The effect of food and water deprivation on the peripheral blood parameters of the mouse

Various peripheral blood and bone marrow parameters were determined during food and water deprivation and during food deprivation alone in order to obtain base lines that may be used to make comparisons with similar data from irradiated mice. The peripheral blood parameters following food and water deprivation were similar to those following food deprivation alone. The mean survival time was about 5 days and the weight loss 40% of the control weight. There was an absolute decrease in the total circulating lymphocyte and platelet counts, while the total granulocyte count remained unchanged or increased. The blood volume decreased, while the hematocrit and specific gravity of the blood increased. The bone marrow parameters following food and water deprivation showed that erythropoiesis was more markedly depressed than myelopoiesis. The tritiated thymidine labeling index for granulopoietic cells and megakaryocytes decreased progressively during starvation. The variations in the white blood count and the bone marrow parameters are not comparable with those found in irradiated mice having the G.I. syndrome; the changes in mean survival time, weight loss, hematocrit, and blood volume are similar.     

Modulation of murine hemopoiesis in vivo by a synthetic hemoregulatory pentapeptide (HP 5b)

A synthetic pentapeptide analogous to an inhibitory factor associated with human granulocytes was tested in vivo on female C3H mice. The relative and absolute numbers of myelopoietic and erythropoietic cells in the bone marrow were measured following injections as well as the continuous infusion of the pentapeptide in dose ranges between 10(-8)M and 10(-4)M (0.12 micrograms to 1.2 mg/mouse). In low doses, the pentapeptide reduced the number of myelopoietic cells in the bone marrow, and this was accompanied by reduced numbers of granulocytes and monocytes and peripheral blood. Elevated doses also decreased erythropoiesis. In contrast, continuous infusion of 14 micrograms/h for 19 days seemed to make the myelopoietic cells refractory to further action. A regulatory function of the pentapeptide is proposed.     

Are endogenous sex hormones related to DNA damage in paradoxically sleep-deprived female rats?

The aim of this investigation was to evaluate overall DNA damage induced by experimental paradoxical sleep deprivation (PSD) in estrous-cycling and ovariectomized female rats to examine possible hormonal involvement during DNA damage. Intact rats in different phases of the estrous cycle (proestrus, estrus, and diestrus) or ovariectomized female Wistar rats were subjected to PSD by the single platform technique for 96 h or were maintained for the equivalent period as controls in home-cages. After this period, peripheral blood and tissues (brain, liver, and heart) were collected to evaluate genetic damage using the single cell gel (comet) assay. The results showed that PSD caused extensive genotoxic effects in brain cells, as evident by increased DNA migration rates in rats exposed to PSD for 96 h when compared to negative control. This was observed for all phases of the estrous cycle indistinctly. In ovariectomized rats, PSD also led to DNA damage in brain cells. No significant statistically differences were detected in peripheral blood, the liver or heart for all groups analyzed. In conclusion, our data are consistent with the notion that genetic damage in the form of DNA breakage in brain cells induced by sleep deprivation overrides the effects related to endogenous female sex hormones.     

Hematopoietic augmentation by a β-(1,4)-linked mannan

 CARN 750 (injectable acemannan) is a polydispersed β-(1,4)-linked acetylated mannan isolated from the Aloe barbadensis plant. It has multiple therapeutic properties including activity in wound repair and as a biological agent for the treatment of neoplasia in animals as well as the ability to activate macrophages. We report herein that CARN 750 directly or indirectly has significant hematoaugmenting properties. We observed that the subcutaneous administration of CARN 750 significantly increases splenic and peripheral blood cellularity, as well as hematopoietic progenitors in the spleen and bone marrow as determined by the interleukin-3-responsive colony-forming unit culture assay and the high-proliferative-potential colony-forming-cell (HPP-CFC) assay (a measure of primitive hematopoietic precursors) in myelosuppressed (7 Gy) C₅₇BL/6 mice. The greatest hematopoietic effect was observed following sublethal irradiation in mice receiving 1 mg CARN 750/animal, with less activity observed at higher or lower doses. Further, CARN 750, following daily injection, has activity equal to or greater than the injection of an optimal dose of granulocyte-colony-stimulating factor (G-CSF) in myelosuppressed mice. In this comparison, significantly greater activity was observed in the splenic and peripheral blood cellularity, and in the frequency and absolute number of splenic HPP-CFC as compared to the mice receiving G-CSF at 3 μg/animal. CARN 750, when administered to myelosuppressed animals. decreased the frequency of lymphocytes with a concomitant significant increase in the frequency of polymorphonuclear leukocytes (PMN). However, owing to the increased cellularity, a significant increase in the absolute number of PMN, lymphocytes, monocytes and platelets was observed, suggesting activity on multiple cell lineages. The latter is the primary difference in activity as compared to G-CSF which has activity predominantly on PMN. Received: 21 November 1995 / Accepted: 13 September 1996     

Pathophysiological Scenario of Hematopoietic Disorders: A Comparative Study of Aplastic Anemia,Myelodysplastic Syndrome and Leukemia in Experimental Animals

The conversion of physiology to pathophysiology in hematological disorders viz: aplastic anemia, myelodysplastic syndrome (MDS) and leukemia in murine models was the subject of study in the present programme. Peripheral blood hemogram, spleno-somatic index, bone marrow smear study, cytochemical staining of marrow, cell release kinetics study during marrow explants culture, hematopoietic niche assessment, chromosomal aberration study, plasma membrane stability study of marrow cells, lysosomal membrane and mitochondrial membrane stability study and innate immune parameters were performed in the aplastic anemia, leukemia and MDS mouse model. In bone marrow aplasia, peripheral blood pancytopenia, marrow hypocellularity, decreased marrow cellular viability, deterioration of bone marrow hematopoiesis as well as hematopoietic microenvironment and extramedullary hematopoiesis were noticed. In addition, disruption of mitochondrial and lysosomal membrane integrity along with reduction of innate immune parameters were found in the hematopoietic suppressed condition. Surprisingly, no noticeable chromosomal aberration was found in the aplastic condition. Ineffective marrow hematopoiesis together with the disruption of hematopoietic microenvironment was observed in MDS. Also, extramedullary hematopoiesis, increased marrow cellular death, chromosomal aberration and loss of innate immunity were the common events. During leukemia, the number of functionally and structurally immature cells in the peripheral blood and bone marrow was increased together with malignant conversion of hematopoietic cells in the presence of malignancy supportive stromal microenvironment. Chromosomal aberration, decrease of cell mediated immunity with least mitochondrial apoptotic damage were also found in leukemic condition as well.     

Physical separation of colony stimulating cells from in vitro colony forming cells in hemopoietic tissue

Hemopoietic colony formation in agar occurred spontaneously in mass cultures of marrow cells obtained from a number of species (guinea pig, rat, lamb, rabbit, pig, calf, human and Rhesus monkey). This contrasted with the observation that colony formation by mouse bone marrow exhibited an absolute requirement for an exogenous source of a colony stimulating factor. Analysis of spontaneous colony formation in Rhesus monkey marrow cultures revealed the presence of a cell type in hemopoietic tissue, capable of elaborating colony stimulating factor when used to condition media or as feeder layers. Equilibrium density gradient centrifugation separated colony stimulating cells from in vitro colony forming cells in monkey bone marrow. Separation studies on spleen, blood and marrow characterized the stimulating cells as of intermediate density, depleted or absent in fractions enriched for cells of the granulocytic series and localized in regions containing lymphocytes and monocytes. Adherence column separation of peripheral blood leukocytes showed the stimulating cells to be actively adherent, unlike the majority of lymphocytes, and combined adherence column and density separation indicated that stimulating cells were present in hemopoietic tissue within the population of adherent lymphocytes or monocytes.     

小鼠骨髓造血干细胞、外周血组成随年龄的变化趋势及其相关性分析

造血干细胞是具有自我更新能力并能分化为血液中各种血细胞组分的多能干细胞。近来研究显示,不同造血干细胞表面标志物标记的造血干细胞具有分化为不同血细胞的趋势,但是这种分化的内在关系仍不清楚。对小鼠CD34~-/Sca-1~+骨髓造血干细胞、外周血组成随小鼠年龄增长的变化情况进行了分析,结果显示:随着年龄的增长,骨髓中的CD34~-/Sca-1~+骨髓造血干细胞比率显著增加;而外周血各组分则随年龄变化呈现不同的趋势。对不同年龄段小鼠的骨髓造血干细胞及其他组分与外周血组分的同步分析发现,外周血中血小板密度变化趋势与CD34~-/Sca-1~+骨髓造血干细胞变化情况相关系数为0.804 8;外周血中淋巴细胞密度变化趋势与CD34~+/Sca-1~-骨髓细胞的变化情况相关系数为0.947 97;外周血中白细胞密度变化趋势与CD34~+/Sca-1~+骨髓细胞变化情况相关系数为0.763 1(大于0.9为极度相关,0.7到0.9为高度相关)。     

A model of Ph' positive chronic myeloid leukemia-blast crisis cell line growth in immunodeficient SCID mice.

A human Philadelphia-chromosome positive chronic myeloid leukemia-blast crisis (CML-BC) cell line BV173 proliferated in the hematopoietic tissues, infiltrated various organs and caused the death of immunodeficient SCID mice. Leukemia spreading was assessed with diminished number of bone marrow cells and caused splenomegaly. The leukemic colonies grew from single cell suspension of bone marrow, spleen and peripheral blood. Bcr-abl m-RNA was detectable in bone marrow, spleen, peripheral blood, liver, lungs and brain. Dying mice demonstrated severely hypoplastic bone marrow, splenomegaly and massive metastases in the liver and kidneys. The survival time of animals was dependent on the number of inoculated leukemia cells.     

Expression pattern of a hematopoietic proteoglycan core protein gene during human hematopoiesis

Expression of the hematopoietic proteoglycan core protein (HpPG) gene was examined in normal peripheral blood, normal bone marrow, and leukemic peripheral blood leukocytes samples to assess the expression pattern of the HpPG gene in these cells and to ascertain points of regulation of this gene during hematopoiesis. In situ hybridization to normal bone marrow and peripheral blood leukocytes demonstrated that the gene was expressed in the promyelocytes at a approximately two fold greater level than in the segmented neutrophils and the expression decreased as the granulocytes matured. The ratio of expression in the other leukocytes to expression in the segmented neutrophils were as follows: eosinophils/basophils approximately 7; monocytes approximately 2; lymphocytes less than 1. Expression of the HpPG gene during myeloblast differentiation was assessed by Northern blot analysis of acute myelogenous leukemia (AML) RNA samples. The expression of this gene, when compared to the levels in HL-60 cells, was approximately ten fold lower in the poorly differentiated blast cells obtained from three AML patients classified M"0". Conversely, the expression in the more differentiated blast cells obtained from 10 of 11 AML patients classified as M1 and M2 were at levels similar to the levels in HL-60 cells. The expression level found in eight lymphoid leukemias was approximately ten fold or more lower than in HL-60 cells. Gene copy number determination confirmed that the HpPG gene is present in one copy per haploid genome. Thus the HpPG gene's expression pattern denotes a single copy gene being differentially expressed during hematopoiesis with initial regulation occurring very early in this developmental process and an additional up-regulatory event occurring during granule genesis.     

Mobilization of human hematopoietic stem/progenitor-enriched CD34+ cells into peripheral blood during stress related to ischemic stroke

The bone marrow-derived stem/progenitor cells were demonstrated to play an important role in a regeneration of damaged tissue. Based on these observations we asked whether the stroke-related stress triggers mobilization of stem/progenitor cells from the bone marrow into the peripheral blood, which subsequently could contribute to regeneration of damaged organs. To address this issue, the peripheral blood samples were harvested from patients with ischemic stroke during the first 24 hrs as well as after the 48 (2nd day) and 144 hrs (6th day) since the manifestation of symptoms. In these patients we evaluated the percentage of hematopoietic stem/progenitor-enriched CD34+ cells by employing flow cytometry and the number of hematopoietic progenitor cells for the granulocyto-monocytic (CFU-GM) and erythroid (BFU-E)-lineages circulating in peripheral blood. We concluded that stress related to ischemic stroke triggers the mobilization of hematopoietic stem/progenitor cells from the bone marrow into peripheral blood. These circulating stem/progenitor cells may play an important role in the process of regeneration of the ischemic tissue.     

IL-33转基因小鼠骨髓造血干／祖细胞向髓系细胞分化的能力增强

目的：利用IL-33转基因小鼠研究IL-33对造血干/祖细胞的增殖和分化影响。方法利用流式细胞仪分析IL-33转基因小鼠及同窝野生对照小鼠的外周血、脾脏、骨髓细胞的免疫表型及造血干细胞分化不同阶段细胞的数量变化;利用体外成克隆实验和细胞周期分析研究IL-33对于造血干细胞增殖能力的影响。结果与野生型小鼠相比,IL-33转基因小鼠B细胞和T细胞在外周血中都明显降低,粒细胞在外周血和骨髓中都有明显增加;IL-33转基因小鼠的骨髓造血干细胞和多能祖细胞数量减少,共同淋系祖细胞数量减少,共同髓系祖细胞和粒单系祖细胞数量增加;IL-33转基因小鼠的造血干细胞处于S-G2-M的细胞增多;体外单克隆实验发现IL-33转基因小鼠造血干细胞形成的集落数增加。结论 IL-33转基因小鼠造血干细胞增殖能力增强,更易向髓系细胞分化。     

T-lymphocyte subpopulations in the peripheral blood and bone marrow of patients with aplastic anemia

A decrease in the absolute number of total lymphocytes, OKT3+ and OKT4+ lymphocytes, and a normal number of OKT8+ lymphocytes were found in the peripheral blood of patients with aplastic anemia. The OKT4:OKT8 ratio was decreased in patients due to a reduction in the percentage of OKT4+ cells and 3 out of 18 patients had a ratio less than 1. The values of the OKT4:OKT8 ratio were not associated either with the severity of the disease or with treatment with androgens. There was no correlation between the OKT4:OKT8 ratio and the number of transfusions received by patients. On the other hand, studies performed with bone marrow lymphocytes showed that the OKT4:OKT8 ratio for both patients and controls was lower than that of the peripheral blood. Since the ratio of OKT4:OKT8 cells in aplastic and control bone marrow was similar no direct pathogenic role can be assigned to the marrow for the imbalance detected in the peripheral blood.     

A new approach to evaluate the total reserve of hematopoietic progenitors after acute irradiation

Based on the capacity of certain hematopoietic growth factors to mobilize the hematopoietic progenitors from bone marrow to peripheral blood, we have investigated whether the number of progenitors that can be mobilized to peripheral blood after irradiation correlates with the radiation dose and reflects the total reserve of bone marrow progenitors that survive the exposure. In three different mouse strains, a close relationship was observed between the number of G-CSF mobilized progenitors and the radiation dose received by the animals. When G-CSF was replaced by one single injection of SD01 plus thrombopoietin, a similar relationship between the two parameters was observed, which fitted to the multitarget theoretical model. This treatment also promoted 50% survival in mice receiving a lethal dose of 9 Gy. The estimation of the total number of CFU-GM progenitors in the irradiated mice also allowed us to establish a good relationship between the number of progenitors that were mobilized to peripheral blood with respect to the global reserve of surviving progenitors. These results suggest that the quantification of mobilized hematopoietic progenitors would predict the severity and reversibility of the hematopoietic syndrome of irradiated victims, based on direct estimations of their global reserve of hematopoietic progenitors and stem cells.     

长期和短期限制性束缚对小鼠造血干细胞的不同影响

慢性心理应激会导致机体多种功能紊乱,其中包括免疫力下降。慢性心理应激能够负调节免疫系统,但机制尚未完全阐明。免疫细胞,包括髓系来源细胞,由骨髓造血干细胞(hematopoietic stem cell, HSC)分化而来,在机体免疫中发挥重要作用。本实验采用短期高强度束缚和长期温和束缚慢性心理应激小鼠模型,探讨不同限制性束缚模式对小鼠骨髓HSC和髓系来源细胞的影响。长期温和束缚模型中,对小鼠连续束缚4周,每天束缚2次,每次束缚2 h (9:00到17:00间完成);短期高强度束缚模型中,小鼠连续束缚5天,每天束缚16 h (当日17:00到次日9:00)。束缚完成后,取小鼠骨髓和外周血进行白细胞计数,流式细胞术检测小鼠骨髓HSC (Lin^-CD117^+Sca1^+CD150^+CD48^-)和髓系细胞(CD11b^+Ly6C^+)的比例和绝对数,以及外周血髓系细胞的比例和绝对数,BrdU掺入实验检测小鼠HSC增殖能力。实验结果表明,长期温和应激导致小鼠骨髓HSC的比例和绝对数增加,而短期高强度应激导致小鼠骨髓HSC的绝对数下降,伴有HSC的增殖下降。两种束缚模式都使小鼠骨髓和外周血CD11b^+Ly6C^+细胞的总数增加或呈现增加的趋势。综上所述,长期温和应激和短期高强度应激对小鼠HSC的比例和绝对数影响不同;两种应激模型都可以使CD11b^+Ly6C^+细胞总数增加,HSC增多可能并不是CD11b^+Ly6C^+细胞增多的主要机制。     

Transplantation of hematopoietic stem cells from the peripheral blood

Hematopoietic stem cells can be collected from the peripheral blood. These hematopoietic stem cells (HSC), or better progenitor cells, are mostly expressed as the percentage of cells than react with CD34 antibodies or that form colonies in semi-solid medium (CFU-GM). Under steady-state conditions the number of HSC is much lower in peripheral blood than in bone marrow. Mobilization with chemotherapy and/or growth factors may lead to a concentration of HSC in the peripheral blood that equals or exceeds the concentration in bone marrow. Transplantation of HSC from the peripheral blood results in faster hematologic recovery than HSC from bone marrow. This decreases the risk of infection and the need for blood-product support. For autologous stem-cell transplantation (SCT), the use of peripheral blood cells has completely replaced the use of bone marrow. For allogeneic SCT, on the other hand, the situation is more complex. Since peripheral blood contains more T-lymphocytes than bone marow, the use of HSC from the peripheral blood increases the risk of graft-versus-host disease after allogeneic SCT. For patients with goodrisk leukemia, bone marrow is still preferred, but for patients with high-risk disease, peripheral blood SCT has become the therapy of choice.     

Clinical assessment of blood leukocytes, serum cytokines, and serum immunoglobulins as responses to sleep deprivation in laboratory rats

The specific systems and mechanisms affected by sleep deprivation that may perpetuate disease processes in humans still are speculative. In laboratory rats, prolonged sleep deprivation induces a state marked by abnormal control over indigenous bacteria that results in transient infections of internal tissues and eventual lethal septicemia. The present studies investigated changes in blood, serum, and bone marrow parameters that may provide diagnostic clues to immunopathology. Prolonged sleep deprivation was produced in rats by the disk-over-water method, a well-established and selective means that does not interfere with normal waking behaviors. Measurements included bone and blood differential white blood cell counts, multiple serum cytokines and chemokines, several major Ig classes and subclasses, and serum endotoxin concentrations. The results indicated mild, regenerative neutrophilia in sleep-deprived rats, initially accompanied by immature neutrophils and later by monocytosis. The corresponding serum cytokine profile revealed an evolving proinflammatory state, particularly by high incidence of interleukin-1beta, implicating mononuclear phagocytes and resident tissue cells as main intermediary sources. In addition, multiple serum Ig classes were increased by sleep deprivation without experimental administration of an exogenous antigen. Despite this immune activation, there was failure to eradicate invading bacteria and toxins, suggesting competing anti-inflammatory processes or interference with immune effector functions during sleep deprivation. Nearly all of the immune-related events that emerged as responses to sleep deprivation have been implicated as etiological or provocative factors in other disease processes and may provide means by which sleep deprivation as a risk factor in disease may become understood.     

Assessment of hemapoietic system reaction of mice when exposed to pulsed magnetic field

The focus of the study was on the reaction of by the haemopoietic system of mice subjected to impulse magnetic field. The source of the impulse magnetic field was the Shakhparonov's generator. The animals used in the experiments were mice of two strains--CBA, C57B1/6 and white non-inbred mice. These animals were exposed to impulse magnetic field during 1, 3 and 7 days. Animals were examined twice: immediately after the termination of exposure and 24 h later. The following effects were observed in the course of the experiments: an increase in the number of bone marrow cells right after the exposure termination; an increase in the number of proliferation pool cells with the increase in their mitotic activity; 1 day after the exposure termination the number of bone marrow cells was restored to the initial values, or even it decreased; the above listed bone marrow changes led to the increase in the number of peripheral blood leucocytes in 1 day after the termination of exposure. The increase of leukocyte counts was not accompanied with changes in peripheral blood cell composition. It was suggested that exposure to impulse magnetic field increases the rates of cell cycle, the cell differentiation and the maturation.     

Dietary antioxidants protect hematopoietic cells and improve animal survival after total-body irradiation

The purpose of this study was to determine whether a dietary supplement consisting of L-selenomethionine, vitamin C, vitamin E succinate, alpha-lipoic acid and N-acetyl cysteine could improve the survival of mice after total-body irradiation. Antioxidants significantly increased the 30-day survival of mice after exposure to a potentially lethal dose of X rays when given prior to or after animal irradiation. Pretreatment of animals with antioxidants resulted in significantly higher total white blood cell and neutrophil counts in peripheral blood at 4 and 24 h after 1 Gy and 8 Gy. Antioxidants were effective in preventing peripheral lymphopenia only after low-dose irradiation. Antioxidant supplementation was also associated with increased bone marrow cell counts after irradiation. Supplementation with antioxidants was associated with increased Bcl2 and decreased Bax, caspase 9 and TGF-beta1 mRNA expression in the bone marrow after irradiation. Maintenance of the antioxidant diet was associated with improved recovery of the bone marrow after sublethal or potentially lethal irradiation. Taken together, oral supplementation with antioxidants appears to be an effective approach for radioprotection of hematopoietic cells and improvement of animal survival, and modulation of apoptosis is implicated as a mechanism for the radioprotection of the hematopoietic system by antioxidants.     

Ganoderma spore lipid protects mouse bone marrow mesenchymal stem cells and hematopoiesis from the cytotoxicity of the chemotherapeutic agent

Cancer chemotherapeutic agents are frequently toxic to bone marrow and impair bone marrow functions. It is unclear whether ganoderma spore lipid (GSL) can protect bone marrow cells from the cytotoxicity of chemotherapy. To investigate the protective effects of GSL on bone marrow mesenchymal stem cells (MSCs) and hematopoiesis, we examined the effects of GSL on MSCs in vitro and hematopoiesis in vivo after treatment with the chemotherapeutic agent cyclophosphamide. MSCs and peripheral blood cells were isolated and counted from the bone marrow of normal mice were pre-treated with GSL before CTX treatment or co-treated with GSL and CTX, followed by examining the changes in phenotype, morphology, proliferation, apoptosis, and differentiation potentials. The results showed that GSL could reduce the CTX-induced changes in the phenotype of MSCs and maintain the elongated fibroblast-like morphology. MTT and annexin V/propidium iodide (PI) analyses found that GSL pre-treatment and co-treatment increased the proliferation and decreased the apoptosis in CTX-treated MSCs. Furthermore, GSL improved the osteogenic and adipogenic differentiation potentials of CTX-treated MSCs. In vivo, GSL treatment increased the number of peripheral blood cells including white blood cells (WBC) and platelets (PLT) in the CTX-treated mice and enhanced the in vitro formation of hematopoietic lineage colonies (erythrocyte colony forming unit, CFU-E; erythroid burst-forming units, BFU-E; and granulocyte macrophage colony-forming units, CFU-GM) from bone marrow cells in these mice. These findings suggest GSL could protect MSCs and hematopoiesis from the cytotoxicity of CTX and might become an effective adjuvant to attenuate side effects of chemotherapy during cancer treatment.     

Prostaglandin E2 enhances human cord blood stem cell xenotransplants and shows long-term safety in preclinical nonhuman primate transplant models

Hematopoietic stem cells (HSCs) are used in transplantation therapy to reconstitute the hematopoietic system. Human cord blood (hCB) transplantation has emerged as an attractive alternative treatment option when traditional HSC sources are unavailable; however, the absolute number of hCB HSCs transplanted is significantly lower than bone marrow or mobilized peripheral blood stem cells (MPBSCs). We previously demonstrated that dimethyl-prostaglandin E2 (dmPGE2) increased HSCs in vertebrate models. Here, we describe preclinical analyses of the therapeutic potential of dmPGE2 treatment by using human and nonhuman primate HSCs. dmPGE2 significantly increased total human hematopoietic colony formation in?vitro and enhanced engraftment of unfractionated and CD34(+) hCB after xenotransplantation. In nonhuman primate autologous transplantation, dmPGE2-treated CD34(+) MPBSCs showed stable multilineage engraftment over 1 year postinfusion. Together, our analyses indicated that dmPGE2 mediates conserved responses in HSCs from human and nonhuman primates and provided sufficient preclinical information to support proceeding to an FDA-approved phase 1 clinical trial.