Preclinical ex vivo expansion of peripheral blood CD34+ selected cells from cancer patients mobilized with combination chemotherapy and granulocyte colony-stimulating factor.

BACKGROUND AND OBJECTIVES: Ex vivo peripheral blood progenitor cell (PBPC) expansion has been proposed as a strategy to increase the number of haematopoietic progenitors available for cell transplantation. We have expanded CD34+ cells from PBPCs obtained from four patients with haematological malignancies and one patient with an Ewing's sarcoma. MATERIALS AND METHODS: Cells were expanded in the Dideco 'Pluricell system'. After 12 days in culture, we evaluated cell phenotype, total nucleated cells, CD34+ fold increase, cell apoptosis and colony assay of expanded cells. Cell engraftment has been evaluated by transplanting two groups of irradiated non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice with expanded and non-expanded cell populations. RESULTS: Total nucleated cells and CD34+ cells increased 59.5 and 4.0 times, respectively. The expanded cells were mainly constituted of myeloid and megakaryocytic cells. A significant increase in the number of colony-forming unit-granulocyte macrophage (CFU-GM) was observed in the CFU assay. Ten mice transplanted with expanded cells showed a best overall survival (80%) compared to 10 mice transplanted with non-expanded cells (20%). Human CD45+ cells were detected by flow cytometry and polymerase chain reaction in bone marrow and spleen of transplanted animals. The relative low engraftment level obtained with the expanded cells suggests a loss of SCID repopulating cells maybe due to cell differentiation during expansion. CONCLUSIONS: We have demonstrated the feasibility of the ex vivo expansion of mobilized PBPCs from cancer patients, evidencing a clonal expansion of CFUs and the ability of the expanded cells to engraft the bone marrow and spleen of immunosuppressed mice. The differentiation of the CD34+ stem cell compartment could be further minimized by ameliorating the expansion conditions.

Associations of the plasma interleukin 6 (IL-6) levels with disability and mortality in the elderly in the Treviso Longeva (Trelong) study.

IL-6 expression is regulated by the interplay of several transcriptional and hormonal factors, including sex steroids and glucocorticoids. In late life IL-6 expression increases as a result from loss of the normally inhibiting sex steroids. IL-6 is one of several proinflammatory cytokines. It has been proposed that many chronic inflammatory diseases are the result of a dysregulation of IL-6 expression. In this work we demonstrate that increased IL-6 levels in elderly are associated with higher disability and mortality, also independently of age and comorbidity.

The Treviso Longeva (Trelong) study: a biomedical, demographic, economic and social investigation on people 70 years and over in a typical town of North-East of Italy.

Longevity is a complex process resulting from genetic and environmental factors, as well as their interaction. These factors are poorly understood, and the comparison among health status, socio-economics, demographic and other characteristics of the elderly people can help in understanding these complex interactions. Such an interdisciplinary approach is necessary to allow an appropriate evaluation of longevity. Here we report the methodology and the first results of a representative study performed in 2003-2004 on people of 70 years and over, living in a typical town of North-East of Italy. In the research we collected biomedical, demographic, socio-economic and quality of life (QoL) data.

Alterations in erythrocyte morphology induced by blood pumps.

A scanning electron microscopy was used after in vitro and in vivo tests to investigate any alterations caused by the peristaltic roller pump in erythrocyte morphology. The electron micrographs of samples were examined as follows: 1) by image analyser; 2) by applying Bessis's classification for the qualitative study of crenated red blood cells (RBCs). The in vitro test was repeated four times using blood from healthy donors. Each basal blood sample was divided into 250 ml portions, each of which was recirculated for 12 minutes at different flow rates. In order to verify any persistent erythrocyte damage caused by the peristaltic pump, 15 minutes after recirculation at 450 ml/min, another sample was prepared using the blood remaining from the last test. A statistically significant direct correlation was found between blood flow (Qb) increase and the percentage of morphologically altered RBCs, when either using an image analyser (r = 0.97; p < 0.05) or Bessis's classification (r = 0.95; p < 0.05). However, neither method showed any statistically significant difference between the percentage of deformed RBCs, determined in the basal sample, or in the percentage found at the end of the 450 ml/min test after standing 15 minutes at room temperature. The in vivo test was carried out on 6 patients over 2 dialysis sessions, which differed only for the Qb: 250 versus 400 ml/min. The two dialysis sessions gave comparable results when using both study methods regarding the presence of deformed RBCs.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological features and kinetics of primary cultures of BPH tissue supported by TV1 medium.

222 human prostatic biopsies were used to prepare cell cultures by means of a medium--colony formation permissive--containing fetal calf serum, called TV1. After 7, 14 and 21 days, the cultures were examined by optical and scanning electron microscopy. TV1 medium induces the formation and growth of two types of colonies, one mainly composed of epithelioid cells and distinguished by early growth; the second one made up exclusively of fibroblastoid cells which appear later in the culture. Epithelioid colonies, comprising three different cell types, appear to be arranged as a growth halo concentric to the bioptic fragment with a large central area, formed by a monolayer, and a pluristratified edge. Fibroblastoid cells weakly adhere to the substrate and form "satellite growth halos" separated from the primitive bioptic fragment. All the epithelioid cells were positive to cytokeratin LP34 Mab and negative to anti-smooth muscle-actin and anti-proline-4-hydroxylase antibodies. Fibroblastoid cells were only anti-proline-4-hydroxylase positive. The cell kinetics of epithelioid cells were also studied, revealing an extension of the S phase, in contrast to what happened with WAJC 404, and consequently a reduction of the percentage of cells entering mitosis. For this reason, the addition of fetal serum to the culture medium does not allow the use of prostate primary cultures for more than 14 days.

Relationship between the proliferation of keratinocytes cultured in vitro and prostaglandin E2.

In the growth of keratinocytes "in vitro", PGE2 seems to play an important role. We have shown that in fibroblast-keratinocyte co-cultures, indomethacin, employed at concentrations which inhibit the PGE2 synthesis, reduced the proliferation of epidermal cells. This effect was reversed by an exogenous PGE2 addition to the culture media. To better understand the relationship between keratinocytes and the autacoid, we have tested PGE2 at various concentrations in different cultural conditions, that is, epidermal cells were grown on a 3T3-J2 feeder layer, without fibroblasts and with a 3T3-J2 conditioned medium. We observed an increase in keratinocyte proliferation induced by the autacoid alone in the presence of fibroblasts, while a severe inhibitory effect was relieved when dermal cells or the conditioning medium were absent. The lack of fibroblasts and their products in the culture medium modified the morphology of keratinocytes cultured in vitro. PGE2 induced significant morphological and morphometrical variations only if added to the conditioning medium. The autacoid decreased the expression of 66 kDa protein, if cells were grown in the presence of fibroblasts or with conditioning medium, whereas it completely inhibited this keratin and those of 60, 54 kDa if cells were cultured only with a basal medium. From morphometrical and electrophoretical data we can suppose that PGE2 inhibits cell differentiation. Thus PGE2 action on keratinocytes seems to be strictly related to the presence of dermal cells.

Production of epidermal growth factor in human prostatic cells cultured in vitro.

The Epidermal Growth Factor (EGF) plays an important role in the regulation of in vitro growth of prostate cells inducing a strong mitogenic effect. Nevertheless in our previous study we observed that the treatment of human hypertrophic prostate cell line U285 with exogenous EGF produces a restricted effect on the cellular growth rate. This phenomenon could be due to the capacity of the cells to produce EGF. In this study we aimed to verify this hypothesis by evaluating the presence of mRNA of EGF and EGF receptor (EGF-R) and of their translation products in U285 cells, before and after the treatment with suramin and exogenous EGF. Moreover we studied the effects exerted by these substances on the proliferative rate of the cells U285 after different treatment protocols. The presence in the cells of mRNA for EGF and EGF-R and of their translation products was demonstrated by means of reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemical methods respectively. The modification of growth rate induced by these drugs was studied by FRAME Cytotoxicity Test. The operative modalities adopted to carry out these growth assays tended to 1) focus the effects of suramin in relation to in vitro cellular growth phase; 2) verify the reversibility of its effects; 3) ascertain if it was possible to antagonize the action of suramin by adding exogenous EGF. The results obtained from the RT-PCR showed the presence, in the control cells and in the treated ones, of mRNA coding for EGF and EGF-R. The immunocytochemical analysis indicated that 20% of the control cells are EGF positive, and 83% are EGF-R positive, confirming the results obtained with RT-PCR. Moreover, these stainings showed that the treatment with EGF does not significantly modify the percentage of cells marked by the anti-EGF antibody, while treatments with suramin and suramin plus EGF double this percentage. None of the treatments modifies the percentage of EGF-R positive cells. The growth assays showed that the exposition to highest doses of suramin in the first 24 h of cultures causes a decrease (p < 0.05) of the cellular proliferation during the following 48 h and 72 h and that these effects are irreversible. Moreover, a contemporaneous exposition of the cells to EGF and suramin at seeding strengthens the cytotoxic action of the last drug. To sum up, the demonstration of the presence in the U285 cells of mRNA coding for EGF and EGF-R and of the corresponding proteins, confirms the hypothesis that these cells can produce EGF. Moreover, the cytotoxicity experiments allowed a focusing of the role of the endogenous EGF in the regulation of the U285 cells proliferation and confirmed the importance of biological events that take place in U285 cells during the first 24 h of culture.

Primary cultures of human hypertrophic prostate tissue in WAJC 404 medium: a study of cell morphology and kinetics.

222 biopsy fragments of human hypertrophic prostate tissue were cultured in WAJC 404 serum-free medium for three weeks. Growth halos were examined after 7, 14 and 21 days of culture by optical and scanning electron microscopy. Colonies formed of two concentric areas showed in the inner halo elementary pseudo-lobular morphological units similar to the prostate structure. The cell morphological patterns of the halo turned out to be four in number. Every cell pattern was defined morphologically, morphometrically and phenotypically. Results indicate that all morphological differences must be attributed to the various phases of cell life, as all cell types were positive to cytokeratin. The nonconstant display of PSAP and PSA showed a moderate tendency to cell differentiation in WAJC 404 medium. Cell kinetics were also studied and revealed a decrease in proliferation after 14 days of culture. Primary cultures from biopsy fragments of human hypertrophic prostate tissue may be used as an experimental model up to the 14th day of culture.

Effects of DHT and EGF on human hyperplastic prostate cells cultured in vitro: growth, morphology and phenotype characterisation.

This work studies the effects of dihydrotestosterone (DHT) and epidermal growth factor (EGF) on the growth, morphology and phenotype characterisation of the U285 line obtained from human prostate hyperplastic tissue. Modifications of growth rate induced by these two substances have been evaluated by means of the neutral red assay formulated by Borenfreund and Puerner (1985) as well as by means of Kenacid blue assay described by Knox et al. (1986), culturing cells for 24, 48 and 72 hr with scalar doses of DHT (0.5, 1, 2, 5, 10 microM) and EGF (5, 10, 20, 100 ng/ml). An optical microscope connected to a computer aided system and a scanning electron microscope were used to monitor morphological changes induced by DHT and EGF. The immunophenotype characterisation of the treated and control cells was carried out by using a monoclonal antibody panel. Our results show that the expression of anti-cytokeratin 5+6+18, anti-cytokeratin 8+18+19 and anti-proline-4-hydroxylase antibodies varied in relation to the type of treatment undergone by the cells. Moreover, exogenous DHT does provoke a flattening of the U285 cells without modifying their rate of growth, while EGF both shortens the lag phase reactivating the quiescent cells and regulates the subsequent log growth phase, thus causing no cellular overgrowth.

[Effects of mepartricin on the growth of hypertrophic prostatic tissue in primary culture. Creation of the experimental model and preliminary results]. / Gli effetti della mepartricina sulla crescita del tessuto prostatico ipertrofico in coltura primaria. Creazione del modello sperimentale e risultati preliminari.

The aim of this study was to standardise a method for the in vitro culture of hypertrophic prostatic tissue, to assay the morphological type of isolated cells, to evaluate the degree of proliferation and to identify their differentiated iter. In addition, the effects of mepartricina on growth was also studied obtaining preliminary results. Of a total of 40 biopsies used in this experiment, 30 were placed directly in culture using Freshney's method, whereas the remaining 10 were used in a method involving the primary culture of the dispersed cells obtained from enzymatically disaggregated tissue. In vitro proliferation was analysed using optic microscopy, histological and histochemical techniques, and a scanning electron microscope. Cellular kinetics were also studied by bromodeoxyuridine marking. Using these tests it was found that it is possible to obtain the development and growth in this form of culture of fibroblastic-type colonies of epithelial origin characterised by different morphologies and growth curves. In addition, cells from the epithelioid colonies are characterised by a high level of proliferative activity and a low degree of differentiation. Mepartricina appears, on the other hand, to inhibit the in vitro growth of hypertrophic prostatic tissue.

[Multitest evaluation of the immune status of patients with bronchogenic carcinoma before and after radiotherapy]. / Valutazione con multitest dello stato immunitario nei pazienti con carcinoma broncogeno prima e dopo la radioterapia.

Sixty-two patients with lung cancer underwent the multitest before and after radiotherapy so as to assess the initial immune state and modifications induced by radiation therapy. In cancer patients, a significantly smaller number of positive skin reactions were encountered than in the controls. No statistically significant differences emerged between patients grouped on the basis of histotype, clinical stage and performance or otherwise of surgery. In living patients, higher values were observed than in patients who died. After radiotherapy, multitest values underwent a very slight decrease.

Middle-term expansion of hematopoietic cord blood cells with new human stromal cell line feeder-layers and different cytokine cocktails.

Cord blood (CB) is a source of hematopoietic stem cells (HSCs) and is an alternative to bone marrow for allogenic transplantation in patients with hematological disorders. The improvement of HSC in vitro expansion is one of the main challenges in cell therapy. Stromal components and soluble factors, such as cytokines, can be useful to induce in vitro cell expansion. Hence, we investigated whether feeder-layers from new stromal cell lines and different exogenous cytokine cocktails induce HSC expansion in middle-term cultures. CB HSC middle-term expansion was carried out in co-cultures on different feeder-layers exposed to three different cytokine cocktails. CB HSC expansion was also carried out in stroma-free cultures in the presence of different cytokine cocktails. Clonogenic tests were performed, and cell growth levels were evaluated. Moreover, the presence of VCAM-1 mRNA was assessed, and the mesenchymal cell-like phenotype expression was detected. All feeder-layers were able to induce a significant clonogenic growth with respect to the control culture, and all of the cytokine cocktails induced a significant increase in CB cell expansion indexes, even though no potential variation dependent on their composition was noted. The modulative effects of the different cocktails, exerted on each cell line used, was dependent on their composition. Finally, all cell lines were positive for CD73, CD117 and CD309, similar to mesenchymal stem cells present in adult bone marrow and in other human tissues, and negative for the hematopoietic markers. These data indicate that our cell lines have, not only a stromal cell-like phenotype, but also a mesenchymal cell-like phenotype, and they have the potential to support in vitro expansion of CB HSCs. Moreover, exogenous cytokines can be used in synergism with feeder-layers to improve the expansion levels of CB HSCs in preparation for their clinical use in allogenic transplantation.

Growth, morphology, and morphometry of human hypertrophic prostate cells treated with suramin in vitro.

This work studies the effects of suramin on the growth and morphology of cell strain U285, obtained from human prostate hypertrophic tissue and cultured in vitro. The FRAME cytotoxicity test was performed to evaluate the inhibition of growth induced by suramin. Cells were exposed to suramin at the time of seeding and 24 hours later; neutral red was added with and without suramin. An optical microscope connected to a computer-aided system and a scanning electron microscope were used to study morphological changes induced by suramin. Growth inhibition depends on drug concentration and exposure period. Moreover, the effect of suramin on neutral red uptake is reversible. Suramin 1,000 microM causes the cells to become spheroid, and they fail to form a monolayer. Our data indicate that the addition of suramin during the lag phase decreases the rate of cell proliferation.

Cellular pharmacology of idarubicinol in multidrug-resistant LoVo cell lines.

Idarubicin (IDA) is a daunorubicin (DAU) analog that is being used to treat a variety of human malignancies. The major circulating metabolite of IDA is idarubicinol (IDOL). After administration of IDA to patients, the systemic exposure to IDOL is greater than IDA. We investigated the cytotoxic effect of IDOL in the LoVo human colon-carcinoma cell line and its derivative multidrug-resistant (MDR) sub-lines. In LoVo-sensitive cells, the extracellular IDOL concentration inhibiting cell growth by 50% (IC50) was about 2-fold higher than IDA IC50 but lower than DAU IC50. After continuous exposure of the LoVo parental cells to 20 nM IDOL, 5 drug-resistant clones were obtained. All these clones exhibited an MDR phenotype, indicating that IDOL is involved in multidrug resistance. The resistance index (RI) to IDOL was investigated in LoVo MDR sub-lines obtained by IDOL (LoVo-IDOL-1), IDA (LoVo-IDA-1) and DOX (LoVo-DOX-1) selection. In spite of the drug used for their selection, all the MDR sub-lines exhibited an RI to IDOL lower than DAU and only 2-fold higher than IDA. In LoVo-IDOL-1 cells the RI was 5, 11 and 32 for IDA, IDOL and DAU respectively. Differences in the RI were explained by the greater intracellular tolerance exhibited by MDR cells to DAU than to IDOL and IDA. In the LoVo-IDOL-1 sub-line, the intracellular drug concentration inhibiting cell growth by 50% (IC50int) was higher than in the sensitive cells by 11.4-, 4.7- and 2.8-fold for DAU, IDOL, and IDA respectively. Differences in the intracellular tolerance were explained by the different intracellular distribution of DAU compared with IDOL and IDA. While DAU had a higher nuclear location in LoVo-sensitive cells than in resistant cells, IDOL and IDA maintained the same distribution both in sensitive and in resistant cells. In conclusion, contrary to what has been observed for other derivative metabolites of anthracyclines, the metabolism of IDA to IDOL must not be considered an inactivation pathway. IDOL is a potent inhibitor of cell growth and retains good activity in MDR cells.

[Effects of radiotherapy on lymphocyte populations in lung cancer]. / Effetti della radioterapia sulle popolazioni linfocitarie nel cancro del polmone.

The authors report on the results of the immune monitoring of a study population of 31 patients with lung cancer who were treated with radiotherapy. A synthetic thymic pentapeptide, thymopentin, was employed whose effect was evaluated on the immunological parameters analyzed. After radiotherapy, a considerable and homogeneous decrement was observed in several lymphocytic subsets (less sensible in activated T-cells), together with a progressive decrement in the helper/suppressor ratio, in the long run. Monocytes and null cells showed more radioresistance. Thymopentin had no influence on the tested immunological parameters up to 6 months after radiotherapy; later on, a slightly more balanced helper/suppressor ratio could be noticed in the surviving patients who had been treated with thymopentin.

Effects of benzopsoralen derivatives on HL60 and HeLa cells.

The effect of 4-hydroxymethyl-6,7,8,9-tetrahydro-2H-benzofuro-[3,2-g]-1-benzo piran-2-one (compound 1) and 4-hydroxymethyl-2H-benzofuro-[3,2g]-1-benzopiran-2-one (compound 2), two new benzopsoralen derivatives, was tested on HL60 and HeLa cell lines in the dark and by UVA irradiation; 8-methoxypsoralen was used as a reference compound. The action of the compounds was evaluated by means of the neutral red uptake assay, by means of ultrastructure, morphometry and interaction with human erythrocytes membrane. In both HL60 and HeLa cell lines benzopsoralen derivatives showed more antiproliferative activity after UVA irradiation, however less than 8-methoxypsoralen. Compound 1 was more effective than compound 2 both in the dark and after UVA irradiation. The ultrastructure showed a morphological rank damage caused by these compounds: compound 2 induced slight modifications in the cytoplasm organization, compound 1 induced some vacuolizations and 8-methoxypsoralen generated plenty of vacuoles and an empty space around the nucleus. Morphometrical data in HL60 cells turned out to be in accordance with the different action mechanisms existing between 8-methoxypsoralen and the two benzopsoralen derivatives; in HeLa cells we noted an increase in the nuclear area induced by all the three compounds. Only compound 1 caused the formation of echinocytes both in the dark and after UVA irradiation, suggesting the involvement of a mechanism not strictly related to DNA interaction and singlet oxygen production.

Uptake and intracellular distribution of idarubicin in secondary cultures of normal and neoplastic urothelium.

This study analyzes the uptake and endocellular distribution of idarubicin (IDA) in normal and neoplastic urothelial secondary cultures in relation to the changes in concentration and time of exposure. The urothelial lines were isolated by Freshney's method from biopsy fragments taken from five patients with superficial bladder cancer. Pharmacological experiments were carried out on subcultures previously immunophenotypically characterized and did not exceed ten passages. The uptake and endocellular distribution of IDA was analyzed by densitometric image analysis on cells treated for 10, 20, 30 and 60 min and 2 h with scalar dosages from 10 ng/ml to 2430 ng/ml. Microscopic observations and densitometric analyzes revealed that in the cells treated with IDA, fluorescence was higher in the cytoplasm compared to the nucleus and increased with the change in dosage. Moreover, densitometric data showed that IDA uptake in the first 20 min was higher in the neoplastic cells, but after that period its behavior became heterogeneous at 30 and 60 min, while at 2 h there was an inversion of the trend. These results suggest that the in vitro cytotoxicity should be evaluated in order to verify whether the elevated uptake of IDA in the first 20 min of treatment is really correlated to a more elevated toxicity in the neoplastic cells with respect to the normal cells. This is presently under investigation.

The effects of prolonged cardiopulmonary bypass on cell-mediated immunity.

Studies of T-cell subsets (CD3+, CD4+, CD8+, CD8+ CD57+ cells), lymphocyte response to concanavalin A (Con A), phytohaemoagglutinin (PHA) and the alterations of white cell membranes shown by scanning electronic microscope (SEM) in 51 patients who underwent cardiac operation were performed. Out of these 51 unselected patients, for 16, duration of CPB was < or = 110 min (group A), while for the other 35 (group B) it was prolonged (> 110 minutes). Although variations of the lymphocyte subset observed between groups A and B were slightly significant (p < 0.05 before CPB and on postoperative day 7), the T-cell reactivity in group B in comparison to that of group A did not normalize by postoperative day 7 regardless of stimulation with PHA or with Con A. With the use of the SEM, the folded aspect of lymphocyte surface decreased after surgery in about 71% (group A) and 78% (group B) of the observed cells. The outcome of the immunological effects given by our studies could have been due to an elongated CPB even if there need to be taken into consideration multifactorial influences, i.e. biological, pharmacological and hormonal hypotheses, and rapid changes in CPB-micro-environment.