Gene recombination in postmitotic cells. Targeted expression of Cre recombinase provokes cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo.

Mouse models of human disease can be generated by homologous recombination for germline loss-of-function mutations. However, embryonic-lethal phenotypes and systemic, indirect dysfunction can confound the use of knock-outs to elucidate adult pathophysiology. Site-specific recombination using Cre recombinase can circumvent these pitfalls, in principle, enabling temporal and spatial control of gene recombination. However, direct evidence is lacking for the feasibility of Cre-mediated recombination in postmitotic cells. Here, we exploited transgenic mouse technology plus adenoviral gene transfer to achieve Cre-mediated recombination in cardiac muscle. In vitro, Cre driven by cardiac-specific alpha-myosin heavy chain (alphaMyHC) sequences elicited recombination selectively at loxP sites in purified cardiac myocytes, but not cardiac fibroblasts. In vivo, this alphaMyHC-Cre transgene elicited recombination in cardiac muscle, but not other organs, as ascertained by PCR analysis and localization of a recombination-dependent reporter protein. Adenoviral delivery of Cre in vivo provoked recombination in postmitotic, adult ventricular myocytes. Recombination between loxP sites was not detected in the absence of Cre. These studies demonstrate the feasibility of using Cre-mediated recombination to regulate gene expression in myocardium, with efficient induction of recombination even in terminally differentiated, postmitotic muscle cells. Moreover, delivery of Cre by viral infection provides a simple strategy to control the timing of recombination in myocardium.

Adenoviral delivery of E2F-1 directs cell cycle reentry and p53-independent apoptosis in postmitotic adult myocardium in vivo.

Irreversible exit from the cell cycle precludes the ability of cardiac muscle cells to increase cell number after infarction. Using adenoviral E1A, we previously demonstrated dual pocket protein- and p300-dependent pathways in neonatal rat cardiac myocytes, and have proven that E2F-1, which occupies the Rb pocket, suffices for these actions of E1A. By contrast, the susceptibility of adult ventricular cells to viral delivery of exogenous cell cycle regulators has not been tested, in vitro or in vivo. In cultured adult ventricular myocytes, adenoviral gene transfer of E2F-1 induced expression of proliferating cell nuclear antigen, cyclin-dependent protein kinase 4, cell division cycle 2 kinase, DNA synthesis, and apoptosis. In vivo, adenoviral delivery of E2F-1 by direct injection into myocardium induced DNA synthesis, shown by 5'-bromodeoxyuridine incorporation, and accumulation in G2/M, by image analysis of Feulgen-stained nuclei. In p53(-)/- mice, the prevalence of G1 exit was more than twofold greater; however, E2F-1 evoked apoptosis and rapid mortality comparably in both backgrounds. Thus, the differential effects of E2F-1 on G1 exit in wild-type versus p53-deficient mice illustrate the combinatorial power of viral gene delivery to genetically defined recipients: E2F-1 can override the G1/S checkpoint in postmitotic ventricular myocytes in vitro and in vivo, but leads to apoptosis even in p53(-)/- mice.

A mouse model of arterial gene transfer: antigen-specific immunity is a minor determinant of the early loss of adenovirus-mediated transgene expression.

We developed a murine model of arterial gene transfer and used it to test the role of antigen-specific immunity in the loss of adenovirus-mediated transgene expression. Adenoviral vectors encoding either beta-galactosidase (beta-gal) or green fluorescent protein were infused to the lumen of normal common carotids of CD-1 and C57BL/6 mice and atherosclerotic carotids of Apoe(-/-) mice. At 3 days after gene transfer, significant reporter gene expression was detected in all strains. Transgene expression was transient, with expression undetectable at 14 days. Next, a beta-gal-expressing vector was infused into carotids of ROSA26 mice (transgenic for, and therefore tolerant of, beta-gal) and RAG-2(-/-) mice (deficient in recombinase-activating gene [RAG]-2 and therefore lacking in antigen-specific immunity). beta-Gal expression was again high at 3 days but declined substantially (>90%) by 14 days. In vivo labeling with bromodeoxyuridine revealed that carotid endothelial proliferation was increased dramatically by the gene-transfer procedure alone, likely leading to the loss of episomal adenoviral DNA. Gene transfer to normal and atherosclerotic mouse carotids can be accomplished; however, elimination of antigen-specific immune responses does not prevent the early loss of adenovirus-mediated transgene expression. Efforts to prolong adenovirus-mediated transgene expression in the artery wall must be redirected. These efforts will likely include strategies to avoid the consequences of increased cell turnover. Nevertheless, despite the brevity of expression, this mouse model of gene transfer to normal and severely atherosclerotic arteries will likely be useful for investigating the genetic basis of vascular disease and for developing gene therapies.

Cardiovascular overexpression of transforming growth factor-beta(1) causes abnormal yolk sac vasculogenesis and early embryonic death.

Transforming growth factor-beta(1) (TGF-beta(1)) is expressed in the adult and embryonic vasculature; however, the biological consequences of increased vascular TGF-beta(1) expression remain controversial. To establish an experimental setting for investigating the role of increased TGF-beta(1) in vascular development and disease, we generated transgenic mice in which a cDNA encoding a constitutively active form of TGF-beta(1) is expressed from the SM22alpha promoter. This promoter fragment directs transgene expression to smooth muscle cells of large arteries in late-term embryos and postnatal mice. We confirmed the anticipated pattern of SM22alpha-directed transgene expression (heart, somites, and vasculature of the embryo and yolk sac) in embryos carrying an SM22alpha-beta-galactosidase transgene. SM22alpha- beta-galactosidase transgenic mice were born at the expected frequency (13%); however, nearly all SM22alpha-TGF-beta(1) transgenic mice died before E11.5. SM22alpha-TGF-beta(1) transgenic embryos identified at E8.5 to E10.5 had growth retardation and both gross and microscopic abnormalities of the yolk sac vasculature. Overexpression of TGF-beta(1) from the SM22alpha promoter is lethal at E8.5 to E10.5, most likely because of yolk sac insufficiency. Investigation of the consequences of increased vascular TGF-beta(1) expression in adults may require a conditional transgenic approach. Moreover, because the SM22alpha promoter drives transgene expression in the yolk sac vasculature at a time when embryonic survival is dependent on yolk sac function, use of the SM22alpha promoter to drive expression of "vasculoactive" transgenes may be particularly likely to cause embryonic death.

Circulation research online only : october 29, 1999UltraRapid CommunicationsA mouse model of arterial gene TransferAntigen-specific immunity is a minor determinant of the early loss of adenovirus-mediated transgene expression

We developed a murine model of arterial gene transfer and used it to test the role of antigen-specific immunity in the loss of adenovirus-mediated transgene expression. Adenoviral vectors encoding either beta-galactosidase (beta-gal) or green fluorescent protein were infused to the lumen of normal common carotids of CD-1 and C57BL/6 mice and atherosclerotic carotids of Apoe(-/-) mice. At 3 days after gene transfer, significant reporter gene expression was detected in all strains. Transgene expression was transient, with expression undetectable at 14 days. Next, a beta-gal-expressing vector was infused into carotids of ROSA26 mice (transgenic for, and therefore tolerant of, beta-gal) and RAG-2(-/-) mice (deficient in recombinase-activating gene [RAG]-2 and therefore lacking in antigen-specific immunity). beta-Gal expression was again high at 3 days but declined substantially (>90%) by 14 days. In vivo labeling with bromodeoxyuridine revealed that carotid endothelial proliferation was increased dramatically by the gene-transfer procedure alone, likely leading to the loss of episomal adenoviral DNA. Gene transfer to normal and atherosclerotic mouse carotids can be accomplished; however, elimination of antigen-specific immune responses does not prevent the early loss of adenovirus-mediated transgene expression. Efforts to prolong adenovirus-mediated transgene expression in the artery wall must be redirected. These efforts will likely include strategies to avoid the consequences of increased cell turnover. Nevertheless, despite the brevity of expression, this mouse model of gene transfer to normal and severely atherosclerotic arteries will likely be useful for investigating the genetic basis of vascular disease and for developing gene therapies. The full text of this article is available at http://www.circresaha. org.

Successful therapy of natural killer-resistant pulmonary metastases by the synergism of gamma-interferon with tumor necrosis factor and interleukin-2 in mice.

The problem associated with lymphokine (gamma-interferon, interleukin-2, and tumor necrosis factor) therapy in cancer is that high toxic doses of these lymphokines must be administered for any significant antitumor results. Therefore this study was undertaken to investigate the antitumor efficacy of these lymphokines in a disseminated pulmonary metastasis when used in combination with each other at dosages well below the toxic level. The role of the immune system of the host, sequencing of lymphokines, and histopathological aspects of lymphokine therapy were also investigated.

Synergistic antitumor effects of interleukin 2 and the monoclonal Lym-1 against human Burkitt lymphoma cells in vitro and in vivo.

Interleukin 2 (IL-2) regulates immune responses by inducing proliferation and differentiation of T-cells into cytotoxic cells, inducing lymphokine activated killer activity and enhancing antibody dependent cellular cytotoxicity (ADCC). Lym-1, a monoclonal antibody, recognizes a membrane antigen present on the surface of B-lymphoma cells and can be used for ADCC. We therefore used Raji (human Burkitt lymphoma) cells to study the efficacy of combination therapy with IL-2, lymphokine activated killer activity, and Lym-1. In vitro ADCC assays using Lym-1 showed that preincubation of peripheral blood lymphocytes with IL-2 had a synergistic antitumor effect. The maximum synergism was achieved when peripheral blood lymphocytes were incubated with IL-2 for 3 days as compared to 1 or 2 days, with the optimal concentration of IL-2 being 1000 units/ml. This effect was specific for Lym-1 as demonstrated by experiments using an irrelevant (antimelanoma) monoclonal antibody or an irrelevant target cell (A375). The ADCC was blocked by an anti-Fe receptor antibody (3G8). In vivo experiments performed by growing Raji tumors in nude mice also demonstrated the increase in ADCC and the synergism between IL-2 and Lym-1 in terms of decreased tumor size and growth. The mechanism of this synergy is probably from activation of cells mediating ADCC. This raises the possibility that treatment of patients with low doses of IL-2 in combination with Lym-1 may enhance immune responses and thereby antitumor activity.

Potent graft antitumor effect in natural killer-resistant disseminated tumors by transplantation of interleukin 2-activated syngeneic bone marrow in mice.

The current study is a continuation of our previous work showing that bone marrow activated in interleukin 2 has antitumor and antiviral activity in vitro. The antitumor efficacy of IL-2-activated bone marrow cells in vivo was assessed here. Our results indicated that bone marrow cells activated in IL-2 for 3 days (ABM) have antitumor activity in vivo and cause significant tumor regression in mice being treated with ABM and concurrent i.p. administration of IL-2. In mice also bearing larger tumor burdens, those receiving ABM and i.p. IL-2 showed the most significant tumor regression. The ABM seem to be more potent than conventional IL-2-activated spleen lymphokine-activated killer cells. In studies done using lower dosages of IL-2 or log lower number of cells, the ABM caused more significant tumor regression than lymphokine-activated killer cells. We also assessed the antitumor efficacy of short term (1 day) IL-2-activated bone marrow, the short term-activated bone marrow being preferred in bone marrow, transplantation because of the minimum amount of cells lost due to its shorter incubation period. We also showed that short term-activated bone marrow caused tumor regression similar to ABM and could reconstitute lethally irradiated mice similar to fresh bone marrow. Therefore, the biomodulation of bone marrow cells could be used as an active therapeutic tool in autologous bone marrow transplantation, producing graft versus tumor effects without any graft versus host effect.

Interaction of various cytokines with interleukin 2 in the generation of killer cells from human bone marrow: application in purging of leukemia.

We have shown that incubation of bone marrow (BM) with interleukin 2 (IL-2) generates activated bone marrow cells (ABM) with potent tumoricidal activity in vitro and in vivo. The present study was carried out to define the interaction of other cytokines with IL-2 in generation of ABM. Our data show that interleukin 1 (IL-1), interferon (IFN)- both gamma and alpha, and tumor necrosis factor (TNF-alpha) significantly increased the cytolytic potential of ABM. Interleukin 3, interleukin 4, transforming growth factor-beta and adherent cells were reduced, while granulocyte-macrophage colony-stimulating factor had no influence on the generation of cytolytic activity. IL-1 was enhanced while TNF-alpha depressed the BM progenitor cell activity in vitro. The IL-2-induced purging ability of BM contaminated with leukemic cells was increased by IL-1, TNF-alpha and IFN-gamma. This study shows that biomodulation of BM with combination of cytokines in vitro can be useful in purging a large leukemic burden.

A novel approach to purging of leukemia by activation of bone marrow with interleukin 2.

The cytotoxic potential of interleukin 2 (IL-2) activated bone marrow (ABM) was compared with that of IL-2 activated peripheral blood lymphocytes (LAK cells) against three hematologic tumor cell lines (K-562, CEM, Daudi) and fresh lymphoid blasts in short-term chromium release assays. ABM was found to be superior to LAK cells against all tumor cells tested. The recovery of bone marrow (BM) cells dropped with passage of time in culture but their clonogenic potential was not impaired (with or without IL-2). BM contaminated with CEM cells and treated with IL-2 showed significant ability to purge itself of the leukemic cells in semisolid agar culture; the purging ability of 3- and 1-day ABM was comparable. IL-2 alone or BM alone had no influence on the growth of CEM cells. This study suggests that BM can be activated with IL-2 in vitro to generate the ability to eliminate contaminating leukemic cells without affecting its progenitor cell function in vitro.

Interleukin-2 (IL-2) and IL-2-activated bone marrow in transplantation: evaluation from a clinical perspective.

Incubation of bone marrow (BM) with interleukin-2 (IL-2) in vitro results in generation of killer cells providing a tool for enhancing the graft-versus-tumor effect in transplantation. We have evaluated the influence of IL-2 on the progenitor cell activity (PCA), homing pattern of BM and hemopoiesis in a syngeneic bone marrow transplantation (BMT) model in mice. The PCA index and homing pattern of BM activated with IL-2 in vitro for 24 h (ABM) were similar to those of fresh bone marrow (FBM). In vitro culture of BM for more than 1 day resulted in progressive decline in its PCA index; this was not related to the presence or absence of IL-2 in the culture medium. Toxicity of IL-2 was related to the dose and not the time of institution of IL-2 therapy after BMT. Maximum tolerated dose of IL-2 instituted immediately after BMT was 10 times higher than the dose in a non-BMT setting. The pattern of marrow reconstitution following BMT with ABM was comparable to that with FBM. This study shows that BMT with BM activated with IL-2 for 24 h results in normal hemopoiesis, and IL-2 therapy instituted immediately after BMT with ABM does not cause additional toxicity.

Angioedema: the role of ACE inhibitors and factors associated with poor clinical outcome.

OBJECTIVE: We sought to study the prevalence of angiotensin-converting enzyme (ACE) inhibitors, a cause of angioedema, and investigate any association between clinical findings at the time of presentation and clinical outcome. DESIGN AND SETTING: Retrospective review of the charts of all patients presenting with angioedema to the emergency department at our tertiary referral teaching hospital or clinics over a 4-year period. The charts were reviewed for documentation of chief complaint(s), physical findings, medical treatment, need for laryngoscopy and/or endotracheal intubation, triage, and probable etiology. RESULTS: Of the 40 patients presenting with angioedema in this study, 15 cases were caused by ACE inhibitors. They were the most common cause of angioedema, accounting for 38% of all cases. The incidence of ACE inhibitor-induced angioedema is estimated to be 0.14%. More patients with angioedema secondary to ACE inhibitors had complaints of odynophagia (p < 0.02), whereas only patients with non-ACE inhibitor causes of angioedema presented with pruritus (p < 0.02). Furthermore, patients presenting with an acute reaction within 24 h of exposure to the causative agent were more likely to require inpatient monitoring (p < 0.05). Both odynophagia and edema of the tongue were significant predictors for undergoing laryngoscopy (p < 0.001 and p < 0.02, respectively) and admission to the hospital (p < 0.05). CONCLUSION: ACE inhibitors are the number one cause of acute angioedema in this tertiary referral teaching hospital. Odynophagia and tongue swelling at the time of presentation had significant implications for diagnostic intervention and admission to the hospital.

Dynamics of temperature dependent optical properties of tissue: dependence on thermally induced alteration.

Thermal damage in heated bovine myocardial tissue is assessed from measured changes in total reflection and transmission of light. Mathematical expressions, based on random walk analysis of light propagation within tissue slabs, are used to relate the diffuse reflection and transmittance to the absorption coefficient, mu a, and effective scattering coefficient, mu's, for samples of myocardial tissue which were subjected to rapid step changes in temperature. Time-dependent changes in mu's, indicate two processes, one with a fast and temperature-dependent rate the other with a slow and apparently temperature-independent rate. For final temperatures above 56.8 degrees C and for the first 500 s after the temperature change, the optical parameters are well fit by exponential forms that exhibit temperature-dependent time constants as predicted by Arrhenius reaction rate theory of thermal damage. The scattering changes are associated with an apparent activation energy, delta E, of 162 kJ/mole and a frequency constant, A, of 3 x 10(23) s-1. This method provides a means for estimating optical coefficients which are needed to assess laser tissue dosimetry.

Fatal cerebral venous thrombosis as the initial manifestation of the antiphospholipid syndrome.

We describe a patient with cerebral venous thrombosis (CVT) who presented with thrombocytopenia and persistent headache. The etiology of her CVT was determined to be the antiphospholipid syndrome (APLS) based on a prolonged dilute Russell viper venom test and elevated anti-cardiolipin IgG antibody. CVT has rarely been reported as the initial manifestation of the APLS. Despite supportive measures and anticoagulation, the patient expired. Clinicians should consider the possibility of CVT when coagulation abnormalities consistent with APLS are combined with neurologic symptoms. Early detection and treatment are crucial for a favorable outcome.

The potential usefulness of interleukin-2 activated bone marrow cells as an active therapeutic tool against cytomegalovirus infection in a bone marrow transplantation setting.

Bone marrow transplantation (BMT) has been used in recent years for the treatment of immunodeficiency diseases, aplastic anemia, and leukemia. However, there are a number of serious problems and limitations associated with autologous or allogeneic BMT. One of these is an increase in opportunistic infections, of which cytomegalovirus (CMV) infection is one of the most important. Cytomegalovirus has been associated with more frequent deaths than any other single agent, with no reproducibly successful or therapy currently available. Recently usage of interleukin-2 or immunomodulation has been suggested as a powerful modality to combat infectious disease. In this study we showed that bone marrow activated in interleukin-2 for 2 days has the ability to lyse spleen cells infected for 3 days with murine CMV (acute infection model) or salivary gland cells infected for 7 days (chronic infection model), while nonactivated bone marrow or natural killer (NK) cells showed no such lysis. The majority of activated cells involved in lysis were antiasialo GM1-, Thy-1+/-, indicating a population of cells other than the natural killer-cell population involved.

Lysosome rich cells contain the lytic activity of lymphokine-activated killer cell populations.

Little is known regarding the effectors of lymphokine-activated killer activity. Lysosomotropic agents such as quinacrine can be used to positively sort for lysosome rich cells in natural killer (NK) cell populations. We therefore decided to use this agent to sort lymphokine-activated killer (LAK) cells to characterize their lysosomal content. We found that the positively sorted population contained all the LAK activity, i.e., lysis of NK-resistant tumor cells (B16 melanoma cell line), with the negatively sorted cells having no killing activity. Therefore separation of interleukin-2-incubated cells for LAK activity could be accomplished using sorting after quinacrine staining. The treatment of positively sorted LAK cell populations with L-leucine methyl ester, a lysosomotropic dye which inhibits killing by lysosome rich cells, caused abrogation of killing of the B16 tumor by the treated populations. Single cell conjugate assays were also done on these sorted cells, with positively sorted cells forming the highest and negatively sorted cells the lowest percent of conjugates. Our data therefore indicates the important role of lysosome rich cells in the LAK cell population in the murine system.

Differential lysis of tumor target cells displayed by lymphokine activated killer (LAK) cell clones.

Lymphokine-activated killer (LAK) cells exhibit major histocompatibility complex (MHC) unrestricted cytolysis against a wide variety of fresh and cultured tumor cells. Because previous work from our laboratory suggested that trypsin treatment of unseparated populations of LAK cells had a differential effect on lysis of different tumors, in this report we analyzed the lytic specificity of LAK cell clones against a panel of three different targets: MCA, B16 and YAC-1. We found that 21 out of the 24 analyzed murine spleen and bone marrow clones killed a combination of two, but not all three, of these tumor cells. Determinations of the phenotype of 10 LAK cell clones showed six with rearrangements for the T cell receptor (TCR) beta chain gene, suggesting a T cell origin, and four with germ line configurations for the TCR beta and delta chain genes, a result consistent with a non-T cell lineage. This cloning procedure provided an experimental tool to develop new procedures of adaptive immunotherapy.

Heterogeneity of cell surface structures involved in cytotoxicity mediated by lymphokine activated killer cells.

Lymphokine activated killer (LAK) cells mediate the lysis of a variety of histologically distinct tumor targets. We investigated the nature and diversity of the structures involved in the recognition phenomenon by evaluating the effects of treating effector and target cells with trypsin and chymotrypsin, enzymes that disrupt surface protein molecules. Chymotrypsin and trypsin treatment of B16 target cells, a murine melanoma cell line, significantly abolished killing by LAK cells. Alternatively, neither of these treatments in P815 cells, a murine mastocytoma cell line, affected killing by LAK cells. Moreover, we found a differential effect of both these enzymes on YAC-1 cells, a murine leukemia cell line, with trypsin having a less inhibitory effect on cytolysis than chymotrypsin. The nature of the LAK cell receptor that presumably plays a role in binding target antigen was also investigated. Treatment of LAK cells with chymotrypsin significantly reduced lysis of the B16 and YAC-1 target cell types. However, trypsin treatment of the effectors only inhibited killing of the B16 tumor cell line. Cytotoxicity exerted against YAC-1 remained unaltered upon trypsinization of LAK cells. These cumulative results indicate heterogeneity of both the receptors on the LAK cells and the surface antigen molecules recognized on these targets. The use of YAC-1 as a target provided us with a tool to compare the LAK with the natural killer (NK) systems. The overall effect of proteolytic enzyme treatment in reducing cell lysis was more pronounced in the NK than in the LAK system.(ABSTRACT TRUNCATED AT 250 WORDS)

Granulocyte-macrophage colony-stimulating factor-induced antibody-dependent cellular cytotoxicity in bone marrow macrophages: application in bone marrow transplantation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been reported to induce antitumor activity in peripheral blood monocytes. We examined the role of GM-CSF on bone marrow (BM) macrophages in inducing antibody-dependent cellular cytotoxicity (ADCC) against murine and human tumor cells in vitro and in vivo with the aim of applying this approach in an autologous bone marrow transplantation (BMT) setting. GM-CSF induced a potent ADCC in BM macrophages against a murine melanoma in vitro. Treatment with GM-CSF alone or with antibody alone had no effect, whereas therapy with combination of both these agents resulted in a significant reduction in dissemination of melanoma both in a nontransplant as well as in BMT settings, with results being more optimal in the latter setting. Adoptive transfer of BM macrophages harvested from mice undergoing therapy with GM-CSF plus antibody significantly reduced the dissemination of melanoma in secondary recipients but only after irradiation, not in intact mice. GM-CSF also induced significant ADCC in human BM macrophages against a melanoma and a lymphoma in vitro and against a lymphoma implanted in nude mice in vivo. Again, these effects were more optimal after chemotherapy. These data suggest that treatment with GM-CSF plus tumor-specific monoclonal antibodies after BMT may induce an antitumor effect and help eradicate the minimal residual disease.