Germline incorporation of a replication-defective adenoviral vector in mice does not alter immune responses to adenoviral vectors.

The utility of adenoviral vectors is limited by immune responses to adenoviral antigens. We sought to develop immune-competent mice in which the immune response to adenoviral antigens was selectively absent. To do so, we generated mice that were transgenic for a replication-defective vector. Adenoviral antigens might be seen as self-antigens by these mice, and the mice could exhibit immunologic tolerance after postnatal exposure to adenoviral vectors. In addition, characterization of these mice could reveal potential consequences of germline transmission of an adenoviral vector, as might occur in a gene therapy trial. Injection of a "null" (not containing a transgene) E1, E3-deleted vector genome into mouse zygotes yielded five founders that were capable of transmitting the vector genome. Among offspring of these mice, transgenic pups were significantly underrepresented: 108 of 255 pups (42%) were transgenic (P<0.02 versus expected frequency of 50%). Postnatal transgenic mice, however, had no apparent abnormalities. Persistence of an adenoviral vector after intravenous injection was equivalent in livers of transgenic mice and their nontransgenic littermates. Transgenic and nontransgenic mice also had equivalent humoral and cellular immune responses to adenoviral vector injection. Mice that are transgenic for an E1, E3-deleted adenoviral genome can be easily generated; however, they are not tolerant of adenovirus. Moreover, germline transmission of an adenoviral vector genome does not prevent generation of a robust immune response after exposure to adenoviral antigens.

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.

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.

Overexpression of transforming growth factor beta1 in arterial endothelium causes hyperplasia, apoptosis, and cartilaginous metaplasia.

Uninjured rat arteries transduced with an adenoviral vector expressing an active form of transforming growth factor beta1 (TGF-beta1) developed a cellular and matrix-rich neointima, with cartilaginous metaplasia of the vascular media. Explant cultures of transduced arteries showed that secretion of active TGF-beta1 ceased by 4 weeks, the time of maximal intimal thickening. Between 4 and 8 weeks, the cartilaginous metaplasia resolved and the intimal lesions regressed almost completely, in large part because of massive apoptosis. Thus, locally expressed TGF-beta1 promotes intimal growth and appears to cause transdifferentiation of vascular smooth muscle cells into chondrocytes. Moreover, TGF-beta1 withdrawal is associated with regression of vascular lesions. These data suggest an unexpected plasticity of the adult vascular smooth muscle cell phenotype and provide an etiology for cartilaginous metaplasia of the arterial wall. Our observations may help to reconcile divergent views of the role of TGF-beta1 in vascular disease.

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.

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.

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.

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.

Rate process model for arterial tissue thermal damage: implications on vessel photocoagulation.

A numerical model for thermal damage to human arterial tissue is presented, based on protein denaturation kinetics. The model involves determination of coefficients of rate processes A & delta E, which are tissue type-dependent (arterial tissue in this study), and definition of threshold damage. A feedback-controlled constant surface temperature device was used to induce 80 coagulative lesions of arterial human tissue ranging in temperature from 66 degrees C to 76 degrees C and in duration from 15 to 1,500 seconds. The measured coefficients were determined to be A = 5.6 x 10(63) s-1 and delta E = 430 KJ mole-1. These numerical values closely approximate the coefficients of the rate process for denaturation of collagen molecules. These and other histological observations strongly suggest collagen to be the primary coagulating component of arterial tissue at the onset of thermal coagulative damage. The ability of this model to predict onset of tissue coagulation during laser coagulation was studied using 10 postmortem human arterial samples exposed to argon laser irradiation.

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.

Synergism of interleukin-2 and cyclosporine A in induction of a graft-versus-tumor effect without graft-versus-host disease after syngeneic bone marrow transplantation.

Interleukin-2 (IL-2) therapy generates killer cells with major histocompatibility complex (MHC)-unrestricted cytotoxicity against most tumors but not normal tissues. Cyclosporine A (CsA) has been reported to break tolerance to self and to induce killer cells with specificity against class II MHC (Ia) antigens both on the host and the tumor cells, resulting in a mild graft-versus-host disease (GVHD) in an autologous bone marrow transplantation (BMT) setting in the rat. We used these two agents in a syngeneic BMT model in a strain of mice that does not develop GVHD with CsA. Therapy with either agent alone was ineffective, whereas a combination of CsA plus IL-2 after BMT induced a potent graft-versus-tumor (GVT) effect against a melanoma and an acute myeloid leukemia. The antitumor effect could be adoptively transferred by infusing spleen cells harvested from mice treated with CsA plus IL-2 into secondary recipients that received chemoradiotherapy. The cytotoxicity of these cells was not influenced by treatment of tumor cells with gamma-interferon or Ia antibody. The cytotoxic effect was mediated by Thy 1+ and asialo GM 1+ cells. There was no GVHD either in the primary recipients of CsA and IL-2 or in those receiving the adoptively transferred spleen cells. Our findings show that combination therapy with CsA and IL-2 after syngeneic BMT induces a potent GVT effect in a non-MHC-restricted manner, and point to the existence of differences between the mechanisms of GVT and GVHD.

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.

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.

Adoptive transfer of anti-cytomegalovirus effect of interleukin-2-activated bone marrow: potential application in transplantation.

This work is a continuation of our studies that showed that interleukin-2 (IL-2)-activated murine bone marrow (ABM) cells have potent cytotoxic potential against murine cytomegalovirus (MCMV)-infected targets in vitro, without loss of reconstitutive ability in vivo. Our data show that ABM cells lyse the MCMV-infected cells in vitro, at both acute and chronic stages of infection; this lysis is specific for the MCMV-infected cells. ABM cells supplemented with IL-2 therapy virtually eradicated the viral infection and prolonged the survival of MCMV-infected Balb/c mice, whether or not they were immunocompromised by irradiation (P less than .001 in both situations). Efficacy of ABM cells alone or IL-2 alone was less than the combination of ABM cells and IL-2. The efficacy of combination treatment with ABM cells and IL-2 in improving the survival of MCMV-infected mice was comparable, whether used in a preventive or a therapeutic setting. Therapy with ABM plus IL-2 also prevented the reactivation of chronic MCMV infection after irradiation. Preliminary findings indicate that Thy-1+ and asialo GM1+ cells limited the MCMV proliferation by approximately 30% and 80%, respectively, while BM macrophages limited the proliferation of MCMV by 100%. These results suggest that BM transplantation (BMT) with ABM cells followed by IL-2 therapy may constitute a novel strategy to improve the host resistance against cytomegalovirus infection after BMT.

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.