Hematological and biochemical profile of BALB/c nude and C57BL/6 SCID female mice after ovarian xenograft.

Hematological and biochemical profile studies help to evaluate functional changes of animals used in experiments. The aim of this study was to determine the hematological and biochemical profile of immunosuppressed BALB/c nude and C57BL/6 SCID mice after bovine ovarian xenotransplantation. Therefore, a total of 74 female mice were divided into four groups: non-xenotransplanted animals, xenotransplanted animals, xenotransplanted animals treated with eCG and xenotransplanted animals treated with FSH + LH. After anesthesia, blood samples were collected and hematologic and biochemical values were evaluated. The results showed no significant differences (p ≤ 0.05) for hematological parameters between the control group and the treatment groups of both strains. However, considering the biochemical profile, it was observed an increase of AST concentrations (p ≤ 0.05) in both strains and a decrease of ALT concentrations (p ≤ 0.05) only in C57BL/6 SCID strain of the groups subjected to hormonal treatment compared with those non subjected. Additionally, the values of the renal enzymes, urea and creatinine, did not differ (p ≤ 0.05) between the groups. Our findings suggest that the xenotransplantation procedure as well as the hormonal dosages had no significant effect on the well-being of the animals considering the evaluated hematological and biochemical profile.

In vivo sequestration of Plasmodium falciparum-infected human erythrocytes: a severe combined immunodeficiency mouse model for cerebral malaria.

Cerebral malaria is a fatal complication of infection by Plasmodium falciparum in man. The neurological symptoms that characterize this form of malarial disease are accompanied by the adhesion of infected erythrocytes to the vasculature of the brain. To study this phenomenon in vivo, an acute phase severe combined immunodeficiency (SCID) mouse model was developed in which sequestration of P. falciparum-infected human erythrocytes took place. During acute cerebral malaria in humans, the expression of intercellular adhesion molecule-1 (ICAM-1) is induced in vascular endothelium by inflammatory reactions. Acute phase ICAM-1 expression can also be obtained in SCID mice. The endothelium of the midbrain region was the most responsive to such inflammatory stimulus. It is noteworthy that the reticular formation in the midbrain controls the level of consciousness, and loss of consciousness is a symptom of cerebral malaria. We found that infected human erythrocytes were retained 24 times more than normal erythrocytes in ICAM-1-positive mouse brain. Sequestration to the brain was reduced by anti-ICAM-1 antibodies. These in vivo results were confirmed by the binding of P. falciparum-infected erythrocytes to the ICAM-1-positive endothelium in tissue sections of mouse brain. We conclude that the SCID mouse serves as a versatile in vivo model that allows the study of P. falciparum-infected erythrocyte adhesion as it occurs in human cerebral malaria. Upregulation of ICAM-1 expression in the region of the midbrain correlates with increased retention of malaria-infected erythrocytes and with the symptoms of cerebral malaria.

Background data on NOD/Shi-scid IL-2Rγnull mice (NOG mice).

To obtain background data of NOD/Shi-scid IL-2Rγnull (NOG) mice, severely immunedeficient mice, a total of 120 animals were examined at 7, 26 and 52 weeks-old (20 mice/sex/group). The survival rate at 52 weeks-old was 95% (19/20) in both sexes. Clinically, circling behavior in one direction along the cage wall was observed in males after 8 weeks and females after 47 weeks-old, and hunchback position was found in males after 32 weeks-old. Hematologically, lymphocyte count markedly decreased at all ages, while white blood cell count increased in several mice at 52 weeks-old. Blood chemistry results revealed high values of aspartate aminotransferase, lactate dehydrogenase and creatine phosphokinase in some females at 26 weeks-old, without any related histological change. Histologically, lymphoid hypoplasia characterized by severe lymphocyte depletion with poorly developed tissue architectures was observed. In addition, spongiotic change in the nerve tissue was observed in both sexes at 7 and 26 weeks-old, and intracytoplasmic materials known as tubular aggregates in the skeletal muscles were found in males terminated at 26 and 52 weeks-old and in females at 52 weeks-old. Malignant lymphoma was found in one female euthanized at 20 weeks-old. Further, small intestinal adenoma, hepatocellular adenoma, leukemia, cerebral lipomatous hamartoma, Harderian gland adenoma and uterine polyp were also observed, and their incidences were low except for that of uterine polyp. This study provided detailed background data on NOG mice up to 52 weeks-old and provided information on appropriate use of NOG mice in the various research fields.

Humanized severe combined immunodeficient mice as a potential model for the study of tolerance to factor VIII.

A Severe Combined Immunodeficient (SCID) mouse model has been established to evaluate experimental conditions leading to the production of factor VIII (FVIII) autoantibodies. To this end, we humanized 10 groups of 7 mice with peripheral blood mononuclear cells of 10 unrelated healthy blood donors (15 x 10(6) cells/mouse). Mice were injected with saline or immunized i.p. with 50 IU of a plasma derived human FVIII 24 h after reconstitution. Further immunization was made with 25 IU of FVIII every fortnight during 6 weeks and animals were sacrificed after 8 weeks. All reconstituted mice showed a spontaneous production of anti-FVIII antibodies in the absence of immunization with the corresponding antigen. However, no differences were observed regarding the quantity or the quality of these antibodies produced in the immunized or the saline group, indicating that tolerance to FVIII had been transferred with cell reconstitution. Affinity purified FVIII specific antibodies were capable of inhibiting FVIII activity and preventing the binding of FVIII to phospholipids in a dose-dependent manner. Immunoprecipitation experiments showed that the antibodies recognized only the C1 and C2 light chain domains. Since antibodies of interest can be found in the SCID mouse model and, moreover, since they are qualitatively comparable with the source donor's antibodies, this model provides a tool to study the regulation of tolerance against self antigens in normal subjects and in acquired haemophilia patients.

Simultaneous xenotransplantation of human Graves' thyroid tissue and autologous bone marrow cells in severe combined immunodeficient mice: successful reconstitution of human Graves' hyperthyroidism.

Human thyroid xenografts and the autologous bone marrow (BM) cells from five patients with Graves' disease (GD) were simultaneously xenografted into severe combined immunodeficient (SCID) mice to study the role of BM cells for the perpetuation of human GD autoimmunity and hyperthyroidism. All SCID mice engrafted with thyroid tissue (TH) alone, TH + autologous peripheral blood mononuclear cells, and TH + autologous BM cells produced similar amounts of human IgG; however, the production in TH + BM-engrafted mice peaked later than that of mice without BM. Production of thyroperoxidase antibody and thyroglobulin antibody in TH + BM-bearing SCID mice peaked in later weeks after xenografting than in those without BM. Moreover, human Graves' hyperthyroidism was actually reconstituted in TH + BM-transplanted mice; this was confirmed by (A) significantly higher levels and longer periods of secreting thyroid-stimulating antibody than those in mice without BM engraftment. (B) persistent hyperthyroxinemia up to the end of the experiment. (C) extremely high radioidine uptake of the xenografted thyroid tissue, and (D) histological findings of the maintenance of hyperplastic change of the xenografted thyroid epithelial cells. Human BM stem cells (CD34) were identified only in mice with TH + BM xenografts when analyzed by immunohistochemistry. In conclusion, (A) we have developed an animal model for human hyperthyroid GD by simultaneous xenotransplantation of GD thyroid tissue plus autologous BM cells into SCID mice, and (B) BM cells have a crucial role for perpetuating human GD autoimmunity and hyperthyroidism in this system.

Cell counts in peripheral blood and bone marrow of male C.B-17 scid/scid mice.

Blood cell and bone marrow cell counts were carried out for male C.B-17 scid/scid (SCID) mice aged 3, 6, 9, 18 and 26 weeks and the values were compared with those in C.B-17 +/+ (C.B-17) mice. In the peripheral blood, SCID mice had markedly low numbers of leucocytes and lymphocytes throughout the study. In the bone marrow, SCID mice had relatively low levels of erythroblasts at an early age, low levels of lymphocytes and plasma cells were absent.

Pathogenic role of tonsillar lymphocytes in associated with HSP60/65 in Pustulosis palmaris et plantaris.

OBJECTIVE: We aimed to define role of tonsillar lymphocytes (TL) and immune cross-reactivity between bacterial-HSP65 and human-HSP60 in Pustulosis palmaris et plantaris (PPP), an intractable chronic disease characterized with pustules and cornification of palms and soles. METHODS: Two sets of crossover trials were designed by employing SCID mice model. In the first trial, mice were transplanted with tonsillar lymphocytes and skin-grafts from PPP patients (TL group). In the second trial, mice were transplanted with tonsillar lymphocytes from PPP patients and injected with recombinant human HSP60. Control groups were designed for each step. Comparisons were performed for immunologic analyses including infiltration of CD4+ lymphocytes in skin-grafts by immunostaining, and levels of anti-HSP65-IgG and cytokines in mice sera by enzyme-linked immunosorbent assay (ELISA). RESULTS: In TL group, infiltration of CD4+ lymphocytes in skin-grafts were significantly higher than mice transplanted with blood lymphocytes (p<0.05), while anti-HSP65-IgG levels in sera showed non-significant tendency to increase in the TL group. CD4+ cells and anti-HSP65-IgG levels were also well-correlated with each other in TL group (p<0.01). Besides, anti-HSP65-IgG levels were significantly correlated with cytokine levels (IL-6, IFN-gamma) in mice sera (p<0.01). We found strong expression of HSP60 in PPP lesions. Finally, HSP60-stimulation in mice transplanted with TL from PPP patients induced significantly higher anti-HSP65-IgG levels in serum compared to control groups including mice without HSP60-stimulation or peripheral blood lymphocytes-transplanted mice or transplanted with TL from control patients (p<0.05). CONCLUSION: Our results indicate the pathogenic role of TL and immune cross-reaction between human-HSP60 and bacterial-HSP65 in PPP.

Rapid blood clearance of injected mouse IgG2a in SCID mice.

SCID mice were found to have rapid blood clearance of injected mouse IgG2a antibodies (Ab), while IgG1 Ab were cleared normally. This effect varied depending on the strain of SCID mice, being very rapid in most Taconic ICR mice and slower in C.B-17 mice. A similar effect was previously described in nude mice, and shown to be due to the very low concentrations of endogenous IgG2a and IgG2b in these mice. Therefore, IgG2a and IgG2b concentrations were assayed in sera from 30 SCID mice: the concentrations were very low, with one exception. Rapid blood clearance in all strains could be strongly inhibited by injection of large amounts of irrelevant IgG2a. Therefore, the low endogenous IgG2a and IgG2b concentrations are responsible for the rapid blood clearance rate of injected IgG2a in these mice, as in nude mice. However, the relatively slow blood clearance of injected IgG2a in certain SCID mice (C.B-17 mice and rare Taconic ICR mice), was not generally due to higher levels of IgG2a or IgG2b, but rather to some other factor that has not been identified. F(ab')(2) Ab fragments were not cleared rapidly, which supports other evidence that clearance is via the CD64 Fcgamma receptor. This rapid clearance of IgG2a affects the ability of such Ab to target tumors, and was also shown to affect the maximum tolerated dose (MTD) of radiolabeled IgG2a Ab, labeled with either (125)I or (111)In. Mice with faster blood clearance had a lower MTD, probably due to the fact that Ab uptake was in the bone as well as the spleen. This effect must be taken into consideration in experiments in which SCID mice are injected with IgG2a or IgG2b Ab.

Lentiviral gene transfer into primary and secondary NOD/SCID repopulating cells.

The ability of lentiviral vectors to transfer genes into human hematopoietic stem cells was studied, using a human immunodeficiency virus 1 (HIV-1)-derived vector expressing the green fluorescence protein (GFP) downstream of the phosphoglycerate kinase (PGK) promoter and pseudotyped with the G protein of vesicular stomatitis virus (VSV). High-efficiency transduction of human cord blood CD34(+) cells was achieved after overnight incubation with vector particles. Sixteen to 28 percent of individual colony-forming units granulocyte-macrophage (CFU-GM) colonies derived from cord blood CD34(+) cells were positive by polymerase chain reaction (PCR) for the GFP gene. The transduction efficiency of SCID-repopulating cells (SRC) within the cord blood CD34(+) population was assessed by serial transplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. When 400,000 cord blood CD34(+) cells were transplanted into primary recipients, all primary and secondary recipients contained and expressed the transgene. Over 50% of CFU-GM colonies derived from the bone marrow of these primary and secondary recipients contained the vector on average as determined by PCR. Transplantation of transduced cells in limiting dilution generated GFP(+) lymphoid and myeloid progeny cells that may have arisen from a single SRC. Inverse PCR analysis was used to amplify vector-chromosomal junctional fragments in colonies derived from SRC and confirmed that the vector was integrated. These results show that lentiviral vectors can efficiently transduce very primitive human hematopoietic progenitor and stem cells. (Blood. 2000;96:3725-3733)

High marrow seeding efficiency of human lymphomyeloid repopulating cells in irradiated NOD/SCID mice.

Transplantable human hematopoietic stem cells (competitive repopulating units [CRU]) can be quantitated based on their ability to produce large populations of lymphoid and myeloid progeny within 6 weeks in the marrow of intravenously injected, sublethally irradiated NOD/SCID mice. It is shown that the proportions of total injected human fetal liver and cord blood CRU in the marrow of mice 24 hours after transplantation are 5% and 7%, respectively, as determined by limiting-dilution assays in other primary and secondary NOD/SCID mice. The similarity in these 2 seeding efficiency values suggests that mechanisms regulating the ability of human hematopoietic stem cells to enter the marrow from the blood, at least in this xenotransplant model, do not change between fetal life and birth. In addition, it appears that previously reported human stem cell frequencies and their in vivo self-renewal activity measured in NOD/SCID mice have been markedly underestimated. (Blood. 2000;96:3979-3981)

Differential white blood cell counts as a preliminary screen for severe combined immunodeficient congenic mice.

Severe combined immunodeficient (SCID) mice are becoming increasingly popular as research animals; as a consequence, more efforts to produce congenic strains carrying the scid gene are underway. In an attempt to conserve time and resources in this endeavor, we used peripheral blood differential white blood cell counts as a preliminary screen to eliminate the homozygous (+/+) wild type and heterozygous (scid/+) animals from intercross generations. The results of our investigation confirm that blood smears can be used as a screen at four weeks of age to identify animals having an inversion of the granulocyte:mononuclear cell ratio. Mice not having an inversion of this ratio, i.e., mononuclear cells exceeding 50%, can be eliminated from the colony. This screen permits elimination of a large portion of the intercross generation one month earlier than other methods that rely on detection of serum immunoglobulin. The screen is highly sensitive and specific. We do not propose that this screen be used as a definitive test but as a tool to eliminate the majority of animals that are not homozygous at the scid locus.

Response of huSCID mice reconstituted with lymphocytes from anti-idiotype-treated melanoma patients.

Severe combined immune deficient (SCID) mice were reconstituted with peripheral blood mononuclear cells (PBMC) from normal and melanoma patients. The melanoma patients were part of a clinical trial of active immunotherapy with the murine monoclonal anti-idiotypic antibody, IMelpgl. IMelpgl represents an idiotypic mimic of the high-molecular-weight melanoma-associated antigen, HMW-MAA. All of the huSCID mice reconstituted fully as evidenced by their production of human immunoglobulins. Furthermore, approximately half of the huSCID mice reconstituted with PBMC from either normal or melanoma patients were able to mount a secondary response to tetanus toxoid. While all of the huSCID mice reconstituted with PBMC from patients undergoing immunotherapy produced strong HAMA responses, only one huSCID mouse responded idiotypically to IMelpgl immunization. These results demonstrate tht the huSCID mouse can be used as an experimental model to monitor and study the response of human B cells derived from patients undergoing active immunotherapy.

Enhanced clearance of lactic dehydrogenase-5 in severe combined immunodeficiency (SCID) mice: effect of lactic dehydrogenase virus on enzyme clearance.

The lactic dehydrogenase (LDH) level in plasma and the clearance of LDH in C.B-17 scid (severe combined immunodeficiency; SCID) mice were compared with those in C.B-17 or BALB/cCrSlc mice with or without lactic dehydrogenase virus (LDV) infection. The resting enzyme level in SCID mice showed little difference from that in C.B-17 or BALB/cCrSlc mice. The degree of increased plasma LDH level in SCID mice was lower than that in C.B-17 and BALB/cCrSlc mice after LDV infection. To assess the mechanisms of decrease in LDH elevation in SCID mice infected with LDV, virus replication was compared in SCID and BALB/cCrSlc mice. The infectivity titre of plasma in SCID mice was higher (more than 10 times) than that in BALB/cCrSlc mice. Moreover, the percentage of virus antigen positive Kupffer cells was higher in SCID mice than that in BALB/cCrSlc mice. The level of endogenous LDH release as a result of carbon tetrachloride treatment was similar in the SCID and BALB/cCrSlc mice. The clearance rate of endogenous LDH was greater in SCID mice than in BALB/cCrSlc mice with or without LDV infection. The rate of clearance of intravenously injected porcine LDH-5, but not porcine LDH-1, was enhanced in SCID mice as compared with that in BALB/cCrSlc mice. Furthermore, carbon clearance was higher in SCID mice than that in BALB/cCrSlc mice. These results suggest that the smaller increase of plasma LDH after infection might be due, at least in part, to the enhanced LDH-5 clearance function by macrophages in SCID mice.

Hepatitis C virus persistence in human hematopoietic cells injected into SCID mice.

The issue of infection of peripheral blood mononuclear cells (PBMC) by the hepatitis C virus (HCV) has potentially important implications, but is still debated. We have used the severe combined immunodeficiency (SCID) mouse model to test for the persistence of HCV in PBMC. Hematopoietic cells isolated from 14 subjects infected by HCV were inoculated intraperitoneally into SCID mice. Serum and blood cell samples from these mice were obtained with a mean follow-up of 8 weeks. As controls, human fibroblasts and sheep PBMC, preincubated with a human HCV-positive serum, were inoculated concomitantly into mice and analyzed. HCV-RNA positive strands were detected in 7 of 26 serum samples and 8 of 26 cell fractions from SCID mice inoculated with HCV-positive PBMC, after 8 weeks of follow-up. In contrast, no HCV RNA was detectable in the 10 control mice. HCV-RNA negative strands were detected in only 2 of 10 tested samples from 2 mice, and both positive mice had been inoculated with PBMC from HCV-positive subjects with malignant hematopoietic syndrome. Our study offers strong evidence for the persistence of HCV infection in mononuclear cells. Our results are also consistent with a low rate of HCV multiplication. This SCID mouse model might therefore be useful in analyzing the mechanisms of HCV persistence in mononuclear cells.

Deficient cytokine response of human allergen-specific T lymphocytes from humanized SCID mice and reconstitution by professional antigen-presenting cells.

BACKGROUND: Hu-PBL-SCID mice generated by the transfer of PBMCs from atopic individuals may provide a physiologic in vivo model for investigating human responses to allergens and potential approaches toward immunotherapy. OBJECTIVE: This study was undertaken to investigate the functional activity and cytokine profile of human allergen-reactive T lymphocytes isolated from hu-PBL-SCID mice. METHODS: PBMCs from allergic individuals were coinjected with allergen into SCID mice. Human lymphocyte migration and phenotype were established by reverse transcription-PCR and immunohistochemistry, IgE levels in sera were determined, and the frequency of allergen-reactive cytokine-producing T lymphocytes was established. RESULTS: After immunization with allergen, specific IgE levels in hu-PBL-SCID sera were comparable with levels in donor sera. Although the majority of lymphocytes remained in the peritoneum, significant numbers of T lymphocytes were located in the spleen, where human IL-4, IL-5, and IFN-gamma messenger RNA expression was detected after stimulation with PHA and phorbol myristate acetate. Failure to induce cytokine production by human T lymphocytes isolated from the peritoneum and spleen of hu-PBL-SCID mice by allergen was reversed by stimulating with allergen in the presence of exogenously added IL-2 and antigen-presenting cells (APC), particularly CD14(+) monocytes. Under these conditions, allergen-reactive T cells expressed a T(H)2-like phenotype. CONCLUSIONS: These data suggest that, after initial activation and induction of antibody production, human T lymphocytes enter a state of unresponsiveness, arising from a loss of human professional APC, in hu-PBL-SCID mice. The use of hu-PBL-SCID mouse models in studies on therapeutic approaches for allergy may benefit from the additional transfer of human professional APC.

Donor stromal cells from human blood engraft in NOD/SCID mice.

Little is known about the presence, frequency, and in vivo proliferative potential of stromal cells within blood-derived hematopoietic transplants. In this study, nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were injected with human CD34(+) peripheral blood cells (PBCs) or cord blood cells (CBCs, either enriched for CD34 or density-gradient separated mononuclear cells). Flow cytometric analysis 5 to 11 weeks after transplantation revealed the presence of a human lymphomyeloid hematopoiesis within the murine bone marrow. Immunohistochemical staining of bone marrow cell suspensions using human-specific antibodies showed human cells staining positive for human fibroblast markers, human von Willebrand factor (vWF) and human KDR (vascular endothelial growth factor receptor-2) in mice transplanted with CD34(+) PBCs or CBCs, with mean frequencies between 0.6% and 2.4%. In stromal layers of bone marrow cultures established from the mice, immunohistochemical staining using human-specific antibodies revealed flattened reticular cells or spindle-shaped cells staining positive with human-specific antifibroblast antibodies (mean frequency, 2.2%). Cell populations of more rounded cells stained positive with human-specific antibodies recognizing CD34 (1.5%), vWF (2.2%), and KDR (1.6%). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and subsequent complementary DNA sequencing detected transcripts of human KDR (endothelial specific) and human proline hydroxylase-alpha (fibroblast specific) within the bone marrow and spleen of transplanted mice. Analysis of nontransplanted control mice yielded negative results in immunocytochemistry and RT-PCR. Cells expressing endothelial and fibroblast markers were also detected in the grafts before transplantation, and their numbers increased up to 3 log in vivo after transplantation. These results indicate that stromal progenitor cells are present in human cytokine-mobilized peripheral blood or cord blood that engraft in NOD/SCID mice. (Blood. 2000;96:3971-3978)

Transfer of human myocarditis into severe combined immunodeficiency mice.

Severe combined immunodeficiency (SCID) mice possess neither T nor B lymphocytes and are thus suitable recipients for lymphocytes of different species. Because autoimmune mechanisms are suspected in the pathogenesis of myocarditis (MC), we attempted to determine whether peripheral blood lymphocytes (PBLs) from patients with MC could be transferred into SCID mice and whether they had an autoimmunologic effect. Groups of three mice each were injected intraperitoneally with up to 50 million PBLs from five MC patients with autoantibodies against the adenine nucleotide translocator (ANT), a myocardial autoantigen. The PBLs from three healthy blood donors were used as controls. After 60 days, human PBLs could be demonstrated in the peripheral blood of the SCID mice transfused with the PBLs of MC patients, representing up to 9.9% of the peripheral blood mononuclear cells. The transfused SCID mice sera showed human immunoglobulin levels of up to 3 mg/mL, both IgG and IgM. Autoantibodies against ANT were present in the mice receiving PBLs from MC patients but not from the control subjects. In addition, infiltrating human lymphocytes were present in the hearts of the SCID mice transfused with PBLs from MC patients. The presence of an ongoing autoimmune process in the SCID mice transfused with PBLs from MC patients is suggested by increased levels of soluble interleukin-2 receptor in the serum in contrast to SCID mice transfused with PBLs from healthy blood donors. We conclude that the autoimmune reactions seen in human MC can be transferred to SCID mice by the transfer of PBLs from MC patients. These findings stress the significance of autoimmune mechanisms in the pathogenesis of human MC.

Characterization of hu-PBL-SCID mice with high human immunoglobulin serum levels and graft-versus-host disease.

A chimeric model consisting of severe combined immune deficiency (SCID) mice populated with human peripheral blood leukocytes (PBL) has recently been described (bu-PBL-SCID mice). These reports indicated a limited reconstruction of the transferred human immune system and functionality of the human graft. Herein we described modifications of the PBL transfer method that minimize transfer time and cellular manipulations, leading to a more effective population of SCID mouse recipients. Severe combined immune deficiency mice given 15 x 10(6) PBL had human IgG serum levels reaching 2 to 5 g/l, and all mice had detectable human anti-tetanus toxoid antibody levels when they received cells from donors with such levels. These transfers were associated also with clinical and histologic evidence of graft-versus-host disease, suggesting responsiveness of the human graft in the recipients. When Epstein-Barr virus seropositive (EBV+) donors were used, the chimeric mice also showed a high incidence of fatal lymphoproliferative disease 1 to 3 months after transfer of 15 x 10(6) PBL. The high level of immunoglobulin synthesis and immunoresponsiveness of the human cells with this transfer procedure may expand the use of these chimeric mice for the manipulations of human immune cells in vivo.

Two serologic markers to monitor the engraftment, growth, and treatment response of human leukemias in severe combined immunodeficient mice.

We have investigated human lactate dehydrogenase (LDH) isoenzymes and human nuclear matrix protein 41/7 (NMP 41/7) as potential serologic markers to monitor the course of human leukemia in severe combined immunodeficient (SCID) mice. Following the transplantation of 10(6) human acute lymphoblastic leukemia (ALL) Nalm-6 cells, human specific LDH isoenzymes were measurable in the serum of SCID mice as early as 7 days after transplantation, although serum total LDH increased in some animals as early as 5 days after transplantation. Human NMP 41/7 was measurable in all animals at day 15 after leukemia cell injection. Serum levels of total LDH, human specific LDH and NMP 41/7 increased progressively over time, reaching total LDH levels as high as 50,000 U/L at day 25 after transplantation. To determine whether the levels of LDH and NMP 41/7 in serum were a reflection of human tumor burden, we studied these serologic markers in SCID mice bearing measurable subcutaneous human neuroblastoma tumors, or compared the serum levels of these markers with the number of human leukemia CD10+ cells in the bone marrow of the SCID mice. The serum levels of total LDH, human specific LDH isoenzymes, and NMP 41/7 correlated well with tumor burden, and they drastically decreased or disappeared from serum after the human leukemia or neuroblastoma cells were selectively killed with a single intravenous (IV) injection of 1 to 3 micrograms diphtheria toxin (DT) (the cellular receptor for DT is present on human cells, but not on mouse cells). Paraplegic mice with central nervous system leukemia completely recovered after DT treatment. We conclude that measurements of serum levels of total LDH, human LDH isoenzymes, and NMP 41/7 are sensitive, quantitative, rapid, and easy to perform serologic methods useful to monitor the engraftment, progression, and treatment response of human leukemia in SCID mice.

Stromal-derived factor 1 inhibits the cycling of very primitive human hematopoietic cells in vitro and in NOD/SCID mice.

Stromal-derived factor 1 (SDF-1) is a -CXC- chemokine that plays a critical role in embryonic and adult hematopoiesis, and its specific receptor, CXCR4, has been implicated in stem cell homing. In this study, it is shown that the addition of SDF-1 to long-term cultures (LTCs) of normal human marrow can selectively, reversibly, and specifically block the S-phase entry of primitive quiescent erythroid and granulopoietic colony-forming cells (CFCs) present in the adherent layer. Conversely, addition of anti-SDF-1 antibody or SDF-1(G2), a specific CXCR4 antagonist, to preactivated human LTCs prevented both types of primitive CFCs from re-entering a quiescent state, demonstrating that endogenous SDF-1 contributes to the control of primitive CFC proliferation in the LTC system. Interestingly, SDF-1 failed to arrest the proliferation of primitive chronic myeloid leukemia CFCs in the adherent layer of LTCs containing normal marrow stromal cells. In vivo, injection of SDF-1 arrested the cycling of normal human LTC-initiating cells as well as primitive CFCs in the marrow of nonobese diabetic/severe combined immunodeficient mice engrafted with human cord blood cells. Conversely, injection of the antagonist, SDF-1(G2), reactivated the cycling of quiescent primitive human CFCs present in the marrow of mice engrafted with human marrow cells. These studies are the first to demonstrate a potential physiological role of SDF-1 in regulating the cell-cycle status of primitive hematopoietic cells and suggest that the deregulated cycling activity of primitive chronic myeloid leukemia (CML) cells is due to the BCR-ABL-mediated disruption of a pathway shared by multiple chemokine receptors.