Pseudo-Wellens syndrome, acute pancreatitis, and an anomalous coronary artery: a case report.

BACKGROUND: Chest pain associated with transient electrocardiogram changes mimicking an acute myocardial infarction have been described in acute pancreatitis. These ischemic electrocardiogram changes can present a diagnostic dilemma, especially when patients present with concurrent angina pectoris and epigastric pain warranting noninvasive or invasive imaging studies. CASE PRESENTATION: A 45-year-old African-American man with a history of alcohol use disorder presented to the emergency department of our institution with 36 hours of concurrent epigastric pain and left-sided chest pain radiating to his left arm and associated with nausea and dyspnea. On physical examination, he was afebrile; his blood pressure was elevated; and he had epigastric tenderness. His laboratory test results were significant for hypokalemia, normal troponin, and elevated serum lipase and amylase levels. Serial electrocardiograms for persistent chest pain showed ST-segment elevations with dynamic T-wave changes in the right precordial electrocardiogram leads, consistent with Wellens syndrome. He was immediately taken to the cardiac catheterization laboratory, where selective coronary angiography showed normal coronary arteries with an anomalous origin of the right coronary artery from the opposite sinus. Given his elevated lipase and amylase levels, the patient was treated for acute alcohol-induced pancreatitis with intravenous fluids and pain control. His chest pain and ischemic electrocardiogram changes resolved within 24 hours of admission, and coronary computed tomography angiography showed an interarterial course of the right coronary artery without high-risk features. CONCLUSIONS: Clinicians may consider deferring immediate cardiac catheterization and attribute electrocardiogram changes to acute pancreatitis in patients presenting with angina pectoris and acute pancreatitis if confirmed by normal cardiac enzymes and elevated levels of lipase and amylase. However, when clinical signs and electrocardiogram findings are highly suggestive of myocardial ischemia/injury, immediate noninvasive coronary computed tomography angiography may be the best approach to make an early diagnosis.

Membrane Mucin Muc4 promotes blood cell association with tumor cells and mediates efficient metastasis in a mouse model of breast cancer.

Mucin-4 (Muc4) is a large cell surface glycoprotein implicated in the protection and lubrication of epithelial structures. Previous studies suggest that aberrantly expressed Muc4 can influence the adhesiveness, proliferation, viability and invasiveness of cultured tumor cells, as well as the growth rate and metastatic efficiency of xenografted tumors. Although it has been suggested that one of the major mechanisms by which Muc4 potentiates tumor progression is via its engagement of the ErbB2/HER2 receptor tyrosine kinase, other mechanisms exist and remain to be delineated. Moreover, the requirement for endogenous Muc4 for tumor growth progression has not been previously explored in the context of gene ablation. To assess the contribution of endogenous Muc4 to mammary tumor growth properties, we first created a genetically engineered mouse line lacking functional Muc4 (Muc4ko), and then crossed these animals with the NDL (Neu DeLetion mutant) model of ErbB2-induced mammary tumorigenesis. We observed that Muc4ko animals are fertile and develop normally, and adult mice exhibit no overt tissue abnormalities. In tumor studies, we observed that although some markers of tumor growth such as vascularity and cyclin D1 expression are suppressed, primary mammary tumors from Muc4ko/NDL female mice exhibit similar latencies and growth rates as Muc4wt/NDL animals. However, the presence of lung metastases is markedly suppressed in Muc4ko/NDL mice. Interestingly, histological analysis of lung lesions from Muc4ko/NDL mice revealed a reduced association of disseminated cells with platelets and white blood cells. Moreover, isolated cells derived from Muc4ko/NDL tumors interact with fewer blood cells when injected directly into the vasculature or diluted into blood from wild type mice. We further observed that blood cells more efficiently promote the viability of non-adherent Muc4wt/NDL cells than Muc4ko/NDL cells. Together, our observations suggest that Muc4 may facilitate metastasis by promoting the association of circulating tumor cells with blood cells to augment tumor cell survival in circulation.

The ER stress transducer IRE1ß is required for airway epithelial mucin production.

Inflammation of human bronchial epithelia (HBE) activates the endoplasmic reticulum (ER) stress transducer inositol-requiring enzyme 1 (IRE1)α, resulting in IRE1α-mediated cytokine production. Previous studies demonstrated ubiquitous expression of IRE1α and gut-restricted expression of IRE1ß. We found that IRE1ß is also expressed in HBE, is absent in human alveolar cells, and is upregulated in cystic fibrosis and asthmatic HBE. Studies with Ire1ß(-/-) mice and Calu-3 airway epithelia exhibiting IRE1ß knockdown or overexpression revealed that IRE1ß is expressed in airway mucous cells, is functionally required for airway mucin production, and this function is specific for IRE1ß vs. IRE1α. IRE1ß-dependent mucin production is mediated, at least in part, by activation of the transcription factor X-box binding protein-1 (XBP-1) and the resulting XBP-1-dependent transcription of anterior gradient homolog 2, a gene implicated in airway and intestinal epithelial mucin production. These novel findings suggest that IRE1ß is a potential mucous cell-specific therapeutic target for airway diseases characterized by mucin overproduction.

Endoplasmic reticulum stress in chronic obstructive lung diseases.

Chronic airway inflammation characterizes several airway diseases, including cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). The altered airway milieu that results from the pathogenic processes in these disorders affects the airway epithelia, leading to an up-regulation of their innate defense. In human airway epithelia, luminal inflammatory stimuli induce an adaptation characterized by an expansion of the endoplasmic reticulum (ER) and its Ca(2+) stores. This epithelial adaption mediates Ca(2+)-dependent "hyperinflammatory" responses, and recent studies have shown that activation of the unfolded protein response (UPR) by ER stress is involved in the process. The UPR is also known to be activated by cigarette smoke, the primary trigger for development of COPD. These studies illustrate the functional role of UPR pathways during airway inflammation and suggest that targeting the UPR may be a therapeutic strategy for obstructive airway diseases. This article reviews the link between airway epithelial inflammation and activation of the UPR, and discusses how UPR activation might be relevant for CF and COPD airways disease.

Mucus clearance, MyD88-dependent and MyD88-independent immunity modulate lung susceptibility to spontaneous bacterial infection and inflammation.

It has been postulated that mucus stasis is central to the pathogenesis of obstructive lung diseases. In Scnn1b-transgenic (Scnn1b-Tg⁺ mice, airway-targeted overexpression of the epithelial Na⁺ channel ß subunit causes airway surface dehydration, which results in mucus stasis and inflammation. Bronchoalveolar lavage from neonatal Scnn1b-Tg⁺ mice, but not wild-type littermates, contained increased mucus, bacteria, and neutrophils, which declined with age. Scnn1b-Tg⁺ mice lung bacterial flora included environmental and oropharyngeal species, suggesting inhalation and/or aspiration as routes of entry. Genetic deletion of the Toll-interleukin-1 receptor adapter molecule MyD88 in Scnn1b-Tg⁺ mice did not modify airway mucus obstruction, but caused defective neutrophil recruitment and increased bacterial infection, which persisted into adulthood. Scnn1b-Tg⁺ mice derived into germ-free conditions exhibited mucus obstruction similar to conventional Scnn1b-Tg⁺ mice and sterile inflammation. Collectively, these data suggest that dehydration-induced mucus stasis promotes infection, compounds defects in other immune mechanisms, and alone is sufficient to trigger airway inflammation.

Transgenic hCFTR expression fails to correct ß-ENaC mouse lung disease.

The relationships between airway epithelial Cl(-) secretion-Na(+) absorption balance, airway surface liquid (ASL) homeostasis, and lung disease were investigated in selected transgenic mice. 1) To determine if transgenic overexpression of wild-type (WT) human CFTR (hCFTR) accelerated Cl(-) secretion and regulated Na(+) absorption in murine airways, we utilized a Clara cell secretory protein (CCSP)-specific promoter to generate mice expressing airway-specific hCFTR. Ussing chamber studies revealed significantly (∼2.5-fold) elevated basal Cl(-) secretory currents in CCSP-hCFTR transgenic mouse airways. Endogenous murine airway Na(+) absorption was not regulated by hCFTR, and these mice exhibited no lung disease. 2) We tested whether hCFTR, transgenically expressed on a transgenic mouse background overexpressing the ß-subunit of the epithelial Na(+) channel (ß-ENaC), restored ion transport balance and ASL volume homeostasis and ameliorated lung disease. Both transgenes were active in CCSP-hCFTR/ß-ENaC transgenic mouse airways, which exhibited an elevated basal Cl(-) secretion and Na(+) hyperabsorption. However, the airway disease characteristic of ß-ENaC mice persisted. Confocal studies of ASL volume homeostasis in cultured tracheal cells revealed ASL autoregulation to a height of ∼6 µm in WT and CCSP-hCFTR cultures, whereas ASL was reduced to <4 µm in ß-ENaC and CCSP-hCFTR/ß-ENaC cultures. We conclude that 1) hCFTR overexpression increases basal Cl(-) secretion but does not regulate Na(+) transport in WT mice and 2) transgenic hCFTR produces increased Cl(-) secretion, but not regulation of Na(+) channels, in ß-ENaC mouse airways and does not ameliorate ß-ENaC mouse lung disease.

Expression of CFTR from a ciliated cell-specific promoter is ineffective at correcting nasal potential difference in CF mice.

Successful gene therapy will require that the therapeutic gene be expressed at a sufficient level in the correct cell type(s). To improve the specificity of gene transfer for cystic fibrosis (CF) and other airway diseases, we have begun to develop cell-type specific promoters to target the expression of transgenes to specific airway cell types. Using a FOXJ1 promoter construct previously shown to direct transgene expression specifically to ciliated cells, we have generated transgenic mice expressing human cystic fibrosis transmembrane conductance regulator (CFTR) in the murine tracheal and nasal epithelia. RNA analysis demonstrated levels of CFTR expression is greater than or equal to the level of endogenous mouse CFTR. Immunoprecipitation and western blotting demonstrated the production of human CFTR protein, and immunochemistry confirmed that CFTR was expressed in the apical region of ciliated cells. The transgenic animals were bred to CFTR null mice (Cftr(tm1Unc)) to determine if expression of CFTR from the FOXJ1 promoter is capable of correcting the airway defects in Cl(-) secretion and Na(+) absorption that accompany CF. Isolated trachea from neonatal CF mice expressing the FOXJ1/CFTR transgene demonstrated a correction of forskolin-stimulated Cl(-) secretion. However, expression of human CFTR in ciliated cells of the nasal epithelia failed to significantly change the nasal bioelectrics of the CF mice.

Bone marrow transplantation from partially HLA-mismatched family donors for acute leukemia: single-center experience of 201 patients.

Between February 1993 and December 1999, 201 patients (1-59 years old, median 23) with acute leukemia (67% not in remission) underwent ex vivo T-cell-depleted (TCD) bone marrow transplants (BMT) from partially mismatched related donors (PMRD; 92% mismatched for 2-3 HLA A, B, DR antigens). Conditioning comprised total body irradiation, cyclophosphamide, cytarabine, etoposide, anti-thymocyte globulin (ATG), and methylprednisolone. Graft-versus-host disease (GVHD) prophylaxis comprised partial TCD with OKT3 (n=143) or T10B9 (n=58), steroids, ATG, and cyclosporine. The engraftment rate was 98%. The cumulative incidences of grades II-IV acute GVHD and chronic GVHD were 13 and 15%, respectively. The 5-year cumulative incidences of relapse and transplant-related mortality (TRM) were 31 and 51%, respectively. The actuarial 5-year overall survival (OS) and disease-free survival (DFS) probabilities were 19 and 18%, respectively. Patient age >15 years, active disease at transplant, donor age >25 years, and 3-antigen donor mismatch (host-versus-graft) affected the outcome adversely. The actuarial 5-year OS of four groups of patients identified based upon these risk factors was 39, 20, 13, and 0%, respectively (P<0.0001). We conclude that PMRD BMT is a potential treatment option for patients with high-risk acute leukemia who require an alternative donor transplant and fall into a group with a reasonable expected outcome.

Cloning and functional characterization of two murine uridine nucleotide receptors reveal a potential target for correcting ion transport deficiency in cystic fibrosis gallbladder.

Extracellular nucleotides regulate transepithelial ion secretion via multiple receptors. The P2Y(2) receptor is the predominant transducer of chloride transport responses to nucleotides in the airways, but the P2 receptors that control ion transport in gastrointestinal epithelia have not been identified. UTP and UDP promote chloride secretion in mouse jejuna and gallbladder epithelia, respectively, and these responses were unaffected by P2Y(2) receptor gene disruption. Pharmacological data suggested the involvement of P2Y(4) and P2Y(6) receptors in gastrointestinal responses. To identify the P2Y receptors responsible for the gastrointestinal actions of UTP and UDP, we have cloned the murine P2Y(4) and P2Y(6) receptors and have stably expressed each in a null cell line to examine the nucleotide-promoted inositol phosphate formation and intracellular Ca(2+) mobilization. The (m)P2Y(4) receptor was potently, but not selectively, activated by UTP (UTP > or = ATP >ITP > GTP > CTP), and it was not activated by UDP or ADP. The (m)P2Y(6) receptor was highly selective for UDP (UDP >> ADP = GDP). The nucleotide selectivities observed with the recombinant (m)P2Y(4) and (m)P2Y(6) receptors resemble those for nucleotide-promoted chloride transport in murine P2Y(2)(-/-) jejuna and gallbladder epithelial cells, respectively. Ion transport responses to nucleotide additions were examined in freshly excised tissues from cystic fibrosis transmembrane regulator-deficient mice. Although the effect of UTP on jejunal short-circuit current (I(sc)) was impaired in the CF mouse, UDP-promoted I(sc) changes were not affected in CF gallbladder epithelium, suggesting that the P2Y(6) receptor is a target for treatment of CF gallbladder disease.

Partially mismatched related donor bone marrow transplantation as salvage for patients with AML who failed autologous stem cell transplant.

Treatment options for patients who relapse are limited and the outcome is dismal. Between August 1993 and January 1999, 17 patients, median age 26 (4-44) years, underwent T cell depleted bone marrow transplant from partially mismatched related donors (PMRD), as a salvage for AML relapsing after an autograft. The median time from auto-transplant to relapse was 7 months (1.5-24) and the interval between transplants was 10 months (3-30). All patients had active leukemia at time of transplant. Donors were siblings (n = 8), parents (n = 2), daughters (n = 4) and others (n = 3), and 82% were > or = 2 major HLA antigen mismatched with the recipient. The conditioning therapy included total body irradiation in 14 patients and was busulfan-based in three. Graft-versus-host disease (GVHD) prophylaxis consisted of partial T cell depletion along with post-transplant immunosuppression. Median day to engraftment was 16 days (12-20). Acute GVHD was seen in six patients, and chronic GVHD in four of 13 surviving beyond 100 days. Ten patients died of non-relapse causes, at 1-588 (median 77) days. Two patients relapsed at 3 and 4 months. Five patients (29%) are surviving leukemia-free 42-84 months post transplant (median 68 months). A short interval between transplants was predictive of early relapse but not mortality. Age <18 and <2 organ toxicities were marginally predictive of better survival. We conclude that BMT from PMRD is a reasonable option for patients with refractory AML post autograft.

Multiple advantages of alpha-fetoprotein as a marker for in vivo gene transfer.

The development of improved gene transfer vectors has been hampered by the lack of a nonimmunogenic reporter gene that can be serially quantified in the serum or from other sites. In response to the need to develop a new reporter protein, we have evaluated alpha-fetoprotein (AFP) as a potential candidate. A first-generation E1/E3-deleted adenoviral vector expressing human AFP (hAFP) was generated as a preliminary tool to evaluate AFP as a reporter. Using both mouse and baboon models, hAFP expression was evaluated in serum after intravenous delivery and in serum and bronchioalveolar lavage (BAL) fluid after delivery to the lung. In immunocompetent animals, intravenous delivery of the hAFP adenoviral vector resulted in hAFP expression in the serum early after injection, which declined rapidly over time. Disappearance of hAFP from the serum was complete by 3-4 weeks after administration and was accompanied by robust antibody responses to hAFP and loss of infected cells. After lung delivery, hAFP could be detected in both serum and BAL. This allowed the analysis of the kinetics of gene expression in the lung without sacrificing the animals. In both liver and lung, immunohistochemical analysis correlated well with hAFP levels as detected in serum or BAL, indicating that serum levels were a reliable marker of tissue expression. Preliminary results with a mouse AFP expressed in a helper-dependent adenoviral vector indicate that use of a species-specific version of AFP will eliminate the complication of antibody development. These initial evaluations suggest that AFP is useful as a reporter gene to evaluate gene expression of therapeutic cassettes in multiple tissues, and it should be considered for use in human subjects.

Evaluation of the half-life of intravenous human cytomegalovirus immune globulin in patients receiving partially mismatched related donor bone marrow transplantation.

STUDY OBJECTIVE: To evaluate the pharmacokinetics and use of intravenous human cytomegalovirus immune globulin (CytoGam) in allogeneic bone marrow transplantation (BMT). DESIGN: Prospective, nonrandomized, nonblinded, single-center study. SETTING: University teaching hospital. PATIENTS: Five consecutive patients with hematologic malignancies receiving partially mismatched related donor BMT with a uniform conditioning regimen including total body irradiation and chemotherapy. INTERVENTION: Serum immunoglobulin and cytomegalovirus (CMV) titers were measured before and 24 hours after the first CytoGam infusion on day -6 during the conditioning regimen. MEASUREMENTS AND MAIN RESULTS: These levels were measured every 5 days, and a second dose was administered when the CMV titer returned to 25-50% of the 24-hour level. The half-life of CytoGam was approximately 7 days. CONCLUSION: We believe this is the first report of CytoGam's half-life in allogeneic BMT. The information may prove vital in a future study in which the agent's potential beneficial effects can be maximized.

Toxicity associated with repeated administration of first-generation adenovirus vectors does not occur with a helper-dependent vector.

BACKGROUND: Certain gene therapy protocols may require multiple administrations of vectors to achieve therapeutic benefit to the patient. This may be especially relevant for vectors such as adenoviral vectors that do not integrate into the host chromosome. Because immunocompetent animal models used for gene transfer studies develop neutralizing antibodies to adenoviral vectors after a single administration, little is known about how repeat administrations of vectors might affect transgene expression and vector toxicity. MATERIALS AND METHODS: We used mice deficient in the membrane spanning region of immunoglobulin (IgM), which do not develop antibodies, to evaluate the effect of repeated intravenous administration of first-generation and helper-dependent adenoviral vectors expressing human alpha 1-antitrypsin (hAAT). The duration and levels of transgene expression were evaluated after repeated administration of vectors. Toxicity was assessed by measuring the level of liver enzymes in the serum and the degrees of hepatocyte hypertrophy and proliferation. RESULTS: We found that previous administration of first-generation adenoviral vectors can alter the response to subsequent doses. These alterations included an increase in transgene expression early (within 1 and 3 days), followed by a rapid drop in expression by day 7. In addition, previous administrations of first-generation vectors led to an increase in toxicity of subsequent doses, as indicated by a rise in liver enzymes and an increase in hepatocyte proliferation. In contrast to first-generation vectors, use of the helper-dependent adenovirus vector, Ad-STK109, which contained no viral coding regions, did not lead to increased toxicity after multiple administrations. CONCLUSIONS: We conclude that the response of the host to adenoviral vectors can be altered after repeated administration, compared with the response after the initial vector dose. In addition, these experiments provide further evidence for the relative safety of helper-dependent adenoviral vectors for gene therapy, compared with first-generation vectors.

Expression of CD134 (0X-40) on T cells during the first 100 days following allogeneic bone marrow transplantation as a marker for lymphocyte activation and therapy-resistant graft-versus-host disease.

CD134 (OX-40) is an activation-associated antigen which functions as a costimulatory receptor for CD4+ T cells. In order to determine the expression of CD134 during immune recovery following allogeneic bone marrow transplantation (BMT), we measured its expression on T cells and T cell subsets during the first 100 days following BMT in 26 patients. CD4+CD134+ T could be seen approximately 14 days following BMT cells in patients who did not develop GvHD which required therapy (n = 20). The percentage of CD4+CD134+ cells continued to increase up to the fourth week following BMT to a maximum of 40-50% of CD4+ T cells (normal = 1-8%). Two patients who developed Grade I-II GvHD and who responded to treatment with pulsed high-dose methylprednisolone (MPD) showed a decline of approximately 40% in CD4+CD134+ T cells was seen within 48 hours of treatment. Four patients who developed GvHD that was not responsive to MPD and who later developed high IV GvHD showed increasing CD4+CD134+ T cells up to 85% of the CD4+ T cells. Additionally, rapid increases in CD134+ T cells following antibody-based T cell therapy were associated with GvHD recurrence. In no cases was the percentage of CD134+ CD4+ T cells predictive of clinical GvHD. In this exploratory study, we have shown that CD134, although not predictive of the initial onset of GvHD, may be a useful tool for monitoring the response to early GvHD therapy and identification of patients at risk for reemergence of GvHD who may benefit from anti-T cell therapy. Cytometry (Comm. Clin. Cytometry) 38: 238-243, 1999.

Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons.

The efficiency of first-generation adenoviral vectors as gene delivery tools is often limited by the short duration of transgene expression, which can be related to immune responses and to toxic effects of viral proteins. In addition, readministration is usually ineffective unless the animals are immunocompromised or a different adenovirus serotype is used. Recently, adenoviral vectors devoid of all viral coding sequences (helper-dependent or gutless vectors) have been developed to avoid expression of viral proteins. In mice, liver-directed gene transfer with AdSTK109, a helper-dependent adenoviral (Ad) vector containing the human alpha(1)-antitrypsin (hAAT) gene, resulted in sustained expression for longer than 10 months with negligible toxicity to the liver. In the present report, we have examined the duration of expression of AdSTK109 in the liver of baboons and compared it to first-generation vectors expressing hAAT. Transgene expression was limited to approximately 3-5 months with the first-generation vectors. In contrast, administration of AdSTK109 resulted in transgene expression for longer than a year in two of three baboons. We have also investigated the feasibility of circumventing the humoral response to the virus by sequential administration of vectors of different serotypes. We found that the ineffectiveness of readministration due to the humoral response to an Ad5 first-generation vector was overcome by use of an Ad2-based vector expressing hAAT. These data suggest that long-term expression of transgenes should be possible by combining the reduced immunogenicity and toxicity of helper-dependent vectors with sequential delivery of vectors of different serotypes.

NSP4 elicits age-dependent diarrhea and Ca(2+)mediated I(-) influx into intestinal crypts of CF mice.

Homologous disruption of the murine gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to the loss of cAMP-mediated ion transport. Mice carrying this gene defect exhibit meconium ileus at birth and gastrointestinal plugging during the neonatal period, both contributing to high rates of mortality. We investigated whether infectious mammalian rotavirus, the recently characterized rotaviral enterotoxin protein NSP4, or its active NSP4(114-135) peptide, can overcome these gastrointestinal complications in CF (CFTR(m3Bay) null mutation) mice. All three agents elicited diarrhea when administered to wild-type (CFTR(+/+)), heterozygous (CFTR(+/-)), or homozygous (CFTR(-/-)) 7- to 14-day-old mouse pups but were ineffective when given to older mice. The diarrheal response was accompanied by non-age-dependent intracellular Ca(2+) mobilization within both small and large intestinal crypt epithelia. Significantly, NSP4 elicited cellular I(-) influx into intestinal epithelial cells from all three genotypes, whereas both carbachol and the cAMP-mobilizing agonist forskolin failed to evoke influx in the CFTR(-/-) background. This unique plasma membrane halide permeability pathway was age dependent, being observed only in mouse pup crypts, and was abolished by either the removal of bath Ca(2+) or the transport inhibitor DIDS. These findings indicate that NSP4 or its active peptide may induce diarrhea in neonatal mice through the activation of an age- and Ca(2+)-dependent plasma membrane anion permeability distinct from CFTR. Furthermore, these results highlight the potential for developing synthetic analogs of NSP4(114-135) to counteract chronic constipation/obstructive bowel syndrome in CF patients.

Toxicological comparison of E2a-deleted and first-generation adenoviral vectors expressing alpha1-antitrypsin after systemic delivery.

Second-generation adenoviral vectors, mutated in E2a, have been proposed to decrease host immune responses against transduced cells, reduce toxicity, and increase duration of expression as compared with first-generation vectors deleted only in E1. To test these hypotheses further, we have developed an E2a-deleted adenoviral vector expressing human alpha1-antitrypsin (hAAT). Toxicity of first-generation and E2a-deleted vectors, as determined by hematological indices, liver function tests, and histological analyses, was evaluated in C3H mice for 21 days after vector administration at increasing doses starting at 1 x 10(12) particles/kg. Both vectors induced dose-dependent abnormalities including transient thrombocytopenia, elevated ALT levels in serum, and increased hepatocyte proliferation followed by inflammation and then hypertrophy. Differences in the ratio of particles to plaque-forming units among vector preparations led to differences in hAAT expression at similar particle doses. There were no differences in toxicity between the two vectors when measured at matching levels of hAAT expression. However, the E2a-deleted vector was demonstrated to have slightly reduced hepatocyte toxicity at an intermediate particle dose. This suggests that hepatocyte toxicity is related primarily to viral entry and expression, rather than to the presence of noninfectious particles, and implies that vectors with complete elimination of viral gene expression, such as vectors with all viral coding sequences deleted, are likely to have substantial advantages in terms of safety and toxicity.

Immune responses to reporter proteins and high viral dose limit duration of expression with adenoviral vectors: comparison of E2a wild type and E2a deleted vectors.

Experiments designed to evaluate the effect of deletion of E2a on duration of expression using adenoviral vectors led to a series of observations regarding host responses to adenoviral vectors and reporter proteins. In studies using human alpha1-antitrypsin (hAAT) as a reporter gene, we found that the duration of expression is very brief for C3H/J and CBA/J mice but is prolonged for C57BL/6J mice, that disappearance of hAAT from the blood is correlated with the appearance of antibodies, and that immunization against hAAT can prevent appearance of the protein in the blood after administration of an adenoviral vector. Deletion of E2a in hAAT vectors did not prolong expession in C3H/J or CBA/J mice and did not shorten duration of expression in C57BL/6J mice. Using similar vectors expressing Escherichia coli beta-galactosidase (beta-Gal) in immunocompetent mice, short duration of expression with a beta-Gal reporter was remarkably different from the long expression with an identical vector expressing hAAT in C57BL/6J. In the case of vectors expressing hAAT, adenoviral sequences persisted in the liver, and inflammatory responses were minimal compared to vectors expressing beta-Gal, where adenoviral sequences disappeared from the liver concomitant with a prominent inflammatory response. The duration of expression of beta-Gal in hepatocytes was increased in transgenic mice expressing the reporter in keratinocytes, indicating that host immune responses to the reporter can limit duration of expression. Dosage studies indicated that persistence of expression of hAAT can be markedly decreased by administration of high doses of vector in a manner consistent with a nonimmune-mediated toxicity following injection. These experiments indicate that host responses to reporter genes rather than host responses to adenoviral proteins can be the primary determinant of duration of expression under many experimental conditions.