Autologous CD133+ cells augment the effect of portal embolization.

AIM: The standard to treat liver tumors is a resection. When the future liver remnant (FLRV) is below 30% (healthy livers) or 40% (cirrhotic livers or previous chemotherapy), surgery carries the risk of severe complications. Portal vein embolization (PVE) gained a worldwide diffusion as a tool to augment the FLRV. Cell therapies are recent players at the frontiers of medicine. This study presents a clinical experience to evaluate the synergistic effect of combined PVE and autologous CD133+ cells coadministration. METHODS: Sixteen patients have been enrolled in the study up today. Inclusion criteria were: primary or metastatic liver malignancy with a FLRV<30% or 40%. A baseline volumetric CT-scan was obtained. CD34+ were mobilized to the blood stream by G-CSF administration and collected by immunomagnetic separation. Simultaneously with PVE, cells were administered to the non occluded liver segments. Follow-up CT scans were taken at 30th post treatment day. RESULTS: The patients (N.=6) showed an increased volume gain (Mann-Whitney test P<0.001, two sided) compared to a set of cases whose treatment was PVE only (N.=10). DISCUSSION: The use of autologous stem cells as an augmenter of liver regeneration has a clinical potential to improve the resectability of liver tumors.

Assessment of the causes of Hg bioaccumulation in the fish of a Mediterranean lagoon subject to environmental management interventions.

In a Mediterranean lagoon characterized by high levels of Hg in the sediment, Hg content in commercial fish species was determined, and, in order to establish Hg concentration in the water column, Diffusive Gradient in Thin films technique (DGT) was used. The results allowed to state (1) the disturbance of the surface sediment and its resuspension in the water column did not cause detectable increase in Hg releases, (2) the East basin acted more than the West one in contamination of fish species, (3) the small-sized sea-breams (<400 g) were more contaminated than larger sizes ones (>400 g). We concluded: (1) fish contamination may also depend on direct contact with releases of the contaminant and small, gregarious and less sedentary subjects are more likely to incur releases of Hg; (2) Hg releases can be greater in areas with a relatively low sedimentary organic matter load compared to areas subjected to high density macroalgal mat, regardless of the amount of Hg present in the sediments; (3) wind transport of plant masses in low energy storage areas, may constitute an increase factor of Hg in the sediment. A critical review of the results was made in comparison with the available literature reports and some hypotheses were proposed regarding the possible dynamics and availability of the contaminant.

Enhanced levels of oxidized low-density lipoprotein prime monocytes to cytokine overproduction via upregulation of CD14 and toll-like receptor 4 in unstable angina.

OBJECTIVES: The purpose of this study was to establish whether oxidized low-density lipoprotein (oxLDL) contributes to cytokine overproduction via upregulation of CD14 and toll-like receptor-4 (TLR-4) expression on circulating monocytes of unstable angina (UA) patients. METHODS AND RESULTS: Expression of CD14 and TLR-4 on circulating monocytes, and the concentration of plasma oxLDL, (interleukin [IL])-6, IL-1 beta, IL-8, tumor necrosis factor (TNF)-alpha, monocyte chemoattractant protein-1 (MCP-1) were measured in 27 control (C) subjects, 29 patients with stable angina (SA), and 27 with UA. CD14 and TLR-4 expression on monocytes and circulating IL-6, IL-1 beta, and oxLDL were higher in UA than in SA and C subjects (P<0.001). In in vitro experiments, oxLDL increased CD14 and TLR-4 expression (P<0.001) in control monocytes as well as IL-6, IL-1 beta, and at a lower extent TNF-alpha and MCP-1 levels in the supernatant (P from <0.05 to <0.001). The preincubation of sera derived from UA patients but with control monocytes also induced a significant increase of CD14 and TLR-4 expression (P<0.001) and of IL-6 and IL-1 beta production (P<0.001) in the supernatant. CONCLUSIONS: In UA patients oxLDL may contribute to monocyte overproduction of some cytokines by upregulating CD14 and TLR-4 expression.

Calcium and polyphosphoinositides: their distribution in relation to the membrane changes occurring in the head of boar spermatozoa.

Ejaculated boar spermatozoa, previously incubated in a rigorously Ca++-free medium, were exposed to Ca++ for different incubation times and processed for the detection of Ca++ localization by a pyroantimonate technique. The distribution of polyphosphoinositides, anionic phospholipids natural constituents of membrane known to bind Ca++, was investigated using a specific cytochemical probe, i.e., neomycin conjugated with horseradish peroxidase. The in situ localizations thus obtained revealed: short exposure to Ca++ ions (10 min) evocated a Ca++-induced release of calcium from the nonmitochondrial intracellular store, i.e., the outer acrosomal membrane; a more prolonged exposure (20 min) triggered the occurrence of fusional and exocytotic events, that appeared to be morphologically related to the acrosome reaction; the outer acrosomal membrane, which is the fusigenic sperm membrane, was the elective site of the neomycin/peroxidase labeling. When assayed for the presence of a phospholipase C-like activity, the detergent extract obtained from boar spermatozoa exhibited substantial amount of p-nitrophenyl-phosphorylcholine hydrolyzing activity. The results, on the whole, allow us to suggest a relationship between Ca++ and polyphosphoinositides turnover in the events triggering the acrosome reaction, the exocytotic process peculiar to mammalian spermatozoa.

Identification of sulfurtransferase enzymes in Azotobacter vinelandii.

Rhodanese and 3-mercaptopyruvate sulphurtransferase have been identified in A. vinelandii. Two distinct active fractions of the two sulphur transferases were obtained after FPLC ion-exchange chromatography of material partially purified from crude extracts. Rhodanese has been purified to homogeneity, and it consists of one polypeptide chain of Mr ca 25,000. A partial purification of 3-mercaptopyruvate sulphurtransferase was obtained.

A new human growth hormone production process using a recombinant Bacillus subtilis strain.

We constructed a series of hybrid plasmids which directed the synthesis of different human growth hormone (hGH) precursor sequences in Bacillus subtilis. In addition to the 191 amino acids of the hormone, the precursors had in common an amino-terminal extension characterized by the presence of a methionine at position 1 and of the tetrapeptide Ile-Glu-Gly-Arg preceding the first residue (Phe) of hGH. The sequence between the methionine and the tetrapeptide was specific for each precursor and, because of the presence of charged residues, conferred particular properties to the molecules. Long homopolymeric tail-containing precursors such as MRRRRRRIILM-IEGR appeared insoluble whereas shorter sequences of the type MRR-IEGR and MEELM-IEGR augmented the solubility of the precursors with respect to Met-hGH. The soluble precursors could be easily purified from the bulk proteins taking advantage of the charged residues present on the N-terminal tail. After purification, the natural hGH was obtained by treating the precursors with the protease Factor Xa which cleaves after the arginine residue of the tetrapeptide IEGR. A protocol for the production and purification of authentic hGH from a strain expressing one of these soluble precursors is reported.

Cytogenetic monitoring of fishermen with environmental mercury exposure.

Due to high mercury levels in many Mediterranean aquatic organisms, people who live in this area and consume large amounts of seafood are exposed to a toxicological hazard. A group of 51 fishermen exposed to mercury through eating contaminated seafood from the northern Tyrrhenian Sea underwent cytogenetic monitoring. This work is part of a research project consisting of the evaluation of micronuclei (MN), chromosomal aberrations (CA) and sister-chromatid exchanges (SCE) in peripheral blood lymphocytes. Here we present data on mercury levels in blood and on micronucleus frequencies in peripheral blood lymphocytes of fishermen. The range of mercury concentrations in blood was 10.08-304.11 ng/g fresh weight, the average was 88.97 +/- 54.09 ng/g. Micronucleus frequency was defined with at least 2000 binucleated cells scored for each person; the average was 8.74 +/- 2.56 expressed on 1000 binucleated cells. A statistical correlation was found between MN frequency and total mercury concentration in blood (p = 0.00041, r = 0.674), as well as between MN frequency and age (p = 0.017). No other parameters taken into account correlated with MN frequency.

Evidence that a Ca2+ chelator and a calmodulin blocker interfere with the structure of inter-Sertoli junctions.

Ca2+ dependence of tight junction structure has been well documented in cultured epithelial tissues, and regulatory mechanisms have been identified. To analyse the possible control exerted on inter-Sertoli junctions, we exposed guinea-pig seminiferous tubules to the presence of a Ca2+ chelator (EGTA) and to a calmodulin blocker (Trifluoperazine, TFP) in vitro, for times ranging from 30 to 120 min. We observed the morphology of junctional complexes and the basal cytoplasmic regions in sections and replicas. Sertoli cell response to Ca2+ depletion involved several events: retraction of cells toward the base of the tubule and a consequent stretching of the points of fusion, augmented density of the cytoplasm, and destabilization of the array of intramembrane particles. Exposure of tubules to TFP resulted in disruption of the interactions between actin filaments and membrane junctional specialization, as well as a disorganization of other cytoskeletal elements. Thus, in vitro, junction integrity appears to be related to Ca2+ level, and Ca2+ depletion apparently interferes with Ca2+ distribution inside the cell and on microfilaments involved in junction regulation. Our results do not provide direct evidence for any particular mechanism of action of TFP, but a multiple effect is evident. TFP, which affects Ca2+ regulation and membrane fluidity, probably acts indirectly on junction-associated filaments. Both the experimental conditions tested suggest a Ca2+-mediated regulatory role of microfilaments of this complex junction.

Polychlorinated biphenyl congeners, hexachlorobenzene and DDTs in human milk in central Italy.

The concentration of organochlorine pesticides (hexachlorobenzene, p,p'-DDT and p,p'-DDE) and polychlorinated biphenyl (PCB) congeners in human milk from 56 donors in the hospitals of two Italian towns (Certaldo and Grosseto) are reported. The highest levels found were those of p,p'-DDE, which reached a mean of 2276 ng g-1 at Certaldo and 2270 ng g-1 at Grosseto, p,p'-DDT levels were about an order of magnitude less, with means of 350 and 298 ng g-1, respectively. The DDE/DDT ratio was approximately 8, which is much higher than that found in previous studies. Mean total PCBs were 451 and 295 ng g-1, respectively. About 70% of the PCB residues in the sample analysed consisted of only three congeners: 2,2',4,4',5,5' (IUPAC No. 153), 2,2',3,4,4',5' (138) and 2,2',3,4,4',5,5' (180).

Biomarker responses at different levels of biological organisation in crabs (Carcinus aestuarii) experimentally exposed to benzo(alpha)pyrene.

The aim of this study was to validate a multi-trial biomarker approach for the evaluation of toxicological risk due to benzo(alpha)pyrene. Carcinus aestuarii, exposed to increasing concentrations of B(alpha)P in the water, was used as the bioindicator organism. A set of biomarkers were tested in order to: identify biological materials for biomarker and residue analysis; determine a group of sensitive techniques for the assessment of PAH contamination; investigate correlation between responses at different levels of biological organisation. The results underlined that BPMO activities in hepatopancreas and gills were a good biomarker of exposure to PAH-type compounds. B esterases activities in hemolymph and porphyrin patterns in excreta could be proposed as a non-destructive approach for evaluating chemical exposure in this species.

[Critical evaluation of the causes of death in thalassemic subjects]. / Valutazione critica delle cause di morte nei soggetti talassemici.

We discuss 25 cases of death observed from 1971 to 1983 in a casistic of 155 patients with Thalassaemic Syndrome. Anemia as a cause of death is disappearing, new triggers are involved such as Yersinia Enterocolitica who can cause severe sepsis even in non-splenectomized patients. Iron overload appears to be the most severe complication in the second decade.

[Preliminary data on the endocrinologic evaluation of thalassemic adolescents]. / Dati preliminari sulla valutazione endocrinologica di adolescenti talassemici.

Endocrine disfunction are well known in young patients with thalassaemia major and are mainly due to iron overload. Gonadotrophin releasing hormone (GnRH) was administered to 13 boys aging more than 14 years and to 13 girls aging more than 13 years to assess pituitary function. Human Chorionic gonadotrophin (HCG) was also administered to 13 boys to evaluate their gonadal function. Most of the patients had evidence of pituitary hypofunction and in 4 boys there was evidence of gonadic failure. This study is mandatory for a correct therapeutic approach.

Arsenic-resistant bacteria isolated from contaminated sediments of the Orbetello Lagoon, Italy, and their characterization.

AIMS: The aim of this study was to isolate arsenic-resistant bacteria from contaminated sediment of the Orbetello Lagoon, Italy, to characterize isolates for As(III), As(V), heavy metals resistance, and from the phylogenetic point of view. METHODS AND RESULTS: Enrichment cultures were carried out in the presence of 6.75 mmol l(-1) of As(III), allowing isolation of ten bacterial strains. Four isolates, ORAs1, ORAs2, ORAs5 and ORAs6, showed minimum inhibitory concentration values equal or superior to 16.68 mmol l(-1) and 133.47 mmol l(-1) in the presence of As(III) and As(V), respectively. Isolate ORAs2 showed values of 1.8 mmol l(-1) in the presence of Cd(II) and 7.7 mmol l(-1) of Zn(II), and isolate ORAs1 pointed out a value of 8.0 mmol l(-1) in the presence of Cu(II). Analysis of 16S rRNA gene sequences revealed that they can be grouped in the three genera Aeromonas, Bacillus and Pseudomonas. Phylogenetic analysis of the four more arsenic-resistant strains was also performed. CONCLUSION: Isolates are highly resistant to both As(III) and As(V) and they could represent good candidates for bioremediation processes of native polluted sediments. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides original results on levels of resistance to arsenic and to assigning genera of bacterial strains isolated from arsenic-polluted sediments.

Morphological evidence for calcium stores at Sertoli-Sertoli and Sertoli-spermatid interrelations.

Potassium pyroantimonate technique has been employed to localize calcium ultrastructurally at Sertoli-Sertoli and Sertoli-spermatid junctional specializations. Identification of Ca++ as the major cation precipitated was performed by EGTA sensitivity and X-ray microprobe analysis. Ca++ deposits have been demonstrated in the endoplasmic reticulum cisternae underlying junctional complexes and along the plasma membranes of both Sertoli and germ cells.

Structure and possible functional role of septate junctions in the ovotestis of a pond snail: inter-Sertoli junctions.

Junctions between Sertoli cells, which form a continuous layer at the periphery of the 'acinus' in the ovotestis of Biomphalaria glabrata (mollusc gastropod), have been studied with conventional sections, lanthanum impregnation, replicas and scanning analysis of unstained sections. Extensive undulating septate junctions link Sertoli cells, which surround the various stages of spermatid differentiation. The morphology of the pleated septate junctions here presented indicates a possible role as a permeability barrier, which might result from the peculiar organization of the septa. The junctional complex is formed of areas where septa are parallel and regularly spaced, alternating with areas where septa separate and assemble with others, thus creating a complex system of channels, completely compatible with the sealing properties expected for these junctions. The morphology of septa, their possible anchorage within the membranes, and the interconnections linking the closely parallel septa are presented.

Ultrastructure of acrosomal malformations in men with obstructive azoospermia.

Testicular biopsies from ten infertile men with obstructive azoospermia were evaluated by light and electron microscopy. Light microscopic analysis revealed normal testicular pattern with active spermatogenesis and many spermatozoa. At ultrastructural level the majority of early spermatids presented acrosomal abnormalities that become evident during the approach of the acrosomal vesicle to the anterior pole of the nucleus, and in subsequent maturational stages; these spermatids continue their development until mature spermatozoa with malformed acrosomes. The type of malformation was very similar in all cases observed and was associated with the same type of seminiferous tubule pattern in patients with the same type of azoospermia. The abnormalities at acrosomal level were related to an abnormal differentiation rather than to degenerative changes. Acrosomal disturbance did not influence nucleus morphology or chromatin condensation.

Testicular biopsy of secretory azoospermia: electron and light microscopic analysis.

The histological and ultrastructural features of testicular cells were examined in testes with secretory azoospermia in seven patients with germinal cell arrest, two with Sertoli-cell-only; and one with tubular hyalinization. Germinal cell arrest was characterized by the presence of spermatogonia AD, AP, B, and some primary spermatocytes: these cells had similar ultrastructural features typical of the adult normal testis. Sertoli cells contained large number of various lipid inclusions and lipofuscin bodies. The multilayered peritubular wall presented increased collagen fibers. The cytoplasm of Leydig cells was filled with dilated vesicles of smooth endoplasmic reticulum and precursors of Reinke crystalloids. A blockage in the seminiferous tubules occurred after puberty, when tubular components, peritubular wall and Leydig cells had reached maturity.

Permeability to lanthanum of blood testis barrier in human germinal aplasia.

The permeability of Sertoli tight junctions to lanthanum administrated during fixation is demonstrated in biopsies of patients with partial germinal aplasia. In freeze-fracture replicas the number of fibrils is not significantly different from the data obtained in normal testis. Thus, in these pathological conditions junctional permeability is not related solely to the complexity of the network revealed by freeze-fracture.

Mercury levels along the food chain and risk for exposed populations.

Mercury was not regarded as a pollutant of primary importance until many deaths due to mercury poisoning occurred in the 1950s. More recently, adverse health effects have been documented at relatively low exposure levels, and monitoring data must now be interpreted in this light. The Mediterranean basin has been studied in great detail over the past 20 years because of the anomalous natural presence of mercury. Marine animals of this basin have higher mercury body burdens than the same (or similar) species in the Atlantic. The mercury found in marine organisms is mainly in the form of methyl mercury. Long-term and frequent intake of seafood with high mercury levels by populations living in coastal fishing villages is associated with a toxic risk, especially in pregnant women. High blood and hair concentrations of mercury have repeatedly been found in fishermen of Tyrrhenian coastal villages. In some cases these concentrations have been associated with an increase in DNA damage in blood cells. High mercury levels in hair and blood of people from a fishing village of Madeira have also been found. This information deserves renewed scrutiny with regard to preventive efforts needed.

Ca++ localization in boar spermatozoa by the pyroantimonate technique and X-ray microanalysis.

Intracellular, loosely bound Ca++ has been localized electron microscopically in freshly ejaculated boar spermatozoa by in situ precipitation with potassium antimonate. Ca++ was identified as the cation precipitated by testing the EGTA-sensitivity of the precipitates and by X-ray microprobe analysis. The data obtained revealed that the outer acrosomal membrane is the preferential site for Ca++ precipitation in the sperm head.