Differential effects of interleukin-15 and interleukin-2 on differentiation of bipotential T/natural killer progenitor cells.

Bipotential T/natural killer (NK) progenitor cells are destined to differentiate mainly into T cell receptor (TCR) alpha beta and TCR gamma delta cells in a thymic microenvironment, whereas extrathymically they selectively develop into NK cells. The exact environmental conditions that are required for differentiation into these three leukocyte populations are largely unknown. In this report, we have investigated and compared the effect of interleukin (IL)-15 and IL-2 in this process. The IL-15 receptor is composed of the gamma and beta chains of the IL-2 receptor (IL-2R gamma and IL-2R beta) and of a specific alpha chain (IL-15R alpha). Here, it is shown that IL-15 mRNA is mainly expressed in thymic epithelial stromal cells, whereas IL-2 mRNA is exclusively expressed in thymocytes. IL-2R beta-expressing cells were present in the fetal thymus with a CD25-CD44+Fc gamma R+HSA-/low TCR- phenotype, which is characteristic of progenitor cells. These cells also expressed IL-15R alpha messenger RNA. Sorted IL-2R beta + TCR- cells differentiated into TCR alpha beta and TCR gamma delta cells after transfer to alymphoid thymic lobes, whereas culture of the same sorted cells in cell suspension in the presence of IL-15 resulted in the generation of functional NK cells. This shows that IL-2R beta +TCR- cells of the fetal thymus contain bipotential T/NK progenitors. Addition of low concentrations of IL-15 to fetal thymic organ culture (FTOC) resulted in an increase of all T cell subpopulations. The largest expansion occurred in the TCR gamma delta compartment. In contrast, low concentrations of IL-2 did not result in a higher total cell number and did not induce outgrowth of TCR gamma delta cells. High concentrations of IL-15 blocked TCR alpha beta development and shifted differentiation towards NK cells. Differentiation towards TCR gamma delta cells still proceeded. High concentrations of IL-2 similarly induced development into NK cells, but the cell number was fourfold lower than in IL-15 cultures. Importantly, blocking of IL-2R alpha in IL-2-treated FTOC resulted in a drastic increase in cell number, indicating that IL-2R alpha negatively regulates cell expansion. Collectively, these experiments provide direct evidence that IL-15 and IL-2 differentially affect the differentiation of bipotential T/NK progenitors.

Intrathymic differentiation of V gamma 3 T cells.

Whereas there is considerable information on the phenotypic and functional maturation of T cell receptor (TCR) alpha/beta thymocytes, comparatively little is known of the maturational processes that affect development of TCR-gamma/delta thymocytes. One class of gamma/delta T cells, those bearing the V gamma 3 gene product, are generated only during the early fetal stages of thymic development, and then migrate to the skin. Here we examine the intrathymic differentiation of these V gamma 3+ cells. The earliest V gamma 3 cells to appear in the thymus expressed low levels of TCR (V gamma 3low) and high levels of heat stable antigen (HSA). Over the next few days, V gamma 3+ thymocytes appeared which expressed high levels of TCR (V gamma 3high) and very low levels of HSA. The antigens CD5, CD45RB, and MEL14 were also differentially expressed on V gamma 3low versus V gamma 3high thymocytes, but the shift in expression was the opposite as compared with immature and mature TCR-alpha/beta thymocytes. Transfer experiments of sorted V gamma 3low/HSAhigh thymocytes to SCID thymic lobes showed that these cells were indeed the precursors of V gamma 3high/HSAlow thymocytes. The phenotype of the V gamma 3high thymocytes was similar to that of the postthymic V gamma 3+ cells found in the skin of adult mice. The differentiation of V gamma 3low in V gamma 3high thymocytes was also observed in fetal thymic organ culture. Addition of cyclosporin A (CsA) to these cultures had little effect on the appearance of V gamma 3low/HSAhigh cells, but blocked the appearance of V gamma 3high/HSAlow cells. These results show that, like alpha/beta T cells, V gamma 3+ thymocytes differentiate from TCRlow precursors to cells with a mature phenotype and that CsA inhibits this transition.

T-, B- and NK-lymphoid, but not myeloid cells arise from human CD34(+)CD38(-)CD7(+) common lymphoid progenitors expressing lymphoid-specific genes.

Hematopoietic stem cells in the bone marrow (BM) give rise to all blood cells. According to the classic model of hematopoiesis, the differentiation paths leading to the myeloid and lymphoid lineages segregate early. A candidate 'common lymphoid progenitor' (CLP) has been isolated from CD34(+)CD38(-) human cord blood cells based on CD7 expression. Here, we confirm the B- and NK-differentiation potential of CD34(+)CD38(-)CD7(+) cells and show in addition that this population has strong capacity to differentiate into T cells. As CD34(+)CD38(-)CD7(+) cells are virtually devoid of myeloid differentiation potential, these cells represent true CLPs. To unravel the molecular mechanisms underlying lymphoid commitment, we performed genome-wide gene expression profiling on sorted CD34(+)CD38(-)CD7(+) and CD34(+)CD38(-)CD7(-) cells. Interestingly, lymphoid-affiliated genes were mainly upregulated in the CD7(+) population, while myeloid-specific genes were downregulated. This supports the hypothesis that lineage commitment is accompanied by the shutdown of inappropriate gene expression and the upregulation of lineage-specific genes. In addition, we identified several highly expressed genes that have not been described in hematopoiesis before.

Homeobox gene expression profile in human hematopoietic multipotent stem cells and T-cell progenitors: implications for human T-cell development.

Class I homeobox (HOX) genes comprise a large family of transcription factors that have been implicated in normal and malignant hematopoiesis. However, data on their expression or function during T-cell development is limited. Using degenerated RT-PCR and Affymetrix microarray analysis, we analyzed the expression pattern of this gene family in human multipotent stem cells from fetal liver (FL) and adult bone marrow (ABM), and in T-cell progenitors from child thymus. We show that FL and ABM stem cells are similar in terms of HOX gene expression, but significant differences were observed between these two cell types and child thymocytes. As the most immature thymocytes are derived from immigrated FL and ABM stem cells, this indicates a drastic change in HOX gene expression upon entry into the thymus. Further analysis of HOX-A7, HOX-A9, HOX-A10, and HOX-A11 expression with specific RT-PCR in all thymocyte differentiation stages showed a sequential loss of 3' region HOX-A cluster genes during intrathymic T-cell development and an unexpected expression of HOX-A11, previously not recognized to play a role in hematopoiesis. Also HOX-B3 and HOX-C4 were expressed throughout thymocyte development. Overall, these data provide novel evidence for an important role of certain HOX genes in human T-cell development.

Developmentally regulated responsiveness to transforming growth factor-beta is correlated with functional differences between human adult and fetal primitive hematopoietic progenitor cells.

Important functional differences exist between primitive CD34++ CD38- hematopoietic progenitor cells derived from human fetal liver (FL) and adult bone marrow (ABM). FL progenitors are known to have higher proliferative capacities and lower cytokine requirements than their ABM counterparts. In this study, we isolated FL and ABM CD34++ CD38- cells and used a two-stage culture system to investigate the effects of transforming growth factor-beta (TGF-beta) and blocking anti-TGF-beta antibodies (anti-TGF-beta) on these cells. First, we demonstrate that FL progenitors are significantly less sensitive to the inhibitory effects of TGF-beta than ABM cells. Second, whereas ABM cells are significantly stimulated by anti-TGF-beta, only very limited effects are seen on FL cells. Third, we show that the effect of anti-TGF-beta is mainly situated at the level of the initial cell cycles of very primitive progenitor cells with a high proliferation potential. Fourth, we demonstrate that blocking the effects of endogenous TGF-beta reduces the growth factor requirements of ABM cells in order to proliferate and differentiate. Based on these data, we hypothesize that at least part of the functional differences that exist between adult and fetal stem cells can be accounted for by a developmental different responsiveness to TGF-beta.

CD34++ CD38- and CD34+ CD38+ human hematopoietic progenitors from fetal liver, cord blood, and adult bone marrow respond differently to hematopoietic cytokines depending on the ontogenic source.

CD34++ CD38- and CD34+ CD38+ hematopoietic progenitor cells (HPCs) from human fetal liver (FL), cord blood (CB), and adult bone marrow (ABM) were isolated and investigated for their growth characteristics, cytokine requirements and response to two modulators of early hematopoiesis, interferon (IFN)-gamma and macrophage inflammatory protein (MIP)-1alpha. We observed first that a significantly lower percentage of CD34++ cells were CD38- in ABM than in FL and CB. Second, the functional differences between CD34++ CD38- and CD34+ CD38+ cells were less pronounced in FL and CB than in their ABM counterparts. Third, an inverse correlation was found between growth factor response and the ontogenic age of HPCs, and a direct correlation was noted between cytokine requirements and the ontogenic age of HPCs. Fourth, spontaneous colony formation in a classic semisolid culture system was reproducibly obtained only in the ontogenically earliest cells, that is, in FL but not in CB and ABM, in which no such spontaneous colony formation was observed. Fifth, the modulatory effects of IFN-gamma and MIP-1alpha were qualitatively different depending on the ontogenic age of the progenitor source: whereas IFN-gamma was only a selective inhibitor of primitive CD34++ CD38- ABM progenitor cells, it inhibited both CD34++ CD38- and CD34+ CD38+ FL and CB cells to the same extent. In contrast to the effects of MIP-1alpha on ABM, we could not find any consistently stimulatory or inhibitory effects on FL and CB progenitors. In conclusion, important functional and biologic differences exist between FL, CB, and ABM progenitor cells, and these differences could have major implications for the use of these cell populations in preparative protocols of ex vivo expansion, transplantation strategies, or gene transfer experiments.

Detection of IL-2 receptor positive tonsillar lymphocytes by monoclonal antibody and protein A coated erythrocytes.

A sensitive rosette method combining staphylococcal protein A coated rabbit red blood cells and the monoclonal antibody anti-Tac was used to search for the presence of interleukin 2 (IL-2) receptor-bearing T lymphocytes in tonsils from patients with chronic tonsillitis. This method revealed the presence of Tac positive T lymphocytes in the tonsils whereas a conventional immunofluorescence technique was unable to do so. To demonstrate that Tac rosette formation resulted in a selective enrichment of IL-2 receptor-bearing T cells, we measured the proliferative response of these cells to exogenous IL-2. The response of Tac rosette positive cells to recombinant IL-2 was always higher than that of the Tac rosette negative or unselected cells, indicating that this rosette method specifically selects T cells expressing IL-2 receptor.

Interleukin-2 stimulated T cell receptor V gamma 3 positive thymocytes do not migrate to the skin.

T cell receptor (TcR) V gamma 3+ thymocytes, which only develop in the fetal thymus, migrate to the skin. IL-2 stimulation of fetal day 18 murine thymocytes results in a cell population of which 45% of the cells express the TcR V gamma 3. In this study, we describe that those IL-2 cultured TcR V gamma 3+ thymocytes have the killing capacity of lymphokine activated killer cells: NK-susceptible as well as NK-resistant tumor cell lines were killed in an MHC-unrestricted manner. Because of these findings, IL-2-expanded TcR V gamma 3+ thymocytes could have a potential use in adoptive immunotherapy for skin-located tumors. Therefore, we analyzed the migration pattern of IL-2-cultured TcR V gamma 3+ thymocytes upon i.v. injection. We describe their initial entrapment in the lungs and subsequent accumulation in the liver. Localization in the skin was practically absent, and did not differ from that of IL-2 cultured adult thymocytes (mainly TcR alpha beta +). The migration pattern was identical in adult and newborn normal mice, and in adult nude mice. Analysis of the expression of asialo-GM1 revealed that it increased strongly after IL-2 culture. The relevance of this change in asialo-GM1 expression with reference to the migration upon i.v. injection is discussed. This study indicates that an improved understanding of the determinants of in vivo localization of IL-2 cultured cells may lead to improved strategies for adoptive immunotherapy of cancer.

LDH isoenzyme distribution in human T gamma lymphocytes.

The lactate dehydrogenase (LDH) isoenzyme distribution was measured in T gamma+ lymphocytes from normal individuals. T gamma+ lymphocytes were obtained from purified T lymphocytes by ox-IgG rosette sedimentation. The B:A subunit ratio was clearly lower in the T gamma+ lymphocytes. Phenotyping of the T gamma+ lymphocytes showed a vast majority of OKT11+, OKM1+, OKT3- lymphocytes. In one experiment, T gamma+ lymphocytes were enriched by OKT3 depletion of T lymphocytes. The low B:A ratio was also found in these cells indicating that the LDH pattern is not the consequence of an in vitro activation by immune complexes. As the T gamma+ lymphocytes were considerably enriched in cells having the characteristics of NK cells according to their phenotyping, morphology and NK cell activity, we may assume that NK cells have a low B:A ratio.

Fetal cardiac output estimated by Doppler echocardiography during mid- and late gestation.

Fetal cardiac output was quantitated by 2-dimensional (2-D) Doppler echocardiography. Circulatory dynamics in the fetus differ from circulatory dynamics after birth because right and left ventricles work in parallel. Therefore, volume flow was estimated over the mitral and tricuspid orifice separately. Serial 2-D echo Doppler studies were performed in 28 normal fetuses at 4-week intervals from 15 to 18 weeks of gestation to the time of parturition. Measurement of blood flow velocities through mitral and tricuspid valve orifices and measurement of the diameter of these valve orifices were obtained from apical 4-chamber views of the fetal heart. Angle-corrected mean temporal blood flow velocities increased linearly with gestational age, whereas the area of blood flow and calculated right and left ventricular output increased exponentially. The right ventricular blood flow velocities, areas of tricuspid orifice and calculated right ventricular outputs were significantly higher than those for the left ventricle when analyzed by paired t test (0.005 greater than 2p greater than 0.001). The ratio of right to left ventricular output decreased from 1.34 +/- 0.28 (+/- standard deviation) at 15 weeks to 1.08 +/- 0.28 at 40 weeks. The combined ventricular output of the fetus near term is approximately 1,735 ml/min, whereas flow indicated for estimated fetal weight is constant at a mean value of 553 +/- 153 ml/min/kg. Thus, 2-D echo Doppler provided a means of estimating fetal cardiac blood flow at the atrioventricular orifices, and blood flow measured at these orifices increased exponentially during gestation, with a larger output from the right ventricle than from the left ventricle.

Human T lymphopoiesis. In vitro and in vivo study models.

Successive steps in T lymphocyte differentiation and T potential of human stem cells (HSC) can be tested in the following models: (a) the infusion of cells in NOD-SCID mice, (b) the injection of cells in renconstituted SCID/hu mice, (c) the differentiation of cells in fetal thymus organ culture (FTOC), and (d) on thymic stromal layers. Using mixed human-murine FTOC, we showed (a) TCR alpha beta, TCR gamma delta lymphocytes, NK cells, and dendritic cells complete their differentiation, (b) IL-7R alpha signaling and IL-7 are essential, (c) a detailed phenotypic and functional analysis of discrete successive steps of positively selected thymocytes, (d) an efficient transduction of genes in HSC with persistent gene expression throughout the T-lymphocyte differentiation, and (e) adaptation to submerging high oxygen culture increases the test sensitivity to a clonal assay. Other approaches are the in vivo SCID/hu reconstitution model. With this method small fragments of human fetal liver and thymus are implanted under the kidney capsule of an adult SCID mouse with result in an impressive human thymus organ, six months after transplantation. We use this model to study thymus T-cell developmental kinetics, development of gene-marked precursor cells and thymic homing of precursor cells.

Phenotyping of mononuclear cells from tonsils and corresponding biopsies using a cytofluorimeter. A comparative study.

A series of B, T, natural killer cell (NK) and monocyte-specific monoclonal antibodies was used to determine the distribution of lymphocyte subpopulations in cell suspensions obtained from whole tonsils as well as their corresponding biopsies. The majority of the mononuclear cells stained with OKB-7 and anti-Dr. A consistent lower percentage of the B lymphocytes stained with OKB-2. The T lymphocytes were composed of a majority of OKT4+ lymphocytes. Only a minority of the lymphocytes stained with Leu-7, whereas Leu-llb+ lymphocytes were virtually absent. A significant proportion of the mononuclear cells stained with OKM-1. There was a highly significant correlation between the distribution of the cells stained with the different monoclonal antibodies obtained from the whole tonsil and the corresponding biopsy (r2 = 0.95). This study shows that biopsies offer a reliable source to study the lymphocyte subsets in tonsils and that cytofluorimetry can be applied to study the lymphocyte distribution in this organ.

Improvement of the quality and comparability of causes-of-death statistics inside the European Community. EUROSTAT Task Force on "causes of death statistics".

BACKGROUND: Cause-of-death statistics are widely used for comparing health characteristics of European Community (EC) countries. Before attempting to interpret between-country differences, it is essential to assess the biases affecting the comparability of the data. EUROSTAT decided to address globally this problem with the objective to improve the quality and comparability of cause-of-death data within the EC. METHODS: The material is based on a review of results of international comparative cause-of-death studies and on specific inquiries among EC. Both cause-of-death certification and codification practices are analysed. Certification is studied comparing the models of death certificates, the type of information captured, certifiers training and querying practices. The different coding systems are analysed (International classification of diseases (ICD) in use, interpretation of the ICD rules, implementation of automated coding systems). RESULTS: International studies on comparability of certification and coding practices between countries are rare. These studies are based on certification of cases histories and recoding of samples of death certificates. Recent studies on respiratory diseases, cancers and diabetes outline differences that influenced on the reported level of mortality. The specific EUROSTAT investigation (1997) outline general discrepancies: models of death certificates, nature and amount of information entered, way to establish the diagnosis, degree of consistency of the certification process, autopsy practices, certifiers practices, implementation of ICD-10 and implementation of automated coding systems. CONCLUSION: EUROSTAT studies are now focused on causes of death requiring special attention for comparability (e.g. suicide, accidental deaths, drug and alcohol related deaths, unknown and ill-defined causes), on procedures to improve the homogeneity of certifiers training and querying practices, on the effect of the transition to ICD-10. The international model of death certificate recommended by the World Health Organization should be adopted as widely as possible. Uniform complementary information (e.g. surgery, pregnancy, autopsy, place of occurrence of accidental deaths, work accident) should also be adopted. The EUROSTAT investigations must result in definitions of common recommendations and guidelines to EC.

Corneal opacifications in a low high density lipoprotein syndrome: suspicion of fish eye disease: a case report.

A 49 year old patient with progressive massive bilateral corneal opacifications associated with a HDL (high-density-lipoprotein) deficiency is described. The opacifications started at the age of twenty and progressed slowly. They were found diffusely over the cornea, though more in the corneal periphery. A penetrating keratoplasty at the right eye was performed. The diagnosis of Fish Eye Disease was put forward on the basis of the ophthalmological, clinical, biochemical and pathological appearance. After 2 year follow-up, the graft was clear. The final distance and near vision of the right eye was 8/10 and Snellen 1 respectively.

[Anterior cruciate ligament prosthesis. Analysis of a failure]. / Les prothèses du ligament croisé antérieur: analyse d'un échec.

Prosthetic ligaments of the knee have been very popular in the eighties. It took a number of years to become aware of the fact they are unable to provide long term stability to the knee joint. This report reviews the development of these prostheses, and tries to analyze the reasons for their failure and to find out why it took such a long time to demonstrate this failure, in order to draw a lesson from the past.

[Synovial cyst of the hip: a rare cause of iliac vein compression]. / Le kyste synovial de la hanche: une cause rare de compression de la veine iliaque.

A case of synovial cyst of the hip presenting as a venous compression in the lower limb is reported. From a review of about 100 cases reported in the literature, the pathogenesis, diagnosis and treatment are discussed.

[A case of a aggressive giant-cell tumor with multiple bone metastases]. / A propos d'un cas de tumeur à cellules géantes d'évolution rapide avec métastases osseuses multiples.

PURPOSE OF THE STUDY: Giant-cell tumor are known for local recurrence. Metastases are rare (2 p. 100), and generally located in the lung. We present a case with multiple bone metastases and rapid course. MATERIAL, METHODS AND RESULTS: A 45-year-old man presented a 5 cm giant-cell tumor of his right distal tibia and two other localisation in the fifth and sixth cervical vertebral bodies. He underwent a resection of the distal tibia and reconstruction with a controlateral free vascularized fibula. The bodies of the fifth and sixth cervical vertebral were resected and replaced by an iliac crest graft. Other localisations appeared in iliac right crest, in the posterior wall of the cotyle and in the second, third and fourth cervical vertebral bodies. Chemotherapy was administered and clinical signs regressed but eight months later the patient presented a recurrence of his tumor in the distal tibia with new localisation in the left fifth rib, in the right clavicle, in the frontal bone, in right ischio-pubal branch and in the right proximal femur. A new chemotherapy was performed but had little effect and probably hasved dubic death 13 month after the first symptom. DISCUSSION: The present observation is characterised by the large number of localisations (13; the highest number found in the literature was 11 localisations) and by the rapidity of the clinical course. Low grade giant-cell tumors generally give benign metastase with a course lasting several years. The chemotherapy helpt us to slow the progression of the lesions but was unable to prevent the development of new localisation and the recurrence in the distal tibia. More over, its toxicity could have caused the death of the patient. CONCLUSION: We present the case of one patient who presented a giant-cell tumor with unusual presentation: multiple bony metastases with rapidly fatal outcome.

Human intrathymic development: a selective approach.

Human T lymphocytes can be generated from CD34 progenitor cells from different sources. This can be obtained in an in vivo model wherein human thymic tissue and fetal liver is transplanted in an immunodeficient mouse. However, human T cells are also generated in immunodeficient mice without co-transplantation of human thymus or in in vitro hybrid human-mouse fetal thymus organ culture. This shows that xenogeneic mouse thymus tissue supports human T cell differentiation. Finally, human T cells are generated on co-culture with murine stromal cells that express the Delta-like1 ligand for the Notch receptor. How these different environments influence the human T cell repertoire is reviewed and discussed.