Poloxamines for deswelling of organ-cultured corneas.

BACKGROUND: Poloxamines are amphiphilic tetrofunctional block copolymers composed of four polyoxyethylene-polyoxypropylene arms joined to a central ethylene diamine bridge. Their safe profile allows diverse pharmaceutical and biomedical applications. AIM: To assess their use for corneal deswelling using a porcine model of organ culture (OC). METHODS: Five poloxamines (T90R4, T904, T908, T1107 and T1307) were dissolved in a standard commercial OC medium (control) to reach 350 mosm kg(-1). In vitro cytotoxicity was tested using MTT assay on human corneal epithelial and endothelial cell (EC) lines and on primary human corneal fibroblasts. Paired porcine corneas stored in OC for 3 days were assigned for 48 h to a poloxamine medium or to a standard deswelling medium containing 5% dextran T500. Corneal EC density, morphometry, mortality, stromal thickness and transparency were evaluated before and after deswelling. Post-deswelling, EC viability/mortality was determined using a fluorescent live/dead assay. RESULTS: Besides similar corneal thickness reduction and transparency improvement, T908, T1107 and T1307 decreased EC loss (5.4 ± 1.7% vs. 9.9 ± 2.6% in controls (p < 0.001)) and mortality, improved EC morphometry and reduced endothelial lesions compared to dextran. CONCLUSION: On this porcine model, poloxamines T908, T1107 and T1307 appear as good candidates to replace dextran for the deswelling. Experiments on human corneas are now necessary to confirm their efficiency and safety profile in OC.

Multivariate classification and modeling in surface water pollution estimation.

The present study deals with the application of self-organizing maps (SOM) and multiway principal-components analysis to classify, model, and interpret a large monitoring data set for surface water quality. The chemometric methods applied made it possible to reveal specific quality patterns of the chemical and biological parameters used to monitor the water quality (relation between water temperature, turbidity, hardness, colibacteria), seasonal impacts during the long period of observation and the relative independence on the spatial location of the sampling sites (water supply sources for the City of Trieste).

Levels of cadmium and zinc in hepatopancreas of reared Mytilus galloprovincialis from the Gulf of Trieste (Italy).

Cadmium and zinc concentrations were determined in hepatopancreatic tissues of Mytilus galloprovincialis, reared in three non-contaminated hatcheries of the Gulf of Trieste (Northern Adriatic Sea). Mussels were collected in 13 sampling exits with monthly frequency for verifying possible seasonal variations. Freeze dried hepatopancreatic tissues were dissolved by nitric acid procedure using a microwave system; analysis of Zn and Cd followed by AAS technique. Metal concentrations were below the critical values for mollusc soft tissues, but xenobiotic cadmium had values requiring monitoring attention. Cadmium and zinc showed moderate variations along the months, with a slight winter maximum followed by a summer pre-spawning minimum, matching the seasonal trends of temperature/salinity. The estuarine site appears to have the best quality of the examined area in terms of metal contents. The results can constitute a guideline for the water quality in the Northern Adriatic Sea, and permit comparisons with the quality of other areas of the Mediterranean Sea.

Case study of groundwater pollution in a critical area of the Southern-Friuli exposed to agricultural and landfill pressures.

Groundwater of the Southern-Friuli displays high levels of agricultural pollutants, such as nitrates and triazinic herbicides not only in the surficial layers, but also in the deeper ones, below 150 m. Some wells of the district of Gonars was monitored. The examined waters, used for irrigation but also for drinkable use, are exposed to environmental risk due to both agricultural practices and presence of many waste disposal sites. Heavy metals, nitrates and triazinic herbicides were measured in samples taken at four wells in three periods having different rain conditions. We found that groundwater quality is affected mainly by agricultural practices: nitrates and triazines are present at levels very near as well as superior to the maximum concentration allowable by Italian law. These agricultural contaminants have similar levels at all sampled sites: no difference was detected between dry periods and rain ones. Heavy metal contents are negligible in all cases; this fact suggests that ion-exchange, sorbing and complexing properties of the soils hinder the way of the metal leachates towards underlying groundwater. Zinc constitutes an exception; it is found at levels near or superior to the maximum allowable concentration (CMA), and the highest contents are observed in rain periods; different sites display different zinc levels, suggesting that this metal could have various point sources. Nitrates fertilisers were found in all sites at similar levels, very near to CMA (50 mg/L). Triazines are specific herbicides for corn growing, highly diffused here: their use in recent years is forbidden by Italian law, but the presence in groundwater of parent triazines and metabolites is a persistent problem of this area. The Italian law indicates a CMA of 0.10 microgram/L for the sum of atrazine and desethylatrazine, but we found that desethylatrazine by itself exceeds largely CMA in all sites.

Expression and activity of caspases 1 and 3 in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are characterized by abnormal growth of committed progenitors in clonogenic assay, with reduced number of colonies and decreased colony/cluster ratio. It has been suggested that excessive apoptosis is the cause of marrow failure in MDS. We studied the expression of caspase-1 (interleukin-1beta-converting enzyme, ICE) and caspase-3 (CPP32/apopain) in marrow mononuclear cells, and the growth pattern of committed progenitors in a series of 83 MDS cases. The percentage of apoptotic cells as detected by TUNEL technique, and the percentage of caspase-3-positive cells were significantly higher in refractory anemia (RA) and RA with ringed sideroblasts (RAS) than in chronic myelomonocytic leukemia (CMML), refractory anemia with excess of blasts (RAEB) and RAEB in transformation (RAEB-T). Spontaneous growth of CFU-GM was associated with a higher percentage of blasts, and with a lower expression of caspase-3 and caspase-1. The yield of CFU-E, BFU-E, and CFU-GM (in the presence of growth factors) was decreased by comparison to normal marrow, but large individual differences were observed in all cytological categories. Inhibition of caspase-1 and caspase-3 activities by specific inhibitors resulted in a significant increase of the production of all types of colonies (up to 50-fold of control). In the presence of caspase-3 inhibitor, the number of BFU-E and CFU-E was in the range of normal values in most cases of RA and RAS. In addition, caspase-1 and -3 protease activities were detectable by fluorogenic assay in all cases studied. Western blot analysis confirmed the expression of caspase-3, including the cleaved (activated)-p17 form in most cases of RA/RAS analyzed. It is concluded that caspase-3 is implicated in the increased apoptosis observed in MDS and that inhibition of its activity can restore at least partially the growth of committed progenitors.

Detecting and characterising sources of persistent organic pollutants (PAHs and PCBs) in surface sediments of an industrialized area (harbour of Trieste, northern Adriatic Sea).

A sediment sampling based on a two-dimensional mapping was performed in the harbour of Trieste (northern Adriatic Sea), considering 28 sites exposed to pollutant inputs from harbour and industrial activities. Polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) were determined in surface sediments, because these very persistent pollutants seem to be responsible for the depletion of benthic populations observed in this area. The correlation matrix indicates that PAHs and PCBs are non-correlated, and probably have different sources. Both cluster analysis performed on the sampling sites and graphical drawing of the PAH sediment contents make it possible to locate along the shoreline a band of more polluted sediments, clustered around a site facing a steelmaking factory, to be considered as the main source point for PAHs. The evaluation of phenanthrene to anthracene (P/AN) and fluoranthene to pyrene (FL/PY) ratios permits the assessment of the pyrolytic, industrial origin of these PAHs, rejecting a second possible source of hydrocarbons (i.e., an oil-pipeline terminal, situated near the steelmaking factory). Graphical drawing of the total PCB iso-concentrations reveals a different source-point for this other category of very persistent pollutants.

Selection of BCR/ABL-negative stem cells from marrow or blood of patients with chronic myeloid leukemia.

Philadelphia (Ph) or BCR/ABL-negative cells with immature phenotype (CD34-positive, DR-negative) can be recovered from patients with chronic myeloid leukemia (CML) in chronic phase. We used the technique described by Berardi et al (Science 1995; 267: 104-108) to select stem cells from marrow or blood of CML patients at diagnosis or during treatment with alpha-interferon. Mononuclear cells (MNC), and in some experiments CD34+ cells, were maintained for 7 days in the presence of 5-fluorouracil (5-FU), stem cell factor and interleukin-3. The number of viable cells recovered after culture was between 7.4 and 70.2 for 10(6) cells plated. These cells exhibited the following phenotype: CD34+, CD117+, CD38-, lineage-, and were able to generate cobblestone areas and secondary colonies in long-term culture (LTC), with a frequency similar to that of cells selected from normal marrow. Study by fluorescence in situ hybridization of LTC cells or secondary colonies showed no evidence of BCR/ABL rearrangement. Reverse transcriptase polymerase chain reaction studies on pooled LTC cells or secondary colonies were also negative. By contrast, LTC cells or secondary colonies obtained from CML CD34+ cells without culture in the presence of 5-FU were always positive for BCR/ABL rearrangement. Finally, 5-FU selected cells were able to engraft NOD/SCID mouse, as human cells were detected in blood and marrow 10 weeks post transplantation, which were BCR/ABL negative by RT-PCR. This method of culture makes it possible to select constantly BCR/ABL-negative cells with capacities of development in LTC assay and of NOD/SCID mouse engraftment.