Host Connective Tissue Research Articles

Host–parasite–environment relationship, morphology and molecular analyses of Henneguya eirasi n. sp. parasite of two wild Pseudoplatystoma spp. in Pantanal Wetland, Brazil

A new myxosporean species, Henneguya eirasi n. sp., is described parasitizing the gill filaments of Pseudoplatystoma corruscans and Pseudoplatystoma fasciatum (Siluriformes: Pimelodidae) caught in the Patanal Wetland of the state of Mato Grosso, Brazil. The parasite formed white, elongated plasmodia measuring up to 3 mm. Mature spores were ellipsoidal in the frontal view, measuring 37.1 ± 1.8 μm in total length, 12.9 ± 0.8 μm in body length, 3.4 ± 0.3 μm in width, 3.1 ± 0.1 μm in thickness and 24.6 ± 2.2 μm in the caudal process. Polar capsules were elongated and equal in size, measuring 5.4 ± 0.5 μm in length and 0.7 ± 0.1 μm in width. Polar filaments had 12–13 coils. Histopathological analysis revealed that the parasite developed in the sub-epithelial connective tissue of the gill filaments and the plasmodia were surrounded by a capsule of host connective tissue. The plasmodia caused slight compression of the adjacent tissues, but no inflammatory response was observed in the infection site. Ultrastructure analysis revealed a single plasmodial wall connected to the ectoplasmic zone through numerous pinocytotic canals. The plasmodial wall exhibited numerous projections and slightly electron-dense material was found in the ectoplasm next to the plasmodial wall, forming a line just below the wall. Partial sequencing of the 18S rDNA gene of H. eirasi n. sp. obtained from P. fasciatum resulted in a total of 1066 bp and this sequence did not match any of the Myxozoa available in the GenBank. Phylogenetic analysis revealed the Henneguya species clustering into clades following the order and family of the host fishes. H. eirasi n. sp. clustered alone in one clade, which was the basal unit for the clade composed of Henneguya species parasites of siluriform ictalurids. The prevalence of the parasite was 17.1% in both fish species examined. Parasite prevalence was not influenced by season, host sex or host size.

Destructive effects of smoking on molecular and genetic factors of periodontal disease

Many epidemiological evidences have proven the association between smoking and periodontal disease. The causality can be further established by linking findings of traditional epidemiological studies with the developments in molecular techniques that occurred in the last decade. The present article reviews recent studies that address the effect of smoking on molecular and genetic factors in periodontal disease. Most findings support the fact that tobacco smoking modulates destruction of the periodontium through different pathways: microcirculatory and host immune systems, connective tissue, and bone metabolism. Although smokers experience an increased burden of inflammatory responses to microbial challenges compared to non-smokers, understanding the association between smoking and periodontal diseases involves substantial problems with respect to accuracy of measurements, and particularly, sampling of many subjects. It remains unclear whether genetic susceptibility to periodontal disease is influenced by exposure to smoking or the effect of smoking on periodontal disease is influenced by genetic susceptibility. Employment of molecular techniques may play a key role in further elucidation of mechanisms linking smoking and periodontal destruction, the direct relationship as environmental factors and indirect relationship through genetic factors.

Identification, expression and immunolocalization of cathepsin B3, a stage-specific antigen expressed by juvenile Fasciola gigantica

To identify antigens that could potentially be developed as vaccines against Fasciola gigantica, somatic antigens were analyzed by immunoprecipitation using pooled sera from rats infected with F. gigantica metacercariae. A prominent antigen of the newly excysted juveniles (NEJ), cathepsin B3 protease (FgCatB3), was identified by N-terminal sequencing and PCR screening of a cDNA library. Recombinant FgCatB3 (rFgCatB3) was expressed in Pichia pastoris, and shown to catalyse the digestion of gelatin, the fluorometric substrate Z-Phe-Arg-AMC and native fibronectin, suggesting that this enzyme may be involved in digesting host connective tissues during the fluke's penetration and migration in the host. Rabbit polyclonal sera against rFgCatB3 was produced and used to determine the distribution of the native cathepsin B3 protease in various developmental stages of F. gigantica. By Western blotting, cathepsin B3 was detected in the whole body (WB) extract of metacercariae and NEJ but not in 4-week-old juveniles or adult parasites which confirmed the stage-specific characteristics of cathepsin B3. Immunolocalization of cathepsin B3 protease in each parasite stage showed that high levels of FgCatB3 were present in the caecal epithelium of the metacercariae and NEJ. The differential distribution of FgCatB3 in the different life cycle stages suggests that this protease is functionally important for the juvenile stage of F. gigantica.

Borrelia burgdorferi RevA Antigen Binds Host Fibronectin

Borrelia burgdorferi, the Lyme disease-causing spirochete, can persistently infect its vertebrate hosts for years. B. burgdorferi is often found associated with host connective tissue, where it interacts with components of the extracellular matrix, including fibronectin. Some years ago, a borrelial surface protein, named BBK32, was identified as a fibronectin-binding protein. However, B. burgdorferi BBK32 mutants are still able to bind fibronectin, indicating that the spirochete possesses additional mechanisms for adherence to fibronectin. We now demonstrate that RevA, an unrelated B. burgdorferi outer surface protein, binds mammalian fibronectin in a saturable manner. Site-directed mutagenesis studies identified the amino terminus of the RevA protein as being required for adhesion to fibronectin. RevA bound to the amino-terminal region of fibronectin. RevA binding to fibronectin was not inhibited by salt or heparin, suggesting that adhesin-ligand interactions are primarily nonionic and occur through the non-heparin-binding regions of the fibronectin amino-terminal domains. revA genes are widely distributed among Lyme disease spirochetes, and the present studies determined that all RevA alleles tested bound fibronectin. In addition, RevB, a paralogous protein found in a subset of B. burgdorferi strains, also bound fibronectin. We also confirmed that RevA is produced during mammalian infection but not during colonization of vector ticks and determined that revA transcription is controlled through a mechanism distinct from that of BBK32.

Incorporation by host tissue of two biomaterials used as repair of defects produced in abdominal wall of rats

Biomaterials may be used as treatment of great abdominal wall defects to avoid tension during repair. In the present research we intended to investigate incorporation type by host tissue of membranes of microbial cellulose (MC), produced by the bacteria Zoogloea sp., and of polytetrafluoroethylene (ePTFE) in abdominal wall defects of rats. Sixty male rats Wistar, anesthetized by ketamine (5mg/100g) and xylazine (2mg/100g), were submitted to a rectangular excision (2 x 3cm) of the abdominal wall, including fascia, muscles and peritoneum and further treated with implants of microbial cellulose (MC Group - 30 animals) or expanded polytetrafluoroethylene ( ePTFE Group- 30 animals). Each group was subdivided in 14th DPO, 28th DPO and 60th DPO Subgroups. Incorporation of biomaterials was observed by wrapping and infiltration by host tissue. It has been found that wrapping associated to infiltration of host connective tissue in implants of ePTFE were present in 100% of the observed samples, and this may be responsible for increase resistance to traction. Inversely, wrapping without host tissue infiltration was seen in 100% of examined specimens of MC implants. Wrapping and host tissue infiltration is seen only in ePTFE implants.

Characterization of Tissue Response and in Vivo Degradation of Cholecyst-Derived Extracellular Matrix

A rat subcutaneous implantation model was used to evaluate the in vivo degradation and tissue response of cholecyst-derived extracellular matrix (CEM). This response was compared to that of glutaraldehyde (GA) cross-linked CEM and porcine heart valve (HV), which are designated as GAxCEM and GAxHV, respectively. Tissue composition, inflammatory cell distribution, and angiogenesis at the implant site were quantified using stereological parameters, thickness (Ta), volume fraction (Vv), surface density (Sv), length density (Lv), and radius of diffusion (Rdiff). CEM was completely infiltrated with host tissue at 21 days and resorbed by 63 days. GAxCEM was also infiltrated with host tissue, while GAxHV matrix was impermeable to host tissue infiltration. Both GAxCEM and GAxHV retained their scaffold integrity until 63 days with no apparent degradation. A fibrous tissue of thickness <52 mum, rich in collagen and vasculature, surrounded all scaffolds, and from 21 to 63 days the fibrous tissue showed maturation with a significant increase in their fibrocyte content. No signs of acute inflammatory response were observed in the study period for any of the scaffolds, while the chronic inflammatory response was predominated with macrophages for all scaffolds except for CEM at 63 days. A higher degree of giant cell formation was observed with GA cross-linked scaffolds. From 21 to 63 days, lymphocytic response decreased for CEM, while it increased significantly for GAxHV. Angiogenesis/neo-vascularization was uniform for CEM (reaching the core), significantly lower for GAxCEM within the implant area as compared to CEM, while restricted to the exterior of GAxHV matrix. In summary, CEM was a fast degrading scaffold that induced a transitional inflammatory response accompanied by gradual resorption and replacement by host connective tissue as compared to the very slow degrading GA cross-linked controls, GAxCEM and GAxHV, which caused a sustained chronic inflammatory response and remained at the site of implantation until the end of the study period of 63 days.

Development of porous ultra high molecular weight polyethylene scaffolds for the fabrication of orbital implant

Porous implants having interconnecting channels allow ingrowth of host connective tissue. Complete implant vascularization reduces the risk of infection, extrusion, and other complications associated with nonintegrated implants. Attempts were made to develop 60% and 70% porous ultra high molecular weight polyethylene (UHMWPE) scaffolds and blocks using sodium chloride as channeling agent, which when dissolved in boiling water leaves behind the interconnecting channels. The average diameter of the pores of 60% and 70% porous scaffolds was found to be approx. 170 µm and 210 µm, respectively. Mechanical characterizations of the scaffolds indicated sufficient strength to be used for orbital implant fabracation. Surface roughness of the scaffolds indicated increase in surface roughness with the increase in porosity. The scaffolds developed were found to be hemocompatible with the human blood. Subsequently, the 70% porous scaffold was dip coated with a solution mixture of sodium carboxy methyl cellulose (SCMC)/polyvinyl alcohol (PVA)/hydroxyapatite (HA) which also showed hemocompatibility. Ciprofloxacin release pattern from the membrane was determined. Finally an orbital implant was fabricated from the 70% porous scaffold.

The interaction between the amoebaBalamuthia mandrillarisand extracellular matrix glycoproteinsin vitro

Balamuthia mandrillaris, a soil amoeba, is the causative agent of Balamuthia granulomatous amoebic encephalitis, a life-threatening brain infection. This amoeba is acquired from contaminated soil and may enter the host through cutaneous lesions or through nasal passages, migrating to the lungs or brain. During invasion, B. mandrillaris has access to components of the extracellular matrix (ECM) of the host. Therefore, we investigated the interaction of B. mandrillaris with 3 ECM glycoproteins (collagen-I, fibronectin and laminin-1) that are encountered in host connective tissues and at the basal lamina. Using optical microscopy, amoeba association on ECM-coated surfaces was examined. Binding of amoebae on laminin was greater than that on collagen or fibronectin. Laminin-adhered B. mandrillaris exhibited elongated and spread forms, distinctive from those observed for amoebae on a plastic surface. Collagen and fibronectin-adhered B. mandrillaris presented elongated shapes with cellular expansions. Binding to collagen, fibronectin, or laminin was inhibited when amoebae were pre-treated with sialic acid. Treatment with galactose resulted in diminished binding of amoebae on laminin, while mannose increased binding in all coating conditions tested. Dependence of divalent cations on amoeba binding was demonstrated for laminin-amoeba interaction. Collectively, the results indicate that B. mandrillaris recognizes specific glycoproteins of the mammalian extracellular matrix.

Inhibition of host connective tissue growth factor expression: a novel Trypanosoma cruzi‐mediated response

Connective tissue growth factor (CTGF) is a secreted cytokine that plays a fundamental role in the development of tissue fibrosis by mediating many of the profibrotic effects of TGF-beta. We present the novel finding that the intracellular pathogen Trypanosoma cruzi elicits immediate and sustained repression of basal CTGF expression in dermal fibroblasts, followed by down-regulation of the extracellular matrix proteins, fibronectin, and collagen I alpha1. To address mechanisms underlying this response, the major CTGF-regulating pathways were investigated. We report that both T. cruzi trypomastigotes and secreted parasite factor(s) antagonize TGF-beta-dependent induction of CTGF in fibroblasts. Of the TGF-beta-dependent signaling pathways required for CTGF expression, we demonstrate that T. cruzi interferes with cellular Erk1/2 phosphorylation but not Smad3 signaling. While increased stimulation of Erk phosphorylation alone was insufficient to override the parasite-mediated repression of CTGF, stimulation of fibroblasts with increased concentrations of TGF-beta, which activates both Smad3 and Erk1/2, completely abrogated this inhibition. Together with the finding that T. cruzi-mediated down-regulation of CTGF expression requires de novo host cell protein synthesis, our data indicate that the unique ability of T. cruzi to interfere with the host fibrogenic response is a complex process requiring input from multiple host cell signaling pathways.

Identification and phenotypic characterization of a subpopulation of T84 human colon cancer cells, after selection on activated endothelial cells.

The extravasation of metastatic cells is regulated by molecular events involving the initial adhesion of tumor cells to the endothelium and subsequently the migration of the cells in the host connective tissue. The differences in metastatic ability could be attributed to properties intrinsic of the various primary tumor types. Thus, the clonal selection of neoplastic cells during cancer progression results in cells better equipped for survival and formation of colonies in secondary sites. A cell line (T84SF) exhibiting an altered phenotypic appearance was selected from a colon cancer cell line (T84) by repetitive plating on TNFalpha-activated human endothelial cells and subsequent selection for adherent cells. Cell growth, motility, chemoinvasive abilities, tyrosine phosphorylation signaling, and the metastasis formation in nude mice of the two cell lines was compared. T84SF cells displayed in vitro an higher proliferation rate and a more invasive behavior compared to the parental cells while formed in vivo a greater number of metastatic colonies in nude mice. As concerns the signaling underlying the phenotypes of the selected cells, we examined the general tyrosine phosphorylation levels in both cell lines. Our results indicate that T84SF have an increased basal tyrosine phosphorylation of several proteins among which src kinase was identified. Treatment of cells with a specific inhibitor of src activity caused a greater in vitro inhibition of proliferation and invasive properties of T84 parental cells with respect to T84SF cells and diminished metastasis formation in vivo. Altogether, these data provide evidences that this new cell line may be valuable for identifying molecular mechanisms involved in the metastatic progression of colon cancer.

Mutants of Plasminogen Activator Inhibitor-1 Designed to Inhibit Neutrophil Elastase and Cathepsin G Are More Effective in Vivo than Their Endogenous Inhibitors

Neutrophil elastase and cathepsin G are abundant intracellular neutrophil proteinases that have an important role in destroying ingested particles. However, when neutrophils degranulate, these proteinases are released and can cause irreparable damage by degrading host connective tissue proteins. Despite abundant endogenous inhibitors, these proteinases are protected from inhibition because of their ability to bind to anionic surfaces. Plasminogen activator inhibitor type-1 (PAI-1), which is not an inhibitor of these proteinases, possesses properties that could make it an effective inhibitor of neutrophil proteinases if its specificity could be redirected. PAI-1 efficiently inhibits surface-sequestered proteinases, and it efficiently mediates rapid cellular clearance of PAI-1-proteinase complexes. Therefore, we examined whether PAI-1 could be engineered to inhibit and clear neutrophil elastase and cathepsin G. By introducing specific mutations in the reactive center loop of wild-type PAI-1, we generated PAI-1 mutants that are effective inhibitors of both proteinases. Kinetic analysis shows that the inhibition of neutrophil proteinases by these PAI-1 mutants is not affected by the sequestration of neutrophil elastase and cathepsin G onto surfaces. In addition, complexes of these proteinases and PAI-1 mutants are endocytosed and degraded by lung epithelial cells more efficiently than either the neutrophil proteinases alone or in complex with their physiological inhibitors, alpha1-proteinase inhibitor and alpha1-antichymotrypsin. Finally, the PAI-1 mutants were more effective in reducing the neutrophil elastase and cathepsin G activities in an in vivo model of lung inflammation than were their physiological inhibitors.

Synthetic elastin hydrogels derived from massive elastic assemblies of self-organized human protein monomers

A key objective of bioengineering is the development of new scaffolding biomaterials with appropriate mechanical and biological properties such as strength, elasticity and biocompatibility that mimic the native host connective tissue. Here we describe the production and properties of massive synthetic elastin assemblies formed by chemically cross-linking recombinant human tropoelastin with bis(sulfosuccinimidyl) suberate, permitting the construction of elastic sponges, sheets and tubes. The innate characteristics of synthetic elastin constructs are common with those of native elastin. The Young's Modulus ranged from 220 to 280 kPa with linearity of extension to at least 150%. Synthetic elastin was extensible by 200–370%. The constructs behaved as hydrogels and displayed stimuli-responsive characteristics towards temperature and salt concentrations. Intrinsic fluorescence spectroscopy demonstrated that the elastin fluorophore is a feature of the polypeptide. Scanning electron microscopy allowed us to construct a model of elastin assembly that was driven by the lateral association of small twisted rope-like fibrils. FT–Raman spectra at 100% strain gave amide I and III peaks that correlated with a stretch-dependent increase in α-helical content. Growth and proliferation of cells were supported in vitro while in vivo implants were well tolerated. We conclude that synthetic elastin has potential as a novel biomaterial that can be easily molded into a variety of shaped tissue substrates and has a range of properties that are required for elastic, cell-interacting and compliant applications. Furthermore, its in vitro construction provides a powerful tool to probe the early stages of elastin assembly and the molecular basis for its elasticity.

Secondary and primary murine alveolar echinococcosis: combined albendazole/nitazoxanide chemotherapy exhibits profound anti-parasitic activity

In this study, the efficacies of chemotherapy employing nitazoxanide (NTZ), albendazole (ABZ), and a NTZ/ABZ-combination against alveolar echinococcosis (AE) were investigated in an experimental murine model. Following secondary infection, meaning i.p. injection of 20 Echinococcus multilocularis metacestodes, the drugs were administered by intragastric inoculation on a daily bases for a period of 5 weeks. Treatment was started either immediately on the day of infection, or at 2 months p.i., respectively. Application of the NTZ/ABZ-combination starting at 2 months p.i. was proven to be most effective in terms of reducing parasite weight (from 4.42±1.03 to 1±0.05 g; P=0.001). Inspection of treated parasites by transmission electron microscopy showed that ABZ- and NTZ-treated metacestode tissues, respectively, were heterogeneous in that both largely intact parasites as well as severely altered metacestodes could be observed. NTZ/ABZ-combination treatment induced the most severe ultrastructural alterations, including massive reduction in length and number of microtriches, severely damaged tegumental architecture, and progressive loss of viability of the germinal layer, associated with encapsulation by host connective tissue. A comparative pharmacokinetic study in mice revealed that the application of ABZ and NTZ in combination resulted in a two- to four-fold increase of albendazole sulfoxide serum levels for the period of 4–8 h following drug uptake compared to application of ABZ alone. In a third experiment, mice were orally infected with E. multilocularis eggs, and treated with NTZ starting at 2 months p.i. This resulted in a significantly lower lesion number in treated versus untreated mice ( P=0.01). This investigation indicates the potential value for NTZ and/or a combined ABZ/NTZ chemotherapy against AE.

Structure and Flexibility of Streptococcus agalactiaeHyaluronate Lyase Complex with Its Substrate: INSIGHTS INTO THE MECHANISM OF PROCESSIVE DEGRADATION OF HYALURONAN

Streptococcus agalactiae hyaluronate lyase degrades primarily hyaluronan, the main polysaccharide component of the host connective tissues, into unsaturated disaccharide units as the end product. Such function of the enzyme destroys the normal connective tissue structure of the host and exposes the tissue cells to various bacterial toxins. The crystal structure of hexasaccharide hyaluronan complex with the S. agalactiae hyaluronate lyase was determined at 2.2 A resolution; the mechanism of the catalytic process, including the identification of specific residues involved in the degradation of hyaluronan, was clearly identified. The enzyme is composed structurally and functionally from two distinct domains, an alpha-helical alpha-domain and a beta-sheet beta-domain. The flexibility of the protein was investigated by comparing the crystal structures of the S. agalactiae and the Streptococcus pneumoniae enzymes, and by using essential dynamics analyses of CONCOORD computer simulations. These revealed important modes of flexibility, which could be related to the protein function. First, a rotation/twist of the alpha-domain relative to the beta-domain is potentially related to the mechanism of processivity of the enzyme; this twist motion likely facilitates shifting of the ligand along the catalytic site cleft in order to reposition it to be ready for further cleavage. Second, a movement of the alpha- and beta-domains with respect to each other was found to contribute to a change in electrostatic characteristics of the enzyme and appears to facilitate binding of the negatively charged hyaluronan ligand. Third, an opening/closing of the substrate binding cleft brings a catalytic histidine closer to the cleavable substrate beta1,4-glycosidic bond. This opening/closing mode also reflects the main conformational difference between the crystal structures of the S. agalactiae and the S. pneumoniae hyaluronate lyases.

Mechanism of Hyaluronan Degradation byStreptococcus pneumoniae Hyaluronate Lyase: STRUCTURES OF COMPLEXES WITH THE SUBSTRATE

Hyaluronate lyase enzymes degrade hyaluronan, the main polysaccharide component of the host connective tissues, predominantly into unsaturated disaccharide units, thereby destroying the normal connective tissue structure and exposing the tissue cells to various endo- and exogenous factors, including bacterial toxins. The crystal structures of Streptococcus pneumoniae hyaluronate lyase with tetra- and hexasaccharide hyaluronan substrates bound in the active site were determined at 1.52- and 2.0-A resolution, respectively. Hexasaccharide is the longest substrate segment that binds entirely within the active site of these enzymes. The enzyme residues responsible for substrate binding, positioning, catalysis, and product release were thereby identified and their specific roles characterized. The involvement of three residues in catalysis, Asn(349), His(399), and Tyr(408), is confirmed, and the details of proton acceptance and donation within the catalytic machinery are described. The mechanism of processivity of the enzyme is analyzed. The flexibility (allosteric) behavior of the enzyme may be understood in terms of the results of flexibility analysis of this protein, which identified two modes of motion that are also proposed to be involved in the hyaluronan degradation process. The first motion describes an opening and closing of the catalytic cleft located between the alpha- and beta-domains. The second motion demonstrates the mobility of a binding cleft, which may facilitate the binding of the negatively charged hyaluronan to the enzyme.

E-selectin modulates the malignant properties of T84 colon carcinoma cells

The extravasation of metastatic cells is regulated by molecular events involving the initial adhesion of tumor cells to the endothelium and subsequently the migration of cells in the host connective tissue. E-selectin on endothelial cells and sialyl Lewis X carbohydrate component on tumor cells are mainly involved in the adhesion of colon carcinoma cells to the endothelium of target organ. Interaction of T84 colon cancer cells to purified E-selectin in vitro caused an increase in the tyrosine phosphorylation of a number of proteins as well as the modulation of cellular properties correlated to the metastatic phenotype. Specifically, E-selectin-stimulated actin reorganization, increased collagenase secretion, and induced cell migration. Treatment of T84 cells with herbimycin A inhibited cell adhesion as well as selectin-induced increase of cell migration, and cytoskeleton assembly. Our data demonstrate that binding of cancer cells to E-selectin starts signal transduction pathways which may affect the tumor metastatic abilities.

Changes in host and parasite-derived cellular and extracellular matrix components in developing cysts of Myxobolus pendula (Myxozoa).

Cysts of Myxobolus pendula from the gill arch of creek chub (Semotilus atromaculatus) were examined at various stages of development using light and electron microscopy. The subepithelial host connective tissue underwent dramatic changes, including degradation and remodelling of collagen and vascularisation, in response to the infection. Inflammatory cells lay in a fluid-filled space beneath the host's connective tissue and surrounded a distinctive parasite-derived matrix, composed of collagen fibril bundles embedded in cellular processes of the underlying secretory cells. This collagen matrix was resistant to degradation and invasion by leukocytes. Secretion of a matrix by M. pendula as a structural support, and a protective barrier against the host inflammatory cells is a novel observation for cyst-forming Myxosporea.

SEM AND TEM STUDY OF THE INTEGUMENT OF OPHIOIKA SP. (CRUSTACEA, COPEPODA)

Abstract The ultrastructure of the integument in a parasitic copepod Ophioika sp., which may be a new species, is described using SEM and TEM of specimens both in situ and dissected out of their brittlestar host, Ophiocoma scolopendrina. The parasite is found embedded in host connective tissue at the base of the arm. The surface of the parasite's integument facing the host has knob-like projections, some of which have long microvilli-like endings. Whether these structures have an absorptive function and play a role in the parasite's feeding is discussed.

High molecular mass glycans are major structural elements associated with the laminated layer of in vitro cultivated Echinococcus multilocularis metacestodes

The laminated layer of the larval stage (metacestode) of the cestode parasite Echinococcus multilocularis is composed largely of carbohydrates, which form a tight microfibrillar meshwork around the entire metacestode. Since this laminated layer is the only parasite structure which is in constant contact with host immune and non-immune cells, and appears largely resistant to physiological and immunological reactions of the host, it most likely carries out important functions with regard to host–parasite interactions. In infected hosts, the metacestode is usually concentrically covered by host connective tissue cells and large amounts of collagen, causing a dense scar-like fibrosis, and it is likely that host-derived components are incorporated into the laminated layer at the host–parasite interface. Therefore, in order to obtain information on the molecular composition of this structure, we used parasite larvae which were generated through in vitro cultivation and thus were largely devoid of interfering host components. Lectin fluorescence on section-labelling of metacestodes embedded in LR-White suggested that the laminated layer is largely composed of N-acetyl- β- d-galactosaminyl, and α- and β- d-galactosyl residues, as well as of the core structure of O-linked carbohydrate chains, N-acetylgalactosamine- β-1.3-galactose, while N-linked glycopeptides and α- d-mannosyl residues and/or glucosyl residues were found mainly within the germinal layer, and within the cellular mass and the surface of developing protoscoleces. Lectin–gold EM confirmed these findings. The laminated layer was isolated from in vitro cultivated metacestodes by urea extraction, and the ultrastructure of the purified laminated layer was assessed comparatively with respect to the laminated layer of intact parasites. The glycan composition was determined using SDS–PAGE and lectin blotting. This work has laid the basis for a more detailed dissection of the molecular composition of the laminated layer.

Dilution of human mesenchymal stem cells with dermal fibroblasts and the effects on in vitro and in vivo osteochondrogenesis.

The stromal elements of human bone marrow include cells, referred to as mesenchymal stem cells (MSCs), that have the potential to differentiate into bone, cartilage, fat, and hematopoietic-supportive stromal tissue. MSCs have been isolated and maintained in culture, and in vivo and in vitro assays have been used to show that these cultured cells possess osteochondral potential. Human mesenchymal stem cells (hMSCs) were combined in a range of proportions with human dermal fibroblasts (hDFs), shown to be devoid of osteochondral potential, and tested in these assays. Results suggest that hMSCs may be intentionally "contaminated" with 25-50% hDFs and still elicit a positive response in alkaline phosphatase and calcium in vitro osteogenic assays, form cartilage in pellet culture conditions, and produce bone when loaded into porous hydroxyapatite-tricalcium phosphate ceramic cubes and then implanted subcutaneously into immunocompromised mice. Although hMSCs can be purified and culture-expanded as a homogeneous subset of marrow cells, the dilution results reported here are encouraging for the prospective use of these cells in clinical applications, where repair grafts that contain 100% hMSCs almost surely will become infiltrated with host connective tissue and vasculature, which will dilute the initial concentration of hMSCs.