PulmoStent: <Emphasis Type="Italic">In Vitro</Emphasis> to <Emphasis Type="Italic">In Vivo</Emphasis> Evaluation of a Tissue Engineered Endobronchial Stent

Currently, there is no optimal treatment available for end stage tumour patients with airway stenosis. The PulmoStent concept aims on overcoming current hurdles in airway stenting by combining a nitinol stent with a nutrient-permeable membrane, which prevents tumour ingrowth. Respiratory epithelial cells can be seeded onto the cover to restore mucociliary clearance. In this study, a novel hand-braided dog bone stent was developed, covered with a polycarbonate urethane nonwoven and mechanically tested. Design and manufacturing of stent and cover were improved in an iterative process according to predefined requirements for permeability and mechanical properties and finally tested in a proof of concept animal study in sheep for up to 24 weeks. In each animal two stents were implanted, one of which was cell-seeded by endoscopic spraying in situ. We demonstrated the suitability of this membrane for our concept by glucose transport testing and in vitro culture of respiratory epithelial cells. In the animal study, no migration occurred in any of the twelve stents. There was only mild granulation tissue formation and tissue reaction; no severe mucus plugging was observed. Thus, the PulmoStent concept might be a step forward for palliative treatment of airway stenosis with a biohybrid stent device.     

Effects of Liposomes of Different Lipid Composition on <Emphasis Type="Italic">In Vitro</Emphasis> Growth of <Emphasis Type="Italic">Mycobacterium Tuberculosis</Emphasis> H37Rv

The growth of M. tuberculosis H37RV in culture medium was studied after addition of liposomes from different lipids (phosphatidylcholine, cardiolipin, and glycosphyngolipids). Addition of phosphatidylcholine into culture medium did not modify the growth and multiplication of mycobacteria. Addition of glycosphyngolipids and their mixture with phosphatidylcholine partially inhibited the growth. Addition of cardiolipin inhibited the growth of mycobacteria and even suppressed it, depending on the dose. Presumably, high concentrations of cardiolipin added into the culture medium, can transfer the mycobacteria into an uncultivable state.     

Comparison of Flow Estimators for Rotary Blood Pumps: An <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis> Study

Various approaches for estimating the flow rate of a rotary blood pump have been proposed for monitoring and control purposes. They have been evaluated under different test conditions and, therefore, a direct comparison among them is difficult. Furthermore, a limited performance has been reported for the areas where the pump flow and motor current present a non-monotonic relationship. In this regard, we selected most approaches that have been presented in literature and added a modified one, resulting in four estimators, which are either non-invasive or invasive, i.e., inlet and outlet pump pressure sensors are used. Data from in vitro and in vivo studies with the Deltastream pump DP2 were used to compare the estimators under the same test conditions. These data included both constant and varying pre- and afterload, contractility, viscosity, as well as pump speed settings. Bland–Altman plots were used to evaluate the performance of the estimators. The mean error of the overall estimated flow in vitro ranged from 0.002 to 0.38 L/min and the limits of agreement (LoA) between?±?2 L/min. During negative flows the mean error decreased by about 25% when the pump inlet pressure was added as an input. In vivo, the mean errors increased, while the LoA remained in the same range. An estimator based on pump pressure difference improves the reliability in areas where flow and current relationship is not monotonic. A trade-off between estimation accuracy and number of sensors was identified. The estimation objective and the potential errors should be considered when selecting an estimation approach and designing the pump systems.     

Carbon Nanotube Reinforced Collagen/Hydroxyapatite Scaffolds Improve Bone Tissue Formation <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis>

Current bone regeneration strategies faced major challenges in fabricating the bionic scaffolds with nano-structure, constituents and mechanical features of native bone. In this study, we developed a new porous scaffold by adding the multi-walled carbon nanotube (MWCNT) into collagen (Col)/hydroxyapatite (HA) composites. Data showed that 0.5%CNT/Col/HA (0.5%CNT) group was approximately tenfolds stiffer than Col–HA, and it was superior in promoting bone marrow mesenchymal stem proliferation and spreading, mRNA and protein expressions of bone sialoprotein (BSP) and osteocalcin (OCN) than Col–HA group. Moreover, we utilized 0.5%CNT composite to repair the rat calvarial defects (8 mm diameter) in vivo, and observed the new bone formation by 3D reconstruction of micro CT, HE and Masson staining, and BSP, OCN by immunohistochemical analysis. Results showed that newly formed bone in 0.5%CNT group was significantly higher than that in Col–HA group at 12 weeks. These findings highlighted a promising strategy in healing of large area bone defect with MWCNT added into the Col–HA scaffold as they possessed the combined effects of mechanical strength and osteogenicity.     

<Emphasis Type="Italic">In Vivo</Emphasis> and <Emphasis Type="Italic">in Vitro</Emphasis> Proliferative and Differentiation Activity of Human Embryonic Retinal Cells

Differentiation of human embryonic retinal cells (20–22 weeks’ gestation) was studied using morphological, immunohistochemical, and biomolecular approaches. The retina included several regions differing by the degree of cell differentiation. Mitoses were rarely found in the marginal zone. This zone contained low differentiated cells. The central retinal area consisted of typical layers with differentiated cells. Culturing was accompanied by the formation of aggregates and neurospheres, where mitoses and progenitor or differentiated cells expressing markers of photoreceptors, neurons, and glia were found.__________Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 2, pp. 103–109, 2005     

<Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">Ex Vivo</Emphasis> Studies of Antioxidant Activity of Carrageenans,Sulfated Polysaccharides from Red Algae

Antioxidant properties of structurally different sulfated polysaccharides (carrageenans) were studied in vitro and ex vivo. Ferric reducing antioxidant activity of carrageenans and their inhibitory effects on hydroxyl radicals and superoxide anion radicals were demonstrated in vitro. Activity of carrageenans depends on the polysaccharide structure. Carrageenans stimulate catalytic activity of SOD from donor erythrocyte.     

The Differential Effect of Endothelial Cell Factors on <Emphasis Type="Italic">In Vitro</Emphasis> Motility of Malignant and Non-malignant Cells

Motility of cancer cells plays a critical role in tumor metastasis, and as such is a target for intervention. The motility of malignant Calu-1 human lung epithelial carcinoma cells is upregulated when placed on a human umbilical vein endothelial cell monolayer, while that of non-malignant L132 human lung epithelial cells is not. To dissect the factor(s) causing such differential behaviors, the motile responses of both cell lines to endothelial cell factors—secreted to the media, on the endothelial cell surface, and secreted to the extracellular matrix—and to individual extracellular matrix proteins were compared. Cell motility was quantified by tracking the cell movement on a surface with time-lapse video microscopy, which was analyzed with the persistent random walk model of motility. None of the factors tested had a remarkable effect on L132 cell motility, but the Calu-1 cell motility was significantly upregulated by endothelial cell extracellular matrix and by laminin, fibronectin, collagen I and collagen VI individually. Flow cytometry analysis revealed significantly higher expression levels of integrin subunits β1, α2, α3, and α6, which are known receptors for these extracellular matrix proteins, on the Calu-1 than L132 cells, implicating a role of these integrins in the observed motile behaviors of these cell lines.     

Neuroprotective Properties of Afobazol <Emphasis Type="Italic">in Vitro</Emphasis>

The effects of a novel selective anxiolytic afobazol on survival of HT-22 neurons were studied in the model of oxidative stress and glutamate toxicity. In both models, the neuroprotective effect of afobazol was established. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 140, No. 8, pp. 161–163, August, 2005     

Potassium Humate Inhibits Complement Activation and the Production of Inflammatory Cytokines <Emphasis Type="Italic">In Vitro</Emphasis>

The effects of brown coal derived potassium humate on lymphocyte proliferation, cytokine production and complement activation were investigated in vitro. Potassium humate increased lymphocyte proliferation of phytohaemaglutinin A (PHA) and pokeweed mitogen (PWM) stimulated mononuclear lymphocytes (MNL) in vitro from concentrations of 20 to 80 μg/ml, in a dose dependant manner. On the other hand potassium humate, at 40 μg/ml, significantly inhibited the release of TNF-α, IL-1β, IL-6 and IL-10 by PHA stimulated MNL. Regarding complement activation it was found that potassium humate inhibits the activation of both the alternative and classical pathways without affecting the stability of the red blood cell membranes. These results indicate that the anti-inflammatory potential of potassium humate could be partially due to the inhibition of pro-inflammatory cytokines responsible for the initiation of these reactions as well as inhibition of complement activation. The increased lymphocyte proliferation observed, might be due to increased IL-2 production as previously been documented.     

Cercarial shedding from <Emphasis Type="Italic">Galba</Emphasis><Emphasis Type="Italic">truncatula</Emphasis> infected with <Emphasis Type="Italic">Fasciola</Emphasis><Emphasis Type="Italic">gigantica</Emphasis> of distinct geographic origins

Galba truncatula snails were experimentally infected with either of two different isolates of Fasciola gigantica, originating from Egypt or China, to determine the influence of these isolates on the characteristics of snail infections. The survival rates of G. truncatula on day 30 post-exposure were 90.0% and 60.2% in the Egyptian and Chinese groups, respectively. The frequency of cercaria-shedding snails within the Egyptian group was 79.8%, whereas in the Chinese group it was 22.4%. The parasite origin had a significant effect on the durations of the prepatent and patent periods. The mean number of cercariae shed from the Egyptian group was significantly greater than that shed from the Chinese group (a mean of 275.5 per cercaria-shedding snail compared with 29.0). These results could be explained by the fact that G. truncatula might be a natural intermediate host for F. gigantica in Egypt, and the greater adaptability of the Egyptian miracidia of F. gigantica to unusual snail hosts. These results demonstrate the influence of the geographic origin of the parasite on the success of trematodes infecting snails.     

Cysteine proteinases from promastigotes of <Emphasis Type="Italic">Leishmania</Emphasis> (<Emphasis Type="Italic">Viannia</Emphasis>) <Emphasis Type="Italic">braziliensis</Emphasis>

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.     

Development of an <Emphasis Type="Italic">In Vitro</Emphasis> 3D Brain Tissue Model Mimicking <Emphasis Type="Italic">In Vivo</Emphasis>-Like Pro-inflammatory and Pro-oxidative Responses

To analyze complex inflammatory responses in an in vitro system, we constructed a new 3D in vitro brain tissue model that exhibits in vivo-like tissue responses (e.g. immune cell phenotypes, and molecular response) to inflammatory stimuli. Finite element modeling of oxygen diffusion and cellular oxygen consumption predicted the oxygen profile within 3D structures, consisting of Type I collagen hydrogel embedded with murine microglia. Viability and cytotoxicity analyses supported the mathematical analysis, determining optimal cell growth conditions for 3D construct development. Real-time RT-PCR and ELISA demonstrated significant up-regulation of pro-inflammatory mediators, such as TNF-α, MCP-1, IL-6 and IL-1β, in lipopolysaccharide (LPS)-stimulated in vitro cell culture (2D and 3D) and in vivo mouse model systems. Interestingly, levels of inflammatory responses from the in vitro 3D model system were more similar to in vivo than in vitro 2D. Additionally, in situ dihydroethidium (DHE) assay and immunofluorescence staining revealed that levels of LPS-stimulated reactive oxygen species (ROS) generation and microglial activation from in vitro 3D model system were closer to in vivo than in vitro 2D. These results demonstrated that an in vitro 3D model provides more physiologically relevant pro-oxidative and pro-inflammatory environments in brain than an in vitro 2D model.     

Expression of<Emphasis Type="Italic"> Tri15</Emphasis> in<Emphasis Type="Italic"> Fusarium sporotrichioides</Emphasis>

In the fungus Fusarium sporotrichioides, biosynthesis of trichothecene mycotoxins requires at least three genetic loci: a core 12-gene cluster, a smaller two-gene cluster, and a single-gene locus. Here, we describe the Tri15 gene, which represents a fourth locus involved in trichothecene biosynthesis. Tri15 is predicted to encode a Cys₂-His₂ zinc finger protein and is expressed in a manner similar to genes in the core trichothecene gene cluster. However, disruption of F. sporotrichioides Tri15 does not affect production of T-2 toxin, the major trichothecene produced by this fungus. This result suggests that Tri15 is not necessary for the production of toxin. Cultures with exogenously added T-2 toxin have high levels of Tri15 expression and no detectable expression of the trichothecene biosynthetic genes Tri5 and Tri6. The expression analysis is consistent with Tri15 being a negative regulator of at least some of the trichothecene biosynthetic genes. In F. graminearum, Tri15 has been mapped to linkage group 2 and is therefore unlinked to the main trichothecene biosynthetic gene cluster.Communicated by U. Kück     

Redescription of <Emphasis Type="Italic">Lemuricola</Emphasis> (<Emphasis Type="Italic">Madoxyuris</Emphasis>) <Emphasis Type="Italic">bauchoti</Emphasis> (Nematoda,Oxyuridae) from <Emphasis Type="Italic">Lemur catta</Emphasis> in Madagascar

Lemuricola (Madoxyuris) bauchoti Chabaud, Brygoo et Petter, 1965 is redescribed from material collected from the ring-tailed lemur, Lemur catta, from the Beza Mahafaly Special Reserve in Madagascar using the scanning electron microscope. This is a new host record and the first oxyurid reported from the ring-tailed lemur. Previously, records of each species of the subgenus Madoxyuris have been restricted to a single host species, but the close relationship between these nematodes and their Strepsirrhini hosts will only be proven when additional records fill in the gaps in their distribution.     

<Emphasis Type="Italic">In vivo</Emphasis> activity of <Emphasis Type="Italic">Sapindus saponaria</Emphasis> against azole-susceptible and -resistant human vaginal <Emphasis Type="Italic">Candida</Emphasis> species

Background  

Study of in vivo antifungal activity of the hydroalcoholic extract (HE) and n-BuOH extract (BUTE) of Sapindus saponaria against azole-susceptible and -resistant human vaginal Candida spp.     

Repeat induced point mutation in two asexual fungi, <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Penicillium </Emphasis><Emphasis Type="Italic">chrysogenum</Emphasis>

Repeat induced point mutation (RIP) is a gene silencing mechanism present in fungal genomes. During RIP, duplicated sequences are efficiently and irreversibly mutated by transitions from C:G to T:A. For the first time, we have identified traces of RIP in transposable elements of Aspergillus niger and Penicillium chrysogenum, two biotechnologically relevant fungi. We found that RIP in P. chrysogenum has affected a large set of sequences, which also contain other mutations. On the other hand, RIP in A. niger is limited to only few sequences, but literally all mutations are RIP-like. Surprisingly, RIP occurred only in transposon sequences that have disrupted open reading frames in A. niger, a phenomenon not yet reported for other fungi. In both fungal species, we identified two sequences with strong sequence similarity to Neurospora crassa RID. RID is a putative DNA methyltransferase and the only known enzyme involved in the RIP process. Our findings suggest that both A. niger and P. chrysogenum either had a sexual past or have a sexual potential. These findings have important implications for future strain development of these fungi.