Herzl Ben-Hur

The chemokine SDF-1 stimulates integrin-mediated arrest of CD34+ cells on vascular endothelium under shear flow

The chemokine SDF-1 plays a central role in the repopulation of the bone marrow (BM) by circulating CD34(+) progenitors, but the mechanisms of its action remain obscure. To extravasate to target tissue, a blood-borne cell must arrest firmly on vascular endothelium. Murine hematopoietic progenitors were recently shown in vivo to roll along BM microvessels that display selectins and integrins. We now show that SDF-1 is constitutively expressed by human BM endothelium. In vitro, human CD34(+) cells establish efficient rolling on P-selectin, E-selectin, and the CD44 ligand hyaluronic acid under physiological shear flow. ICAM-1 alone did not tether CD34(+) cells under flow, but, in the presence of surface-bound SDF-1, CD34(+) progenitors rolling on endothelial selectin rapidly developed firm adhesion to the endothelial surface, mediated by an interaction between ICAM-1 and its integrin ligand, which coimmobilized with SDF-1. Human CD34(+) cells accumulated efficiently on TNF-activated human umbilical cord endothelial cells in the absence of SDF-1, but they required immobilized SDF-1 to develop firm integrin-mediated adhesion and spreading. In the absence of selectins, SDF-1 also promoted VLA-4-mediated, Gi protein-dependent tethering and firm adhesion to VCAM-1 under shear flow. To our knowledge, this is the first demonstration that SDF-1 expressed on vascular endothelium is crucial for translating rolling adhesion of CD34(+) progenitors into firm adhesion by increasing the adhesiveness of the integrins VLA-4 and LFA-1 to their respective endothelial ligands, VCAM-1 and ICAM-1.

Tumor Necrosis Factor Promotes Human T‐Cell Development in Nonobese Diabetic/Severe Combined Immunodeficient Mice

A major problem after clinical hematopoietic stem cell transplantations is poor T‐cell reconstitution. Studying the mechanisms underlying this concern is hampered, because experimental transplantation of human stem and progenitor cells into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice usually results in low T–lymphocyte reconstitution. Because tumor necrosis factor α (TNFα) has been proposed to play a role in T‐lineage commitment and differentiation in vitro, we investigated its potential to augment human T‐cell development in vivo. Administration of TNF to irradiated NOD/SCID mice before transplantation of human mononuclear cells from either cord blood or adult G‐CSF–mobilized peripheral blood (MPBL) led 2–3 weeks after transplantation to the emergence of human immature CD4+CD8+ double‐positive T‐cells in the bone marrow (BM), spleen, and thymus, and in this organ, the human cells also express CD1a marker. One to 2 weeks later, single‐positive CD4+ and CD8+ cells expressing heterogenous T‐cell receptor αβ were detected in all three organs. These cells were also capable of migrating through the blood circulation. Interestingly, human T‐cell development in these mice was associated with a significant reduction in immature lymphoid human CD19+ B cells and natural killer progenitors in the murine BM. The human T cells were mostly derived from the transplanted immature CD34+ cells. This study demonstrates the potential of TNF to rapidly augment human T lymphopoiesis in vivo and also provides clinically relevant evidence for this process with adult MPBL progenitors.

An immunohistochemical study of the secretory immune system in human fetal membranes and decidua of the first trimester of pregnancy.

Problem: We analyzed the presence and distribution of components of the secretory immune system (SIS) in human fetal membranes (amnion, yolk sac, chorion) and decidua from the first trimester of pregnancy.

Cycling G1 CD34+/CD38+ Cells Potentiate the Motility and Engraftment of Quiescent G0 CD34+/CD38−/low Severe Combined Immunodeficiency Repopulating Cells

The mechanism of human stem cell expansion ex vivo is not fully understood. Furthermore, little is known about the mechanisms of human stem cell homing/repopulation and the role that differentiating progenitor cells may play in these processes. We report that 2‐ to 3‐day in vitro cytokine stimulation of human cord blood CD34+‐enriched cells induces the production of short‐term repopulating, cycling G1 CD34+/CD38+ cells with increased matrix metalloproteinase (MMP)‐9 secretion as well as increased migration capacity to the chemokine stromal cell–derived factor‐1 (SDF‐1) and homing to the bone marrow of irradiated nonobese diabetic severe/combined immunodeficiency (NOD/SCID) mice. These cycling G1 cells enhance SDF‐1–mediated in vitro migration and in vivo homing of quiescent G0 CD34+ cells, which is partially abrogated after inhibition of MMP‐2/‐9 activity. Moreover, the engraftment potential of quiescent G0 SCID repopulating cells (SRCs) is also increased by the cycling G1 CD34+/CD38+ cells. This effect is significantly abrogated after incubation of cycling G1 cells with a neutralizing anti‐CXCR4 antibody. Our data suggest synergistic interactions between accessory cycling G1 CD34+/CD38+ committed progenitor cells and quiescent, primitive G0 CD34+/CD38−/low SRC/stem cells, the former increasing the motility and engraftment potential of the latter, partly via secretion of MMP‐9.

The plant lectin FRIL supports prolonged in vitro maintenance of quiescent human cord blood CD34(+)CD38(-/low)/SCID repopulating stem cells.

Ex vivo maintenance of human stem cells is crucial for many clinical applications. Current culture methods rely on optimized combinations of cytokines. Although these conditions provide some level of stem cell support, they primarily induce proliferation and differentiation, resulting in reduced repopulation capacity. The recently identified legume lectin FRIL has been shown to preserve human cord blood progenitors up to a month in suspension culture without medium changes. To test whether FRIL also preserves human SCID repopulating stem cells (SRC), we cultured human CD34(+) cord blood cells in medium containing FRIL, with or without subsequent exposure to cytokines, and tested their repopulating potential. We report that FRIL maintains SRC between 6 and 13 days in culture. Incubation of CD34(+) cells with FRIL results in significantly lower numbers of cycling cells compared with cytokine-stimulated cells. CD34(+) cells first cultured with FRIL for 6 days and subsequently exposed to cytokines for an additional 4 days generated significantly more mononuclear and progenitor cells and higher levels of engraftment in NOD/SCID mice compared with CD34(+) cells cultured with FRIL alone. Similar results were obtained with CD34(+)CD38(-/low) cells, including expansion of SRC that were cultured in FRIL followed by cytokine stimulation. Moreover, CD34(+) cells precultured with FRIL successfully engrafted primary and more importantly secondary recipients with lymphoid and myeloid cells, providing further support that FRIL maintains SRC for prolonged periods.FRILs ability to preserve quiescent primitive cells in a reversible manner may significantly expand the time and range of ex vivo manipulations of human stem cells for clinical applications.

Immunoglobulin A in the epithelium of the respiratory tract and intrahepatic bile ducts of fetuses and newborns with pneumonia and sepsis

The level of different immunoglobulins (IgA, IgG, IgM) in the tissues of 28 late fetuses and newborns was studied with peroxidase-labeled monoclonal antibodies. IgA+ and IgM+ lymphocytes were found in the spleen, lymph nodes and sometimes in the liver. IgG+ lymphocytes were not found. A high level of IgA+ material was found in the epithelium of the trachea, the epithelium and submucosal glands of the bronchi, but not the bronchioles, and in the epithelium of hepatic bile ducts and in their lumina. Such IgA is considered to be secretory--sIgA. Secretory IgA-containing epithelial cells appeared at 20 to 21 weeks of gestation; their number increased from 2.5 cells/10,000 microns2 in 23- to 26-week-old fetuses, to 8 cells/10,000 microns2 in 36- to 40-week-old fetuses. Secretory IgG and IgM were not detected. In fetuses with pneumonia or sepsis, the number of IgM+ and IgA+ lymphocytes increased significantly. IgM+ lymphocytes appeared not only in the spleen and lymph nodes, but also in the lungs. In such cases, the number of sIgA-containing epithelial cells in the trachea, bronchi and intrahepatic bile ducts decreased, sometimes completely disappearing. The amount of IgA+ material in the lumina of these organs increased, reflecting an intensification of sIgA secretion during infections. The presence of a marked amount of sIgA in fetuses from week 20 of gestation is considered to reflect the high importance of this immunoglobulin against normal contamination by microbes after birth, and to evidence the early maturation of the immune system.

Secretory Component, J Chain, and Immunoglobulins in Human Embryos and Fetuses of the First Trimester of Pregnancy: Immunohistochemical Study

nIn our previous studies, we described the development of the secretory (mucosal) immune system (SIS) in human fetuses in the second trimester of pregnancy. In the present study, we examined the presence and distribution of components of this system in human embryos and early fetuses in the first trimester. An immunohistochemical study was performed on 17 embryos and 9 fetuses (4 to 12 wk of development) using antibodies against secretory component (SC), joining (J) chain, immunoglobulins (IgA, IgM, IgG), subsets of T and B lymphocytes, and macrophages. Cells positive for SC, J chain, and IgG were found in epithelial tissues from wk 4 of pregnancy. In the internal organs, such as the myocardium and endocardium, capillary endothelium, epithelium of the kidney tubules and some others, only J chain and immunoglobulins were seen. IgA was weakly reactive in tissues where SC and/or J chain were presented. IgM was very weak or absent. Among the cellular components of the SIS, only macrophages were seen in 4-wk-old embryos. CD3+ and CD20+ lymphocytes were found at wk 7 to 8. IgA- and IgM-positive lymphocytes appeared at the end of wk 9. The SIS is widespread in embryonic and early fetal periods and begins to function before the appearance of the common immune system in the developing organism. The first functional components of the SIS, such as IgG and IgA observed in this study, are most probably of maternal origin.n

Lymphoid-Epithelial Secretory Immune System in Human Fetuses in the Second Trimester of Gestation

The development of the secretory immune system (SIS) in the respiratory, digestive, and urogenital tracts and other organs of fetuses in the second trimester of gestation is described. Tissues of all internal organs of human fetuses (n = 36) that had died between 13 and 25 weeks of gestation were studied immunohistochemically for the presence of secretory component (SC), J chain, IgA, IgM, IgG, macrophages, and different subsets of lymphocytes. We found protein elements of the SIS in fetuses during the entire second trimester in the epithelium of the digestive, respiratory, and urinary tracts; in hepatocytes; in the epithelium of the bile duct, renal tubules, and all the urinary tract; in the salivary glands, pancreas, and thyroid; in the epithelium of the Fallopian tubes and uterus; in the epididymis and the rete testes; in the skin; and in other organs. Immunocompetent cells, including IgA- and IgM-secreting cells, were located in these organs under the epithelium and sometimes between epithelial cells. In fetuses with acute infection, the number of immunocompetent cells was higher, reflecting a whole–immune system reaction, including the SIS. We conclude that the fetal SIS is a ramified, defensive immune system that is distributed throughout most organs of epithelial origin in second-trimester fetuses, and that it reacts against intrafetal infiltration by foreign antigens.

Carcinoma of the Clitoris: A Histologic Study with Cytokeratin Profile

A carcinoma of the clitoris showed a variety of histologic features not usually encountered in squamous cell carcinoma of the vulva. In addition to poorly differentiated nonkeratinizing squamous cell carcinoma, there were large areas showing a transitional cell carcinoma pattern, as well as foci of spindle and anaplastic cells. The clitoral neoplasm also differed in its cytokeratin (CK) profile from that described in invasive squamous cell carcinoma of the vulva, especially with regard to the widespread staining for CK 18 and 19 in the former. Thus, our study suggests that some carcinomas of the clitoris differ from those of ordinary squamous cell carcinomas of the vulva. More cases should be studied using immunohistochemical methods in order to establish the full histopathologic and CK range of the former.

Cytokeratin patterns in the epidermis of human ovarian mature cystic teratomas.

Using a battery of monoclonal antibodies, we investigated the cytokeratin pattern in the epidermides of 12 human ovarian mature cystic teratomas (MCTs) and compared them with those of infant, adult, and fetal skin. Histologically, two types of epidermal layers were identified in the MCTs, a mature layer and an immature layer. The mature layer was similar to the epidermis of infants and adults, while the immature layer resembled stratified nonkeratinizing and metaplastic squamous epithelium. The cytokeratin pattern of the histologically mature epidermis in MCT was either similar to that in infants and adults or was of the fetal type. The cytokeratin expression of the histologically immature epidermis in MCT also showed many similarities to the fetal cytokeratin pattern. We conclude that histologic maturity of the epidermis in MCT is not necessarily expressed by the cytokeratin pattern, which reflects the state of molecular rather than histologic differentiation. Since prognosis in germ cell tumors is usually related to the degree of tissue maturation, our observations raise the possibility that the cytokeratin profile may eventually prove to be a valuable prognostic tool in some of the neoplasms that contain epithelial elements.