Human Bone Fragments Research Articles

Matrix metalloproteinase activity, bone matrix turnover, and tumor cell proliferation in prostate cancer bone metastasis.

The metastasis of prostate cancer to bone is associated with a substantial increase in bone matrix turnover. Matrix metalloproteinases (MMPs) play roles in both normal bone remodeling and invasion and metastasis of prostate cancer. This study was designed to determine the role of MMP activity in prostate cancer that has metastasized to bone. Single human fetal bone fragments were implanted subcutaneously in immunodeficient mice. Four weeks later, PC3 human prostate cancer cells were injected directly into some of the implants, and daily treatment was begun with batimastat (a broad-spectrum MMP inhibitor). There were six mice (i.e., six implants) in each of four experimental arms: bone alone with and without batimastat and bone injected with PC3 cells with and without batimastat. Bone implants were harvested after 14 days of treatment and analyzed for MMP expression, bone histomorphometry, osteoclast counts, blood vessel density, and tumor cell proliferation and apoptosis. Complementary data were obtained from bone biopsy samples from patients and a bone organ coculture system. All statistical tests were two-sided. MMPs were detected in tumor and stromal cells of clinical specimens and experimental bone implants. In vivo, MMP inhibition reduced the number of osteoclasts per millimeter in PC3-injected implants-from 8.2 (95% confidence interval [CI] = 7.9 to 8.5) to 3.0 (95% CI = 2.3 to 3.7) (P =.006). In addition, it prevented degradation of marrow trabeculae within the bone implants (cross-sectional area of implant occupied by mineralized trabeculae: untreated implant = 29.1% [95% CI = 27.1% to 31.1%], PC3-injected implant = 14.0% [95% CI = 10.9% to 17.1%] [P =.005 versus untreated], and batimastat-treated PC3-injected implant = 27.2% [95% CI = 22.4% to 32.0%] [P =.03 versus PC3 injected alone]). MMP inhibition reduced proliferating tumor cells from 20.8% (95% CI = 19.9% to 21.7%) to 7.4% (95% CI = 5.2% to 9.6%) (P =.006), without affecting angiogenesis or apoptosis. In vitro, MMP inhibition had no toxic effect on PC3 cells but prevented calcium release from bone fragments cocultured with PC3 cells. MMP activity appears to play an important role in bone matrix turnover when prostate cancer cells are present in bone. Bone matrix turnover and metastatic tumor growth appear to be involved in a mutually supportive cycle that is disrupted by MMP inhibition.

The Human Remains from HMS Pandora

In 1977 the wreck of HMS Pandora (the ship that was sent to re-capture the Bounty mutineers) was discovered off the north coast of Queensland. Since 1983, the Queensland Museum Maritime Archaeology section has carried out systematic excavation of the wreck.During the years 1986 and 1995-1998, more than 200 human bone and bone fragments were recovered. Osteological investigation revealed that this material represented three males. Their ages were estimated at approximately 17 +/-2 years, 22 +/-3 years and 28 +/-4 years, with statures of 168 +/-4cm, 167 +/-4cm, and 166cm +/-3cm respectively. All three individuals were probably Caucasian, although precise determination of ethnicity was not possible. In addition to poor dental hygiene, signs of chronic diseases suggestive of rickets and syphilis were observed. Evidence of spina bifida was seen on one of the skeletons, as were other skeletal anomalies. Various taphonomic processes affecting the remains were also observed and described. Compact bone was observed under the scanning electron microscope and found to be structurally coherent. Profiles of the three skeletons were compared with historical information about the 35 men lost with the ship, but no precise identification could be made. The investigation did not reveal the cause of death. Further research, such as DNA analysis, is being carried out at the time of publication.Acknowledgements Thanks for assistance provided by the Queensland Museum, the Museum of Tropical Queensland and the Department of Anatomical Sciences at the University of Queensland. Special thanks to the Pandora project leader and senior curator of Maritime Archaeology, Mr Peter Gesner (Museum of Tropical Queensland), Mr Ron Coleman (retired Curator of Maritime Archaeology, Queensland Museum), Associate Professor A.J. Ansford, Leo Freney and Peter Clausen (John Tonge Centre for Forensic Biology) and Dr Ann Kemp for their assistance.

AMS Dating of Human Bone from Palau: New Evidence for a Pre-2000 BP Settlement

Direct dating of a human bone fragment from the Chelechol ra Orrak site (western Micronesia) has yielded one of the earliest dates for Palau thus far. This date compares well with recently collected paleoenvironmental evidence and radiocarbon dates on Babeldaob Island suggesting that settlement of the Palauan archipelago took place much earlier than previously thought.

The Last Domicile of the Iceman from Hauslabjoch: A Geochemical Approach Using Sr, C and O Isotopes and Trace Element Signatures

We present Sr, C and O isotope ratios and quantitative ratios of trace element ratios in the rib and the femur of the Tyrolean Iceman “Ötzi”. The results from the rib and the femur are compared with those obtained from pre-industrial historic human skull bone fragments collected in the late 19th century from charnel houses located on different geological backgrounds throughout North and South Tyrol. Comparison of strontium isotope data from the bone fragments locates the Iceman's domicile, during the last 5 to 10 years of his life, to the crystalline part of the Central Alps. Statistical analysis of the trace element ratios, together with oxygen isotope results, suggests that the Iceman probably spent most of his last years in southern elevated part of the Ötztal. However, the results from Iceman only represent those from a single individual and are not a representative average of a group. It cannot therefore be established with absolute certainty that the original trace element signatures were conserved, especially during the first stage of mummification. Our conclusions should therefore be regarded as tentative, but nevertheless the best possible indication his domicile, and a stimulus for further research.

Dominant expansion of human T cells in non-obese diabetic/severe combined immunodeficiency mice implanted with human bone fragments

Objective To establish an in vivo animal model in which human T cells develop and function normally, a step toward developing new vaccines or chemical compounds that modulate immune functions and toward understanding T-cell immunity in humans. Materials and Methods Human bone fragments were implanted into non-obese diabetes / severe combined immunodeficiency (NOD/SCID) mice. The presence of human blood cells in the peripheral blood of these mice was monitored periodically by immunostaining and fluorescence-activated cell sorting. Results After implantation of bone fragments, dominant expansion of human T lymphocytes, rather than myeloid and B cells, was observed over a 3-month period. In some cases, the proportion of human T cells rose to 40% of the peripheral blood mononuclear cells. These T cells showed CD4/CD8 ratios similar to those observed in human peripheral blood lymphocytes and had a broad repertoire of rearranged T-cell receptor genes. Graft-versus-host reaction was not noted in any organ analyzed. To assess the suitability of NOD/SCID mice implanted with human bone fragments (hu-bone-NOD/SCID mice) as an in vivo model for HIV infection, the mice were infected with a T-lymphotropic strain of HIV-1 (NL4-3) at 7 weeks posttransplant. Serum p24 gag was detected at 2 weeks after inoculation, after which total CD4-positive cell numbers declined, as seen clinically in patients infected with HIV. Conclusion Although the precise mechanism is yet to be determined by which predominant expansion of human T cells occurs in hu-bone-NOD/SCID mice, such mice appear likely to serve as a useful and versatile model for studies involving human T-cell immunity.

Human Remains from the North Gate, Silchester: An 'Early' and a 'Late' Radiocarbon Date from the City

Human Remains from the North Gate, Silchester: an 'Early' and a 'Late' Radiocarbon Date from the City. Michael Fulford writes: Excavations at the North Gate, Silchester in 1991 recovered fragments of human bones from five different locations in and around the gate.40 Apart from two skull fragments which derived from a gully sealed beneath the latest cobbled road surface through the gate, they occurred in unsealed late or post-Roman contexts above or cutting through the latest road surface. Earlier excavations in 1909 had reported the find of a human skull in the 'counterscarp' of the late Roman defensive ditch to the north of the gate.41 Parts of a second cranium were recovered from this location in 1991. Although it was suggested that the crania might originally have been displayed on the gate as trophies, the only close parallel (from Colchester) dated from about the middle of the first century A.D. It was even more difficult to understand the original context of the post-cranial material, of which two fragments appeared to be stratified in late Roman contexts.42 One possibility, that this bone may have been disturbed from a late Roman extra-mural cemetery, was discounted on the grounds that recent fieldwork in the vicinity had encountered no evidence of any human burial in the area between the town wall and the Outer Earthwork.43 In order to obtain independent evidence of date two fragments were selected for AMS radiocarbon dating in 1999: part of the cranium of an adult found in 1991 in a comparable location to that reported from 1909;44 and part of a femur with a cut mark sustained in antiquity from the disturbed contexts of excavation trenches of 1890 and 1909, from which the majority of fragments derived.45 In the case of the latter the inferior trochanter appeared to have been partially chopped off with a sharp implement while the bone was comparatively fresh. This bone yielded a radiocarbon age (OXA 8732) of 2320+/-40 BP, which calibrates to 550-200 B.C. at one standard deviation (750-20 B.C. at two standard deviations).46 The radiocarbon age of the cranium (OXA 8733) was considerably younger at 1600+/-40BP, which calibrates to A.D. 420-540 at one standard deviation, A.D. 340-570 at two standard deviations.47 We are thus presented with two extremes of date: midor early-to-mid-Iron Age on the one hand, and post-Roman on the other. In the light of this evidence it is appropriate to see the human bone from the 1991 excavations at the North Gate as originating in at least two separate periods. While the collection from which the femur was selected may yet prove to contain specimens of different date, the cranium was the only find of its kind from the upper fill of the late Roman ditch. George Boon has drawn together the few objects which attest to activity on the Silchester plateau before the late Iron Age.48 In particular he has noted in the Silchester Collection in Reading Museum 'a coloured glass bead of a kind identical with a pair from a princess's grave at Reinheim, and a small iron cheek-piece with bronze rosettes from a helmet of Montefortino affinity'. On the basis of this evidence he 'suggest(ed) that barrows of the Early Iron Age, about the fourth century B.C., may have been levelled within the walled area'.49 Although we have no idea of the precise provenance within the Roman town of these finds in the Silchester Collection, their age corresponds with that of our femur, which might conceivably have derived from an inhumation beneath a barrow; however, burial in this manner is not well attested in the south of Britain at this time.50 On the other hand there is considerable evidence for less formal disposal of human remains in this period, as evidenced for example at Danebury hillfort, also in Hampshire,5 and elsewhere in Wessex,52 but usually in association with pottery or other material evidence. While the combined evidence of the finds from the early excavations and the radiocarbon-dated bone strengthens the case for

Persistence of human vascular endothelium in experimental human prostate cancer bone tumors.

Using the SCID-human model, we recently found that human circulating prostate cancer cells formed tumors in human bone but not mouse bone (Nemeth et al. Cancer Res 1999; 59: 1987-93). It is possible that this tissue preference was mediated by interaction between human tumor cells and human endothelial cells within the implanted bone tissue. We sought to determine the relative amounts of human and mouse vasculature within human bone implants and resulting prostate cancer bone tumors in the SCID-human model. Paraffin sections of plain bone implants or PC3 or LNCaP human bone tumors were double stained for factor VIII (all vessels) and human CD31 (human vessels) followed by fluorescent secondary reagents. At 4 weeks post implantation (when cancer cells are typically introduced), the vasculature within human bone fragments remained primarily human (84.5%), and this pattern persisted to at least 10 weeks (91.6% human). Injection of PC3 cells into the bone resulted in an increase in mouse-derived vessels, however the majority (58%) of the vessels remained human even after the formation of large bone tumors. LNCaP bone tumors were highly angiogenic, and there was a sharp decline in the proportion of vessels which were antigenically human (36.8%), suggesting recruitment of mouse endothelial cells during the angiogenic process. Nonetheless, the persistence of human vasculature suggests the SCID-human model can be used to study the interaction between bone-seeking tumor cells, such as prostate cancer, and human bone endothelium in vivo, and to test potential therapeutic strategies which may depend on the presence of human vessels.

Ex Vivo Expansion of Autologous Bone Marrow CD34+ Cells With Porcine Microvascular Endothelial Cells Results in a Graft Capable of Rescuing Lethally Irradiated Baboons

Hematopoietic stem cell (HSC) self-renewal in vitro has been reported to result in a diminished proliferative capacity or acquisition of a homing defect that might compromise marrow repopulation. Our group has demonstrated that human HSC expanded ex vivo in the presence of porcine microvascular endothelial cells (PMVEC) retain the capacity to competitively repopulate human bone fragments implanted in severe combined immunodeficiency (SCID) mice. To further test the marrow repopulating capacity of expanded stem cells, our laboratory has established a myeloablative, fractionated total body irradiation conditioning protocol for autologous marrow transplantation in baboons. A control animal, which received no transplant, as well as two animals, which received a suboptimal number of marrow mononuclear cells, died 37, 43, and 59 days postirradiation, respectively. Immunomagnetically selected CD34(+) marrow cells from two baboons were placed in PMVEC coculture with exogenous human cytokines. After 10 days of expansion, the grafts represented a 14-fold to 22-fold increase in cell number, a 4-fold to 5-fold expansion of CD34(+) cells, a 3-fold to 4-fold increase of colony-forming unit-granulocyte-macrophage (CFU-GM), and a 12-fold to 17-fold increase of cobblestone area-forming cells (CAFC) over input. Both baboons became transfusion independent by day 23 posttransplant and achieved absolute neutrophil count (ANC) >500/microL by day 25 +/- 1 and platelets >20,000/microL by day 29 +/- 2. This hematopoietic recovery was delayed in comparison to two animals that received either a graft consisting of freshly isolated, unexpanded CD34(+) cells or 175 x 10(6)/kg unfractionated marrow mononuclear cells. Analysis of the proliferative status of cells in PMVEC expansion cultures demonstrated that by 10 days, 99.8% of CD34(+) cells present in the cultures had undergone cycling, and that the population of cells expressing a CD34(+) CD38(-) phenotype in the cultures was also the result of active cell division. These data indicate that isolated bone marrow CD34(+) cells may undergo cell division during ex vivo expansion in the presence of endothelial cells to provide a graft capable of rescuing a myeloablated autologous host.

Determining the human origin of fragments of burnt bone: a comparative study of histological, immunological and DNA techniques

In situations where badly burnt fragments of bone are found, identification of their human or non-human origin may be impossible by gross morphology alone and other techniques have to be employed. In order to determine whether histological methods were redundant and should be superseded by biomolecular analyses, small fragments of artificially burnt bone (human and non-human) were examined by quantitative and standard light microscopy, and the findings compared with newer biomolecular analyses based on identifying specific human albumin by ELISA and amplifying human mitochondrial DNA by PCR. For quantitative microscopy, reference data were first created using burnt bones from 15 human and 20 common domestic and farm animals. Measured osteon and Haversian canal parameters were analysed using multivariate statistical methods. Highly significant differences were found between values for human and non-human bone, and a canonical discriminant function equation was derived, giving a predicted correct classification of 79%. For the main study, samples of cortical bone were taken from three fresh cadavers, six human skeletons and ten freshly slaughtered animals and burnt by exposure to temperatures ranging from 800 to 1200°C; charred fragments of human cortical bone from two forensic cases were also tested. Quantitative microscopy and canonical discriminant function gave the correct origin of every sample. Standard microscopy falsely assigned burnt bone from one human skeleton and one forensic case to a non-human source, but otherwise gave correct results. Human albumin was identified in five individuals, including one of the forensic cases, but mitochondrial DNA could not be amplified from any of the human bone. No false positive test results were seen with either biomolecular method; and human albumin and mitochondrial DNA were correctly identified in all unburnt control specimens. It was concluded that histological methods were not redundant and that quantitative microscopy provided an accurate and consistent means of determining the human or non-human origin of burnt bone and was more reliable than standard microscopy or the newer immunological and DNA techniques tested here.

Administration of Flk2/Flt3 ligand induces expansion of human high-proliferative potential colony-forming cells in the SCID-hu mouse

The effects of Flk2/Flt3 ligand (FL) administration on human hematopoiesis were investigated using SCID-hu mice transplanted with human fetal bone fragments. Treatment with recombinant human FL induced significant increases in the frequencies of the high-proliferative potential colony-forming cells and low-proliferative potential colony-forming cells in steady-state human bone marrow. FL also promoted the expansion of high-proliferative potential colony-forming cells and low-proliferative potential colony-forming cells in the human bone marrow during the recovery phase after irradiation, which was evident in increases in the frequencies as well as in the absolute numbers of colony-forming cells. Furthermore, higher percentages of CD33 + CD15 − cells were found in the marrows treated with FL as compared to that of controls, indicating that FL hastened the recovery of at least some aspect of myelopoiesis after irradiation. These results indicate that FL induces the expansion of primitive hematopoietic progenitor cells in vivo and, therefore, may be useful in treating patients to promote an early hematopoietic recovery after cytoablative therapies.

Transplantation of stromal cells transduced with the human IL3 gene to stimulate hematopoiesis in human fetal bone grafts in non-obese, diabetic-severe combined immunodeficiency mice.

The non-obese diabetic-severe combined immunodeficiency (NOD-SCID) mouse is a convenient host for human hematopoietic tissues and cells. Human fetal bone fragments engrafted subcutaneously in NOD-SCID mice sustain human hematopoiesis for several months. MS5 murine bone marrow stromal cells were transfected by electroporation with a plasmid containing the human interleukin-3 gene. As expected, stably transfected hu-IL3-MS5 cells supported human hematopoiesis in vitro more efficiently than MS5 cells. hu-IL3-MS5 cells were then injected intravenously into hu-NOD-SCID mice to test their ability to home to the mouse and/or human bone marrow, and to evaluate the role of hu-IL3 secretion on human hematopoiesis in vivo. hu-IL3 was detected in the mouse serum for up to an observation time of 8 weeks. hu-IL3-MS5 cells engrafted the bone marrow, spleen, liver and lungs of the mice but also the human bone graft. The presence of hu-IL3-MS5 cells in the human bone significantly stimulated local human hematopoiesis. This setting could be used to model the bone marrow homing of intravenously injected stromal cells or stromal cell precursors. The same experimental principle could also be applied in a therapeutic perspective to malignant human bone marrow hematopoiesis.

Resistance of human hematopoietic stem cells to a monoclonal antibody recognizing CD43.

Hematopoietic progenitor cells (HPC) interact with bone marrow stroma by adhesion molecules which are thought to be critically important to the regulation of hematopoiesis. The specific roles of individual adhesion molecules involved in these interactions remain poorly understood. A monoclonal antibody (mAb) recognizing CD43, an adhesion molecule highly expressed by HPC, induces apoptosis in CD34hiLin- marrow cells. This process operates at a single-cell level, and the initiation of apoptosis requires crosslinking of surface CD43 and the presence of cytokines. In contrast to HPC, more differentiated hematopoietic cells do not undergo apoptosis in response to the CD43-mediated stimulation. Not all progenitor cells undergo apoptosis upon stimulation of CD43. Dividing progenitor cells are most affected, whereas more primitive, quiescent cells survive anti-CD43 mAb treatment. These surviving cells: A) are enriched for cobblestone area-forming cells; B) repopulate fragments of human fetal bone implanted into CX.B-17 severe combined immunodeficiency (SCID/hu) mice; C) have a potential to differentiate in vivo to myeloid and lymphoid cells, and D) have a high proliferative potential in long-term stromal cell-free liquid culture. These data indicate tha cells with hematopoietic stem cell characteristics are relatively resistant to CD43-mediated apoptosis compared to HPC and that CD43 may function as a negative regulator of early events occurring during hematopoiesis.

Phenotypic and Functional Evidence for the Expression of CD4 by Hematopoietic Stem Cells Isolated From Human Fetal Liver

AbstractExpression 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.

The Final Days of the Franklin Expedition: New Skeletal Evidence

In 1992, a previously unrecorded site of Sir John Franklin's last expedition (1845-1848) was discovered on King William Island in the central Canadian Arctic. Artifacts recovered from the site included iron and copper nails, glass, a clay pipe fragment, pieces of fabric and shoe leather, buttons, and a scatter of wood fragments, possibly representing the remains of a lifeboat or sledge. Nearly 400 human bones and bone fragments, representing a minimum of 11 men, were also found at the site. A combination of artifactual and oxygen isotope evidence indicated a European origin for at least two of these individuals. Skeletal pathology included periostitis, osteoarthritis, dental caries, abscesses, antemortem tooth loss, and periodontal disease. Mass spectroscopy and x-ray fluorescence revealed elevated lead levels consistent with previous measurements, further supporting the conclusion that lead poisoning contributed to the demise of the expedition. Cut marks on approximately one-quarter of the remains support 19th-century Inuit accounts of cannibalism among Franklin's crew.

Human Hematopoiesis in SCID Mice Implanted With Human Adult Cancellous Bone

The persistence of hematopoietic cells from human adult cancellous bone fragments implanted subcutaneously into CB-17 scid/scid mice was studied. Recipient mice received either no pretreatment (control group) or pretreatment with 3 Gy total-body irradiation and anti-asialo GM1 sera (ASGM1; pretreated group) before implantation. Pretreated severe combined immunodeficient (SCID) mice implanted with human bone were subsequently given ASGM1 every 7 days for the duration of the experiments. At 12 weeks postimplantation, flow cytometry of cells from pretreated and control animal tissues detected human CD45+ cells in the mouse spleen (mean, 7.8% and 3.4% positive cells, pretreated and control animals, respectively), bone marrow (BM; mean, 16.5% and 4.8% positive cells, respectively), and blood (mean, 5.5% and < 2% positive cells, respectively), and in the implanted human bone (73% and 8.9% positive cells, respectively). At 12 weeks, pretreated mice had human granulocyte-macrophage colony-forming cells (GM-CFC) and burst-forming units-erythrocyte (BFU-E) in the implanted human bone in the murine BM and in some of the spleens. The spleens also had extensive infiltration of human B cells and macrophages. Mean serum levels of human IgG in pretreated animals were 14 micrograms/mL during weeks 6 to 12, compared with trace levels (< 1 microgram/mL) in control mice. Bone from patients with acute myeloblastic leukemia (AML) was also implanted in pretreated SCID mice, and retrieved at 8 weeks for analysis. Comparison of preimplantation and implanted samples showed that the original histology was maintained, and massive infiltration of human CD68+ cells was observed in the mice spleens and BM. Implantation of AML bone in SCID mice facilitates analysis of in situ AML cell interaction with stromal cells in the leukemic state, and therapies against AML can be tested in this system, especially the selective killing of AML cells in the presence of other BM cells. Furthermore, this model requires no exogenous administration of cytokines to maintain human hematopoiesis with both normal or AML bone. Because the structure and function of both normal and diseased human adult bone is maintained, this animal model should facilitate investigation of both normal human hematopoiesis and hematopoietic malignancies.

Hematopoietic Potential of Cryopreserved and Ex Vivo Manipulated Umbilical Cord Blood Progenitor Cells Evaluated In Vitro and In Vivo

The hematopoietic potential of cryopreserved and ex vivo manipulated umbilical cord blood (UCB) samples was evaluated in vitro and in vivo. Phenotypic analysis shows that approximately 1% of cord blood mononuclear cells express high levels of CD34 antigen on their surface (CD34hi), but none of a panel of lineage antigens (Lin-), suggesting that they are hematopoietic progenitor cells that have not yet committed to a specific lineage. Approximately 1% of CD34hi/Lin- cells are primitive hematopoietic progenitors that produce B lymphoid and multiple myeloid progeny for up to 7 weeks in stromal cell cultures. Twenty-one percent (+/- 13%) of CD34hi/Lin- cells also express low levels of the Thy-1 antigen and are threefold to fourfold enriched over CD34hi/Lin- cells in primitive hematopoietic potential as measured by long-term culture and phenotypic analysis. One-week liquid cultures of CD34-enriched UCB progenitor cells in the presence of interleukin (IL)-3, IL-6, and stem cell factor (SCF) results in a two-fold to threefold expansion of progenitors capable of reinitiating long-term stromal cell cultures. Only the CD34hi/Thy-1+/Lin- cell population was capable of maintaining progenitors with secondary transfer potential in long-term stromal cell cultures and is thus postulated to contain all of the primitive hematopoietic stem cells in UCB. The in vivo transplantation potential of UCB was also measured. Ex vivo manipulated UCB progenitor cells were used to engraft irradiated human thymus fragments implanted in severe combined immunodeficiency (SCID) mice. Thymic engraftment with >5% donor-derived cells and a normal CD4/CD8 distribution was observed in 19 of 23 tissues tested. UCB cells from in vitro expansion cultures engrafted with efficiencies comparable to nonexpanded cells. Similar results were obtained for UCB engraftment of human bone fragments implanted in SCID mice. In all cases, engraftment was achieved in competition with endogenous competitor stem cells and across major histocompatibility barriers. Taken together, this data demonstrates that human UCB is a rich source of multipotent hematopoietic progenitors that can be cryopreserved, enriched by physical methods, and expanded in a limited fashion without measurable loss of long-term culture or in vivo engrafting potential as measured in these assays.

IL-4 induces human B cell maturation and IgE synthesis in SCID-hu mice. Inhibition of ongoing IgE production by in vivo treatment with an IL-4/IL-13 receptor antagonist.

The effect of cytokine treatment on the in vivo maturation and Ig isotype switching of human B cells was studied in a modified SCID-hu mouse model. SCID mice, subcutaneously cotransplanted with small fragments of fetal human thymus and bone (SCID-hu BM/T mice) generated all human leukocyte lineages including T and B lymphocytes, macrophages, and granulocytes. All SCID-hu BM/T mice spontaneously produced human IgM and IgG, whereas IgE and IgA were detected in 37 and 80% of the mice, respectively, indicating that productive human T-B cell interactions resulting in Ig isotype switching occur in these mice. Administration of IL-4 to SCID-hu BM/T mice enhanced human B cell maturation, as judged by the increase in the percentages of CD45+, CD19+ bone marrow B cells expressing CD20, CD23, CD40, sIgM, and sIgD. Furthermore, these cells were also functionally more mature because they spontaneously produced human IgG/IgG4 in vitro and could be induced to secrete human IgE by addition of anti-CD40 mAb alone. In contrast, B cells isolated from PBS-treated mice only produced significant Ig levels after stimulation with anti-CD40 mAb in the presence of exogenous IL-4. IL-4 administration also induced human IgE synthesis in 44% of the mice, which had no serum IgE before treatment. More importantly, ongoing human IgE synthesis in SCID-hu BM/T mice was suppressed by > 90% following administration of an IL-4 mutant protein, which acts as an IL-4 and IL-13 receptor antagonist. These results suggest that IL-4/IL-13 receptor antagonists have potential clinical utility in treating human atopic diseases associated with enhanced IgE production.

Delineation of T-Progenitor Cell Activity Within the CD34+ Compartment of Adult Bone Marrow

T-cell production is largely dependent on the presence of a thymus gland where CD34+ precursors mature into T lymphocytes. Prethymic stages of T-cell development are less defined. Therefore, this study aims to delineate T-progenitor cell potential within the CD34+ Lineage--(Lin-) cell compartment of adult bone marrow (ABM). Fractionation of CD34+ Lin- ABM cells with CD45RA, Thy-1, CD38, and HLA-DR failed to absolutely segregate T-cell reconstituting ability, indicating broad distribution of T-progenitor cell potential. Titration experiments showed that low numbers of CD34+ Lin- CD45RA+ (RA+) cells had greater thymus repopulating ability than CD34+ Lin- CD45RA- cells (RA-). The great majority (> 95%) of RA+ cells expressed CD38, HLA-DR and 70% to 90% of RA+ cells lacked Thy-1 surface expression. RA+ cells contained colony-forming unit granulocyte-macrophage (CFU-GM) progenitor cells but were depleted of erythroid potential, did not provide hematopoietic reconstitution of human bone fragments implanted into SCID mice, and did not efficiently maintain CD34+ cells with secondary clonogenic potential in bone marrow cultures. Thus, RA+ cells are oligopotent (nonprimitive) CD34+ progenitors with T-cell reconstituting ability. In contrast, these same assays indicated that CD34+ Lin- CD45RA- cells (RA- cells) comprised hematopoietic stem cells (HSC) with primitive multilineage (T, B, myeloid, and erythroid) hematopoietic potential. It was confirmed that HSC-containing populations, such as CD34+ Lin- CD45RA- Thy-1+ cells had thymus repopulating ability. Culture of RA- cells on murine bone marrow stromal cells in the presence of interleukin (IL)-3, IL-6, and leukemia inhibitory factor (LIF) generated CD34+ CD45RA+ progeny engrafting in a secondary severe combined immunodeficiency (SCID)-hu thymus assay. Altogether, our results underscore the fact that T-cell reconstituting potential can be dissociated from HSC activity. Furthermore, we speculate that HSC might develop into the T lineage indirectly, via differentiation into an intermediate oligopotent CD34+ CD45RA+ stage. Finally, T-progenitor cells can be cultured in vitro.

Differential Survival of Albumin in Ancient Bone

The identification of blood proteins in ancient bone is important (1) because detection of simple proteins can give information on species specificity and evolution, and (2) because the methods used provide a sound technical basis for investigating the survival of more informative proteins, such as HLA and blood groups, which would shed light on genetic profiles and disease predispositions of ancient populations. Our previous studies used a sensitive and specific enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies and showed that albumin can be identified in human skeletal remains at least 4000 years old. However, the factors which affect survival and diagenesis of proteins within ancient bone remain largely unresolved. In the present studies, the results obtained after testing extracts of ancient bovine and human bone for albumin with the ELISA have been compared. The human extracts were prepared from intact (“closed”) vertebrae from formal burial sites. Well-preserved bovine material, on the other hand, could not easily be found and most of the bovine extracts had to be prepared from assemblages of bone which were “open” (i.e. the spongy interior was exposed), either because of butchering or weathering. Although albumin could be detected in both bovine and human bones which were several thousand years old, the survival pattern between the species was strikingly different (chi-square=11·7, P<0·001); albumin was identified in 23 of the 31 human extracts but in only two of the 14 bovine ones. The assay for bovine albumin was as sensitive as that for human albumin and had been equally successful in identifying blood stains on buried samples of gauze. As part of the present studies, a modern experiment was also performed in which “open” fragments of bovine and human bone were buried in a suburban garden exposed to high rainfall and exhumed and tested at set intervals over a 26-month period. The results showed a pattern of albumin survival similar to that observed with the ancient bovine bone, i.e. survival was much poorer than with the intact “closed” bone from the ancient human burials. It was concluded that bone integrity is of great importance for protein survival and that exposure of marrow and trabecular bone to the physical and chemical effects of water is the most significant single factor in the production of diagenetic changes. In this respect, water appears to have a far greater effect than age, soil content, pH or heat.

Engraftment of human hematopoietic precursor cells with secondary transfer potential in SCID-hu mice

Human fetal bone fragments implanted subcutaneously in immunodeficient (SCID) mice maintain active human hematopoiesis. In this study, we show that this human hematopoietic microenvironment supports the engraftment and differentiation of HLA-mismatched, CD34+ primitive hematopoietic progenitor cells isolated from fetal and adult human bone marrow (BM). The BM CD34+ cells were depleted of CD2, CD14, CD15, CD16, glycophorin A, and CD19 lineage-committed cells (CD34+Lin-). Donor cell engraftment was manifested by the presence of B (CD19+) and myeloid (CD33+) cells of donor HLA phenotype. Successful engraftment was observed as early as 4 weeks after fetal BM donor cell injection and sustained for at least 12 weeks, with engraftment success rates of 100% (11/11 grafts) and 92% (11/12 grafts) at 8 and 12 weeks, respectively. Mixed BM chimerism of donor and endogenous cells was consistently observed in SCID-hu bones successfully engrafted with HLA-mismatched CD34+Lin- donor cells. Preconditioning of the SCID-hu bone with a single dose of sublethal (350 rad) whole body irradiation (WBI) immediately before cell injection enhanced the repopulation of the bone grafts with donor cells and, in some instances, resulted in complete repopulation. After WBI, as few as 500 fetal bone marrow CD34+Lin- cells injected in the human bone grafts resulted in donor-derived hematopoiesis. Donor progenitor cells recovered from the SCID-hu bone grafts 8 weeks postinjection had the capacity to repopulate secondary groups of HLA-disparate fetal human bones in SCID-hu mice with B and myeloid cells as well as CD34+ cells in some recipients. In addition, these cells repopulated fetal human thymus fragments in SCID mice with donor thymocytes including immature CD4+CD8+ and mature CD4+CD8- as well as CD4-CD8+ subsets. These results indicate that the fetal human bone implants of SCID-hu mice can support the maintenance of a cell population that has both multilineage potential and repopulating potential for periods of time as long as 16 weeks. The SCID-hu bone model consistently supported the engraftment of both fetal and adult CD34+Lin- cells without the administration of exogenous human cytokines to these animals. This model is currently being used to permit the isolation and characterization of candidate human hematopoietic stem cells (HSCs) and provide important information critical for human HSC therapy in humans.