Features, reason for testing, and changes with time of 583 paroxysmal nocturnal hemoglobinuria clones from 529 patients: a multicenter Italian study.

In this study, we aimed at disclosing the main features of paroxysmal nocturnal hemoglobinuria (PNH) clones, their association with presentation syndromes, and their changes during follow-up. A large-scale, cooperative collection (583 clones from 529 patients) of flow cytometric and clinical data was entered into a national repository. Reason for testing guidelines were provided to the 41 participating laboratories, which followed the 2010 technical recommendations for PNH testing by Borowitz. Subsequently, the 30 second-level laboratories adopted the 2012 guidelines for high-resolution PNH testing, both upon order by the local clinicians and as an independent laboratory initiative in selected cases. Type3 and Type2 PNH clones (total and partial absence of glycosyl-phosphatidyl-inositol-anchor, respectively) were simultaneously present in 54 patients. In these patients, Type3 component was sevenfold larger than Type2 (p < 0.001). Frequency distribution analysis of solitary Type3 clone size (N = 442) evidenced two discrete patterns: small (20% of peripheral neutrophils) and large (> 70%) clones. The first pattern was significantly associated with bone marrow failure and myelodysplastic syndromes, the second one with hemolysis, hemoglobinuria, and thrombosis. Pediatric patients (N = 34) showed significant preponderance of small clones and bone marrow failure. The majority of PNH clones involved neutrophils, monocytes, and erythrocytes. Nevertheless, we found clones made exclusively by white cells (N = 13) or erythrocytes (N = 3). Rare cases showed clonal white cells restricted only to monocytes (6 cases) or neutrophils (3 cases). Retesting over 1-year follow-up in 151 cases showed a marked clone size increase in 4 cases and a decrease in 13, demonstrating that early breaking-down of PNH clones is not a rare event (8.6% of cases). This collaborative nationwide study demonstrates a clear-cut difference in size between Type2 and Type3 clones, emphasizes the existence of just two classes of PNH presentations based on Type3 clone size, depicts an asymmetric cellular composition of PNH clones, and documents the possible occurrence of changes in clone size during the follow-up.

Adrenomedullin and endothelin-1 stimulate in vitro expansion of cord blood hematopoietic stem cells.

The improvement of techniques for in vitro expansion of cord blood (CB) hematopoietic stem cells (SCs) is, at present, one main task of tissue engineering. Hence, we investigated whether endothelin-1 (ET-1) and adrenomedullin (AM), two regulatory peptides exerting growth promoting action on several cell systems, favor the in vitro expansion of CB SCs in liquid culture. CB hematopoietic cell middle-term expansion was carried out in a stroma-free liquid culture medium in the presence of ET-1, AM and three different cytokine combinations. After two weeks of incubation, aliquots of expanded-cell suspension were seeded on semisolid medium and clonogenic tests were carried out by counting the number of colony forming units (CFUs) after 14 days of culture. Neither ET-1 nor AM (2.5 x 10(-8) M) were per se able to significantly increase the CFU number, but both peptides magnified the pro-expansive effects of some cytokine cocktails. In light of these findings, we conclude that ET-1 and AM are to be considered novel promising molecules that, in association with cytokines, can be utilized as pro-expansive factors of CB SCs in prevision of their clinical use in allogeneic transplantation.

New experimental models for the in vitro reconstitution of human bladder mucosa.

This study describes two experimental models for the in vitro reconstitution of the human bladder mucosa (neo-bladder): human urothelial stabilized cell lines were cultured on three-dimensional matrices, collagen or platelet-fibrin gels, containing murine fibroblast 3T3-J2 cells. Low-density seeding (2x10(4) cells/ml) of both normal (TCA-48) and neoplastic cell lines (TCA-47) on collagen matrix gave rise to isolated papillar colonies, while high-density seeding (3.75x10(6) cells/ml) led to the formation of wide pluristratified epithelial sheets, resembling the normal transitional epithelium. In contrast, high-density seeding (5x10(5) cells/ml) on platelet-fibrin matrix did not allow the formation of epithelial sheets: only isolated voluminous colonies of normal TCA-48 cells, and sparse and small colonies of neoplastic TCA-47 could be observed. Growth assays and cytotoxicity reduction tests showed that the growth inhibitory effect of platelet-fibrin gel on urothelial cells was probably due to the aspecific activation of the complement contained in the plasmatic fraction, whose precipitation forms fibrin-glue. Collectively, these findings allow us to draw the following conclusions: i) neobladders obtained by culturing urothelial cells on collagen matrix reproduce normal bladder mucosa and could be utilized in pharmacological studies; and ii) platelet-fibrin gels, that specifically inhibit neoplastic urothelial cell growth, could be used as scaffolds in surgical bladder reconstitution.

Effect of cryopreservation on in vitro clonal growth of cordonal blood cells.

Cordonal blood (CB) is today recognized as a potentially important source of hematopoietic stem cells (SCs) for allogeneic transplantation, and to this task it would be of extreme importance to have the possibility of using cryopreserved CB units. Hence we investigated whether freezing and thawing alter the viability of CB hematopoietic cells. Mononuclear cells, recovered from fresh CB units by density-gradient centrifugation, were partly frozen and then thawed and partly immediately utilized for clonogenic tests. The cells were cultured in H4330 (C group) or H4434 (H4330 added with SC clonogenic growth factors) (C+ group) semisolid media added or not with serum-free Dulbecco modified Eagle medium (DMEM/sf). Cells seeded on H4330 were exposed to the conditioned supernatants from two human-embryo liver cell lines, which have been previously found to stimulate clonal growth of fresh CB hematopoietic cells. As expected, the number of colony-forming units (CFU) was higher in C+ than C group, and was not influenced by the addition of DMEM/sf. CFU number was higher in cryopreserved than fresh cells in both C and C+ culture groups. Conditioned supernatants from both cell lines stimulated clonal growth in both fresh and cryopreserved cell cultures. These findings indicate that cryopreservation and thawing do not alter the viability of CB SCs, but, on the contrary, improve their basal and cytokine-stimulated clonal growth, probably by negatively selecting SCs among the mononuclear CB cell population.

Adrenomedullin is expressed in cord blood hematopoietic cells and stimulates their clonal growth.

Adrenomedullin (AM) is a hypotensive peptide, which originates from the proteolytic cleavage of pro(p)AM and acts via AM22-52-sensitive receptors. Reverse transcription polymerase chain reaction allowed the detection of the specific mRNAs of pAM in the mononuclear hematopoietic cells of the cord blood and immunocytochemistry demonstrated their abundant AM-immunoreactivity. AM (10(-8) M) markedly enhanced clonal growth of cord blood hematopoietic cells cultured on semisolid media added with stem cell-growth promoting cytokines, and this effect was abolished by AM22-52 (10(-6) M). Collectively, these findings indicate that AM is expressed in and stimulates the proliferation of cord blood hematopoietic stem cells. Cord blood has been proposed as a source of stem cells alternative to bone marrow for allogeneic transplantation, and our study suggests that AM may be used, in addition to the classic cytokines, to expand in vitro cord blood stem cells in advance of their clinical use.

New human embryo liver cell lines obtained by stabilization and immortalization enhance in vitro clonal growth of cordonal blood cells.

We developed two new cell lines derived from embryo liver and tested their inductive capacity on in vitro clonal growth of cordonal blood (CB) hematopoietic cells. One line was stabilized and named BAEP2-WILD (W), and the other one was immortalized by retroviral transduction with SV40 Large T antigen and called BAEP2-SV40. Southern blot analysis demonstrated the integration of the Large T antigen gene in the BAEP2-SV40 cell genome, but this line did not display the expected growth arrest at the non-permissive temperature of 39 degrees C. Immunocytochemistry showed that BAEP2-SV40 cell line was positive for several cytokeratins and stromal markers (vimentin, desmin and laminin), as well as for epidermal growth factor (EGF), fibroblast growth factor (FGF) and their receptors (Rs). In contrast, BAEP2-W evidenced positivity only for cytokeratin-7 and laminin, and low positivity to EGF, EGF-R, FGF and FGF-R. BAEP2-SV40 cell line, but not BAEP2-W, expressed interleukin (IL)-1, IL-6, granulocyte macrophage-colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor, stem cell factor and vascular-cell adhesion molecule-1 mRNAs, and secreted IL-6 and GM-CSF. Taken together, these findings could suggest that BAEP2-W cell line possesses the phenotype of fetal hepatocytes, while BAEP2-SV40 cell line has that of stromal cells. The supernatants conditioned by both cell lines stimulated the clonal growth of CB hematopoietic cells cultured on semisolid media deprived of growth factors and cytokines, the inductive capacity of the BAEP2-SV40 cell line being markedly higher than that of its wild counterpart, conceivably due to its ability to produce cytokines. Our study indicates that these two new cell lines, and especially BAEP2-SV40 one could be used in co-culture systems as feeder-layers for hematopoietic CB SC expansion in vitro.