Src activation generates reactive oxygen species and impairs metabolism-secretion coupling in diabetic Goto-Kakizaki and ouabain-treated rat pancreatic islets.

AIMS/HYPOTHESIS: Na(+)/K(+)-ATPase inhibition by ouabain suppresses ATP production by generating reactive oxygen species (ROS) and impairs glucose-induced insulin secretion from pancreatic islets. To clarify the signal-transducing function of Na(+)/K(+)-ATPase in decreasing ATP production by the generation of ROS in pancreatic islets, the involvement of Src was examined. In addition, the significance of Src activation in diabetic islets was examined. METHODS: Isolated islets from Wistar rats and diabetic Goto-Kakizaki (GK) rats (a model for diabetes) were used. ROS was measured by 5-(and 6)-chloromethyl-2',7'-dichlorofluorescein fluorescence using dispersed islet cells. After lysates were immunoprecipitated by anti-Src antibody, immunoblotting was performed. RESULTS: Ouabain caused a rapid Tyr(418) phosphorylation, indicating activation of Src in the presence of high glucose. The specific Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) restored the ouabain-induced decrease in ATP content and the increase in ROS production. Both PP2 and ROS scavenger restored the impaired insulin release and impaired ATP elevation in GK islets, but had no such effect in control islets. PP2 reduced the high glucose-induced increase in ROS generation in GK islet cells but had no effect on that in control islet cells. Moreover, ouabain had no effect on ATP content and ROS production in the presence of high glucose in GK islets. CONCLUSIONS/INTERPRETATION: These results indicate that Src plays a role in the signal-transducing function of Na(+)/K(+)-ATPase, in which ROS generation decreases ATP production in control islets. Moreover, ROS generated by Src activation plays an important role in impaired glucose-induced insulin secretion in GK islets, in which Src is endogenously activated independently of ouabain.

Measurement of colonic mucosal concentrations of 5-aminosalicylic acid is useful for estimating its therapeutic efficacy in distal ulcerative colitis: comparison of orally administered mesalamine and sulfasalazine.

OBJECTIVES: Oral 5-aminosalicylic acid (5-ASA) preparations have been used frequently in the treatment of ulcerative colitis. However, there have been few reports investigating the relationship between colonic mucosal concentrations of 5-ASA and its clinical efficacy when oral sulfasalazine or 5-ASA compounds were administered. The aim of this study is to compare the mucosal concentrations of 5-ASA ensured by sulfasalazine or mesalamine, and to define the clinical significance of the measurement of 5-ASA concentrations in the treatment of distal ulcerative colitis. MATERIALS AND METHODS: Biopsies were taken from the rectum and sigmoid colon of the oral sulfasalazine group (n = 13) and the slow-release 5-ASA (mesalamine) group with (n = 5) or without (n = 11) rectal administration of 5-ASA. High-pressure liquid chromatography was used to measure the tissue concentrations of 5-ASA and its metabolites. We compared the 5-ASA concentrations of the sulfasalazine group with the mesalamine group. Furthermore, we analyzed the relationship between tissue 5-ASA concentrations and the Disease Activity Index (DAI). RESULTS: The concentrations of 5-ASA and acetyl-5-ASA in the sulfasalazine group were higher than those in the group taking oral mesalamine alone (p < 0.01). The concentration of 5-ASA was much higher in the patients who received oral and rectal mesalamine in an enema than in the patients who had oral mesalamine alone. There was a significant inverse correlation between the DAI and concentrations of 5-ASA in the rectum (r = 0.712, p < 0.001). CONCLUSIONS: We demonstrated that the colonic mucosal concentration of 5-ASA was significantly higher in the sulfasalazine group than in the mesalamine group. Furthermore, the concentrations of mucosal 5-ASA may be a good marker for the estimation of its efficacy in the treatment of ulcerative colitis.

[Efficacy of mesalamine enema in the treatment of steroid-resistant or dependent distal ulcerative colitis].

The efficacy and safety of mesalamine enema were examined in 20 patients with steroid-resistant or dependent, distal ulcerative colitis. Rectal bleeding disappeared in 3 (18%). 8 (50%) of 16 patients within 2 weeks and 4 weeks after the start of mesalamine enema treatment, respectively. Mean clinical activity index (CAI) score after the treatment was significantly reduced (8.1-->3.6, p < 0.001). Furthermore, Mean doses of oral corticosteroid after the treatment (7.3 mg) were also significantly lower than those before the treatment (12.8 mg) (p < 0.01). Four patients dropped out. Three patients could not retain the enemas because of abdominal discomfort and one patient had fever and rash. There were no significant differences in age, gender, disease duration, disease type, and mean doses of oral corticosteroid before the treatment between the response group (n = 8) and the non-response group (n = 8). However, clinical and endoscopic activities before mesalamine enema treatment in the non-response group (CAI 9.8, Matts score 8.0) were higher than those in the response group (CAI 6.4, Matts score 5.5). These results suggest that mesalamine enema is useful for mildly to moderately active distal ulcerative colitis by improving clinical symptoms and reducing corticosteroid.

Powerful Claisen condensation and Claisen-aldol tandem reaction of alpha,alpha-dialkylated esters promoted by ZrCl4-iPr2NEt.

Powerful Claisen ester condensations of alpha,alpha-dialkylated esters mediated by ZrCl4-iPr2NEt were performed to give the corresponding thermodynamically unfavorable alpha,alpha-dialkylated beta-ketoesters, and Claisen-aldol tandem reactions between an intermediary Zr-enolate of a alpha,alpha-dialkylated beta-ketoester and aldehydes also proceeded.

Defensins act as potent adjuvants that promote cellular and humoral immune responses in mice to a lymphoma idiotype and carrier antigens.

Defensins released by neutrophils are able to kill a broad spectrum of microbes. They also induce leukocyte migration in vitro and elicit inflammatory leukocyte responses at s.c. injection sites in mice. In vitro experiments showed that human defensins enhanced concanavalin A-stimulated murine spleen cell proliferation and IFN-gamma production. This led us to examine the effects of human defensins on specific immune responses in vivo. BALB/c mice were immunized with 50 microg of keyhole limpet hemocyanin (KLH) adsorbed to aluminum hydroxide and administered with defensins in aqueous solution. Intraperitoneal administration of defensins significantly increased the production of KLH-specific IgG1, IgG2a and IgG2b antibodies 14 days after immunization. In vitro splenic KLH-specific proliferative responses were higher in mice treated with KLH and defensins than in those treated with KLH alone. Increased IFN-gamma and, to a lesser extent, IL-4 production were also detected in the supernatants of ex vivoKLH-activated spleen cells from mice treated with defensins. Finally, defensins significantly enhanced the antibody response to a syngeneic tumor antigen, lymphoma Ig idiotype and also augmented resistance to tumor challenge. These results indicate that defensins act as potent immune adjuvants by inducing the production of lymphokines, which promote T cell-dependent cellular immunity and antigen-specific Ig production. Thus, defensins appear to function as neutrophil-derived signals that promote adaptive immune responses.

CD40 stimulation promotes human secondary immunoglobulin responses in HuPBL-SCID chimeras.

Antibodies to CD40 have been demonstrated to promote B-cell growth and differentiation in vitro. In order to determine if CD40 stimulation could promote antigen-specific human immunoglobulin (Ig) production in vivo, we examined the effects of anti-human CD40 MoAb in an in vivo system where human peripheral blood lymphocytes (huPBL) were engrafted into mice with severe combined immune deficiency (SCID). The huPBL-SCID mice were then given various doses of diphtheria-tetanus toxoid (DT) vaccine and were examined for the presence of human DT-specific antibodies by ELISA. Surprisingly, treatment with anti-CD40 significantly lowered background DT responses versus untreated chimeras in unimmunized huPBL-SCID mice. However, after immunization, huPBL-SCID mice treated with anti-CD40 MoAb responded to a significantly greater extent in response to the vaccine compared with control huPBL-SCID mice, although total Ig levels were sometimes lower in anti-CD40-treated mice. The predominant Ig isotype induced after immunization was IgG. Thus, CD40 stimulation promotes human secondary IgG responses in huPBL-SCID mice. These data demonstrate that CD40 stimulation is capable of promoting antigen-specific human B-cell responses in vivo.

Role of daunorubicin in the induction therapy for adult acute myeloid leukemia.

PURPOSE: To evaluate the relationship of total-dose of daunorubicin (DNR) to the induction therapy and treatment outcome, we have administered individualized doses of DNR during induction treatment to patients with acute myelogenous leukemia (AML). PATIENTS AND METHODS: Ninety-two previously untreated adult patients with AML who entered our hospital were analyzed for the dose of DNR required to achieve complete remission (CR), the CR rate, disease-free survival (DFS), and overall survival (OS). Induction therapy consisted of DNR 40 mg/m2 daily intravenously from day 1 until the marrow was hypoplastic, cytarabine (Ara-C), prednisolone (PRD), and/or 6-thioguanine (6-TG). RESULTS: Eighty-three of 92 patients with adult AML were assessable for this study. Sixty-three (76%) patients achieved CR. Fifty-two of 63 CR patients achieved the CR in the first course of induction therapy, and 11 patients required the second course of induction therapy. The 5-year and 10-year DFS rates were 31.2% and 5-year and 10-year OS rates were 45.1% and 42.3%, respectively. The median total dose of DNR in the induction therapy was 280 mg/m2 (120 to 480 mg/m2). DNR dose did not influence the response to therapy and was not influenced by the initial WBC count or French-American-British (FAB) system classification. CONCLUSION: These results indicated that when the dose was linked to observed tumor response, the optimal dose of DNR in the induction therapy was approximately 280 mg/m2 (40 mg/m2 for 7 days), which is greater than the conventional dose of 40 to 60 mg/m2 for 3 days.

Recombinant human growth hormone promotes hematopoietic reconstitution after syngeneic bone marrow transplantation in mice.

Recombinant human growth hormone (rhGH) was administered to mice after syngeneic bone marrow transplantation (BMT) to determine its effect on hematopoietic reconstitution. BALB/c mice were given 10 microg intraperitoneal injections of rhGH every other day for a total of 10 injections following syngeneic BMT. Mice that received rhGH exhibited significant increases in total hematopoietic progenitor cell content (colony-forming unit-culture) in both bone marrow and spleen. Erythroid cell progenitor content (burst-forming unit-erythroid) was also significantly increased after rhGH treatment. Analysis of peripheral blood indicated that administration of rhGH resulted in significant increases in the rate of white blood cell and platelet recovery. Granulocyte marker 8C5+ cells were also increased in the bone marrow and spleens of treated mice. Red blood cell, hematocrit, and hemoglobin levels were increased at all time points after rhGH treatment. No significant pathologic effects or weight gain were observed in mice receiving repeated injections of 10 microg rhGH. Thus, rhGH administration after syngeneic BMT promoted multilineage hematopoietic reconstitution and may be of clinical use for accelerating hematopoiesis after autologous BMT.

Engraftment of ex vivo expanded and cycling human cord blood hematopoietic progenitor cells in SCID mice.

The ability of human hematopoietic cells to engraft SCID mice provides a useful model in which to study the efficiency of retroviral gene transfer and expression in primitive stem cells. In this regard, it is necessary to determine whether SCID mice can be engrafted by cycling human hematopoietic progenitor cells. Human cord blood cells from 12 different donors were cultured in vitro for 6 days with interleukin-3 and stem cell factor. Phenotypic analysis indicated that hematopoietic cells were induced to cycle and the number of progenitors was expanded, thus making them targets for retroviral gene transfer. The cells were then transferred to SCID mice. Human hematopoietic progenitor cell engraftment was assessed up to 7 weeks later by growth of human progenitor cells in soft agar. After in vitro culture under conditions used for retroviral gene transfer, human cord blood hematopoietic cells engrafted the bone marrow and spleen of SCID mice. Interestingly, cultured cord blood cells engrafted after intraperitoneal but not after intravenous injection. Furthermore, engraftment of cord blood cells was observed in mice receiving no irradiation before transfer of the human cells, suggesting that competition for space in the marrow is not a limiting factor when these cells have been cultured. Administration of human cytokines after transfer of human cord blood cells to SCID mice was also not required for engraftment. Thus, engraftment of SCID mice with human hematopoietic cells cultured under conditions suitable for gene transfer may provide an in vivo assay for gene transfer to early human hematopoietic progenitor cells.

Antitumor effects of nonconjugated murine Lym-2 and human-mouse chimeric CLL-1 monoclonal antibodies against various human lymphoma cell lines in vitro and in vivo.

Lym-2 is a murine monoclonal antibody (MoAb) directed towards a human class II molecule variant reactive with both normal and neoplastic human B lymphocytes. Previous studies have shown that signals transmitted by class II molecules that stimulate normal lymphocytes can be inhibitory for B-cell lymphoma growth by signaling activation-induced cell death. Therefore, we sought to evaluate the effects of nonconjugated murine Lym-2 and a human-mouse chimeric Lym-2 (chCLL-1; with murine variable regions and human constant regions) MoAb on the growth of various human lymphomas by using both in vitro and in vivo assays. Cell lines derived from Burkitt's lymphomas, diffuse large cell B-cell lymphomas, anaplastic large-cell lymphomas, and Epstein-Barr virus-induced B-cell lymphomas were incubated with Lym-2 or chCLL-1 in vitro, and effects on proliferation were determined by [3H]-thymidine incorporation. The effects of Lym-2 in vitro were also compared with those of Lym-1, which is a similar MoAb that has been evaluated clinically. After immobilization, which enhances crosslinking of the MoAbs, both Lym-2 and chCLL-1 were capable of directly inhibiting the growth of various lymphoma lines in vitro. These human lymphomas were then transferred into mice with severe combined immunodeficiency to evaluate the efficacy of these MoAbs in vivo. Treatment with either murine Lym-2 or the chimeric chCLL-1 were significantly effective in improving the survival of tumor-bearing mice. These results indicate that stimulation by nonconjugated chCLL-1 may offer a biological approach to the treatment of various human lymphomas.

Conventional and high-yield synthesis of DTPA-conjugated peptides: application of a monoreactive DTPA to DTPA-D-Phe1-octreotide synthesis.

Successful imaging of somatostatin receptor-positive tumors with 111In-DTPA-D-Phe1-octreotide has stimulated development of peptide radiopharmaceuticals using DTPA as the chelating agent. However, use of cyclic DTPA dianhydride (cDTPA) resulted in low synthetic yields of DTPA-peptide by either solution or solid-phase syntheses. This paper reports a novel high-yield synthetic procedure for DTPA-D-Phe1-octreotide that is applicable to other peptides of interest using a monoreactive DTPA derivative. A monoreactive DTPA that possesses one free terminal carboxylic acid along with four carboxylates protected with tert-butyl ester (mDTPA) was synthesized. Fmoc-Thr(tBu)-ol, prepared from Fmoc-Thr(tBu)-OH, was loaded onto 2-chlorotrityl chloride resin. After construction of the peptide chains by Fmoc chemistry, mDTPA was coupled to the alpha amine group of the peptide on the resin in the presence of 1,3-diisopropylcarbodiimide and 1-hydroxybenzotriazole. Treatment of the mDTPA-peptide-resin with trifluoroacetic acid-thioanisole removed the protecting groups and liberated [Cys(Acm)2,7]-octreotide-D-Phe1-DTPA from the resin. Iodine oxidation of the DTPA-peptide, followed by the reversed-phase HPLC purification, produced DTPA-D-Phe1-octreotide in overall 31.8% yield based on the starting Fmoc-Thr(tBu)-ol-resin. The final product gave a single peak on analytical HPLC, and amino acid analysis and mass spectrometry confirmed the integrity of the product. 111In radiolabeling of the product provided 111In-DTPA-D-Phe1-octreotide with > 95% radiochemical yield, as confirmed by analytical reversed-phase HPLC, TLC, and CAE. These finding indicated that use of mDTPA during solid-phase peptide synthesis greatly increased the synthetic yield of DTPA-D-Phe1-octreotide, due to the absence of nonselective reactions that are unavoidable when cDTPA is used. These results also suggested that mDTPA would be a versatile reagent to introduce DTPA with high yield into peptides of interest.

Immunologic and hematopoietic effects of CD40 stimulation after syngeneic bone marrow transplantation in mice.

CD40 is a molecule present on multiple cell types including B lymphocyte lineage cells. CD40 has been shown to play an important role in B cell differentiation and activation in vitro, although little is known concerning the effects of CD40 stimulation in vivo. We therefore examined the effects of CD40 stimulation in mice using a syngeneic bone marrow transplantation (BMT) model in an effort to augment B cell recovery after high dose therapy with hematopoietic reconstitution. After the BMT, mice were treated with or without 2-6 microg of a soluble recombinant murine CD40 ligand (srmCD40L) given intraperitoneally twice a week. A significant increase in B cell progenitors (B220+/ surface IgM-) was observed in the bone marrow of mice receiving the srmCD40L. The treated recipients also demonstrated improved B-cell function with increases in total serum immunoglobulin and increased splenic mitogen responsiveness to LPS being noted. Additionally, srmCD40L treatment promoted secondary lymphoid organ repopulation, accelerating germinal center formation in the lymph nodes. Total B cell numbers in the periphery were not significantly affected even with continuous srmCD40L administration. Lymphocytes obtained from mice treated with the ligand also had increases in T cell mitogen and anti-CD3 mAb responsiveness and acquired the capability to produce IL-4. Surprisingly, treatment with srmCD40L also produced hematopoietic effects in mice, resulting in an increase of BM and splenic hematopoietic progenitor cells in the mice after BMT. Treatment with srmCD40L significantly increased granulocyte and platelet recovery in the peripheral blood. Incubation of BMC with srmCD40L in vitro also resulted in increased progenitor proliferation, demonstrating that the hematopoietic effects of the ligand may be direct. Thus, stimulation of CD40 by its ligand may be beneficial in accelerating both immune and hematopoietic recovery in the setting of bone marrow transplantation.

Effects of CD40 stimulation in the prevention of human EBV-lymphomagenesis.

CD40 is a molecule present on B lineage cells, both normal and neoplastic. Signalling through CD40 has been demonstrated to promote B cell growth and differentiation in vitro. In contrast to its effects on normal B cells, we have found that CD40 stimulation can inhibit the growth of various aggressive histology human B cell lymphomas both in vitro and in vivo. Moreover, using a human/mouse chimera model in which human EBV-induced B cell lymphomas can spontaneously arise, we have found that CD40 stimulation an prevent the occurrence of this human lymphoma in mice. However, normal human B cell engraftment and function was not adversely affected in these mice by CD40 stimulation. This indicates that CD40 stimulation is selective in its effects on aggressive histology B cell lymphomas. Thus, CD40 stimulation either by antibody or a recombinant soluble ligand, may be of potential clinical use, not only in the treatment of EBV-induced B cell lymphomas, but also in their prevention.

Slow axonal transport: the subunit transport model.

A central problem concerning slow transport of cytoskeletal proteins along nerve axons is where they are assembled and the form in which they are transported. The polymer and subunit transport models are the two major hypotheses. Recent developments using molecular and cellular biophysics, molecular cell biology and gene technology have enabled visualization of moving forms of cytoskeletal proteins during their transport. Here, we argue that these studies support the subunit transport theory.

Differential in vitro and in vivo antitumor effects mediated by anti-CD40 and anti-CD20 monoclonal antibodies against human B-cell lymphomas.

The antitumor effects of CD40 and CD20 monoclonal antibodies (mAbs) were compared on various human B-cell lymphomas by using both in vitro and in vivo assays. Anti-CD40 directly inhibited the proliferation of human B-cell lymphomas in vitro, whereas anti-CD20 exerted no inhibitory effects on the growth of any lymphoma tested. These lymphomas were then injected into immunodeficient mice to examine the antitumor efficacy of these unconjugated mAbs in vivo. This xenogeneic model was used in the evaluation of various potential therapeutic agents against human cancers in an in vivo setting. Surprisingly, in contrast to its negligible effects on lymphoma growth in vitro, anti-CD20 was more efficacious than anti-CD40 in promoting the survival of mice bearing some but not all lymphoma lines. To determine whether the antitumor effects of these mAbs were direct or indirect in vivo, we concurrently treated tumor-bearing mice with mAbs to the murine Fc receptor to block antibody-dependent cell-mediated cytotoxicity (ADCC). When these neutralizing antibodies against Fc receptors were administered at the same time as mAb treatment, the antitumor effects of anti-CD20 in vivo were completely abrogated, whereas anti-CD40 treatment, although also diminished, still provided significant antitumor effects. These results indicate that the in vivo antitumor activity of the murine anti-human CD20 mAb was primarily due to ADCC by murine effector cells, which may not translate into comparable effects in humans. By contrast, anti-CD40 may be of potential clinical use in the treatment of lymphomas in humans because of its additional direct anti-proliferative effects. The results also demonstrate a possible difficulty in accurately evaluating the potential clinical efficacy of murine antibodies against human tumors in a human/mouse model system. Murine monoclonal anti-human antibodies may produce greater effects in human/mouse xenogeneic models, in which they are more likely to elicit host effector systems than when used in vivo in humans.

Chemokines and T lymphocyte activation: II. Facilitation of human T cell trafficking in severe combined immunodeficiency mice.

Previous studies from this laboratory have demonstrated that the chemokines RANTES (recombinant human regulated upon activation, normally T cell expressed and presumably secreted), macrophage chemotactic peptide-1, recombinant human macrophage inflammatory protein-1 alpha (rhMIP-1 alpha) IL-8, and IP-10 are capable of inducing human T cell infiltration into the injection site of severe combined immunodeficiency (SCID) mice reconstituted with human PBL. However, the ability of these chemokines to facilitate T cell homing into various lymphoid tissues has not been examined. Initial studies focused on the ability of rhMIP-1 beta to induce human T cell infiltration into injection sites in human PBL-SCID mice. SCID mice received s.c. injections of rhMIP-1 beta or PBS (1 microgram/injection) in the hindflank for 4 h or sequential injections for 3 days. Biopsies of the MIP-1 beta injection site revealed the presence of significant mononuclear cell accumulation 72 h after injection. Immunohistologic evaluation determined that significant numbers of human CD3+ T cells were recruited in response to MIP-1 beta injections, and this infiltration could be specifically blocked by co-administration of anti-MIP-1 beta antiserum. We subsequently examined these chemokine-injected mice for the effect of trafficking of human T cells to peripheral lymphoid organs. Flow cytometric analysis of the thymus in human PBL-SCID mice revealed that treatment with rhMIP-1 beta or rhRANTES, but not platelet factor-4, resulted in improved thymic homing of the human T cells after 72 h. This trafficking effect was shown to be direct, as pretreatment of the human T cells with the chemokines in vitro also improved peripheral lymphoid trafficking of the human cells. In addition, co-injection of rhMIP-1 beta with anti-1 beta antiserum abrogated the increase in T cell homing to the thymus. These data demonstrate that MIP-1 beta and RANTES directly augment human T cell trafficking to peripheral murine lymphoid tissues. Chemokines may, therefore, under either isogeneic or xenogeneic conditions, play a role in normal lymphocyte recirculation and homing, and may be of potential clinical use in promoting immune cell trafficking and function.

In vivo antitumor effects of unconjugated CD30 monoclonal antibodies on human anaplastic large-cell lymphoma xenografts.

CD30 is a M(r) 120,000 surface antigen identified originally by the Ki-1 monoclonal antibody (moAb) against primary and cultured Reed-Sternberg cells present in Hodgkin's disease and anaplastic large-cell lymphomas (ALCLs). Examination of two ALCL cell lines (Karpas 299 and Michel) demonstrated cell surface expression of CD30. Incubation of these lymphomas with two anti-CD30 moAbs that recognize the ligand-binding site (M44 or HeFi-1) resulted in significant growth inhibition in vitro, with significant decreases in cell viability. Another anti-CD30 moAb, Ber-H2, which recognizes a determinant not involved in ligand binding, had no effect on ALCL growth in vitro. When these human ALCL lines were transferred i.v. into mice with severe combined immune deficiency, the mice developed extensive metastasis in the s.c., brain, or eye tissues. The treatment of mice with either M44 or HeFi-1 anti-CD30 moAbs resulted in significant increases in survival, with some mice remaining disease free for more than 100 days. Thus, anti-CD30 treatment is efficacious for CD30+ ALCL cell lines in vivo, and unconjugated anti-CD30 moAbs may be of potential clinical use.

De Novo synthesis and posttranslational processing of L-histidine decarboxylase in mice.

We purified L-histidine decarboxylase from mouse mastocytoma cells and cloned mouse HDC cDNA, and found that the primary translated product (74 kD) is posttranslationally processed in its C-terminal region to yield a native HDC subunit (53 kD). Recombinant 74-kD, but not 53-kD HDC species was present mainly in the particulate fraction of Sf9 cells. The particulate 74-kD recombinant HDC was cleaved by porcine pancreatic elastase, and a homodimer of a 53-kD subunit having the identical catalytic properties to those of native HDC was solubilized. The particulate HDC from mouse stomach was partially purified and it was solubilized by porcine pancreatic elastase to yield the 53-kD subunit of HDC. We identified endogenous proteolytic activity, which converts the particulate recombinant 74-kD HDC to the soluble 53-kD HDC in the supernatant of mouse stomach. In mastocytoma cells, we demonstrated that the induction of HDC activity and HDC mRNA synergistically occurred upon treatment with dexamethasone + TPA, and also with cAMP + Ca2+. On a genomic DNA cloning, we found that two upregulations occurred via the involvement of the regulatory elements binding to the sequences from -132 to -53 and -267 to -53, respectively.

Engraftment of SCID mice by human bone marrow hematopoietic cells cultured in vitro: an in vivo model for human gene transfer.

Demonstration of the ability of fresh human hematopoietic cells to engraft severe combined immuno-deficient (scid) mice has provided an in vivo assay for expansion and maturation of early human progenitor cells. However, engraftment of cultured hematopoietic cells has been difficult to achieve. We wished to further develop this model as an in vivo assay for efficiency of retroviral gene transfer and expression in the differentiated progeny of adult human bone marrow progenitor cells. Human bone marrow cells were cultured in vitro for six days under conditions suitable for infection by retroviral vectors prior to transfer to irradiated scid mice. Cultured human bone marrow cells introduced by both intravenous (i.v.) and intraperitoneal (i.p.) injection persisted in the bone marrow, spleen and peritoneum of recipient animals up to four weeks after transfer. Following irradiation scid mice receiving cultured human bone marrow cells by either i.v. or i.p. routes demonstrated engraftment of the bone marrow and spleen as determined by the growth of human hematopoietic progenitors in soft agar. By flow cytometric analysis human cells were also detected in the peritoneum of mice receiving cultured human bone marrow cells i.p. These results suggest that the transfer of cultured human bone marrow cells to scid mice with the subsequent engraftment of these cells in the bone marrow, spleen and peritoneum of recipients can routinely occur. This provides an in vivo model for retroviral gene transfer to human cells.