Human hepatocytes in mice receiving pre-immune injection with human cord blood cells

It is well established that certain subpopulations of human adult stem cells can generate hepatocyte-like cells when transplanted into adult immunosuppressed mice. In the present study, we wanted to explore whether xeno-transplantation of human cord blood CD34(+) (hCBCD34(+)) cells during pre-immune stages of development in immunocompetent mice might also lead to human-mouse liver chimerism. Freshly isolated hCBCD34(+) cells were xeno-transplanted into non-immunosuppressed mice by both intra-blastocyst and intra-fetal injections. One and four weeks after birth, immunostaining for different human-specific hepatocyte markers: human hepatocyte-specific antigen, human serum albumin, and human alpha-1-antitrypsin indicated the presence of human hepatocyte-like cells in the livers of transplanted animals. Detection of human albumin mRNA further corroborated the development of pre-immune human-mouse chimeras. The current report, besides providing new evidence of the potential of hCBCD34(+) cells to generate human hepatocyte-like cells, suggests novel strategies for generating immunocompetent mice harboring humanized liver.     

Evaluation of MT expression and detection of apoptotic cells in LEC rat kidneys

To confirm our previous observations on the effectiveness of long term treatment with Zn on Long-Evans Cinnamon (LEC) rats, we extended these studies determining the effects of Zn on trace elements, metallothionein (MT) concentrations and immunolocalization, and on the levels of both MT-1 and MT-2 mRNAs in the LEC rat kidneys. We also localized the renal cells that had chromatin condensation and nuclear fragmentation typical of apoptosis. The results demonstrate that the amount of Zn increased in the treated rats with respect to both untreated and basal rats. In the treated rats the amount of Cu and Fe was similar to that of the basal rats. MT concentrations did not change either with or without Zn treatment, but were higher than the basal group. However, if we consider the percentage of oxidized MT (MTox), we note that Zn treatment is very effective in reducing this value. MTox is not able to bind metals, so it does not perform a "scavenger" function. Moreover, quantification of mRNA indicates that the MT-1 isoform was significantly higher than the MT-2 isoform following Zn treatment. Untreated group sections showed a confocal fluorescent signal that highlighted the irregular nuclei and small apoptotic bodies. The intensity and quantity of fluorescence decreased in the treated group sections. These findings suggest that, in LEC rats, Zn may contribute to cytoprotection through the regulation of MT expression which may provide a cellular defence strategy in response to DNA damage.     

Bioprocess-centered molecular design (BMD) for the efficient production of an interfacially active peptide

The efficient expression and purification of an interfacially active peptide (mLac21) was achieved by using bioprocess-centered molecular design (BMD), wherein key bioprocess considerations are addressed during the initial molecular biology work. The 21 amino acid mLac21 peptide sequence is derived from the lac repressor protein and is shown to have high affinity for the oil-water interface, causing a substantial reduction in interfacial tension following adsorption. The DNA coding for the peptide sequence was cloned into a modified pET-31(b) vector to permit the expression of mLac21 as a fusion to ketosteroid isomerase (KSI). Rational iterative molecular design, taking into account the need for a scaleable bioprocess flowsheet, led to a simple and efficient bioprocess yielding mLac21 at 86% purity following ion exchange chromatography (and >98% following chromatographic polishing). This case study demonstrates that it is possible to produce acceptably pure peptide for potential commodity applications using common scaleable bioprocess unit operations. Moreover, it is shown that BMD is a powerful strategy that can be deployed to reduce bioseparation complexity.     

Skeletal muscle differentiation potential of human adult bone marrow cells

Murine bone marrow (BM) cells have been shown to undergo myogenic differentiation and participate in muscle repair in different muscle regeneration models. In the present paper, we report on a subset of cells (CD45+/desmin+) with myogenic potential being present at very low frequencies in human adult BM. By a simple culture method, we were able to obtain in vitro multinucleated myotubes in up to 20% of the cultures. Myotubes were generated using both BM flushed from rib fragments obtained during thoracotomy and BM derived from iliac crest aspirates. Cells of the different adherent and non-adherent fractions expressed numerous muscle specific markers by immunocytochemistry, real-time RT-PCR, flow cytometry, and Western blot analyses. Moreover, direct injection of whole BM into the right tibialis anterior muscle of immunodeficient mice (NOD/RAG) that had previously been treated with cardiotoxin to induce muscle degeneration, showed a variable but significant level of human cell engraftment (from 0.06 to 0.26% Dys+/FISH+ fibers). These data suggest that cells with skeletal muscle differentiation potential are present in adult human BM can differentiate in vitro and give rise to myogenic cells in vivo in immunodeficient mice after muscle damage. Further improvements might allow new approaches to cell-mediated therapies for muscular diseases.     

The epigenome network of excellence

An initiative funded by the European Union is building a collaborative network of established and younger research groups to tackle key questions in epigenetics.     

Characterization of depolarization and repolarization phases of mitochondrial membrane potential fluctuations induced by tetramethylrhodamine methyl ester photoactivation

Depolarization and repolarization phases (D and R phases, respectively) of mitochondrial potential fluctuations induced by photoactivation of the fluorescent probe tetramethylrhodamine methyl ester (TMRM) were analyzed separately and investigated using specific inhibitors and substrates. The frequency of R phases was significantly inhibited by oligomycin and aurovertin (mitochondrial ATP synthase inhibitors), rotenone (mitochondrial complex I inhibitor) and iodoacetic acid (inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase). Succinic acid (mitochondrial complex II substrate, given in the permeable form of dimethyl ester) abolished the rotenone-induced inhibition of R phases. Taken together, these findings indicate that the activity of both respiratory chain and ATP synthase were required for the recovery of the mitochondrial potential. The frequency of D phases prevailed over that of R phases in all experimental conditions, resulting in a progressive depolarization of mitochondria accompanied by NAD(P)H oxidation and Ca2+ influx. D phases were not blocked by cyclosporin A (inhibitor of the permeability transition pore) or o-phenyl-EGTA (a Ca2+ chelator), suggesting that the permeability transition pore was not involved in mitochondrial potential fluctuations.     

New basic discoveries and frontiers in diagnosis, prognosis and prediction of response to therapy in human thyroid, urinary bladder, and prostate tumors

The fourth edition of this workshop mainly focused on three different human oncotypes, which included thyroid, urinary bladder, and prostate tumors as clinical models to gain new basic knowledge on tumor diagnosis, prognosis, and treatment. At the previous editions (Giordano et al., 2000, J Cell Physiol 183:284-287; Giordano et al., 2001, J Cell Physiol 188:274-280; Giordano et al., 2002, J Cell Physiol 191:362-365), leaders in the fields of pathology, clinical oncology, and basic research presented and discussed the most recent and prevalent findings in such neoplasms from a basic and clinical perspective. A concept that has been widely proposed is that the analysis of intrinsic biological factors displayed by primary tumors may be a valid method for diagnosing different neoplasias and for measuring both their aggressiveness and response to therapy. To date, however, no single prognostic factor, such as oncogenes, suppressor genes, or genes involved in the control of the cell cycle and/or apoptosis has yet proven to be potent enough to be used in clinical practice as a prognostic and predictive factor. The new possibility to simultaneously analyze the expression of the complete repertoire of human genes and a large number of proteins could offer a new scenario in tumor classification, allowing for the formulation of a list of genes able to define a "signature" of tumor outcome. Moreover, starting from data obtained from biomolecular tumor analyses, it has been demonstrated that with this approach, it is also possible to design future therapeutic strategies.     

Placental uptake and transport of ACP, a neutral nonmetabolizable amino acid, in an ovine model of fetal growth restriction

Reductions in fetal plasma concentrations of certain amino acids and reduced amino acid transport in vesicle studies suggest impaired placental amino acid transport in human fetal growth restriction (FGR). In the present study, we tested the hypothesis of an impairment in amino acid transport in the ovine model of hyperthermia-induced FGR by determining transplacental and placental retention and total placental clearance of a branched-chain amino acid (BCAA) analog, the nonmetabolizable neutral amino acid aminocyclopentane-1-carboxylic acid (ACP), in singleton control (C) and FGR pregnancies at 135 days gestation age (dGA; term 147 dGA). At study, based on the severity of the placental dysfunction, FGR fetuses were allocated to severe (sFGR, n = 6) and moderate FGR (mFGR, n = 4) groups. Fetal (C, 3,801.91 +/- 156.83; mFGR, 2,911.33 +/- 181.35; sFGR, 1,795.99 +/- 238.85 g; P < 0.05) and placental weights (C, 414.38 +/- 38.35; mFGR, 306.23 +/- 32.41; sFGR, 165.64 +/- 28.25 g; P < 0.05) were reduced. Transplacental and total placental clearances of ACP per 100 g placenta were significantly reduced in the sFGR but not in the mFGR group, whereas placental retention clearances were unaltered. These data indicate that both entry of ACP into the placenta and movement from the placenta into fetal circulation are impaired in severe ovine FGR and support the hypothesis of impaired placental BCAA transport in severe human FGR.     

Animal resources and subsistence strategies

Several faunal assemblages excavated in deposits of different antiquity (from Lower Paleolithic to Bronze Age), located in Northern, Central and Southern Italy, were studied from the archeozoological and taphonomic point of view. Data obtained by different Authors allow reconstruction of subsistence strategies adopted by prehistoric humans in these areas and through time, in particular as far as the exploitation of animal resources is concerned. The following assemblages were considered: Isernia La Pineta (Molise; Lower Paleolithic), Grotta Breuil (Latium; Middle Paleolithic), Grotta della Ghiacciaia (Verona; Middle Paleolithic), Riparo di Fumane and Riparo Tagliente (Verona; Middle and Upper Paleolithic), Riparo Mochi (Liguria; Upper Paleolithic), Grotta della Continenza (L'Aquila; Upper Paleolithic and Mesolithic), Grotta dell'Edera (Trieste; Mesolithic and Neolithic), Grotta della Cala at Marina di Camerota (Salerno; Eneolithic), Contraguda (Sassari; Neolithic), Castellaro Lagusello (Mantova; Bronze Age). Exploitation of the vegetal resources has been analyzed in the Neolithic sites of Colle Santo Stefano (Fucino), Settefonti (L'Aquila) and Catignano (Pescara).