the interaction of L. mexicana with components of the fibrinolytic system is involved in the virulence of the parasite.     

Interaction of Leishmania gp63 with Cellular Receptors for Fibronectin

The most abundant protein on the surface of the promastigote form of the protozoan parasites Leishmania spp. is a 63-kDa molecule, designated gp63 or leishmanolysin. Because gp63 has been shown to possess fibronectin-like properties, we examined the interaction of gp63 with the cellular receptors for fibronectin. We measured the direct binding of Leishmania to human macrophages or to transfected mammalian cells expressing human fibronectin receptors. Leishmania expressing gp63 exhibited modest but reproducible adhesion to human macrophages and to transfected CHO cells expressing alpha4/beta1 fibronectin receptors. In both cases, this interaction depended on gp63 but occurred independently of the SRYD sequence of gp63, because parasites expressing gp63 with a mutated SRYD sequence bound to macrophages and alpha4/beta1 receptor-expressing cells as well as did wild-type parasites. The contribution of gp63 to parasite adhesion was more pronounced when the assays were performed in the presence of complement, suggesting that the receptors for complement and fibronectin may cooperate to mediate the efficient adhesion of parasites to macrophages. The interaction of gp63 with fibronectin receptors may also play an important role in parasite internalization by macrophages. Erythrocytes to which gp63 was cross-linked were efficiently phagocytized by macrophages, whereas control erythrocytes opsonized with complement alone bound to macrophages but remained peripherally attached to the outside of the cell. Similarly, parasites expressing wild-type gp63 were rapidly and efficiently phagocytized by resting macrophages, whereas parasites lacking gp63 were internalized more slowly. This rapid internalization of gp63-expressing parasites was dependent on the beta1 integrins, because pretreatment of macrophages with monoclonal antibodies to the beta1 integrins decreased the internalization of gp63-expressing parasites. These observations indicate that complement receptors are the primary mediators of parasite adhesion; however, maximal parasite adhesion and internalization may require the participation of the beta1 integrins, which recognize fibronectin-like molecules such as gp63 on the surface of the parasite.     

Electrochemical Surface Plasmon Resonance and Field‐Enhanced Light Scattering: Monomer Copolymerization with a Polysiloxane‐Conjugated Polythiophene Network Precursor

The in situ electrochemical and dielectric film properties of monomer co‐electropolymerization with a CPN precursor was investigated. A polysiloxane/polythiophene precursor was electropolymerized with thiophene monomer at different composition ratios using cyclic voltammetry. This was investigated in situ by EC‐SPR spectroscopy and EC‐SPFELS. The characteristic oscillations corresponded to distinct transitions in film deposition. The resonance coupling of the SPR decreased with higher thiophene monomer content. Increasing the amount of thiophene monomer increased the RMS roughness of the film as observed by AFM, which corresponded to an increase in scattered light intensity with EC‐SPFELS.

     

Paracoccidioides brasiliensis Enolase Is a Surface Protein That Binds Plasminogen and Mediates Interaction of Yeast Forms with Host Cells

Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis, is a disseminated, systemic disorder that involves the lungs and other organs. The ability of the pathogen to interact with host components, including extracellular matrix (ECM) proteins, is essential to further colonization, invasion, and growth. Previously, enolase (EC 4.2.1.11) was characterized as a fibronectin binding protein in P. brasiliensis. Interaction of surface-bound enolase with plasminogen has been incriminated in tissue invasion for pathogenesis in several pathogens. In this paper, enolase was expressed in Escherichia coli as a recombinant glutathione S-transferase (GST) fusion protein (recombinant P. brasiliensis enolase [rPbEno]). The P. brasiliensis native enolase (PbEno) was detected at the fungus surface and cytoplasm by immunofluorescence with an anti-rPbEno antibody. Immobilized purified rPbEno bound plasminogen in a specific, concentration-dependent fashion. Both native enolase and rPbEno activated conversion of plasminogen to plasmin through tissue plasminogen activator. The association between PbEno and plasminogen was lysine dependent. In competition experiments, purified rPbEno, in its soluble form, inhibited plasminogen binding to fixed P. brasiliensis, suggesting that this interaction required surface-localized PbEno. Plasminogen-coated P. brasiliensis yeast cells were capable of degrading purified fibronectin, providing in vitro evidence for the generation of active plasmin on the fungus surface. Exposure of epithelial cells and phagocytes to enolase was associated with an increased expression of surface sites of adhesion. In fact, the association of P. brasiliensis with epithelial cells and phagocytes was increased in the presence of rPbEno. The expression of PbEno was upregulated in yeast cells derived from mouse-infected tissues. These data indicate that surface-associated PbEno may contribute to the pathogenesis of P. brasiliensis.Microbial adhesion to host tissues is the initial event of most infectious process (39). Interaction with extracellular matrix (ECM) proteins has been correlated with the invasive abilities of different organisms (28, 40). ECM underlines epithelial and endothelial cells and surrounds connective tissues, and its major components are the collagens, laminin, fibronectin, and proteoglycans (52). After adherence, the next step must be to overcome the barriers imposed by epithelial tissues and ECM. The proteolytic activity achieved by subversion of host proteases by pathogens, such as plasmin, has been shown to be important during the process of many types of infections (47, 51).Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis (PCM), a human systemic mycosis that constitutes a major health problem in South America (44). Clinical manifestations of PCM are related to chronic granulomatous reactions with involvement of the lungs and the reticuloendothelial system, as well as mucocutaneous areas and other organs (22). In the soil, the fungus grows as saprobic mycelium, resulting in the formation of infectious propagules. After penetrating the host, the fungus differentiates into its yeast form, a fundamental step for the successful establishment of the disease (46).Although P. brasiliensis is not traditionally considered a typical intracellular pathogen, independent studies have demonstrated that P. brasiliensis yeast cells have the capacity to adhere and invade host cells (4, 24, 31). P. brasiliensis may actively penetrate the mucocutaneous surface and parasitize epithelial cells, thus evading the host defenses and reaching deeper tissues.Fungal ECM-binding adhesins have been characterized in different models, including P. brasiliensis. Vicentini et al. (49) showed specific binding of the protein gp43 to laminin, which is correlated to the adhesiveness of the fungus in vitro as well as to an enhancement of pathogenic potential. We have been systematically searching for new adhesion proteins in P. brasiliensis that have the potential to play roles in the fungal virulence, and proteins such as P. brasiliensis malate synthase (PbMLS) (34), PbDfg5p (defective for filamentous growth protein) (10), triosephosphate isomerase (PbTPI) (41), and glyceraldehyde-3-phosphate dehydrogenase (PbGAPDH) (4) were found to associate with ECM components. In particular, enolase from P. brasiliensis (PbEno) is a fibronectin-binding protein, as characterized by affinity ligand assays (17).The importance of plasminogen in infectious diseases is supported by the fact that many pathogens manifest the ability to bind plaminogen (47, 13). Plasminogen is a single-chain glycoprotein with a molecular mass of 92 kDa. Its protein structure comprises an N-terminal preactivation peptide, five consecutive disulfide-bonded triple-loop kringle domains, and a serine-protease domain containing the catalytic triad (48). The kringle domains of plasminogen mediate its attachment to cells surfaces by binding proteins with accessible carboxyl-terminal or internal lysine residues. The plasminogen system displays a unique role in the host defense by dissolving fibrin clots and serving as an essential component to maintain homeostasis (43). Activation of the fibrinolytic system is dependent on the conversion of plasminogen to the serine-protease plasmin by the physiological activators urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) (9). Plasmin is involved in fibrinolysis homeostasis and degradation of the extracellular matrix and basement membrane. The mammalian plasminogen-plasmin proteolytic system plays a crucial role in extracellular matrix degradation which is exploited by invasive pathogens, including fungi (25, 47). Microbe-derived plasminogen conversion to plasmin may promote dissemination of the pathogen within the host (1).Among several proteins, enolase has been found to play a major role in microbial recruitment of plasminogen (32). By serving as a key surface receptor for plasminogen recruitment, enolase has been shown to function as mediator of microbial virulence (6, 15). The potential of P. brasiliensis to recruit human plasminogen for invasion and virulence has not been studied until now. In this study, we demonstrated for the first time that P. brasiliensis is capable of recruiting plasminogen and activating the plasminogen fribrinolytic system in a process, at least in part, mediated by the cell wall-localized enolase. Furthermore, recombinant PbEno (rPbEno) promoted an increase in the adhesion/invasion of P. brasiliensis in in vitro models of infection, a process that seems to be associated with the enolase ability of modifying the surface of host cells. These data suggest that PbEno may play a role in mediating the P. brasiliensis recruitment of plasminogen as well as in attachment and internalization of the fungus to host tissues, potentially playing a role in the establishment of PCM.     

Interaction of Leishmania parasites with dendritic cells and its functional consequences

Interaction between dendritic cells (DC) and T cells is essential for the generation of cell mediated immunity and thus DC play a critical role in the initiation of immune responses against Leishmania parasites. Although macrophages (Mphi) are the major targets of all species of Leishmania, a number of studies demonstrated the infection of DC by Leishmania. DC specific intracellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), has been reported to be the receptor for Leishmania amastigotes. The functional consequences in DC after Leishmania infections appear to depend on species of Leishmania. Some species of Leishmania enhance the surface expression of co-stimulatory molecules and CD40-ligand-induced IL-12 production in DC. On the other hand other species down-regulate co-stimulatory molecules and inhibit IL-12 production. The intrinsic differences among Leishmania species with regard to alteration of cell surface molecules and IL-12 production in DC may contribute to the healing and non-healing forms of the disease.     

肺癌细胞与胞外基质选择裱衬表面粘附力学的研究

肿瘤细胞与细胞外基质的粘附特性与肿瘤的侵袭转移密切相关,作者力图揭示人肺癌细胞相应的生物力学和生物流变学特征,采用微管吸吮技术定量测定体外培养的低转移人肺腺癌（PAa_)细胞和高转移人肺巨细胞癌（PG）细胞与细胞外基质重要组份胶原蛋白IV和层粘连蛋白（LN）的粘附力学特性。结果表明：与胶原蛋白IV裱衬在的粘附力,在PA,aPG细胞均较裱衬前增加,但在较低胶原浓度（1．00ug/ml,2.00ug/ml)时PAa细胞的粘附力增加幅度大于PG细胞,与LN和固定浓度（2．00ug/ml)胶原蛋白IV的复合裱衬面的粘附力为PAa细胞大于PG细胞,在较低LN浓度,PAa(0.625ug/ml),PG(0.625ug/ml,1.25ug/ml)细胞粘附力相对降低,尤以PG细胞中的降低程度和幅度为大,因此,癌细胞与细胞外基质的粘附和去粘附行为主要通过膜受体介导,从而影响癌细胞生物学特性及侵润转移能力。     

Interaction of Pneumocystis carinii with Host Lungs: an Ultrastructural Study

Pneumocystis carinii pneumonia was produced in rats by the administration of corticosteroids, low (8%) protein diet, and tetracycline in the drinking water. The rats were sacrificed at weekly intervals, and their lungs were examined by electron microscopy. For the first 6 weeks, few alterations were noted in host pulmonary tissue, except a close attachment of P. carinii trophozoites to the type I pneumocytes. At 7 to 8 weeks, when the infection reached the peak intensity on light microscopy, degenerative changes occurred in the type I pneumocyte, beginning with subepithelial bleb formation and followed by denudation of the basement membrane. This denuded surface appeared to be the site both of exudation of serum and tissue fluid into the alveolar space and of spread of P. carinii into the interstitium. There was hypertrophy of type II pneumocytes, which also occurred in uninfected control rats ingesting tetracyclines. With tapering of the corticosteroid dose, P. carinii was slowly cleared from the lungs, but latent infection persisted for at least 21 weeks. The host response to the corticosteroid dose tapering included increased prominence of alveolar macrophages and progressive interstitial lymphocytic infiltrate and fibrosis. Thus, P. carinii interacts with, and is associated with damage to, specific host cells. This interaction is important in the host-parasite relationship in this infection.     

Investigation and Functional Enrichment Analysis of the Human Host Interaction Network with Common Gram-Negative Respiratory Pathogens Predicts Possible Association with Lung Adenocarcinoma

Haemophilus influenzae (Hi), Moraxella catarrhalis (MorCa) and Pseudomonas aeruginosa (Psa) are three of the most common gram-negative bacteria responsible for human respiratory diseases. In this study, we aimed to identify, using the functional enrichment analysis (FEA), the human gene interaction network with the aforementioned bacteria in order to elucidate the full spectrum of induced pathogenicity. The Human Pathogen Interaction Database (HPIDB 3.0) was used to identify the human proteins that interact with the three pathogens. FEA was performed via the ToppFun tool of the ToppGene Suite and the GeneCodis database so as to identify enriched gene ontologies (GO) of biological processes (BP), cellular components (CC) and diseases. In total, 11 human proteins were found to interact with the bacterial pathogens. FEA of BP GOs revealed associations with mitochondrial membrane permeability relative to apoptotic pathways. FEA of CC GOs revealed associations with focal adhesion, cell junctions and exosomes. The most significantly enriched annotations in diseases and pathways were lung adenocarcinoma and cell cycle, respectively. Our results suggest that the Hi, MorCa and Psa pathogens could be related to the pathogenesis and/or progression of lung adenocarcinoma via the targeting of the epithelial cellular junctions and the subsequent deregulation of the cell adhesion and apoptotic pathways. These hypotheses should be experimentally validated.     

杜氏利什曼原虫表膜的制备及保护性McAb识别膜抗原的研究

本文用匀浆及超声波破碎杜氏利什曼原虫四川犬分离株前鞭毛体,蔗糖密度梯度离心分离其表膜,电镜观察见所得样品是单位膜和膜下微管组成的表膜,测得微管直径平均为23.3nm,微管间距巨平均为15.7nm,经SDS-PAGE分离显示1条主带,次带约15条,再转移到硝化纤维膜上,用单克隆抗体2H6-E3识别,仅见有一条区带,分子量为63kDa,该单克隆抗体是己经被证实定位于前鞭毛体表膜,并具有免疫保护性的。     

Computer Imaging Analysis of Glomerular Extracellular Components in Patients with IgA Nephropathy

Computer imaging analysis was used for quantitative evaluation of the extents, amounts and distributions of glomerular extracellular components, such as the 7S and NC 1 domains of type IV collagen, laminin (LN), fibronectin (FN) and IgA, in glomeruli from patients with IgA nephropathy. Renal biopsy specimens from 13 patients with IgA nephropathy were incubated with mouse monoclonal antibodies against the FN or non collagenous (NC 1) domain of type IV collagen or polyclonal antiserum against the LN or 7S domain of human type IV collagen, and then stained with appropriate dilutions of FITC labeled anti mouse Ig antisera. Marked staining of the 7S or NC 1 domain of type IV collagen, LN or FN was detected in the glomerular capillary walls and/or mesangial areas in patients with IgA nephropathy. In particular, a prominent increase of FN was observed in the subendothelial regions of glomerular capillary walls, i.e. mesangial interposition, in the moderate or advanced stage of IgA nephropathy. Therefore, computer imaging analysis was shown to be useful for the quantitative determination of such components distributed in glomeruli from patients with IgA nephropathy. Acta Pathol Jpn 39: 296 305, 1989.