Confirmatory validation analysis of the PROFFIT questionnaire to assess financial toxicity in cancer patients.

BACKGROUND: The Patient Reported Outcome for Fighting FInancial Toxicity (PROFFIT) questionnaire was developed to measure financial toxicity (FT) and identify its determinants. The aim of the present study was to confirm its validity in a prospective cohort of patients receiving anticancer treatment. PATIENTS AND METHODS: From March 2021 to July 2022, 221 patients were enrolled at 10 Italian centres. Selected items of the EORTC-QLQ-C30 questionnaire represented the anchors, specifically, question 28 (Q-28) on financial difficulties, and questions 29-30 measuring global health status/quality of life (HR-QOL). The study had 80% power to detect a 0.20 correlation coefficient (r) between anchors and PROFFIT-score (items 1-7, range 0-100, 100 indicating maximum FT) with bilateral alpha 0.05 and 80% power. Confirmatory factor analysis was conducted. FT determinants (items 8-16) were described. RESULTS: Median age of patients was 65 years, 116 (52.5%) were females, 96 (43.4%) had low education level. Confirmatory factor analysis confirmed goodness of fit of the PROFFIT-score. Significant partial correlation of PROFFIT-score was found with Q-28 (r = 0.51) and HR-QOL (r = -0.23). Mean (SD) PROFFIT-score at baseline was 36.5 (24.9); it was statistically significantly higher for patients living in South Italy, those with lower education level, those who were freelancer/unemployed at diagnosis and those who reported significant economic impact from the COVID-19 pandemic. Mean (SD) scores of determinants ranged from 17.6 (27.1) for item 14 (support from medical staff) to 49.0 (36.3) for item 10 (expenses for medicines or supplements). PROFFIT-score significantly increased with worsening response to determinants. CONCLUSIONS: External validation of PROFFIT-score in an independent sample of patients was successful. The instrument is now being used in clinical studies.

Mindfulness-based stress reduction in cancer patients: impact on overall survival, quality of life and risk factor.

Mindfulness-based stress reduction, a complementary and alternative therapy, is able to decrease cancer-related fatigue, and stress and to improve the quality of life in cancer patients. Some studies evaluated if mindfulness-based stress reduction could improve some cardiometabolic and cancer risk factors, including systemic chemokines, growth factors, and pro-inflammatory biomarkers (e.g., C-reactive protein, Interleukin-1). In this narrative review, we highlight the pleiotropic beneficial effects of mindfulness-based stress reduction and its clinical impact on cardiovascular and cancer risk factors among patients with cancer in different stages. Moreover, improvements in the overall quality of life, sleep quality, and immune functions [changes in plasma levels of interleukin-4 (IL-4), interferon-γ (INF-γ), and interleukin-10 (IL-10)] will also be discussed. Albeit few clinical studies available in the literature, evidenced the beneficial effects of mindfulness-based stress reduction on the immune and cardiometabolic profile in cancer patients, providing important insights into the closest collaboration between psycho-oncologists, oncologists, and cardiologists.

Measurement of pulmonary gas exchange variables and lactic anaerobic capacity during field testing in elite indoor football players.

AIM: The aims of this study were: 1) to examine the gas exchange responses of elite indoor football players to a repeated sprint ability (RSA) test; and 2) to verify whether or not the excess of carbon dioxide production (CO2excess) correlates with blood lactate accumulation during RSA field testing. METHODS: Eleven elite male indoor football players were recruited. A preliminary incremental exercise test on a treadmill was performed to elicit V'O2max. Then, participants underwent an RSA test consisting in a shuttle running through a course with various changes of direction while wearing a portable gas analyzer able to provide values of oxygen uptake, carbon dioxide production, and CO2excess. BLa concentrations during recovery were also measured. RESULTS: The main results were that: 1) during the RSA test subjects did not reached the V'O2max level achieved in the preliminary test; 2) during the RSA test BLa levels were higher compared with the preliminary test; 3) the peak BLa concentration during recovery was significantly correlated with the average CO2excess CONCLUSION: It was concluded that the RSA test did not appear to be useful to elicit V'O2max. Rather, it seemed suitable to recruit subjects' lactic anaerobic capacity. Moreover, CO2excess appeared suitable for qualitatively estimate BLa accumulation during field testing.

Development of humanized mice for the study of hepatitis C virus infection.

The development of a small animal model for hepatitis C virus (HCV) infection is a critical issue for the development of novel anti-HCV drugs. To this aim, we have tried many different approaches for generating mice carrying humanized liver. Main efforts were focused on the transplantation of human hepatocytes into immunocompromised mice (SCID-/-, Bg-/-) carrying a genetic lethal liver disease (Alb-uPA). Survival of homozygotic animals should largely depend on early transplantation with healthy hepatocytes. In parallel to establishing a colony of Alb-uPA/SCID/Bg mice, we developed a microsurgical procedure for intrasplenic xenotransplantation of healthy hepatocytes in 1-week-old mice. So far, we generated several chimeras by xenotransplanting human hepatocytes in Alb-uPA+/+/SCID-/-/Bg-/- mice at 1 week after birth. In a first step, identification of successfully engrafted animals is possible by quantification of human serum albumin and human alpha 1 antitrypsin in mouse sera. Additional preliminary histomorphological analysis of liver sections from chimeric animals was also carried out. One of the mice was transiently infected with HCV, reaching viremia levels of approximately 10(5) genomes/mL. However, the efficiency of this system to generate chimeric mice is still very limited. We are currently exploring the use of more robust models of hepatic disease. Moreover, we have been also exploring novel strategies for the generation of chimeric mice by xenotransplanting human adult stem cells, instead of human hepatocytes, at preimmune stages of development.

Amplification of T cells from human cord blood in serum-deprived culture stimulated with stem cell factor, interleukin-7 and interleukin-2.

We report the effects exerted by cytokine combinations, including stem cell factor (SCF), interleukin-7, interleukin-4 and interleukin-2, on the amplification of T cells from cord blood (CB) mononuclear cells cultured for 10-11 days under serum-deprived conditions. Of all the combinations investigated, SCF+interleukin-7 sustained the best fold increase (FI) of total nucleated cells (FI=6.4+/-1.17), amplifying preferentially CD4(+) over CD8(+) T-cell subsets (FI=4.72+/-0.79 vs 2.73+/-1.2, respectively, P<0.05). The addition of interleukin-2 to this combination did not significantly increase the total number of cells generated (FI=7.4+/-2.27), but allowed preferential amplification of CD8(+) over CD4(+) T cells (FI=6.04+/-0.14 vs 1.67+/-0.6, respectively, P<0.05). Single-strand conformation polymorphism analysis of the T-cell receptor V(beta)-chain rearrangements expressed by the expanded T cells indicated that the complexity of the T-cell repertoire had increased after 10 days of culture in the presence of SCF and IL-7. Interestingly, a modest expansion (FI=8.67+/-1.5) of myeloid progenitor cells was also observed in these cultures. These results indicate that it is possible to expand specific T-cell subsets for adoptive immunotherapy without losing myeloid progenitor cells necessary for neutrophil recovery after CB transplantation, by modulating the cytokines added to the cultures.

Circulating hematopoietic progenitor cells in a fetus with alpha thalassemia: comparison with the cells circulating in normal and non-thalassemic anemia fetuses and implications for in utero transplantations.

Our aim was to evaluate the number of progenitor cells circulating in an alpha-thalassemic fetus during its infusion in utero with paternal CD34(+) and adult red cells and to compare those values with those circulating in normal and non-thalassemic anemic fetuses of matched gestational age. The treatment of the alpha-thalassemic fetus has been described elsewhere. Fetal blood was obtained from normal and anemic fetuses by fetal blood sampling for diagnostic or therapeutic purposes according to a protocol approved by the human subject committee. The number of progenitor cells in fetal blood was estimated on the basis of the number of colonies they gave rise to in semisolid cultures. The alpha-thalassemic fetus, as did the other fetuses analyzed, contained high numbers (10(6)-10(7) depending on the age) of progenitor cells, values which were higher than the number (10(4)-10(5)) of paternal progenitor cells being transplanted. Progenitor cells with adult characteristics (adult kinetics of differentiation) were detected rapidly (10 min) after the CD34(+) cell infusion, but were not detectable 2-3 weeks after the transplant. These results indicate that adult progenitor cells do not have a numerical advantage when transplanted into alpha-thalassemic fetuses.

Accentuated response to phenylhydrazine and erythropoietin in mice genetically impaired for their GATA-1 expression (GATA-1(low) mice).

The response of mice genetically unable to up-regulate GATA-1 expression (GATA-1(low) mice) to acute (phenylhydrazine [PHZ]-induced anemia) and chronic (in vivo treatment for 5 days with 10 U erythropoietin [EPO] per mouse) erythroid stimuli was investigated. Adult GATA-1(low) mice are profoundly thrombocytopenic (platelet counts [x 10(9)/L] 82.0 +/- 28.0 vs 840 +/- 170.0 of their control littermates, P <.001) but have a normal hematocrit (Hct) (approximately.47 proportion of 1.0 [47%]). The spleens of these mutants are 2.5-fold larger than normal and contain 5-fold more megakaryocytic (4A5(+)), erythroid (TER-119(+)), and bipotent (erythroid/megakaryocytic, TER-119(+)/4A5(+)) precursor cells. Both the marrow and the spleen of these animals contain higher frequencies of burst-forming units-erythroid (BFU-E)- and colony-forming units-erythroid (CFU-E)-derived colonies (2-fold and 6-fold, respectively) than their normal littermates. The GATA-1(low) mice recover 2 days faster from the PHZ-induced anemia than their normal littermates (P <.01). In response to EPO, the Hct of the GATA-1(low) mice raised to.68 proportion of 1.0 (68%) vs the.55 proportion of 1.0 (55%) reached by the controls (P <.01). Both the GATA-1(low) and the normal mice respond to PHZ and EPO with similar (2- to 3-fold) increases in size and cellularity of the spleen (increases are limited mostly to cells, both progenitor and precursor, of the erythroid lineage). However, in spite of the similar relative cellular increases, the increases of all these cell populations are significantly higher, in absolute cell numbers, in the mutant than in the wild-type mice. In conclusion, the GATA-1(low) mutation increases the magnitude of the response to erythroid stimuli as a consequence of the expansion of the erythroid progenitor cells in their spleen.

Establishing an employee baseline purified protein derivative status in the division of immigration health Service Processing Centers.

While the incidence of reportable tuberculosis in United States born persons declined, the number of cases among foreign-born persons increased by 6 percent in 1998. The Immigration and Naturalization Service (INS) processes about 95,000 undocumented aliens annually from countries with a high prevalence of tuberculosis. An effort was made to establish a baseline Purified Protein Derivative (PPD) status of employees of the Division of Immigration Health (DIH) and INS, Services Processing Centers (SPC). This was achieved through a special operation (project) of a one time, two-step, mass PPD testing of all SPC employees on all eleven sites in the United States and Puerto Rico. A reading of > 10mm was considered positive. The operation was optional and open to all SPC employees. Exclusion criteria for the study included a history of PPD skin testing within six months of the operation, past history of positive PPD test and past history of tuberculosis. Preliminary results from El Paso SPC, Texas, which is the largest SPC, showed that of the 148 employees which were tested (67 percent of all employees), 17 (11 percent) were PPD positive. Security officers constituted 100 percent of all positive cases. Eighty-one percent of the employees at El Paso are security officers, eighty-seven percent of whom participated in the study. Only 20 (23 percent) of administrative staff participated in the study. Results from El Paso are suggestive of differences in the pattern PPD positivity among SPC employees. The complete results of the study should provide sound evidence for formulating appropriate policies for establishing an effective employee tuberculosis prevention and surveillance program in the Service Processing Centers.

Standardization of progenitor cell assay for cord blood banking.

Cord blood has proved itself, if correctly stored with rational criteria, an excellent source of stem cells for related and unrelated transplants. It has been recently proven that the factor which predicts the best the speed of engraftment in cord blood transplants in the dose of progenitor cells injected per kg of body weight of the recipient. This result has been obtained thanks to a careful standardization of the neonatal progenitor cell assay. This manuscript describes such a standardization realized as a joined effort by the Istituto Superiore di Sanità, Rome, and the pivotal cord blood bank founded as a feasibility study by the National Institutes of Health, Bethesda at the New York Blood Center.

Erythropoietin-dependent suppression of the expression of the beta subunits of the interleukin-3 receptor during erythroid differentiation.

To clarify how erythroid cells lose their response to interleukin-3 (IL-3), we analyzed the expression of the alpha (alpha(IL-3)) and beta (beta(IL-3)/beta(com)) subunits of its receptor in a panel of murine cell lines immortalized at different stages of hemopoietic differentiation. The panel was composed by the mast cell line 32D and by its granulo-monocytic (32D GM), granulocytic (32D G), and erythroid (32D Epo1.1 and Epo) subclones. The 32D Epo cells grow only in erythropoietin (EPO) while the Epo1.1 subclone grows in either EPO or IL-3. The phenotype of these cells is that of early (expression of globins and erythroid-specific carbonic anhydrase II) and late (also expression of the erythroid-specific band 4.1 mRNA) erythroblasts when they grow in IL-3 or EPO, respectively. All the cell lines expressed comparable levels of alpha(IL-3). In contrast, the expression of beta(IL-3)/beta(com) was restricted to cells growing in IL-3 and was barely detectable in 32D Epo and 32D Epo1.1 cells growing in EPO. When switched from EPO to IL-3, 32D Epo1.1 cells expressed 10 times more beta(IL-3)/beta(com) by rapidly activating (within 1 h) their transcription rate. When reexposed to EPO, 32D Epo1.1 cells first expressed (1-6 h) more beta(IL-3)/beta(com) (2 times) but suppressed such an expression at later time points (by 48 h). The beta(IL-3)/beta(com) mRNA half-life was also different when 32D Epo1.1 cells grew in EPO or IL-3 (2-3 h vs >5 h, respectively). These results indicate that EPO specifically induces transcriptional and posttranscriptional downmodulation of beta(IL-3)/beta(com) expression in late erythroid cells.

A new determinant of endoplasmic reticulum localization is contained in the juxtamembrane region of the ectodomain of hepatitis C virus glycoprotein E1.

Hepatitis C virus glycoproteins E1 and E2 do not reach the plasma membrane of the cell but accumulate intracellularly, mostly in the endoplasmic reticulum. Previous studies based on transient expression assays have shown that the transmembrane domains of both glycoproteins are sufficient to localize reporter proteins in the endoplasmic reticulum and that other localization signals may be contained in the ectodomain of E1 protein. To identify such signals we generated chimeric proteins between E1 and two reporter proteins, the human CD8 glycoprotein and the human alkaline phosphatase, and analyzed their subcellular localization in stable as well as transient transfectants. Our results showed that (i) an independent localization determinant for the endoplasmic reticulum is present in the juxtamembrane region of the ectodomain of E1 protein and (ii) the localization dictated by this determinant is either due to direct retention or to a recycling mechanism from the intermediate compartment/cis-Golgi complex region, which is clearly different from those previously described for other retrieval signals. These results show for the first time in mammalian cells that the localization in the endoplasmic reticulum of transmembrane protein can be determined by specific targeting signals acting in the lumen of the compartment.

Effects of cell banking manipulations on ex vivo amplification of umbilical cord blood.

The small volume of placental/umbilical cord blood (PUCB) collectable restricts the use of these stem cells to pediatric transplantation. To extend the use of PUCB to adult recipients, many laboratories are investigating the feasibility of ex vivo PUCB expansion. The present study analyses the effects that PUCB banking cell manipulations (cell sedimentation, cryopreservation and thawing, mononuclear and CD34+ cell isolation) have on the number, viability and ex vivo expansion potential of PUCB cells. The results presented indicate the necessity of an open discussion on whether procedures used for handling the cells in PUCB banks can be extrapolated or not as such to the clinical use of ex vivo expanded PUCB.

Identification and characterization of a bipotent (erythroid and megakaryocytic) cell precursor from the spleen of phenylhydrazine-treated mice.

We have identified a cell population expressing erythroid (TER-119) and megakaryocyte (4A5) markers in the bone marrow of normal mice. This population is present at high frequency in the marrows and in the spleens involved in the erythroid expansion that occurs in mice recovering from phenylhydrazine (PHZ)-induced hemolytic anemia. TER-119(+)/4A5(+) cells were isolated from the spleen of PHZ-treated animals and were found to be blast-like benzidine-negative cells that generate erythroid and megakaryocytic cells within 24-48 hours of culture in the presence of erythropoietin (EPO) or thrombopoietin (TPO). TER-119(+)/4A5(+) cells represent a late bipotent erythroid and megakaryocytic cell precursors that may exert an important role in the recovery from PHZ-induced anemia. (Blood. 2000;95:2559-2568)

In vivo expansion of purified hematopoietic stem cells transplanted in nonablated W/Wv mice.

We have evaluated the in vivo amplification potential of purified murine hematopoietic stem cells, identified as Wheat Germ Agglutinin+ (WGA+), 15-1.1(-) , Rhodamine 123 Dull (Rho-dull) cells, by serial transplantation into stem cell defective nonmyeloablated W/Wv mice. C57BL Rho-dull cells (250/ 500 cells/mouse) permanently engrafted nonablated W/Wv mice as defined by the presence of > 95% red and > 20% white donor-derived circulating cells for at least 1.5 years following transplantation. At this time, approximately 61% of Rho-dull cells and all the Rho-bright progenitor and colony forming cells of the engrafted mice were found to be donor-derived by c-Kit genotyping and by their response to stem cell factor (SCF). Retransplantation of 250-1000 Rho-dull cells from primary into secondary W/Wv recipients generated C57BL hematopoiesis in 40%-64% of animals revealing the presence of donor derived hematopoietic stem cells (HSC) in the bone marrow of the primary recipients. One and half years after transplantation, the bone marrow of the secondary engrafted animals contained C57BL Rho-dull cells approximately = 51% by genotype), which were capable of reconstituting tertiary W/Wv recipients. In this respect, 25% of tertiary mice expressed C57BL hematopoiesis when transplanted with 250-1000 Rhodull cells purified from secondary W/Wv recipients. On the basis of the number of Rho-dull cells purified from a single mouse, we calculate that approximately 7.3x10(4) Rho-dull cells, which are genotypically and functionally defined as C57BL long-term repopulating stem cells, were generated in the marrow of reconstituted primary W/Wv recipients transplanted 1.5 years earlier with 250-500 C57BL Rho-dull cells. We conclude that murine HSC have extensive amplification capacity in nonmyeloablated animals.

Lineage-restricted expression of protein kinase C isoforms in hematopoiesis.

The pattern of expression of several protein kinase C (PKC) isoforms (alpha, betaI, delta, epsilon, eta, and zeta) during the course of hematopoietic development was investigated using primary human CD34(+) hematopoietic cells and stable cell lines subcloned from the growth factor-dependent 32D murine hematopoietic cell line. Each 32D cell clone shows the phenotype and growth factor dependence characteristics of the corresponding hematopoietic lineage. Clear-cut differences were noticed between erythroid and nonerythroid lineages. (1) The functional inhibition of PKC-epsilon in primary human CD34(+) hematopoietic cells resulted in a twofold increase in the number of erythroid colonies. (2) Erythroid 32D Epo1 cells showed a lower level of bulk PKC catalytic activity, lacked the expression of epsilon and eta PKC isoforms, and showed a weak or absent upregulation of the remaining isoforms, except betaI, upon readdition of Epo to growth factor-starved cells. (3) 32D, 32D GM1, and 32D G1 cell lines with mast cell, granulo-macrophagic, and granulocytic phenotype, respectively, expressed all the PKC isoforms investigated, but showed distinct responses to growth factor readdition. (4) 32D Epo 1.1, a clone selected for interleukin-3 (IL-3) responsiveness from 32D Epo1, expressed the epsilon isoform only when cultured with IL-3. On the other hand, when cultured in Epo, 32D Epo1.1 cells lacked the expression of both epsilon and eta PKC isoforms, similarly to 32D Epo1. (5) All 32D cell lines expressed the mRNA for PKC-epsilon, indicating that the downmodulation of the epsilon isoform occurred at a posttranscriptional level. In conclusion, the PKC isoform expression during hematopoiesis appears to be lineage-specific and, at least partially, related to the growth factor response.

Selection of functional variants of the NS3-NS4A protease of hepatitis C virus by using chimeric sindbis viruses.

The NS3-NS4A serine protease of hepatitis C virus (HCV) mediates four specific cleavages of the viral polyprotein and its activity is considered essential for the biogenesis of the HCV replication machinery. Despite extensive biochemical and structural characterization, the analysis of natural variants of this enzyme has been limited by the lack of an efficient replication system for HCV in cultured cells. We have recently described the generation of chimeric HCV-Sindbis viruses whose propagation depends on the NS3-NS4A catalytic activity. NS3-NS4A gene sequences were fused to the gene coding for the Sindbis virus structural polyprotein in such a way that processing of the chimeric polyprotein, nucleocapsid assembly, and production of infectious viruses required NS3-NS4A-mediated proteolysis (G. Filocamo, L. Pacini, and G. Migliaccio, J. Virol. 71:1417-1427, 1997). Here we report the use of these chimeric viruses to select and characterize active variants of the NS3-NS4A protease. Our original chimeric viruses displayed a temperature-sensitive phenotype and formed lysis plaques much smaller than those formed by wild-type (wt) Sindbis virus. By serially passaging these chimeric viruses on BHK cells, we have selected virus variants which formed lysis plaques larger than those produced by their progenitors and produced NS3-NS4A proteins different in size and/or sequence from those of the original viruses. Characterization of the selected protease variants revealed that all of the mutated proteases still efficiently processed the chimeric polyprotein in infected cells and also cleaved an HCV substrate in vitro. One of the selected proteases was expressed in a bacterial system and showed a catalytic efficiency comparable to that of the wt recombinant protease.

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) and leukemia inhibitory factor (LIF) protect the repopulating ability of purified murine hematopoietic stem cells in serum-deprived cultures stimulated with SCF and IL-3.

The engraftment potential of murine stem cells (HSC) is greatly reduced when these cells are expanded in vitro with stem cell factor and interleukin-3. We have evaluated if the addition of MIP-1 alpha or LIF to these cultures would protect the ability of murine wild type HSC to engraft the stem cell defective W/Wv recipient. In this transplantation model red and white blood cell reconstitution is assessed by hemoglobin electrophoresis and c-kit PCR genotyping, respectively. The results obtained indicate that both MIP-1 alpha and LIF protect, at least transiently, the HSC repopulating ability in vivo in spite of the modest expansion in the number of nucleated and progenitor cells observed.

Stem cell factor induces proliferation and differentiation of fetal progenitor cells in the mouse.

We have investigated the kinetics of the amplification of the progenitor cell compartments (CFC) in haemopoietic organs during murine ontogenesis and compared the growth requirements of fetal and adult CFC. Two haemopoietic phases were recognized in the fetal liver (FL): an exponential growth phase, from 11.5 to 15.5 d post conception (p.c.), during which the mean number of nucleated cells and of CFC in the FL increased from 4.9 x 10(5) to 7.0 x 10(7) and from 4.5 x 10(3) to 2.7 x 10(5), respectively, and a recessive phase after 15.5 d p.c., during which the CFC number in the FL gradually decreased, although some CFC were still detectable in the liver after birth. In serum-deprived cultures, FL and adult marrow (AM) CFC had similar responses to GM-CSF, and did not respond to G-CSF or IL-3. In contrast, FL, but not AM, erythroid colonies grew Epo-independently whereas SCF alone induced formation of maximal numbers of erythroid bursts from FL, but not from AM cells. The proliferative and differentiative effect of SCF alone on fetal cells was confirmed in serum-deprived cultures of purified early progenitor cells isolated by cell sorting on the basis of multiple parameters from FL and AM light-density cells. In culture of purified FL cells, SCF alone induced a similar amplification of total cells (maximal amplification at day 12: 800-300-fold) and total CFC (11-38-fold of maximal amplification at day 6) to the combination of SCF plus IL-3 (1300-800-fold amplification of total cells and 31-88-fold amplification of CFC). In contrast, SCF alone allowed only survival of purified AM early progenitor cells. Therefore FL early progenitor cells have an intrinsic higher potential than their adult counterpart to respond to SCF, confirming the potent role of this growth factor in the development of the murine haemopoietic system.

The making of an erythroid cell. Molecular control of hematopoiesis.

The number of circulating red cells is regulated by the daily balance between two processes: the destruction of the old red cells in the liver and the generation of new cells in the bone marrow. The process during which hematopoietic stem cells generate new red cells is called erythropoiesis. This manuscript will describe the molecular mechanisms involved in the process of erythroid differentiation as we understand them today. In particular it will review how erythroid specific growth factor-receptor interactions activate specific transcription factors to turn on the expression of the genes responsible for the establishment of the erythroid phenotype.

Thymus-independent T-cell differentiation in vitro.

The generation of large quantities of novel human T-cell clones ex vivo would make a wide range of gene- and immuno-therapies for tumours and viral infections possible. Several techniques have been described to generate, in vitro and in vivo (using xenogenic hosts), mature T cells from fetal-neonatal and adult human CD34+ cells. All these techniques are cumbersome and cannot be easily translated into clinical protocols because they involve co-cultivation of CD34+ cells with thymic fragments from either human or murine fetuses. We report that the mononuclear cells of human cord blood contain a cell population that supports the differentiation of CD34+ cells into CD4+ or CD8+ naive T cells in serum-deprived cultures stimulated with stem cell factor and interleukin 7. CD4+ or CD8+ CD45RA+ TCRalphabeta+ T cells were continuously produced in vitro over a period of 20 d under these conditions. The generation of T cells in these cultures was a dynamic process and clones of T cells expressing new T-cell receptor beta-chain rearrangements were generated over time. These results pave the way for the development of very simple culture conditions for ex-vivo production of naive helper or cytotoxic T cells which could be very useful for gene- and immuno-therapy of human diseases.