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A Carè

Publications and source records attributed to A Carè.

At least 19 recordsLinked to original sources
PubMed2006

Unilineage monocytopoiesis in hematopoietic progenitor culture: switching cytokine treatment at all Mo developmental stages induces differentiation into dendritic cells.

We have developed a new culture system whereby human hematopoietic progenitors purified from adult peripheral blood extensively proliferate and gradually differentiate into >95% pure monocytic (Mo) cells. At all developmental stages treatment with interleukin (IL)-4+granulocyte-macrophage colony-stimulating factor or IL-4+c-Kit-ligand+FLT-3 ligand switched the Mo precursors into dendritic cells (DCs). The switching capacity decreased only at the end of the culture, when most Mo cells matured to macrophages. Moreover, the Mo precursors were highly susceptible to transduction with lentiviral vectors: once switched to DCs, they maintained the transgene expression, as well as the phenotype and function of the DC lineage. Our results provide new insight into the potential role of the Mo lineage as a reservoir of DCs in vivo. Furthermore, the methodology for transduction of Mo precursors provides a tool to generate genetically modified, normally functioning DCs potentially useful for immunotherapy.

Cell Lineage↗
PubMed2001

HOXB7: a key factor for tumor-associated angiogenic switch.

We had demonstrated previously a functional bridge between altered homebox (HOX) gene expression and tumor progression through HOXB7 transactivation of basic fibroblast growth factor. Here, we have studied whether HOXB7, in addition to basic fibroblast growth factor, may induce other genes directly or indirectly related to neoangiogenesis and tumor invasion. Parental, beta-galactosidase-transduced, and HOXB7-transduced SkBr3 cell lines were examined for the expression of several growth factors and growth factor receptors involved in the proliferative and angiogenic processes. Vascular endothelial growth factor, melanoma growth-stimulatory activity/growth-related oncogenene alpha, interleukin-8, and angiopoietin-2 were up-regulated by HOXB7 transduction. The exception was angiopoietin-1 expression that was abrogated. Additional analyses included the expression levels of enzymes such as matrix metalloprotease (MMP)-2 and MMP-9 and heparanase, capable of proteolytic degradation of extracellular matrix and basement membranes. Results showed an induction of only MMP-9. The functional implication of such a finding was tested using an in vitro coculture assay in a three-dimensional matrix. A delay of differentiation with persistent nests of proliferating cells was found in endothelial cells cocultured with HOXB7-transduced SkBr3 cells. Tumorigenicity of these cells has been evaluated in vivo. Xenograft into athymic nude mice showed that SkBr3/HOXB7 cells developed tumors in mice, either irradiated or not, whereas parental SkBr3 cells did not show any tumor take unless mice were sublethally irradiated. Comparison of tumor nodules for vascularization by CD-31 and CD-34 immunostaining revealed an increased number of blood vessels in tumors expressing HOXB7. Together, the results indicate HOXB7 as a key factor up-regulating a variety of proangiogenic stimuli. Thus, HOXB7 gene or protein is a target to aim at to inhibit tumor-associated neoangiogenesis, considering the number and the redundancy of proangiogenic molecules that should be targeted one by one to theoretically achieve the same effect.

Adenocarcinoma↗
PubMed1999

Enforced expression of HOXB7 promotes hematopoietic stem cell proliferation and myeloid-restricted progenitor differentiation.

Hematopoietic progenitor/stem cells (HPCs/HSCs) purified from human adult peripheral blood (PB) were triggered into cycling, retrovirally transduced with HOXB7 and then functionally assayed in vitro. HPCs were assayed in multi- and unilineage differentiation cultures in either liquid phase or semisolid medium, primitive HPCs in the high proliferative potential colony-forming cell (HPP-CFC) evaluation system and putative HSCs in Dexter type long-term culture (LTC) as LTC initiating cells (LTC-ICs). Control experiments ensured that the exogenous HOXB7 gene was constantly expressed, while the endogenous one was barely or not transcribed. Enforced expression of the gene markedly modulated the proliferation/differentiation program of the entire HSC/HPC population. Enforced HOXB7 expression exerted a potent stimulatory effect on the proliferation of the primitive HPC and putative HSC subsets, assayed as HPP-CFCs and LTC-ICs respectively. While not modifying the total number of HPCs, exogenous HOXB7 induced an increase of the number of granulo-monocytic (GM) HPCs [colony-forming unit GM (CFU-GM) CFU-GM, CFU-G and CFU-M, as evaluated by clonogenic assays] and markedly amplified the progeny of both CFU-G and CFU-M, which showed a sustained proliferation through at least 1-2 months (as evaluated in liquid suspension culture). The prolonged proliferative stimulus induced by HOXB7 transfer into LTC, primitive and GM oriented HPC culture was characterized by persistent proliferation of a discrete population of blast cells and a large pool of differentiated myeloid precursors. Altogether, these results suggest the hypothesis that the proliferative stimulus exerted by exogenous HOXB7 in primitive and GM-oriented HPCs may represent a preleukemic immortalization step. Consistent with the functional role of HOXB7 in the initial ontogenetic phase, these studies indicate that ectopic HOXB7 expression in early HPCs and HSCs from adult PB stimulates their self renewal, sustained proliferation and myeloid differentiation.

Adult↗
PubMed1998

HIV/gp120 and PMA/ionomycin induced apoptosis but not activation induced cell death require PKC for Fas-L upregulation.

HIV protein gp120 in combination with T cell antigen receptor (TCR) triggering induces apoptosis (gp120-apoptosis) in Th1 cells. Gp120-apoptosis occurs by induction of Fas-L and subsequent triggering of the Fas apoptotic pathway. Here, through the use of several compounds inhibiting induction of Fas-L, we show that, in a Th1 clone, a protein kinase C (PKC) independent pathway activated by TCR stimulation is distinguishible from a PKC dependent pathway activated by either phorbol 12-myristate 13-acetate (PMA)/ionomycin or asynchronous stimulation of TCR and CD4 as occurs in gp120-apoptosis.

Antigens, Surface↗
PubMed1996

HOXB cluster genes in activated natural killer lymphocytes: expression from 3'-->5' cluster side and proliferative function.

The expression of HOXB cluster genes (i.e., B1 through B9) was evaluated in purified IL-2/IL-1 beta-activated NK lymphocytes from normal adult peripheral blood by RNase protection and reverse transcription-PCR. In quiescent NK cells these genes are essentially not expressed. After IL-2/IL-1 beta addition, we observed a coordinate induction wave in the 3'-5' HOXB cluster direction, i.e., from B1 through B9. As notable exceptions, B8 is silent, while B9 RNA is detected starting from 6 h through day 11. Furthermore, the 3' located B2/B3/B4 are expressed earlier and at higher level than the 5' located B5/B6/B7/B8. In IL-2/IL-1 beta-activated NK cells, treatment with antisense oligonucleotides targeting B2 mRNA causes a significant inhibition of both cell proliferation and expression of activation markers (i.e., IL-2R alpha-chain and transferrin receptor). These studies provide novel evidence of the role of HOX genes in adult NK cell proliferation. Thus, 1) a coordinate activation of HOXB genes from the 3'-->5' cluster side apparently underlies IL-2/IL-1 beta-induced NK cell activation. 2) Since NK cell activation and survival induced by IL-12 and c-kit ligand, respectively, are not associated with cell proliferation of HOXB gene expression, it is apparent that HOXB gene induction is specifically associated with IL-2-induced NK cell proliferation. 3) Studies with antisense oligomer targeting HOXB2 mRNA suggest an important role for 82 in NK cell proliferation, possibly in part via the IL-2R.

Adult↗
PubMed1994

Coordinate expression and proliferative role of HOXB genes in activated adult T lymphocytes.

We investigated the expression of HOXB cluster genes in purified phytohemagglutinin (PHA)-activated T lymphocytes from normal adult peripheral blood by reverse transcription PCR and RNase protection. These genes are not expressed in quiescent T cells, except for barely detectable B1 RNA. After the PHA stimulus, HOXB gene activation initiates coordinately as a rapid induction wave in the 3'-->5' cluster direction (i.e., from HOXB1 through B9 genes). Thus, (i) expression of the foremost 3'-located B1 and B2 genes peaks 10 min after PHA addition and then rapidly declines, (ii) activation of B3, B4, and B5 begins 10 min after PHA addition and peaks at later times (i.e., at 120 min for B5), (iii) B6, B7, and B9 are expressed at a low level starting at later times (45 to 60 min), and (iv) B8 remains silent. Treatment of PHA-activated T lymphocytes with antisense oligonucleotides to B2 or B4 mRNA causes a drastic inhibition of T-cell proliferation and a decreased expression of T-cell activation markers (i.e., interleukin 2 and transferrin receptors). Similarly, treatment of CEM-CCRF, Peer, and SEZ627 T acute lymphocytic leukemia cell lines with anti-B4 oligomer markedly inhibits cell proliferation. Finally, T cells stimulated by a low dosage of PHA in the presence of 1 microM retinoic acid show a marked increase of both HOXB expression, particularly B2, and cell proliferation. These studies provide novel evidence on the role of HOX genes in adult cell proliferation. (i) Coordinate, early activation of HOXB genes from the 3'-->5' cluster side apparently underlies T-cell activation. (ii) The expression pattern in adult PHA-activated T cells is strikingly similar to that observed in retinoic acid-induced teratocarcinoma cells (A. Simeone, D. Acampora, L. Arcioni, P. W. Andres, E. Boncinelli, and F. Mavilio, Nature (London) 346:763-766, 1990), thus suggesting that molecular mechanisms underlying HOX gene expression in the earliest stages of development may also operate in activated adult T lymphocytes.

Adult↗
PubMed1994

c-fes expression in ontogenetic development and hematopoietic differentiation.

The c-fes protein (NCP92) is a tyrosine-specific protein kinase, capable of both autophosphorylation and phosphorylation of other substrates. We have analysed c-fes RNA expression in human/murine ontogenetic development and in homogeneous populations of embryonic and adult human hematopoietic cells. c-fes expression has been observed in rapidly proliferating embryonic-fetal tissues originating from different germinal layers, but not in adult non-hematopoietic tissues. In particular, a spatially and temporally regulated transcription was observed in the central nervous system and in developing cartilage. Expression in hematopoietic cells was evaluated in progenitors purified from embryonic-fetal liver and adult peripheral blood differentiating gradually and specifically along the erythroid or granulomonocytic lineage. In both embryonic and adult hematopoietic cells c-fes was abundantly expressed in undifferentiated progenitors of both lineages, as well as in differentiated granulomonocytic precursors, but not in erythroblasts. This expression pattern correlates with that of GM-CSF and in part IL-3 receptors (Testa et al., 1993 and our unpublished results). Altogether, these results suggest a possible role for c-fes in signal transduction, in both embryonic non-hematopoietic tissues and embryonic/adult hematopoietic cells, following interaction of growth factors with their tyrosine-kinase negative receptors (i.e., GM-CSF and IL-3 receptors in adult hematopoietic cells and other hypothetical growth factor(s) receptors during embryonic development.

Adult↗
PubMed1993

Interleukin-6 expression in human neutrophil and eosinophil peripheral blood granulocytes.

Human peripheral blood granulocytes were analyzed for expression of interleukin-6 (IL-6) using reverse-transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization. Neutrophil granulocytes from healthy donors were shown to express variable levels of IL-6. This expression was rapidly down-regulated after the removal of the cells from the circulating blood. In vitro culture of neutrophils abolished IL-6 expression, which could be reactivated by addition of GM-CSF to the culture medium. Constitutive expression of IL-6 was instead demonstrated in eosinophil granulocytes purified from normal donors and from a hypereosinophilic patient. In situ hybridization of unstimulated granulocytes confirmed that IL-6 expression occurs both in eosinophils and in neutrophils from peripheral blood. These findings show that granulocytes can actively contribute to cytokine expression in the peripheral blood and suggest their role in the afferent limb of the immune response, since by IL-6 production they might modulate T- and B-lymphocyte functions, granulocyte self-priming, and endothelial interaction.

Cells, Cultured↗
PubMed1992

Production of hemolymphopoietic cytokines (IL-6, IL-8, colony-stimulating factors) by normal human astrocytes in response to IL-1 beta and tumor necrosis factor-alpha.

Astrocyte-enriched populations were established from human embryonic brain analyzed for their ability to synthesize cytokines potentially relevant for mechanisms of inflammation and immunity in the brain. Unstimulated astrocytes did not secrete significant IL-6, IL-8, macrophage CSF (M-CSF), granulocyte-macrophage CSF (GM-CSF), or granulocyte-CSF (G-CSF), as determined by specific ELISA and/or bioassay. With the exception of M-CSF mRNA, transcripts for the above factors were not detected in unstimulated astrocytes. On exposure of human astrocytes to IL-1 beta, high levels of IL-6, IL-8, M-CSF, G-CSF, and GM-CSF mRNAs were detected; moreover, active secretion of all the above cytokines was demonstrated. TNF-alpha was also able to stimulate IL-6, IL-8, M-CSF, GM-CSF, and G-CSF synthesis and secretion, but was generally less potent than IL-1 beta. No IL-3 mRNA or protein was detected in unstimulated or cytokine-treated astrocytes. IL-1 alpha and IL-1 beta mRNAs and proteins were not detected in unstimulated astrocytes, but were present in very small amounts after stimulation with TNF-alpha/IL-1 beta. No IL-6, M-CSF, GM-CSF, G-CSF, or IL-8 were induced by IL-1 beta or TNF-alpha in early primary cultures, which mainly contain undifferentiated neuronal/glial progenitor cells. These studies demonstrate for the first time the production of multiple cytokines by normal human astrocytes stimulated in culture by IL-1 beta and TNF-alpha. The capacity of human astrocytes to synthesize and release cytokines active on hemolymphopoietic cells supports the concept that these cells play an important role in the regulation of inflammatory and immune responses in a variety of brain pathologies.

Astrocytes↗
PubMed1992

Cell cycle-dependent initiation and lineage-dependent abrogation of GATA-1 expression in pure differentiating hematopoietic progenitors.

The programmed activation/repression of transcription factors in early hematopoietic differentiation has not yet been explored. The DNA-binding protein GATA-1 is required for normal erythroid development and regulates erythroid-expressed genes in maturing erythroblasts. We analyzed GATA-1 expression in early human adult hematopoiesis by using an in vitro system in which "pure" early hematopoietic progenitors are induced to gradual and synchronized differentiation selectively along the erythroid or granulocyte-macrophage pathway by differential treatment with hematopoietic growth factors. The GATA-1 gene, though virtually silent in quiescent progenitors, is activated after entrance into the cell cycle upon stimulation with hematopoietic growth factors. Subsequently, increasing expression along the erythroid pathway contrasts with an abrupt downregulation in the granulocyte-macrophage lineage. These results suggest a microenvironment-directed, two-step model for GATA-1 expression in differentiating hematopoietic progenitors that involves (i) cycle-dependent initiation and (ii) lineage-dependent maintenance or suppression. Hypothetically, on/off switches of lineage-restricted transactivators may underlie the binary fate decisions of hematopoietic progenitors.

Antigens, Surface↗
PubMed1992

Expression of selected human HOX-2 genes in B/T acute lymphoid leukemia and interleukin-2/interleukin-1 beta-stimulated natural killer lymphocytes.

Although the key role of human homeobox (HOX) genes in development is well established, their function in adult cells is still under scrutiny. We have analyzed, in normal adult blood cell subpopulations, acute lymphoid leukemia (ALL) cells lines, and primary blasts, the RNA expression of all HOX-2 cluster genes (5'-2.5, 2.4, 2.3, 2.2, 2.1, 2.6, 2.7, 2.8, 2.9, 3') and nine genes in the HOX-1, -3, and -4 cluster by Northern blotting, RNAse protection, and/or reverse transcriptase polymerase chain reaction (RT-PCR). The analyzed HOX-1, -3, and -4 genes were never expressed in all tested cell populations. Natural killer (NK) cells activated in interleukin-2 (IL-2)/IL-1 beta-treated cultures exhibit a gradually increasing, abundant expression of three HOX-2 genes (2.2, 2.6, 2.8), while three other genes (2.3, 2.1, 2.7) are expressed at a lower level at late culture times. However, no HOX-2 gene is expressed in quiescent lymphocytes (NK, B and T [T-cell receptor (TCR) alpha/beta, gamma/delta lymphocytes, thymocytes] cells), granulocytes, and monocytes. In B- and T-ALL cell lines, HOX-2 genes are expressed according to different patterns: (1) widespread transcription (seven of nine genes, including 2.3 and 2.6) in the Peer line bearing the TCR gamma/delta; (2) expression of 2.5, 2.2, and 2.6 in the SEZ 627 line, which derives from an HTLV-1+ T-helper leukemia; (3) transcription of 2.3 and 2.6 in both the T-ALL CEM line and four B-ALL lines (interestingly, CALLA- B-ALL lines are constantly 2.3/2.6 RNA+); (4) no HOX-2 gene expression was detected in one T- and two B-ALL lines. Primary blasts from five T- and five pre-B-ALL showed selective expression of one or more HOX-2 genes, namely 2.5, 2.2, 2.6, and 2.7. Our data are compatible with the hypothesis that selected HOX-2 genes play a role in the IL-2/IL-1 beta-induced activation and/or proliferation of normal NK lymphocytes and possibly in the oncogenetic process of some T- and B-ALL.

Base Sequence↗
PubMed1992

Cellular and molecular mechanisms in early hematopoietic differentiation.

Recently developed methodology allows virtually complete purification and abundant recovery of hematopoietic progenitors from human adult peripheral blood (PB) (1). We have recently utilized the population of stringently purified progenitors to investigate cellular and molecular mechanisms underlying the early steps of hematopoietic differentiation. Three aspects of these studies are briefly reported here.

Cell Differentiation↗
PubMed1991

Molecular heterogeneity of the 1.0-kb T beta transcript in natural killer and gamma/delta lymphocytes.

The T cell receptor (TcR) beta chain is encoded by a 1.3-kb mRNA which contains variable (V), diversity (D), joining (J) and constant (C) regions. A shorter 1.0-kb transcript with C but no V sequences is found in thymocytes, alpha/beta and gamma/delta T lymphocytes and natural killer cells. The origin, clonal variability and function of this transcript is unknown. We isolated cDNA clones representative of the 1.0-kb mRNA from cDNA libraries of either polyclonal or clonal natural killer and gamma/delta lymphocytes. Ten different types of cDNA clones belonging to three separate groups were identified: (a) "J-C clones" consisting of one of five J beta regions and the corresponding C beta 1 or C beta 2 regions; (b) "D-J-C clones" composed of D beta 1/or D beta 2/J beta rearranged sequences and C beta 1 or C beta 2 sequences; (c) "5'C clones" made up of C beta 1 or C beta 2 regions preceded by the corresponding genomic 5' flanking region. The presence of recombination signal sequences in J-C and D-J-C clones suggests that the first derive from mRNA transcribed from promoters located in the 5' J region and the second from mRNA transcribed from promoters situated in the 5' D region. Nucleotide sequence analysis demonstrated that only three of the ten types of clones isolated had the potential to code a short cytoplasmic T beta protein with a variable D-J beta N terminus. These findings have implications for the mechanisms of T beta germ-line transcription and the function of the T beta transcripts.

Amino Acid Sequence↗
PubMed1990

Natural killer cells carry the germ-line configuration of the T cell receptor delta chain gene and heterogeneously express six distinct delta transcripts.

Investigations on T cell receptor delta chain (TcR delta) gene expression in freshly isolated normal large granular lymphocytes, natural killer (NK) cell clones and NK bulk cultures revealed six T delta transcripts (3.5, 3.1, 2.2, 2.0, 1.5 and 1.3 kb) which varied in number and relative abundance in the different samples. None of the six known T delta variable regions was expressed and the T delta locus was retained in its germ-line configuration. The origin and significance of these NK-associated T delta transcripts are discussed.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1990

Molecular studies on LAK cells.

We have developed a culture system for long-term growth of human LAK cells exhibiting an elevated, wide-spectrum anti-tumor cytotoxicity. The phenotypic and molecular properties of the final LAK cell populations indicated that they consist of three main types: a) NK-like lymphocytes (type I): NKH-1+, Ti alpha/beta-, Ti gamma/delta-, CD3-lymphocytes carrying the germline configuration of all TCR genes and expressing variable amount of the 1.0 beta mRNA and variably sized T delta transcripts; b) gamma/delta-like T lymphocytes (type II) NKH-1+, Ti alpha/beta-, Ti gamma/delta+, CD3+ lymphocytes carrying polyclonal rearrangements of the gamma and delta genes and expressing high levels of mature gamma and delta transcripts; c) alpha/beta-like T lymphocytes (type III): NKH-1+, Ti alpha/beta+, Ti gamma/delta-, CD3+ lymphocytes carrying rearrangements of all TCR genes and expressing high levels of mature alpha and beta transcripts. We took advantage of the high number of available LAK cells to clarify: 1) the origin of the NK-LAK delta transcripts. delta gene expression in LGL, NK clones and type I LAK cultures revealed six delta transcripts (3.5, 3.1, 2.2, 2.0, 1.5 and 1.3 kb), which varied in number and relative abundance in the different samples. None of the six known V delta was expressed and the delta locus was retained in its germline configuration suggesting that the delta expression is due to a partially rearranged or germline transcripts; 2) the origin of the NK-LAK truncated T beta transcript. We isolated two different clone types from a type I LAK cell cDNA library: a) J-C clones consisting of one of three J beta regions and the corresponding C beta 1 or C beta 2 regions; b) X-J-C and C-X clones, containing as yet unidentified (X) sequences. The presence of RSSs in J-C clones suggests that they derive from mRNAs transcribed from a promoter in the 5'J. Nucleotide analysis demonstrated that only one of the isolated clones had the potential to code a short T beta protein.

Amino Acid Sequence↗
PubMed1990

Interleukin 2 in cancer therapy.

The goal of adoptive immunotherapy is the stimulation of host antitumor immunity, either cellular or humoral. The introduction of recombinant human cytokines into clinical oncology represents a new useful tool for the development of new strategies of antitumor immunotherapy. This article describes the current clinical status of IL-2 in the therapy of human cancer as it relates to what is already known about the biology and activity of this lymphokine. All the evidences suggest that the antitumor effect of IL-2 seems to be mediated through complex indirect mechanisms involving different effector cells of the immune system.

Adult↗
PubMed1989

Differential expression of human HOX-2 genes along the anterior-posterior axis in embryonic central nervous system.

We have investigated the structure of the human HOX-2 locus, which encompasses a 90-kb region on chromosome 17q21. Five new human HOX-2 homeoboxes, termed HOX-2.5, 2.4, 2.6, 2.7 and 2.8, have been identified, and their nucleotide sequences are reported. They have the same 5'-3' transcriptional orientation and are clustered with three previously described HOX-2 homeoboxes (5'-2.5-2.4-2.3-2.2-2.1-2.6-2.7-2.8-3'). We have also investigated the region-specific expression of HOX-2 genes in human embryonic-fetal life by Northern-blot analysis. All genes are expressed in whole embryos and fetuses at 5-9 weeks from conception. Their major site of expression lies within the central nervous system (CNS), although they are transcribed at a lower level in body structures other than the CNS. Their relatively abundant expression in CNS has been analyzed along the anterior-posterior axis by dissecting the brain, the medulla oblongata and the spinal cord proper. HOX-2.5, 2.4 and 2.3 transcripts are markedly more abundant in spinal cord than in medulla, whereas 2.2, 2.1, 2.6 and 2.7 mRNAs are progressively more abundant in the medulla. Additionally, expression in brain was detected, although at lower level, for HOX-2.1, 2.6, 2.7, 2.8. Thus, the relative position of HOX-2 homeobox genes along the chromosome in the 5'-3' direction appears to correlate with the relative position of their expression domains along the CNS longitudinal axis in the caudal-cephalic direction.

Amino Acid Sequence↗