A new reference cigarette, the 3R4F, has been developed to replace the depleting supply of the 2R4F cigarette. The present study was designed to compare mainstream smoke chemistry and toxicity of the two reference cigarettes under the International Organization for Standardization (ISO) machine smoking conditions, and to further compare mainstream smoke chemistry and toxicological activity of the 3R4F cigarette by two different smoking regimens, i.e., the machine smoking conditions specified by ISO and the Health Canada intensive (HCI) smoking conditions.The in vitro cytotoxicity and mutagenicity was determined in the neutral red uptake assay, the Salmonella reverse mutation assay, and the mouse lymphoma thymidine kinase assay. Additionally, a 90-day nose-only inhalation study in rats was conducted to assess the in vivo toxicity. The comparison of smoke chemistry between the two reference cigarettes found practically the same yields of total particulate matter (TPM), ‘tar’, nicotine, carbon monoxide, and most other smoke constituents. For both cigarettes, the in vitro cytotoxicity, mutagenicity, and in vivo toxicity showed the expected smoke-related effects compared to controls without smoke exposure. There were no meaningful differences between the 2R4F and 3R4F regarding these toxicological endpoints. The assessments for the 3R4F cigarette by smoking regimen found as a trivial effect, due to the higher amount of smoke generated per cigarette under HCI conditions, an increased yield of toxicant and higher toxicological activity per cigarette. However, per mg TPM, ‘tar’, or nicotine, the amounts of toxicants and the in vitro toxicity were generally lower under HCI conditions, but the in vivo activity was not different between the two machine smoking conditions. Overall, as the main result, the present study suggests equivalent smoke chemistry and in vitro and in vivo toxicity for the 2R4F and 3R4F reference cigarettes.

Polysialic acid (polySia) is a large glycan with restricted expression, typically found attached to the protein scaffold neural cell adhesion molecule (NCAM). PolySia is best known for its proposed role in modulating neuronal development. Its presence and potential functions outside the nervous systems are essentially unexplored. Herein we show the expression of polySia on hematopoietic progenitor cells, and demonstrate a role for this glycan in immune response using both acute inflammatory and tumor models. Specifically, we found that human NK cells modulate expression of NCAM and the degree of polymerization of its polySia glycans according to activation state. This contrasts with the mouse, where polySia and NCAM expression are restricted to multipotent hematopoietic progenitors and cells developing along a myeloid lineage. Sialyltransferase 8Sia IV−/− mice, which lacked polySia expression in the immune compartment, demonstrated an increased contact hypersensitivity response and decreased control of tumor growth as compared with wild-type animals. This is the first demonstration of polySia expression and regulation on myeloid cells, and the results in animal models suggest a role for polySia in immune regulation.

We studied the potential role of the human placenta as a hematopoietic organ during embryonic and fetal development. Placental samples contained two cell populations—CD34++CD45low and CD34+CD45low—that were found in chorionic villi and in the chorioamniotic membrane. CD34++CD45low cells express many cell surface antigens found on multipotent primitive hematopoietic progenitors and hematopoietic stem cells. CD34++CD45low cells contained colony-forming units culture (CFU-C) with myeloid and erythroid potential in clonogenic in vitro assays, and they generated CD56+ natural killer cells and CD19+CD20+sIgM+ B cells in polyclonal liquid cultures. CD34+CD45low cells mostly comprised erythroid- and myeloid-committed progenitors, while CD34− cells lacked CFU-C. The placenta-derived precursors were fetal in origin, as demonstrated by FISH using repeat-sequence chromosome-specific probes for X and Y. The number of CD34++CD45low cells increased with gestational age, but their density (cells per gram of tissue) peaked at 5–8 wk, decreasing more than sevenfold at the onset of the fetal phase (9 wk of gestation). In addition to multipotent progenitors, the placenta contained myeloid- and erythroid-committed progenitors indicative of active in situ hematopoiesis. These data suggest that the human placenta is an important hematopoietic organ, raising the possibility of banking placental hematopoietic stem cells along with cord blood for transplantation.

Summary:Human fetal livers (FL), between 16 and 24 weeks of gestation, were studied for their potential as a source of hematopoietic stem cells for prenatal and postnatal transplantation. In this report we give a quantitative evaluation of human FL as a source of candidate stem cells, and develop a protocol for the isolation of these cells free of microbial contaminants and almost free of mature T cells. Human FLs contained a median 1.9 ؋ 10 9 viable cells and a mean of 1. 2,3 Some promising results were reported with the transplantation of FL cells in humans, either before or after birth.1,4-8 However, factors limiting the use of first trimester FL cells became apparent. Among these are the difficulties in harvesting liver at early gestational ages, the relatively low number of cells available from a single first trimester FL and the risk of microbial contamination of the FL tissues. Nonetheless, we have reported recently on a procedure for obtaining first-trimester FL that addresses the ethical concerns regarding such an endeavor and we also demonstrated that after minimal processing these FLs were free of microbial and specific viral contaminants. Thus, a bank of cryopreserved first-trimester FLs was established, but its utility is limited to pre-natal transplantation because of the small size of the samples. 9 We hypothesized that mid-trimester FLs, between the gestational ages of 16 and 24 weeks, represented an abundant source of HSCs that could be made safe for transplantation. It was the goal of this study to determine the potential yield of progenitors and HSCs that could be harvested from mid-trimester FLs and to estimate the risk of graftversus-host disease from mature T and NK cells. In this study we also define a protocol for the processing of human fetal HSC. The aim was to define a method of processing FLs that ensures the highest yield possible of committed progenitors and HSCs without compromising the quality of the grafts. The magnetic cell separation system, Isolex 50, was chosen for this task. The quality of the processed grafts was measured in terms of bacterial and fungal contamination in addition to the enumeration of T cells, progenitors and HSCs. Materials and methods AntibodiesThe following phycoerythrin (PE)-labeled, or fluorescein isothiocyanate (FITC)-labeled, monoclonal antibodies

The generation of erythroid, myeloid, and lymphoid cells from human fetal liver progenitors was studied in colony-forming cell (CFC) assays. CD38− and CD38+ progenitors that expressed high levels of CD34 were grown in serum-deprived medium supplemented with kit ligand, flk2/flt3 ligand, GM-CSF, c-mpl ligand, erythropoietin, and IL-15. The resulting colonies were individually analyzed by flow cytometry. CD56+ NK cells were detected in 21.9 and 9.9% of colonies grown from CD38− and CD38+ progenitors, respectively. NK cells were detected in mostly large CD14+/CD15+ myeloid colonies that also, in some cases, contained red cells. NK cells were rarely detected in erythroid colonies, suggesting an early split between the erythroid and the NK cell lineages. CD1a+ dendritic cells were also present in three-quarters of the colonies grown from CD38− and CD38+ progenitors. Multilineage colonies containing erythrocytes, myeloid cells, and NK cells were present in 13.7 and 2.7% of colonies grown from CD38− and CD38+ progenitors, respectively. High proliferative-potential CFCs that generated multilineage colonies were also detected among both populations of progenitors. The total number of high proliferative-potential CFCs with erythroid, myeloid, and NK cell potential was estimated to be 2-fold higher in the CD38+ fraction compared with the CD38− fraction because of the higher frequency of CD38+ cells among CD34++ cells. The broad distribution of multipotent CFCs among CD38− and CD38+ progenitors suggests that the segregation of the erythroid, myeloid, and lymphoid lineages may not always be an early event in hemopoiesis. Alternatively, some stem cells may be present among CD38+ cells.

It was reported that the dose of self-antigens can determine the consequence of deletional tolerance and donor T cells are critical for tolerance induction in mixed chimeras. This study aimed at assessing the effect of cell doses and marrow T cells on engraftment and tolerance induction after prenatal bone marrow transplantation. Intraperitoneal cell transplantation was performed in FVB/N (H-2K q ) mice at gestational day 14 with escalating doses of adult C57BL/6 (H-2K b ) marrows. Peripheral chimerism was examined postnatally by flow cytometry and tolerance was tested by skin transplantation. Transplantation of light-density marrow cells showed a dose response. High-level chimerism emerged with a threshold dose of 5.0 × 10 6 and host leukocytes could be nearly replaced at a dose of 7.5-10.0 × 10 6 . High-dose transplants conferred a steady long-lasting donor-specific tolerance but were accompanied by >50% incidence of graft-versus-host disease. Depletion of marrow T cells lessened graft-versus-host disease to the detriment of engraftment. With low-level chimerism, tolerance was a graded phenomenon dependent upon the level of chimerism. Durable chimerism within 6 months required a threshold of ≥ 2% chimerism at 1 month of age and predicted a 50% chance of long-term tolerance, whereas transient chimerism (<2%) only caused hyporesponsiveness to the donor. Tolerance induction did not succeed without peripheral chimerism even if a large amount of injected donor cells persisted in the peritoneum. Neither did an increase in cell doses or donor T-cell contents benefit skin graft survivals unless it had substantially improved peripheral chimerism. Thus, peripheral chimerism level can be a simple and straightforward test to predict the degree of prenatal immune tolerance.

Cytokine combinations were tested for their ability to expand murine bone marrow (BM) progenitors in short-term suspension cultures (delta- cultures) with the aim of providing an enriched source of progenitors for BM transplantation (BMT). In a comparison of the efficacy of the combinations interleukin-1 (IL-1) + IL-3, IL-1 + kit-ligand (KL), and IL-1 + IL-6 + KL, BMT with IL-1 + KL expanded progenitors was found to be most effective in accelerating the recovery of peripheral blood leukocytes, platelets, and erythrocytes in lethally irradiated mice. The ex vivo expansion of BM in IL-1 + KL-stimulated delta-cultures also greatly reduced the number of transplanted cells needed to provide radioprotection. All mice survived at least 30 days when receiving 5 x 10(3) delta-cultured d1 5-fluorouracil (5-FU) BM cells (BM cells harvested 1 day after 5-FU administration), whereas complete survival of mice receiving fresh d1 5-FU BM required BMT with a 200-fold greater number of cells. BMT with expanded BM lead to predominantly donor- derived hematopoietic reconstitution for 280 days postprimary BMT and another 71 days after secondary BMT. The expansion of BM did not adversely effect the proliferative capacity and lineage potential of the stem cell compartment.

Expression of the CD4 antigen was observed on human fetal liver, fetal bone marrow (BM), and umbilical cord blood progenitors expressing high levels of CD34. Using clonal and liquid-culture assays, CD4+ CD34++ Lin− (lineage = CD3, CD8, CD10, CD14, CD15, CD16, CD19, CD20, and glycophorin A) fetal liver progenitors were found to have a greater proliferative potential than CD4− CD34++ Lin− progenitors, whereas the CD4− fraction was more enriched for erythroid progenitors. Both the CD4+ and the CD4− progenitor subpopulations also gave rise to multilineage engraftment upon transplantation into human fetal bone fragments, supportive of B-lymphoid and myeloid growth, or into human fetal thymic fragments, supportive of T-cell growth, implanted in scid/scid (SCID) mice. However, in SCID-hu mice transplanted with graded doses of donor cells ranging from 2.0 × 102 to 2.0 × 104 cells, BM reconstitution by the CD4+ fraction of CD34++ Lin− cells was more frequent than by the CD4− fraction when low numbers of cells were injected. These functional data strongly suggest that stem cells reside among CD4+ CD34++ Lin− fetal liver cells. This hypothesis was further supported by the observations that CD4+ CD34++ Lin− fetal liver cells were enriched for CDw90+ (Thy-1), CD117+ (kit), CD123+, HLA-DR+, CD7−, CD38−, CD45RA−, CD71−, CD115− (fms), and rhodamine 123dull cells, a phenotypic profile believed to represent fetal stem cells. Furthermore, all CD4+ CD34++ Lin− fetal liver cells also expressed CD13 and CD33.