Stefano Buttiglieri

Isolation of Renal Progenitor Cells from Adult Human Kidney

We describe here isolation and characterization of CD133+ cells derived from normal adult human kidney. These cells lacked the expression of hematopoietic markers and expressed PAX-2, an embryonic renal marker, suggesting their renal origin. Renal tissue-derived CD133+ cells and clones of individual cells were capable of expansion and limited self-renewal and differentiated in vitro into epithelial or endothelial cells. On subcutaneous implantation in SCID mice, the undifferentiated cells formed tubular structures expressing renal epithelial markers. At variance, when differentiated in endothelial cells, these cells formed functional vessels. On intravenous injection in SCID mice with glycerol-induced tubulonecrosis, the in vitro expanded renal-derived CD133+ cells homed into the injured kidney and integrated in tubules. We propose that CD133+ cells from kidney represent a multipotent adult resident stem cell population that may contribute to the repair of renal injury.

Isolation and Characterization of a Stem Cell Population from Adult Human Liver

Several studies suggested the presence of stem cells in the adult normal human liver; however, a population with stem cell properties has not yet been isolated. The purpose of the present study was to identify and characterize progenitor cells in normal adult human liver. By stringent conditions of liver cell cultures, we isolated and characterized a population of human liver stem cells (HLSCs). HLSCs expressed the mesenchymal stem cell markers CD29, CD73, CD44, and CD90 but not the hematopoietic stem cell markers CD34, CD45, CD117, and CD133. HLSCs were also positive for vimentin and nestin, a stem cell marker. The absence of staining for cytokeratin‐19, CD117, and CD34 indicated that HLSCs were not oval stem cells. In addition, HLSCs expressed albumin, α‐fetoprotein, and in a small percentage of cells, cytokeratin‐8 and cytokeratin‐18, indicating a partial commitment to hepatic cells. HLSCs differentiated in mature hepatocytes when cultured in the presence of hepatocyte growth factor and fibroblast growth factor 4, as indicated by the expression of functional cytochrome P450, albumin, and urea production. Under this condition, HLSCs downregulated α‐fetoprotein and expressed cytokeratin‐8 and cytokeratin‐18. HLSCs were also able to undergo osteogenic and endothelial differentiation when cultured in the appropriated differentiation media, but they did not undergo lipogenic differentiation. Moreover, HLSCs differentiated in insulin‐producing islet‐like structures. In vivo, HLSCs contributed to regeneration of the liver parenchyma in severe‐combined immunodeficient mice. In conclusion, we here identified a pluripotent progenitor population in adult human liver that could provide a basis for cell therapy strategies.

CD40-dependent Activation of Phosphatidylinositol 3-Kinase/Akt Pathway Mediates Endothelial Cell Survival and in Vitro Angiogenesis

CD40 has been involved in tumor and inflammatory neoangiogenesis. In this study we determined that stimulation of endothelial CD40 with sCD154 induced resistance to apoptosis andin vitro vessel-like formation by human microvascular endothelial cells (HMEC). These effects were determined to be mediated by CD40-dependent signaling because they were inhibited by a soluble CD40-muIg fusion protein. Moreover, apoptosis of HMEC was associated with an impairment of Akt phosphorylation, which was restored by stimulation with sCD154. The anti-apoptotic effect as well as in vitro vessel-like formation and Akt phosphorylation were inhibited by treatment of HMEC with two unrelated pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002. CD40 stimulation induced a rapid increase in Akt enzymatic activity that was not prevented by cycloheximide, an inhibitor of protein synthesis. The enhanced Akt activity induced by stimulation of endothelial CD40 was temporarily correlated with the association of CD40 with TRAF6, c-Cbl, and the p85 subunit of PI3K. Expression of negative-dominant Akt inhibited the activation of endogenous Akt through CD40 stimulation, despite the observation that association of CD40 with TRAF6, c-Cbl, and PI3K was intact. The defective activation of Akt abrogated not only the anti-apoptotic effect of CD40 stimulation but also the proliferative response, the enhanced motility, and the in vitro formation of vessel-like tubular structures by CD40-stimulated HMEC. In conclusion, these results suggest that endothelial CD40, through activation of the PI3K/Akt signaling pathway, regulates cell survival, proliferation, migration, and vessel-like structure formation, all steps considered critical for angiogenesis.

Prolactin in autoimmunity and antitumor defence

Prolactin (PRL) enhances inflammatory and antitumor responses in vitro and thus exhibits Th1-type cytokine-like effects. Evidence from experimental models indicates that inhibition of PRL release by bromocriptine downregulates immune reactions and ameliorates autoimmune diseases in which Th1 responses are predominant. A direct effect of locally produced PRL in some Th1 diseases, such as rheumatoid arthritis, supports this concept. Paradoxically, however, hyperprolactinemia can also be associated with conditions such as pregnancy, where remission of Th1-mediated diseases is known to occur in the context of a Th2-dominated milieu. This reversal of the Th1-promoting effect of PRL may be due to major changes in the levels of other hormones that can annul and/or override the PRL-mediated proinflammatory state. Nevertheless, PRL, as an immunopotentiating agent, may have a powerful therapeutic role in cancer and other immunocompromised patients.

Influence of drug-induced apoptotic death on processing and presentation of tumor antigens by dendritic cells.

Here we have studied the effects of apoptotic cell death induced by chemotherapic agents on tumor phagocytosis by dendritic cells (DC) and presentation of the relevant antigen to T lymphocytes. Annexin‐V‐FITC (Ann‐V) and propidium iodide (PI) staining was used to assess early apoptotic (Ann‐V+/PI−) vs. late apoptotic/secondary necrotic (Ann‐V+/PI+) death after a 24 hr observation of untreated and drug‐treated gastric carcinoma cells. After treatments, the HLA‐A*0201+ tumor cell line KATO III was exposed for 24 hr to allogeneic, HLA‐related GM‐CSF, IL‐4‐driven immature (i) DC. Tumor‐loaded iDC were tested for IL‐12 release in an ELISA assay, incubated with the DC‐maturating factor TNF‐α and used as stimulators for autologous T lymphocytes. Generation of antitumor T response against KATO cells was evaluated in an anti‐MHC class I MAb‐blocked Interferon‐γ ELISPOT assay. After treatment with Cis‐platin (cis), all dying cells were in early apoptosis, whereas secondary necrosis was the prevalent death pattern observed after epirubicin (epi) and doxorubicin (doxo). Doxo and epi increased tumor expression of heat shock protein (hsp) 70 and uptake of tumor cell components by DC, whereas cis treatment had no effect on hsp70 and was associated with poor tumor uptake by DC. Significant upmodulation of IL‐12 was observed by DC that had taken up the doxo‐ and epi‐treated tumors (p< 0.005 and p< 0.01, respectively). Increased IFN‐γ release was also observed after stimulation of T lymphocytes with DC loaded with doxo‐ and epi‐treated (p< 0.02 and p< 0.005, respectively) but not with cis‐treated DC. These data show that the products of early apoptosis cannot efficiently cross‐activate MHC class I‐restricted anti‐tumor lymphocytes even in the presence of DC maturating factors, whereas secondary necrosis is associated with robust T cell response.

PROLACTIN IS AN AUTOCRINE GROWTH FACTOR FOR THE JURKAT HUMAN T-LEUKEMIC CELL LINE

Despite convincing evidence of cooperation between IL-2 and endogenous prolactin (PRL) during T cell activation, the individual role of PRL as a T-cell lineage cytokine remains to be defined. We have examined the production and function of PRL on the Jurkat human T-leukemic cell line, which does not constitutively produce IL-2. The majority of Jurkat cells expressed PRL receptor (R) under standard culture conditions, whereas appearance of the alpha chain of the IL-2-R required PHA-PMA stimulation, as did IL-2 synthesis. Western blotting revealed a predominant band at 23.5 kDa and a weaker band at 25.5 kDa in both Jurkat cell lysates and human (h) pituitary PRL. Metabolic labeling of the cell lysates with 35S-methionine and immunoprecipitation with an antiserum against hPRL showed that both forms of PRL are actively synthesized by the Jurkat cell line. PRL released in the medium was biologically active in the rat Nb2 lymphoma mitogenic assay. Depletion of medium PRL with two polyclonal anti-hPRL antisera inhibited the growth of Jurkat cells in a dose-dependent manner, as evaluated by cell number and 3H-TdR uptake. Purified pituitary or recombinant hPRL at a wide range of concentrations had no significant effect on their growth, but reversed the blocking activity of the anti-hPRL antibody. Recombinant IL-2 had no effect on the antibody-induced growth inhibition. Taken as a whole, these results demonstrate that PRL can act as an autocrine T cell growth factor independently of IL-2 and are the first evidence of its involvement in human leukemic growth and possibly in leukemic transformation.

Minced Umbilical Cord Fragments as a Source of Cells for Orthopaedic Tissue Engineering: An In Vitro Study

A promising approach for musculoskeletal repair and regeneration is mesenchymal-stem-cell- (MSC-)based tissue engineering. The aim of the study was to apply a simple protocol based on mincing the umbilical cord (UC), without removing any blood vessels or using any enzymatic digestion, to rapidly obtain an adequate number of multipotent UC-MSCs. We obtained, at passage 1 (P1), a mean value of 4, 2 × 106 cells (SD 0,4) from each UC. At immunophenotypic characterization, cells were positive for CD73, CD90, CD105, CD44, CD29, and HLA-I and negative for CD34 and HLA-class II, with a subpopulation negative for both HLA-I and HLA-II. Newborn origin and multilineage potential toward bone, fat, cartilage, and muscle was demonstrated. Telomere length was similar to that of bone-marrow (BM) MSCs from young donors. The results suggest that simply collecting UC-MSCs at P1 from minced umbilical cord fragments allows to achieve a valuable population of cells suitable for orthopaedic tissue engineering.

Expression of prolactin and prolactin receptors by non-Hodgkin's lymphoma cells

Prolactin (PRL) interacts with lymphocyte‐signaling molecules and cytokines. Previous work has shown independent and synergistic effects of PRL on the generation of IL‐2‐driven anti‐tumor lymphokine activated killer (LAK) activity by peripheral blood mononuclear cells (PBMC). The potential importance of PRL as a biological immunomodifier, however, is challenged by its ability to influence normal lymphocyte mitogenesis and hence lymphoid tumor growth. Since non‐Hodgkins lymphoma (NHL) cell lines were efficiently killed by LAK generated with native (n) or recombinant (r) human PRL combined with low, per se ineffective doses of IL‐2, we have addressed here the question of whether PRL acts as a growth factor for LAK targets. NHL cells were analyzed for: 1. expression of the PRL receptor (PRL‐R); 2. responsiveness to nPRL or rPRL; 3. constitutive expression and release of PRL; 4. existence of a PRL autocrine loop. PRL‐R, defined by multiple antibodies, was detected in 3 of 12 NHL cell lines. However, nPRL or rPRL, in a wide range of concentrations (0.75–50 ng/ml), were not mitogenic for growth‐arrested, PRL‐R positive NHL cell lines. PRL mRNA was detected by RT‐PCR in 10 of the 12 cell lines examined with a higher frequency among AIDS‐related NHL cell lines. PRL protein in the immunoprecipitate of 35S‐methionine‐labeled cell lysates and supernatants paralleled mRNA expression, and Western blotting analysis showed the presence of the pituitary/lymphocyte non‐glycosylated (23.5 kDa) and glycosylated (25 kDa) isoforms. Experiments with blocking antibodies showed the independence from endogenous PRL for NHL cell growth. Int. J. Cancer 85:124–130, 2000.

Myeloablative doses of yttrium-90-ibritumomab tiuxetan and the risk of secondary myelodysplasia/acute myelogenous leukemia

Because the long‐term toxicity of myeloablative radioimmunotherapy remains a matter of concern, the authors evaluated the hematopoietic damage and incidence of secondary myelodysplastic syndrome and acute myelogenous leukemia (sMDS/AML) in patients who received myeloablative doses of the radiolabeled antibody yttrium‐90 (90Y)‐ibritumomab tiuxetan.

Introduction of a Novel Prototype Bioartificial Liver Support System Utilizing Small Human Hepatocytes in Rotary Culture

The aim of this study was to establish a stand-alone, perfused, rotary cell culture system using small human hepatocytes (SH) for bioartificial liver (BAL) support. SH were grown on cytodex 3 microcarriers (beads) to a maximum density of 1.2 +/- 0.3 x 10(7) cells per mL within 12 days. Size of aggregates formed by up to 15 beads was regulated by rotation speed. Cell function was proven by treatment with ammonia and galactose, and metabolism was analyzed. Treatment strategy was comprised of two phases, namely growth phase and treatment phase. Cells were grown for 6 days and subsequently incubated with ammonia or galactose for 2 days, followed by a 2-day regeneration period and another 2-day treatment phase. Consumption of glucose, release of lactate dehydrogenase, formation of lactate, and production of urea and albumin were determined regularly. Mean galactose consumption was 50 microg per 106 cells per hour, ammonia-induced urea formation was 3.6 microg per 106 cells per hour, and albumin production was 110 ng per 106 cells per hour. All metabolic parameters followed a logarithmic trend and were found to be very stable in the second half of the culture period when cells were treated with ammonia or galactose. Dissolved oxygen (%DO), pH, and temperature were monitored in-stream at intervals of 7 min, and the values were logged. Viability and morphology of cells were monitored via confocal microscopy. Viability was around 95% in controls and 90% during treatment. Promising results were obtained in support of our ongoing efforts to establish a fully autonomous BAL support device utilizing SH as a bridge to transplantation.