ATG-induced accelerated immune senescence: clinical implications in renal transplant recipients.

Persistent ATG-induced CD4(+) T cell lymphopenia is associated with serious clinical complications. We tested the hypothesis that ATG induces accelerated immune senescence in renal transplant recipients (RTR). Immune senescence biomarkers were analyzed at transplant and one-year later in 97 incident RTR -62 patients receiving ATG and 35 receiving anti-CD25 mAb (α-CD25). This consisted in: (i) thymic output; (ii) bone marrow renewal of CD34(+) hematopoietic progenitor cells (CD34(+) HPC) and lymphoid (l-HPC) and myeloid (m-HPC) progenitor ratio; (iii) T cell phenotype; and (iv) measurement of T cell relative telomere length (RTL) and telomerase activity (RTA). Clinical correlates were analyzed with a 3 year follow-up. Thymic output significantly decreased one-year posttransplant in ATG-treated patients. ATG was associated with a significant decrease in l-HPC/m-HPC ratio. Late stage differentiated CD57(+) /CD28(-) T cells increased in ATG-treated patients. One-year posttransplant T cell RTL and RTA were consequently lower in ATG-treated patients. ATG is associated with accelerated immune senescence. Increased frequency of late differentiated CD4(+) T cell frequency at transplantation tended to be predictive of a higher risk of subsequent opportunistic infections and of acute rejection only in ATG-treated patients but this needs confirmation. Considering pretransplant immune profile may help to select those patients who may benefit from ATG to prevent severe infections and acute rejection.

[Stem cell biology and therapeutic hopes: forbidden game?]. / Biologie des cellules souches et perspectives thérapeutiques: enjeux interdits?

Stem cell biology is one of the most exciting subjects in life science nowadays. The major point in stem cell biology is the extraordinary capacity of these cells to self-renew and to give rise to different cell types. Nevertheless, major issues remain to be cleared and very few diseases can actually be cured based on stem cell therapy. Adult stem cells remain difficult to locate, isolate and amplify in a homogeneous fashion and, thus, limit their therapeutic application in clinical trial. Embryonic stem cells could represent a new hope in stem cell therapy but in addition to the scientific difficulties, over ethical and judiciary issues should be addressed. In order to cure routinely patients, controlled conditions for stem cell isolation, amplification, differentiation, and administration must be defined and effective tissue integration have to be established. In this review we will discuss these different aspects of stem cell biology.

Cloning and developmental expression of STAT5 in Xenopus laevis.

Transcription factors of the signal transducer and activator of transcription (STAT) family are required for cellular responses to multiple signalling molecules. After ligand binding-induced activation of cognate receptors, STAT proteins are phosphorylated, hetero- or homodimerize, and enter the nucleus. STAT dimers bind to specific DNA elements and alter the transcriptional activity of the signal-responsive genes. We report the cloning and developmental pattern of expression of XSTAT5, a Xenopus laevis member of the STAT family, closely related to the mammalian STAT5A and STAT5B. XSTAT5 is expressed maternally and zygotically. With the onset of neurulation, XSTAT5 RNA are clearly localized in the anterior neural plate and subsequently in the neural structures of the developing eye, the pineal gland and the cement gland anlage. At late tailbud stages, a faint expression is detected in a ventral location that might correspond to the ventral blood islands.

Outpatient cardiac infusion units: impact in elderly patients with refractory heart failure.

BACKGROUND: The use of intermittent intravenous infusions of inotropic drugs is under evaluation in the management of patients with refractory heart failure. OBJECTIVES: We investigated the impact of intermittent outpatient infusions of inotropes on hospital admissions, emergency room visits, functional class, and symptom-free interval after administration of inotropes to elderly patients with advanced heart failure symptoms. METHODS: This retrospective analysis involved 24 elderly outpatients with a New York Heart Association class of III or IV and refractory heart failure symptoms. RESULTS: Seven patients with class III heart failure (age 74A+/-4 years; left ventricular ejection fraction 27A+/-9%) and 17 patients of class IV (age 73A+/-4 years; LVEF 21A+/-10%) were included. Twenty patients were males. A total of 365 outpatient treatment sessions were administered, with 15A+/-9 sessions per patient (range, 6-43). Eleven patients improved and were discharged; seven patients died; three discontinued treatment; two patients remain on therapy; and one patient required continuous infusion. During this treatment, there were only three emergency room visits and six hospital admissions due solely to heart failure. Fourteen patients required no emergency room visits or hospitalization. Of the patients discharged from the program, the interval without heart failure symptoms ranged from 60-356 days, with an improvement in NYHA class from 3.5A+/-0.6 to 1.4A+/-0.5 and no emergency room visits or hospital admissions. CONCLUSIONS: This type of therapy is well tolerated among elderly patients with refractory heart failure symptoms and its use deserves further investigation.

Mechanically strained cells of the osteoblast lineage organize their extracellular matrix through unique sites of alphavbeta3-integrin expression.

Bone cells transduce mechanical signals into anabolic biochemical responses. However, the mechanisms of mechanotransduction are unknown. To address this issue, we performed studies in primary cells of the human osteoblast lineage grown on collagen/vitronectin-coated supports. We discovered that mechanical strain stimulated a redistribution of the alphavbeta3-integrin to irregular plaque-like areas at the cell-extracellular matrix surface. Proteins involved in integrin-matrix interactions in focal adhesions, vinculin and talin, did not localize to the plaque-like areas of alphavbeta3-expression, but signaling molecules such as focal adhesion kinase (FAK) did. Mechanical strain increased the number and size of the plaques defined by surface expression of alphavbeta3-integrin. Osteopontin was secreted as a cross-linked macromolecular complex, likely through the action of tissue transglutaminase that also was found in the plaques of alphavbeta3-integrin cell-matrix interaction. Mechanical strain increased mineralization of the extracellular matrix that developed in these plaques in alphavbeta3-integrin-dependent manner. Because the plaque-like areas of cell-matrix interaction exhibit macromolecular assembly and mineralization, we conclude that they may represent subcellular domains of bone formation and that alphavbeta3-integrin activation represents one mechanism by which mechanical strain stimulates bone formation.

Intermittent infusion of inotropes in the outpatient setting: one-year post treatment analysis.

The course of patients with New York Heart Association (NYHA) class III and IV and refractory heart failure symptoms is characterized by progressive clinical deterioration and frequent hospital readmissions. The value of intermittent intravenous administration of inotropes in managing this group of patients in the outpatient setting has been controversial. In this study, patients with refractory heart failure symptoms were enrolled to assess the impact of a multidisciplinary outpatient program in terms of on hospital admissions, emergency room visits, and interval free of symptoms after administration of inotropes. This is a retrospective analysis on 41 patients with refractory heart failure treated at our outpatient cardiac infusion unit over a 20 month period. Thirteen patients with a NYHA class III [age 64 +/- 13; LVEF 27 +/- 9%] and 28 patients with a NYHA class IV [age 65 +/- 13 years; LVEF 21 +/- 9%], mostly males, were included. A total of 65 admissions for decompensated HF were recorded in the previous 6-months prior to initiation of the outpatient program; compared to only 4 emergency room visits and 7 hospital admissions after enrollment. Furthermore, 17 patients have been discharged with improvement in NYHA class from 3.5 +/- 0.6 to 1.4 +/- 0.5. On these patients, the interval free of symptoms since the last infusion treatment has ranged from 201 to 489 days, without emergency room visits or hospital admissions for congestive heart failure. The results of this study support the use of intermittent infusion of inotropes in the outpatient setting. Although the natural history for patients with refractory heart failure has been grim; the use of these intermittent infusions may in fact alter the natural course of end stage congestive heart failure patients and deserves further investigation.

Peptidomimetic antagonists of alphavbeta3 inhibit bone resorption by inhibiting osteoclast bone resorptive activity, not osteoclast adhesion to bone.

Osteoclasts are actively motile on bone surfaces and undergo alternating cycles of migration and resorption. Osteoclast interaction with the extracellular matrix plays a key role in the osteoclast resorptive process and a substantial body of evidence suggests that integrin receptors are important in osteoclast function. These integrin receptors bind to the Arg-Gly-Asp (RGD) sequence found in a variety of extracellular matrix proteins and it is well established that the interaction of osteoclast alpha v beta 3 integrin with the RGD motif within bone matrix proteins is important in osteoclast-mediated bone resorption. In this study, we characterized the effects of two synthetic peptidomimetic antagonists of alpha v beta 3, SC-56631 and SC-65811, on rabbit osteoclast adhesion to purified matrix proteins and bone, and on bone resorption in vitro. SC-56631 and SC-65811 are potent inhibitors of vitronectin binding to purified alpha v beta 3. Both SC-56631 and SC-65811 inhibited osteoclast adhesion to osteopontin- and vitronectin-coated surfaces and time-lapse video microscopy showed that osteoclasts rapidly retract from osteopontin-coated surfaces when exposed to SC-56631 and SC-65811. SC-56631 and SC-65811 blocked osteoclast-mediated bone resorption in a dose-responsive manner. Further analysis showed that SC-65811 and SC-56631 reduced the number of resorption pits produced per osteoclast and the average pit size. SC-65811 was a more potent inhibitor of bone resorption and the combination of reduced pit number and size led to a 90% inhibition of bone resorption. Surprisingly, however, osteoclasts treated with SC-65811, SC-56631 or the disintegrin echistatin, at concentrations that inhibit bone resorption did not inhibit osteoclast adhesion to bone. These results suggest that alphavbeta3 antagonists inhibited bone resorption by decreasing osteoclast bone resorptive activity or efficiency but not by inhibiting osteoclast adhesion to bone per se.

TEL-JAK2 transgenic mice develop T-cell leukemia.

We previously reported a fusion between TEL and JAK2 in a t(9;12)(p24;p13) chromosomal translocation in childhood acute T-cell leukemia. This fusion gene encodes a TEL-JAK2 chimeric protein in which the 336 amino-terminal residues of TEL, including its specific self-association domain, are fused to the kinase domain of JAK2. TEL-JAK2 exhibits constitutive activation of its tyrosine kinase activity which, in turn, confers growth factor-independent proliferation to the interleukin-3-dependent Ba/F3 hematopoietic cell line. To elucidate the properties of TEL-JAK2 in primary cells and to create an animal model for TEL-JAK2-induced leukemia, we generated transgenic mice in which the TEL-JAK2 complementary DNA was placed under the transcriptional control of the EmuSRalpha enhancer/promoter. TEL-JAK2 founder mice and their transgenic progeny developed fatal leukemia at 4 to 22 weeks of age. Selective amplification of CD8-positive T cells was observed in blood, lymph nodes, thymus, spleen, and bone marrow. Expression of a tyrosine-phosphorylated TEL-JAK2 protein and activation of STAT1 and STAT5 (signal transducer and activator of transcription) were detected in leukemic tissues. TEL-JAK2 diseased mice also displayed invasion of nonhematopoietic organs, including liver, brain, lung, and kidney, by leukemic T cells. Leukemic organs of founder and transgenic progeny contained a monoclonal/oligoclonal T-cell population as analyzed by the rearrangement of the TCRbeta locus. Transplantation of TEL-JAK2 leukemic cells in nude mice confirmed their invasive nature. We conclude that the TEL-JAK2 fusion is an oncogene in vivo and that its expression in lymphoid cells results in the preferential expansion of CD8-positive T cells. (Blood. 2000;95:3891-3899)

TEL is a sequence-specific transcriptional repressor.

TEL is a gene frequently involved in specific chromosomal translocations in human leukemia and sarcoma that encodes a member of the ETS family of transcriptional regulators. TEL is unusual among other ETS proteins by its ability to self-associate in vivo, a property that is essential to the oncogenic activation of TEL-derived fusion proteins. We show here that TEL is a sequence-specific transcriptional repressor of ETS-binding site-driven transcription of model and natural promoters. Deletion of the oligomerization domain of TEL or its substitution by the homologous region of monomeric ETS1 impaired the ability of TEL to repress. In contrast, substitution of the oligomerization domain of TEL by unrelated oligomerization domains resulted in an active repressor, showing that the ability of TEL to repress depends on its ability to self-associate. The study of the properties of TEL fusions to the heterologous DNA binding domain of Gal4 identified two autonomous repression domains in TEL, distinct from its oligomerization domain, that are essential to the ability of TEL to repress ETS-binding site-containing promoters. These results have implications for the normal function of TEL, its relation to other ETS proteins, and its role in leukemogenesis.

Characteristics of cation binding to the I domains of LFA-1 and MAC-1. The LFA-1 I domain contains a Ca2+-binding site.

The crystal structures of the I domains of integrins MAC-1 (alphaM beta2; CD11b/CD18) and LFA-1 (alphaL beta2; CD11a/CD18) show that a single conserved cation-binding site is present in each protein. Purified recombinant I domains have intrinsic ligand binding activity, and in several systems this interaction has been demonstrated to be cation-dependent. It has been proposed that the I domain cation-binding site represents a general metal ion-dependent adhesion motif utilized for binding protein ligands. Here we show that the purified recombinant I domain of LFA-1 (alphaLI) binds cations, but with significantly different characteristics compared with the I domain of MAC-1 (alphaMI). Both alphaLI and alphaMI bind 54Mn2+ in a conformation-dependent manner, and in general, cations with charge and size characteristics similar to Mn2+ most effectively inhibit 54Mn2+ binding. Surprisingly, however, physiological levels of Ca2+ (1-2 mM) inhibited 54Mn2+ binding to purified alphaLI, but not to alphaMI. Using 45Ca2+ and 54Mn2+ in direct binding studies, the dissociation constants (KD) for the interactions between these cations and alphaLI were estimated to be 5-6 x 10(-5) and 1-2 x 10(-5) M, respectively. Together with the available structural information, the data suggest differential affinities for Mn2+ and Ca2+ binding to the single conserved site within alphaLI. Antagonism of LFA-1, but not MAC-1, -mediated cell adhesion by Ca2+ may be related to the Ca2+ binding activity of the LFA-1 I domain.

A peptidomimetic antagonist of the integrin alpha(v)beta3 inhibits Leydig cell tumor growth and the development of hypercalcemia of malignancy.

The integrin alpha(v)beta3 interacts with the arginine-glycine-aspartic acid (RGD) tripeptide recognition sequence of a variety of extracellular matrix proteins. Recent studies show that alpha(v)beta3 plays an important role in tumor-induced angiogenesis and tumor growth and that antagonists of alpha(v)beta3 inhibit angiogenic processes that include endothelial cell adhesion and migration. Consequently, we reasoned that an RGD-based peptidomimetic antagonist of alpha(v)beta3 might inhibit tumor angiogenesis and tumor growth in vivo. An RGD-peptidomimetic library was screened to identify antagonists of vitronectin binding to alpha(v)beta3, and the compounds chosen were modified to produce selective and potent inhibitors of alpha(v)beta3. One of these compounds, beta-[[2-2-[[[3-[(aminoiminomethyl)amino]-phenyl]carbonyl]amino]ac etyl]amino]-3,5-dichlorobenzenepropanoic acid (SC-68448), inhibited vitronectin binding to both alpha(v)beta3 and the closely related platelet receptor, alpha(IIb)beta3, in a dose-responsive manner. SC-68448 inhibited vitronectin binding to alpha(v)beta3 (IC50, 1 nM) and fibrinogen binding to the platelet receptor alpha(IIb)beta3 (IC50, >100 nM), demonstrating that SC-68448 was 100-fold more potent as an inhibitor of alpha(v)beta3 versus alpha(IIb)beta3. In cell-based studies, SC-68448 inhibited alpha(v)beta3-mediated endothelial cell proliferation in a dose-dependent manner but did not inhibit tumor cell proliferation, suggesting that effects on endothelial cell proliferation were not due to SC-68448-induced cytotoxicity. In accord with these results, SC-68448 inhibited angiogenesis in vivo in a basic fibroblast growth factor-induced rat corneal neovascularization model. A xenogeneic severe combined immune deficiency mouse/rat Leydig cell tumor model was developed for testing SC-68448 as an inhibitor of tumor growth in vivo. Rat Leydig cell tumors grew rapidly in severe combined immune deficiency mice and produced humoral hypercalcemia of malignancy. SC-68448 inhibited the growth of the tumors in mice by up to 80% and completely blocked the development of hypercalcemia. Together, these results demonstrate the feasibility of antitumor therapies based upon the development of nontoxic small molecule pharmacological antagonists of integrin alpha(v)beta3.

A series of function blocking antibodies against the alpha v beta 3 integrin bind allosteric to the ligand binding site and induce ligand dissociation.

The alpha v beta 3 integrin plays a critical role in bone resorption, angiogenesis, and tumor cell invasion. A blockade of this receptor has therapeutic potential in osteoporosis, vascular restenosis, and cancer. In this report, we characterize the mechanism by which six monoclonal antibodies inhibit the function of alpha v beta 3. All six antibodies interact with a common site that is partially comprised of residues 164-202 within the beta 3 subunit. This domain is physically separate from the RGD binding site, and appears to regulate ligand binding allosterically. Thus, the blocking antibodies function, in part, by inducing the dissociation of ligand from alpha v beta 3. Although this family of antibodies is able to virtually abolish alpha v beta 3-mediated cell adhesion, they only block about one-half of soluble ligand binding to the integrin. This observation is consistent with the idea that two functionally distinct populations of alpha v beta 3 are present on the cell surface. The unique mechanism of action of these antibodies provides new insight in the structure-function relationships of alpha v beta 3, and also suggest that such antibodies are likely to behave differently than RGD mimetics if used as drugs.

Four new clerodane diterpenes from the leaves of Casearia guianensis which inhibit the interaction of leukocyte function antigen 1 with intercellular adhesion molecule 1.

Four new clerodane diterpenes, casearinols A and B (1 and 2) and casearinones A and B (3 and 4), were isolated from the leaves of Casearia guianensis. These immunomodulatory compounds have been structurally elucidated primarily on the basis of 2D NMR analysis and spectral data comparison with known compounds. These compounds inhibited the binding of T-cell leukocyte function antigen 1 to intercellular adhesion molecule 1.

Identification of a region in the integrin beta3 subunit that confers ligand binding specificity.

Many integrin adhesion receptors bind ligands containing the Arg-Gly-Asp (RGD) peptide motif. Most integrins exhibit considerable specificity for particular ligands and can distinguish among the many conformations of RGD. In this study we identify the domain of the integrin beta subunit involved in determining ligand binding specificity. Chimeras of beta3 and beta5, the most homologous integrin beta subunits, were expressed with alphav on the surface of human 293 cells. The ligand binding phenotype of each chimera was assessed using the ligands Fab-9 and fibrinogen, both of which have a binding preference for alphavbeta3. The results of the study show that when exons C and D of the beta3 subunit (residues 95-233) are substituted into beta5, the chimera gained the ability to bind Fab-9 with an affinity close to that of wild-type alphavbeta3. This chimera was able to mediate cell adhesion to fibrinogen. Furthermore, the swap of only a 39-residue segment of this larger domain, beta3 residues 164-202, into the backbone of beta5 enabled the chimeric integrin to bind soluble Fab-9. This small domain is highly divergent among the integrin beta subunits, suggesting that it may play a role in determining ligand selection by all integrins.

Low affinity of cell surface lymphocyte function-associated antigen-1 (LFA-1) generates selectivity for cell-cell interactions.

We examined binding of soluble intercellular adhesion molecule-1 (ICAM-1) dimers and a range of ICAM-1-coated particles to activated T cells. Lymphocyte function-associated antigen-1 (LFA-1) on the surface of activated T cells did not bind soluble ICAM-1 dimers with high affinity. In contrast, activated T cells adhered avidly to ICAM-1-coated planar surfaces. Between these two extremes, a range of ICAM-1-bearing particles was tested for binding. Activated T cells bound particles of 1-microm diameter or larger, but did not bind particles of 0.5-microm diameter or smaller. This threshold was eliminated, and all forms of ICAM-1 bound to LFA-1 when LFA-1 was converted to a high affinity form with an activating antibody. We show that high affinity LFA-1 is generated only as a consequence of an initial low affinity interaction of LFA-1 with ICAM-1 under physiological conditions. Therefore, the selectivity of cell surface LFA-1 for cell-sized particles bearing ICAM-1 appears to be due to the maintenance of low affinity LFA-1 on the surface of activated T cells. These findings alter the definition of inside-out signaling for LFA-1.

The TEL gene products: nuclear phosphoproteins with DNA binding properties.

The human TEL gene is involved in several 12p13 chromosomal abnormalities present in various human hematological malignancies, the most frequent being the t(12;21)(p13;q22), specific for childhood acute lymphoblastic leukemia. The predicted product of TEL harbours an amino acid region similar to the ETS DNA binding domain. We now report the isolation of the murine TEL cDNA and the characterization of the human TEL proteins. Human and murine TEL proteins are particularly homologous within their aminoterminal regions and their ETS domains. TEL proteins are nuclear and display specific DNA binding activity toward classical ETS binding sites. In addition, we show that TEL mRNAs initiate translation at either of the two first inframe ATGs (codon 1 and 43) to encode 50 kDa and 57 kDa TEL proteins. In vivo, each of these primary translational products is modified by multiple phosphorylation events.

A domain of TEL conserved in a subset of ETS proteins defines a specific oligomerization interface essential to the mitogenic properties of the TEL-PDGFR beta oncoprotein.

TEL is a novel member of the ETS family of transcriptional regulators which is frequently involved in human leukemias as the result of specific chromosomal translocations. We show here by co-immunoprecipitation and GST chromatography analyses that TEL and TEL-derived fusion proteins form homotypic oligomers in vitro and in vivo. Deletion mutagenesis identifies the TEL oligomerization domain as a 65 amino acid region which is conserved in a subset of the ETS proteins including ETS-1, ETS-2, FLI-1, ERG-2 and GABP alpha in vertebrates and PNTP2, YAN and ELG in Drosophila. TEL-induced oligomerization is shown to be essential for the constitutive activation of the protein kinase activity and mitogenic properties of TEL-platelet derived growth factor receptor beta (PDGFR beta), a fusion oncoprotein characteristic of the leukemic cells of chronic myelomonocytic leukemia harboring a t(5;12) chromosomal translocation. Swapping experiments in which the TEL oligomerization domain was exchanged by the homologous domains of representative vertebrate ETS proteins including ETS-1, ERG-2 and GABP alpha show that oligomerization is a specific property of the TEL amino-terminal conserved domain. These results indicate that the amino-terminal domain conserved in a subset of the ETS proteins has evolved to generate a specialized protein-protein interaction interface which is likely to be an important determinant of their specificity as transcriptional regulators.

Intercellular adhesion molecule-1 dimerization and its consequences for adhesion mediated by lymphocyte function associated-1.

Intercellular adhesion molecule-1 (ICAM-1, CD54) is a ligand for the integrins lymphocyte function associated-1 (LFA-1, CD11a/CD18) and complement receptor-3 (Mac-1, CD11b/CD18) making it an important participant in many immune and inflammatory processes. Modified recombinant soluble ICAM-1 formed dimers. This result indicated that the ectodomain of ICAM-1 contains homophilic interaction sites. Soluble ICAM-1 dimers bind to solid-phase purified LFA-1 with high avidity (dissociation constant [Kd] = 8 nM) in contrast to soluble ICAM-1 monomers whose binding was not measurable. Cell surface ICAM-1 was found to be dimeric based on two distinct criteria. First, a monoclonal antibody specific for monomeric soluble ICAM-1, CA7, binds normal ICAM-1 poorly at the cell surface; this antibody, however, binds strongly to two mutant forms of ICAM-1 when expressed at the cell surface, thus identifying elements required for dimer formation. Second, chemical cross-linking of cell surface ICAM-1 on transfected cells and tumor necrosis factor-activated endothelial cells results in conversion of a portion of ICAM-1 to a covalent dimer. Cell surface ICAM-1 dimers are more potent ligands for LFA-1-dependent adhesion than ICAM-1 monomers. While many extracellular matrix-associated ligands of integrins are multimeric, this is the first evidence of specific, functionally important homodimerization of a cell surface integrin ligand.