Endocytosis and intracellular localisation of type 1 ribosome-inactivating protein saporin-s6.

Saporin-S6 is a single-chain ribosome-inactivating protein (RIP) that has low toxicity in cells and animals. When the protein is bound to a carrier that facilitates cellular uptake, the protein becomes highly and selectively toxic to the cellular target of the carrier. Thus, saporin-S6 is one of the most widely used RIPs in the preparation of immunoconjugates for anti-cancer therapy. The endocytosis of saporin-S6 by the neoplastic HeLa cells and the subsequent intracellular trafficking were investigated by confocal microscopy that utilises indirect immunofluorescence analysis and transmission electron microscopy that utilises a direct assay with gold-conjugated saporin-S6 and an indirect immunoelectron microscopy assay. Our results indicate that saporin-S6 was taken up by cells mainly through receptor-independent endocytosis. Confocal microscopy analysis showed around 30% co-localisation of saporin-S6 with the endosomal compartment and less than 10% co-localisation with the Golgi apparatus. The pathway identified by the immunofluorescence assay and transmission electron microscopy displayed a progressive accumulation of saporin-S6 in perinuclear vesicular structures. The main findings of this work are the following: i) the nuclear localisation of saporin-S6 and ii) the presence of DNA gaps resulting from abasic sites in HeLa nuclei after intoxication with saporin-S6.

Dysfunctional vasa vasorum in diabetic peripheral artery obstructive disease with critical lower limb ischaemia.

OBJECTIVES AND DESIGN: To establish whether in diabetic patients with peripheral artery obstructive disease (PAOD) vasa vasorum (vv) neoangiogenesis is altered with increased arterial damage. MATERIALS: Thirty-three patients with PAOD and critical lower limb ischaemia, 22 with type II diabetes. METHODS: Immunohistochemistry for endothelial cell markers (CD34 and von Willebrand Factor); real-time reverse transcription polymerase chain reaction (RT-PCR) to quantify arterial wall expression of vascular endothelial growth factor (VEGF); enzyme-linked immunosorbent assay (ELISA) to assess blood VEGF; flow cytometry to detect circulating endothelial cells (CECs). RESULTS: Patients with PAOD and diabetes have a higher frequency (60% vs. 45%) of advanced atherosclerotic lesions and a significant reduction (p = 0.0003) in CD34(+) capillaries in the arterial media. Adventitial neoangiogenesis was increased equally (CD34(+) and vWF(+)) in all patients. Likewise, all patients have increased CEC and VEGF concentration in the blood as well as in-situ VEGF transcript expression. CONCLUSIONS: Patients with PAOD have remarkable arterial damage despite increased in-situ and circulating expression of the pro-angiogenic VEGF; a dysfunctional vv angiogenesis was seen in diabetics which also showed a higher frequency of parietal damage; it is suggested that in diabetic arterial wall, injury is worsened by vv inability to finalise an effective VEGF-driven arterial wall neoangiogenesis.

Angiogenic potential of human dental pulp stromal (stem) cells.

Dental pulp is a heterogeneous microenviroment where unipotent progenitor and pluripotent mesenchymal stem cells cohabit. In this study we investigated whether human dental pulp stromal (stem) cells (DP-SCs) committed to the angiogenic fate. DP-SCs showed the specific mesenchymal immunophenotypical profile positive for CD29, CD44, CD73, CD105, CD166 and negative for CD14, CD34, CD45, in accordance with that reported for bone marrow-derived SCs. The Oct-4 expression in DP-SCs, evaluated through RT-PCR analysis, increased in relation with the number of the passages in cell culture and decreased after angiogenic induction. In agreement with their multipotency, DP-SCs differentiated toward osteogenic and adipogenic commitments. In angiogenic experiments, differentiation of DP-SCs, through vascular endothelial growth factor (VEGF) induction, was evaluated by in vitro matrigel assay and by cytometric analysis. Accordingly, endothelial-specific markers like Flt-1 and KDR were basally expressed and they increased after exposure to VEGF together with the occurrence of ICAM-1 and von Willebrand factor positive cells. In addition, VEGF-induced DP-SCs maintained endothelial cell-like features when cultured in a 3-D fibrin mesh, displaying focal organization into capillary-like structures. The DP-SC angiogenic potential may prove a remarkable tool for novel approaches to developing tissue-engineered vascular grafts which are useful when vascularization of ischemic tissues is required.

Isolation of stem/progenitor cells from normal lung tissue of adult humans.

OBJECTIVES: This study aimed to isolate and characterize stem/progenitor cells, starting from normal airway epithelia, obtained from human adults. MATERIALS AND METHODS: Cultures of multicellular spheroids were obtained from human lung tissue specimens after mechanical and enzymatic digestion. Tissue-specific markers were detected on their cells by immunohistochemical and immunofluorescent techniques. Ultrastructural morphology of the spheroids (termed as bronchospheres) was evaluated by electron microscopy, gene expression analysis was performed by reverse transcription-polymerase chain reaction, and gene down-regulation was analysed by an RNA interference technique. RESULTS: Bronchospheres were found to be composed of cells with high expression of stem cell regulatory genes, which was not or was only weakly detectable in original tissues. Morphological analysis showed that bronchospheres were composed of mixed phenotype cells with type II alveolar and Clara cell features, highlighting their airway resident cell origin. In addition to displaying specific pulmonary and epithelial commitment, bronchospheres showed mesenchymal features. Silencing of the Slug gene, known to play a pivotal role in epithelial-mesenchymal transition processes and which was highly expressed in bronchospheres but not in original tissue, led bronchospheres to gain a differentiated bronchial/alveolar phenotype and to lose the stemness gene expression pattern. CONCLUSIONS: Ours is the first study to describe ex vivo expansion of stem/progenitor cells resident in human lung epithelia, and our results suggest that the epithelial-mesenchymal transition process, still active in a subset of airway cells, may regulate transit of stem/progenitor cells towards epithelial differentiation.

Mesenchymal stem cell interaction with a non-woven hyaluronan-based scaffold suitable for tissue repair.

The fabrication of biodegradable 3-D scaffolds enriched with multipotent stem cells seems to be a promising strategy for the repair of irreversibly injured tissues. The fine mechanisms of the interaction of rat mesenchymal stem cells (rMSCs) with a hyaluronan-based scaffold, i.e. HYAFF(R)11, were investigated to evaluate the potential clinical application of this kind of engineered construct. rMSCs were seeded (2 x 10(6) cells cm(-2)) on the scaffold, cultured up to 21 days and analysed using appropriate techniques. Light (LM), scanning (SEM) and transmission (TEM) electron microscopy of untreated scaffold samples showed that scaffolds have a highly porous structure and are composed of 15-microm-thick microfibres having a rough surface. As detected by trypan blue stain, cell adhesion was high at day 1. rMSCs were viable up to 14 days as shown by CFDA assay and proliferated steadily on the scaffold as revealed by MTT assay. LM showed rMSCs in the innermost portions of the scaffold at day 3. SEM revealed a subconfluent cell monolayer covering 40 +/- 10% of the scaffold surface at day 21. TEM of early culture showed rMSCs wrapping individual fibres with regularly spaced focal contacts, whereas confocal microscopy showed polarized expression of CD44 hyaluronan receptor; TEM of 14-day cultures evidenced fibronexus formation. Immunohistochemistry of 21-day cultures showed that fibronectin was the main matrix protein secreted in the extracellular space; decorin and versican were seen in the cell cytoplasm only and type IV collagen was minimally expressed. The expression of CD90, a marker of mesenchymal stemness, was found unaffected at the end of cell culture. Our results show that HYAFF(R)11 scaffolds support the adhesion, migration and proliferation of rMSCs, as well as the synthesis and delivery of extracellular matrix components under static culture conditions without any chemical induction. The high retention rate and viability of the seeded cells as well as their fine modality of interaction with the substrate suggest that such scaffolds could be potentially useful when wide tissue defects are to be repaired as in the case of cartilage repair, wound healing and large vessel replacement.

Fetal calf serum versus human serum: ultrastructural evaluation of protein support influence on human ovarian tissue cryopreservation.

The aim of this study was to examine the effect of the different protein supports in the cryopreservation solution on improving human ovarian tissue preservation after frozen-thawed procedures. Biopsies of ovarian cortical tissue were obtained from 14 subjects. All specimens were cryopreserved using a slow freezing/rapid thawing method in a solution consisting of propanediol and sucrose in different proportions of 3 protein supports: 30% human serum (HS) (solution A), 20% HS (solution B), or 20% fetal calf serum (solution C). After thawing, 191 follicles and a total of 70 samples were analyzed using transmission electron microscopy (TEM). The post-thaw preservation rate of the follicles in solution A was significantly higher with respect to solution C (p < 0.05). Unlike the follicles, the stromal cell morphology was not affected by any of the solutions investigated. By comparing stromal morphology and the patient age, it was found that HS better preserved the tissue in patients over 20 years of age with respect to younger ones, which showed a wider variability in ovarian preservation. TEM evaluation showed that 30% HS is more suitable for human ovarian tissue cryopreservation, and research should be focused on defining cryopreservation protocols specific to young patients.

Monochromatic ultraviolet light induced damage to Photosystem II efficiency and carbon fixation in the marine diatom Thalassiosira pseudonana (3H).

Low light adapted cultures of the marine diatom Thalassiosira pseudonana (3H) were cultured and incubated for 30 min under different ultraviolet (UV) wavelengths of near monochromatic light with and without background photosynthetically active radiation (PAR, 380-700 nm). Maximum damage to the quantum yield for stable charge separations was found in the UVB (280-320 nm) wavelengths without background PAR light while the damage under PAR was 30% less. UV induced damage to carbon fixation in the cells was described by a function similar to non-linear functions of inhibiting irradiance previously published with the exception that damage was slightly higher in the UVA (320-380). Various measurements of fluorescent transients were measured and the results indicate localised damage most likely on the acceptor side of the Photosystem II reaction center. However, dark adapted measurements of fluorescence transients with and without DCMU do not result in similar functions. This is also true for the relationships between fluorescence transients and carbon fixation for this species of marine diatom. The correlation between the weightings varepsilon (H) from measurements of carbon fixation and the quantum yield for stable charge separation as calculated from induction curves with DCMU and without DCMU is R (2) 0.44 and R (2) 0.78, respectively. The slopes of the two measurements are 3.8 and 1.4, respectively. The strong correlation between the weightings of the induction curves without DCMU and carbon fixation are due to a loss of electron transport from the reaction center to plastoquinone. Under these experimental conditions of constant photon flux density (PFD) this is manifested as a strong linear relationship between the decrease in the operational quantum yield of Photosystem II and carbon fixation.

Pharmacokinetics of furosemide in gestosis of pregnancy.

Furosemid 50 mg was administered orally and intravenously to twelve gestotic women for brief periods as a part of a randomized, multicentre clinical trial comparing the efficacy of bed rest and pharmacological treatment. The pharmacokinetic profile was investigated using a gas-liquid chromatographic technique. The plasma half-life after oral and intravenous administration was 115 +/- 37.1 and 71.8 +/- 26.3 min and plasma clearance was 153 +/- 48 and 152 +/- 23 ml/min, respectively (mean +/- SD). Comparative data from healthy pregnant women cannot be obtained for ethical reasons. The results show that gestosis has only a marginal if any effect on the kinetics of furosemide in comparison with published kinetic parameters in healthy volunteers, and patients with renal failure. The new-born babies where checked for side effects according to a protocol in use in a larger regional surveillance programme. No clinical side-effects were attributable to furosemide, but the small size of the group does not permit any definitive conclusions about this aspect.