Comparison of bone marrow cells harvested from various bones of cynomolgus monkeys at various ages by perfusion or aspiration methods: a preclinical study for human BMT

Using cynomolgus monkeys, we have previously established a new method for harvesting bone marrow cells (BMCs) with minimal contamination of the BMCs with T cells from the peripheral blood. We originally conducted this new "perfusion method" in the long bones (the humerus, femur, and tibia) of cynomolgus monkeys. Here, we apply the perfusion method to obtain BMCs from the ilium of cynomolgus monkeys, since BMCs are usually collected from the ilium by the conventional aspiration method in humans. The perfusion method consists of two approaches: transverse iliac perfusion and longitudinal iliac perfusion. BMCs harvested by the perfusion method from the long bones and ilium were compared with those collected from the ilium by the aspiration method. The contamination of BMCs with peripheral blood, determined by the frequencies of CD4+ and CD8+ T cells, was significantly lower in BMCs obtained from the ilium or long bones by the perfusion method (CD4+ plus CD8+ T cells <4%) than in those obtained by the iliac aspiration method (CD4+ plus CD8+ T cells >20%). However, the numbers of immature myeloid cells, such as myeloblasts, promyelocytes, myelocytes, and metamyelocytes, were higher in BMCs obtained by the iliac perfusion method than in those obtained by the iliac aspiration method. The assays for in vitro colony-forming unit in culture revealed that progenitor activity was significantly higher in BMCs obtained by the perfusion method than in those obtained by the aspiration method. These findings suggest that the contamination of BMCs with peripheral blood is much less when using the perfusion method than when using the aspiration method. To determine the best site for harvesting BMCs by the perfusion method, age-dependent changes in BMCs harvested by the perfusion method from the long bones and ilium were examined. The numbers of BMCs varied in the long bones (humerus > femur > tibia) and showed age-dependent decreases, whereas they remained similar in the ilium of cynomolgus monkeys from 3 years to 6 years of age. However, in cynomolgus monkeys, BMC harvesting by the perfusion method from the ilium (but not from the long bones) is found to involve the risk of fat emboli, particularly when the BMCs are quickly perfused under high pressure. These findings suggest, even in humans, that the perfusion method is better than the aspiration method, and that the best site for collection of BMCs is the humerus.     

Extensive studies on perfusion method plus intra-bone marrow-bone marrow transplantation using cynomolgus monkeys

The collection of bone marrow cells (BMCs) using a perfusion method has been advantageous not only because of the low contamination of BMCs with T cells from the peripheral blood but also the enrichment of stromal cells, which support hemopoiesis. Before the application of this new method to humans, its safety needed to be confirmed using cynomolgus monkeys. We therefore performed the perfusion method on more than 100 cynomolgus monkeys using the long bones (such as the humerus and femur) and also the iliac bones (for human application); in the more than 150 trials to date, there have been no accidental deaths. Furthermore, the technical safety of a new method for the intra-bone marrow (IBM) injection of BMCs (termed IBM-bone marrow transplantation) has also been confirmed using 30 monkeys. Disclosure of potential conflicts of interest is found at the end of this article.     

Stem cell transplantation for autoimmune diseases: what can we learn from experimental models?

Using animal models for autoimmune diseases, we have previously shown that allogeneic bone marrow transplantation (allo BMT) can be used to treat autoimmune diseases. Using cynomolgus monkeys, we have recently developed new BMT methods for the treatment of autoimmune diseases. The methods include the perfusion method (PM) for the collection of bone marrow cells (BMCs), and intra-bone marrow (IBM)-BMT for the direct injection of collected whole BMCs into the bone marrow cavity. The PM, in comparison with the conventional aspiration method, can minimize the contamination of BMCs with T cells from the peripheral blood. Therefore, without removing T cells, no graft-versus-host disease (GvHD) develops in the case of the PM. Since BMCs collected by the PM contain not only hemopoietic stem cells (HSCs) but also mesenchymal stem cells (MSCs), the injection of both cells directly into the bone marrow cavity (IBM-BMT) facilitates the engraftment of donor hemopoietic cells. In organ allografts with IBM-BMT, no graft failure occurs even if the radiation dose is reduced. In addition, IBM-BMT is applicable to regeneration therapy and various age-associated diseases such as osteoporosis, since it can efficiently recruit donor-derived normal MSCs. We have also found that IBM-BMT in conjunction with donor lymphocyte infusion can prevent GvHD, but suppress tumor growth. We believe that this strategy heralds a revolution in the field of transplantation (BMT and organ allografts) and regeneration therapy.     

Variants of autophagy-related gene 5 are associated with neuromyelitis optica in the Southern Han Chinese population

Using animal models for autoimmune diseases, we have previously shown that allogeneic bone marrow transplantation (allo BMT) can be used to treat autoimmune diseases. Using cynomolgus monkeys, we have recently developed new BMT methods for the treatment of autoimmune diseases. The methods include the perfusion method (PM) for the collection of bone marrow cells (BMCs), and intra-bone marrow (IBM)-BMT for the direct injection of collected whole BMCs into the bone marrow cavity. The PM, in comparison with the conventional aspiration method, can minimize the contamination of BMCs with T cells from the peripheral blood. Therefore, without removing T cells, no graft-versus-host disease (GvHD) develops in the case of the PM. Since BMCs collected by the PM contain not only hemopoietic stem cells (HSCs) but also mesenchymal stem cells (MSCs), the injection of both cells directly into the bone marrow cavity (IBM–BMT) facilitates the engraftment of donor hemopoietic cells. In organ allografts with IBM–BMT, no graft failure occurs even if the radiation dose is reduced. In addition, IBM–BMT is applicable to regeneration therapy and various age-associated diseases such as osteoporosis, since it can efficiently recruit donor-derived normal MSCs.

We have also found that IBM–BMT in conjunction with donor lymphocyte infusion can prevent GvHD, but suppress tumor growth. We believe that this strategy heralds a revolution in the field of transplantation (BMT and organ allografts) and regeneration therapy.     

Intra-bone marrow injection of donor bone marrow cells suspended in collagen gel retains injected cells in bone marrow, resulting in rapid hemopoietic recovery in mice

We have recently developed an innovative bone marrow transplantation (BMT) method, intra-bone marrow (IBM)-BMT, in which donor bone marrow cells (BMCs) are injected directly into the recipient bone marrow (BM), resulting in the rapid recovery of donor hemopoiesis and permitting a reduction in radiation doses as a pretreatment for BMT. However, even with this IBM injection, some of the injected BMCs were found to enter into circulation. Therefore, we attempted to modify the method to allow the efficient retention of injected BMCs in the donor BM. The BMCs of enhanced green fluorescent protein transgenic mice (C57BL/6 background) were suspended in collagen gel (CG) or phosphate-buffered saline (PBS), and these cells were then injected into the BM of irradiated C57BL/6 mice. The numbers of retained donor cells in the injected BM, the day 12 colony-forming units of spleen (CFU-S) counts, and the reconstitution of donor cells after IBM-BMT were compared between the CG and PBS groups. The number of transplanted cells detected in the injected BM in the CG group was significantly higher than that in the PBS group. We next carried out CFU-S assays. The spleens of mice in the CG group showed heavier spleen weight and considerably higher CFU-S counts than in the PBS group. Excellent reconstitution of donor hemopoietic cells in the CG group was observed in the long term (>100 days). These results suggest that the IBM injection of BMCs suspended in CG is superior to the injection of BMCs suspended in PBS.     

Viral contamination of intradermal skin test syringes

Intradermal skin tests are often performed using a common syringe with multiple needles. Bacterial contamination of intradermal skin test syringes can occur as a result of apparent siphoning caused by needle changing. The bacterial contamination of the syringe can be prevented by flushing the contaminated needle prior to changing. In this study, two different needle changing techniques were examined using a polio virus contaminant. Viral contamination of the syringe was not prevented by flushing the infected needle prior to removal. All syringes were contaminated with virus regardless of needle changing technique. We, therefore, cannot recommend the continued use of a common syringe for intradermal skin tests between patients regardless of needle changing technique.     

Facilitation of hematopoietic recovery by bone grafts with intra-bone marrow-bone marrow transplantation

We have previously shown that T cells can acquire donor-type major histocompatibility complex (MHC) restriction and can interact with both donor-type antigen-presenting cells (APCs) and B cells, when adult donor bones are co-grafted with intravenous (IV) injection of bone marrow cells (BMCs) in order to supply donor bone marrow (BM) stromal cells. We have also found that the direct injection of donor BMCs into recipient BM (intra-bone marrow-bone marrow transplantation: IBM-BMT) produces more rapid reconstitution (including T-cell functions) and higher survival rates than IV injection (IV-BMT) even in chimerism-resistant combinations. In the present study, we show that the co-administration of bones from suckling (2-3 days old) donor mice is also effective in the IBM-BMT system. Even when a relatively low number of BMCs were injected into adult (more than 15 weeks old) mice, complete reconstitution was achieved in the mice that had received IBM-BMT+bone grafts, but not in the mice that had received IBM-BMT alone. Most BM and splenic adherent cells obtained from the recipients that had received IBM-BMT+bone grafts were reconstituted by donor-type cells. Both T-cell proliferation and plaque-forming cell assays indicated that the T cells of such mice showed donor-type MHC restriction. Moreover, the analyses of thymic sections using confocal microscopy revealed that donor BM stromal cells had migrated into the thymus. Thus, the co-administration of donor bones has great advantages for allogeneic BMT in adult mice.     

Immunoglobulin G from anti-glucose-6-phosphate isomerase antibodies positive patient with rheumatoid arthritis induces synovitis in cynomolgus monkeys

Anti-glucose-6-phosphate isomerase (GPI) antibodies (Abs) solely induce arthritis in mice. High titers of anti-GPI Abs are found in some patients with rheumatoid arthritis (RA), but their pathogenic role remains elusive. The aim of this study was to evaluate the pathogenic role of anti-GPI Abs in cynomolgus monkeys. IgG fractions were separated from sera of anti-GPI Abs-positive RA patients and healthy subjects and directly injected into the metacarpophalangeal joints of 4 cynomolgus monkeys. At day 16, the joints were harvested and examined histologically and immunohistochemically. The expression of C5a receptor (C5aR) molecule in the synovium was quantified by real-time PCR using cDNA from monkey joints. In monkey joints, IgG including anti-GPI Abs resulted in recruitment of granulocytes and mononuclear cells, strong deposition of human IgG on the articular surface, and overexpression of C5aR, but no joint swelling. No infiltrated cells or IgG deposition were observed in monkeys injected with IgGs from healthy subjects. Our results suggest that IgG fraction from RA patients including anti-GPI Abs may play a crucial role in the generation of synovitis in monkeys, although the pathogenesis of anti-GPI Abs in RA patients is still uncertain.     

Fine-needle aspiration by vacuum tubes

Fine-needle aspiration of subcutaneous masses, accepted in many parts of Europe and the Americas as a routine diagnostic technique, employs a syringe holder to facilitate the creation of a vacuum to withdraw cells. This investigation demonstrates that a vacuum tube used in venipuncture can be used to supply the negative pressure to suck cells into the needle. This apparatus is more readily available than a syringe holder in hospitals and clinics, and particularly provides the operator with a more dexterous approach to the mass because the fingers holding the needle can be much closer to the mass being immobilized by the other hand.     

Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina

It has recently been shown that bone marrow cells can differentiate into various lineage cells including neural cells in vitro and in vivo. We therefore examined whether bone marrow stem cells can differentiate into retinal neural cells in adult rats. PKH-67-labeled stem cell-enriched bone marrow cells (BMCs) were injected into the vitreous space of eyes in which the retinas had been mechanically injured using a hooked needle. Two weeks after the injection of these cells, immunohistochemical examinations were carried out. The stem cell-enriched BMCs had been incorporated and had differentiated into retinal neural cells in the injured retina. The stem cell-enriched BMCs had accumulated mainly in the outer nuclear layer around the injured sites. The incorporated cells expressed glial fibrillary acidic protein, calbindin, rhodopsin, and vimentin. These results raise the possibility that stem cell-enriched BMCs have the ability to differentiate into retinal neural cells, and that the injection of stem cell-enriched BMCs into the retina would help repair damaged retinal cells.     

Analyses of very early hemopoietic regeneration after bone marrow transplantation: comparison of intravenous and intrabone marrow routes

In bone marrow transplantation (BMT), bone marrow cells (BMCs) have traditionally been injected intravenously. However, remarkable advantages of BMT via the intra-bone-marrow (IBM) route (IBM-BMT) over the intravenous route (IV-BMT) have been recently documented by several laboratories. To clarify the mechanisms underlying these advantages, we analyzed the kinetics of hemopoietic regeneration after IBM-BMT or IV-BMT in normal strains of mice. At the site of the direct injection of BMCs, significantly higher numbers of donor-derived cells in total and of c-kit(+) cells were observed at 2 through 6 days after IBM-BMT. In parallel, significantly higher numbers of colony-forming units in spleen were obtained from the site of BMC injection. During this early period, higher accumulations of both hemopoietic cells and stromal cells were observed at the site of BMC injection by the IBM-BMT route. The production of chemotactic factors, which can promote the migration of a BM stromal cell line, was observed in BMCs obtained from irradiated mice as early as 4 hours after irradiation, and the production lasted for at least 4 days. In contrast, sera collected from the irradiated mice showed no chemotactic activity, indicating that donor BM stromal cells that entered systemic circulation cannot home effectively into recipient bone cavity. These results strongly suggest that the concomitant regeneration of microenvironmental and hemopoietic compartments in the marrow (direct interaction between them at the site of injection) contributes to the advantages of IBM-BMT over IV-BMT. Disclosure of potential conflicts of interest is found at the end of this article.     

灵长类动物子宫内膜异位症病证结合模型构建初探

目的　探索食蟹猴子宫内膜异位症（简称内异症）病证结合模型构建的方法。 方法　（1）6只雌性食蟹猴随机分为实验组5只、对照组1只,实验组开腹行宫颈环扎术,对照组仅开腹。（2）于术前、术中及术后第1、2、3个月运用PSI测量猴舌血液灌注量。（3）术后3个月腹腔镜探查,因均未见内异症病灶,改进建模方法：将猴分为实验组4只、对照组2只,实验组月经期收集子宫内膜组织盆腔内注射,连续使用3个月免疫抑制剂,对照组不作处理。 结果　（1）宫颈环扎后3个月后腹腔镜探查,两组均未见内异症病灶。（2）猴子术后2月舌部血液灌注量明显高于对照组,差异有统计学意义（P<0.05）。（3）每只猴的舌面及舌底血液灌注量变化趋势一致。（4）改进建模方案后3个月腹腔镜探查示,实验组重度盆腔粘连、子宫表面血管增生并充血明显,一只猴可见内异症病灶,对照组则无。 结论　收集子宫内膜盆腔内注射、加用免疫抑制剂可以构建食蟹猴内异症模型。运用PSI测量猴舌部血液灌注量的方法稳定可行,有望成为疾病病证结合模型舌象的量化评价方法。     

灵长类动物子宫内膜异位症病证结合模型构建初探

目的　探索食蟹猴子宫内膜异位症（简称内异症）病证结合模型构建的方法。 方法　（1）6只雌性食蟹猴随机分为实验组5只、对照组1只,实验组开腹行宫颈环扎术,对照组仅开腹。（2）于术前、术中及术后第1、2、3个月运用PSI测量猴舌血液灌注量。（3）术后3个月腹腔镜探查,因均未见内异症病灶,改进建模方法：将猴分为实验组4只、对照组2只,实验组月经期收集子宫内膜组织盆腔内注射,连续使用3个月免疫抑制剂,对照组不作处理。 结果　（1）宫颈环扎后3个月后腹腔镜探查,两组均未见内异症病灶。（2）猴子术后2月舌部血液灌注量明显高于对照组,差异有统计学意义（P<0.05）。（3）每只猴的舌面及舌底血液灌注量变化趋势一致。（4）改进建模方案后3个月腹腔镜探查示,实验组重度盆腔粘连、子宫表面血管增生并充血明显,一只猴可见内异症病灶,对照组则无。 结论　收集子宫内膜盆腔内注射、加用免疫抑制剂可以构建食蟹猴内异症模型。运用PSI测量猴舌部血液灌注量的方法稳定可行,有望成为疾病病证结合模型舌象的量化评价方法。     

Syngenic bone marrow cells restore hepatic function in carbon tetrachloride-induced mouse liver injury

Progenitor cells in bone marrow have been explored for the treatment of liver injury. Stem cell homing to the injured tissue is regulated through stromal cell derived factor-1 (SDF-1) and its receptor CXCR4. We hypothesized that syngenic bone marrow cells (BMCs) would restore hepatic function in the injured liver through the regulation by SDF-1/CXCR4 system. After injecting carbon tetrachloride (CCl(4)), the mice were injected with syngenic BMCs or normal saline. Morphological and functional analysis of the liver was performed. Flow cytometry for the stem cell markers and CXCR4 was done with the liver, BM, and spleen cells from each group. Carboxyfluorescein diacetate succinimidyl ester was used to trace the homing of transplanted BMCs. The SDF-1 expression of the liver was assessed by immunohistochemistry. Hepatosplenomegaly and necrosis of the CCl(4)-injected mouse liver were improved after BMCs transplantation The hepatic enzymes were increased after injury and then decreased after BMCs transplantation. The expression of stem cell markers and CXCR4 was exclusively increased in the damaged liver compared to the BM and spleen, and even more elevated after BMCs transplantation. SDF-1 expression in the liver was observed after CCl(4) injection and it was elevated after BMCs transplantation. The intrinsic and extrinsic BMCs migrate specifically to the injured liver rather than BM or spleen, and the transplanted BMCs contribute to the repair of the damaged liver. SDF-1/CXCR-4 interaction plays a role in stem cell homing toward the damaged organ, and transplanted BMCs are involved in the up-regulated SDF-1 expression seen in the injured liver.     

中国人肝细胞系1运用于生物人工肝的生物代谢功能

背景：前期研究发现,中国人肝细胞系1细胞分化程度高且生物代谢功能良好, 并且中国人肝细胞系1细胞组织学上来源于正常肝组织,较其他来源于肿瘤源性的肝细胞系更为安全。 目的：探讨中国人肝细胞系中国人肝细胞系1细胞在混合型生物人工肝中的生物代谢功能。 方法：15只食蟹猴随机分成对照组(n=5)和治疗组(n=10),均建立急性肝功能衰竭模型,治疗组接受以全接触灌流型生物反应器接种微载体微重力中国人肝细胞系1细胞建立的人源细胞混合型生物人工肝进行治疗。 结果与结论：急性肝功能衰竭食蟹猴血清谷氨酸转氨酶、总胆红素、总胆汁酸、尿素氮、肌酐、血氨均明显上升,而白蛋白、Fischer指数则显著下降;人源细胞混合型生物人工肝治疗后,急性肝功能衰竭食蟹猴血清谷氨酸转氨酶、总胆红素、总胆汁酸、尿素氮、肌酐、血氨和白蛋白均恢复。提示中国人肝细胞系1细胞在混合型生物人工肝中生物代谢功能良好,表现出良好的肝特异性生物合成及生物代谢功能。     

Prospective isolation of murine hematopoietic stem cells by expression of an Abcg2/GFP allele

Stem cells from a variety of tissues can be identified by a side population (SP) phenotype based on Hoechst 33342 dye efflux. The Abcg2 transporter is expressed in hematopoietic stem cells (HSCs) and confers this dye efflux activity. To further explore the relationship among Abcg2 expression, the SP phenotype, and HSC activity, we have generated mice in which a green fluorescent protein (GFP) reporter gene was inserted into the Abcg2 locus. In these mice, the majority of bone marrow (BM) cells that expressed the Abcg2/ GFP allele were Ter119(+) erythroid cells. The Abcg2/GFP allele was also expressed in approximately 10% of lineage-negative (Lin(-)) and in 91% of SP cells using stringent conditions for the SP assay. Flow cytometric sorting was used to isolate various Abcg2/GFP(+) BM cell populations that were then tested for HSC activity in transplant assays. There was significant enrichment for HSCs in sorted Lin(-)/ GFP(+) cells, with a calculated HSC frequency of approximately one in 75. There was no HSC activity detected in Lin(-)/GFP(+) cells. Altogether, these results show that Abcg2 is expressed on essentially all murine BM HSCs and can be used as a prospective marker for HSC enrichment.     

Evaluation of Sca-1 and c-Kit as selective markers for muscle remodelling by nonhemopoietic bone marrow cells

Bone marrow (BM)-derived cells (BMCs) have demonstrated a myogenic tissue remodeling capacity. However, because the myoremodeling is limited to approximately 1%-3% of recipient muscle fibers in vivo, there is disagreement regarding the clinical relevance of BM for therapeutic application in myodegenerative conditions. This study sought to determine whether rare selectable cell surface markers (in particular, c-Kit) could be used to identify a BMC population with enhanced myoremodeling capacity. Dystrophic mdx muscle remodeling has been achieved using BMCs sorted by expression of stem cell antigen-1 (Sca-1). The inference that Sca-1 is also a selectable marker associated with myoremodeling capacity by muscle-derived cells prompted this study of relative myoremodeling contributions from BMCs (compared with muscle cells) on the basis of expression or absence of Sca-1. We show that myoremodeling activity does not differ in cells sorted solely on the basis of Sca-1 from either muscle or BM. In addition, further fractionation of BM to a more mesenchymal-like cell population with lineage markers and CD45 subsequently revealed a stronger selectability of myoremodeling capacity with c-Kit/Sca-1 (p < .005) than with Sca-1 alone. These results suggest that c-Kit may provide a useful selectable marker that facilitates selection of cells with an augmented myoremodeling capacity derived from BM and possibly from other nonmuscle tissues. In turn, this may provide a new methodology for rapid isolation of myoremodeling capacities from muscle and nonmuscle tissues. Disclosure of potential conflicts of interest is found at the end of this article.     

Optical imaging of PKH-labeled hematopoietic cells in recipient bone marrow in vivo

This work describes an optical technique for characterization of the early stages of hematopoietic stem cell (HSC) engraftment under physiological conditions and in real time. Bone marrow cells (BMCs) labeled with PKH membrane linkers were injected into conditioned recipients (B10-->B10.BR mice) preoperated for placement of optical windows over femoral epiphyses. Labeled cells were tracked in vivo by fluorescence microscopy. Cellular adhesion to the BM stroma was tested with laser tweezers, and viability was assayed by the propidium iodide (PI) exclusion test, as determined from energy-transfer measurements of the pair PKH67-PI in freshly excised femurs in situ. At optimal concentrations for in vivo tracking, 1-4 micro M PKH dyes neither impaired the viability of BMCs nor the capacity of allogeneic HSCs to reconstitute hematopoiesis in myeloablated recipients. The optical window allowed in vivo visualization of 23%-26% of the PKH-labeled BMCs in the femur. The homing efficiencies at 16 hours posttransplantation were quantified as 1.77% +/- 0.15% and 0.21% +/- 0.02% for syngeneic and allogeneic BMCs, respectively. In femurs excised 16 hours after transplantation, 70% +/- 9% of the cells were adherent to the BM stroma, and two-thirds of the cells were PI negative (viable). In vivo tracking and in situ assessment of labeled HSCs in recipient BM provide important quantitative and qualitative insights into the early stages of engraftment. Correlation of early events and the efficiency of durable engraftment serve as the basis for a systematic approach toward optimization of the conditions for transplantation.     

Lack of correlation between an assay used to determine early marrow allograft rejection and long-term chimerism after murine allogeneic bone marrow transplantation: effects of marrow dose.

The acute rejection of bone marrow (BM) allografts by host effectors can occur within a short period after BM transplantation (BMT) in lethally irradiated mice. Common assays used to ascertain engraftment/resistance involve measuring the growth of granulocyte/monocyte progenitors (colony-forming unit-granulocyte-macrophage) in vitro or splenocyte proliferation assessed by radioisotope incorporation in vivo 5 to 8 days after BMT. However, the correlation of the long-term outcome of BMT with the kinetics of recovery by using the dose of allogeneic BM cells (BMCs) that leads to early rejection as determined by the in vitro assessment has not been extensively studied. Thus, to investigate whether the early rejection of donor BMCs is an indication of a long-term engraftment failure, C57BL/6 (H2b) mice were lethally irradiated and transplanted with various doses of BALB/c (H2d) BMCs. The short-term engraftment of donor precursors (colony-forming unit-granulocyte-macrophage), the kinetics of hematopoietic cell recovery, the extent of donor chimerism, and the proportion of the recipients with long-term survival were determined. The results show that the kinetics and extent of hematopoietic cell recovery were significantly delayed in mice receiving limiting doses of BMCs that were rejected or severely resisted at day 8 after BMT. However, a proportion of these mice survived up to 98 days after BMT with mixed chimerism or donor chimerism. This study demonstrates that early rejection of BM precursors, as assessed by measurement of myeloid progenitors in the spleen after BMT, does not always correlate with the long-term outcome of the marrow allograft and that significant variability is inherent in the extent of chimerism when threshold amounts of BMCs are used.     

Differential effects of administration of a human anti-CD4 monoclonal antibody, HM6G, in nonhuman primates.

A human sequence IgGkappa anti-CD4 monoclonal antibody (mAb), HM6G, originally isolated from a human immunoglobulin transgenic mouse was specific for and bound with high binding avidity to the CD4 antigen expressed on human, chimpanzee, and cynomolgus monkey T cells. Prior to testing this mAb in human clinical trials, a number of preclinical primate studies were performed. In chimpanzees, HM6G did not deplete circulating CD4(+) T cells and was cleared in a dose-dependent manner. In contrast, this mAb administered to cynomolgus monkeys depleted CD4(+) T cells (albeit only at high doses) and its clearance, which had reached saturation even at very low doses, was much slower. These differences were most likely due to the additional and rather substantial expression of the CD4 antigen on chimpanzee monocytes. In monkeys, the T cell depletion was mitigated by infusing the mAb over 30 min or longer (as opposed to 30 s) while only slightly altering the clearance. As expected, the human mAb did not induce an immune response in chimpanzees, although it did induce a low titer response in monkeys. These disparate pharmacokinetic and pharmacodynamic results suggest prudence when extrapolating results obtained in nonhuman models to humans.