Human IL-12 p40 as a reporter gene for high-throughput screening of engineered mouse embryonic stem cells.

BACKGROUND: Establishing a suitable level of exogenous gene expression in mammalian cells in general, and embryonic stem (ES) cells in particular, is an important aspect of understanding pathways of cell differentiation, signal transduction and cell physiology. Despite its importance, this process remains challenging because of the poor correlation between the presence of introduced exogenous DNA and its transcription. Consequently, many transfected cells must be screened to identify those with an appropriate level of expression. To improve the screening process, we investigated the utility of the human interleukin 12 (IL-12) p40 cDNA as a reporter gene for studies of mammalian gene expression and for high-throughput screening of engineered mouse embryonic stem cells. RESULTS: A series of expression plasmids were used to study the utility of IL-12 p40 as an accurate reporter of gene activity. These studies included a characterization of the IL-12 p40 expression system in terms of: (i) a time course of IL-12 p40 accumulation in the medium of transfected cells; (ii) the dose-response relationship between the input DNA and IL-12 p40 mRNA levels and IL-12 p40 protein secretion; (iii) the utility of IL-12 p40 as a reporter gene for analyzing the activity of cis-acting genetic elements; (iv) expression of the IL-12 p40 reporter protein driven by an IRES element in a bicistronic mRNA; (v) utility of IL-12 p40 as a reporter gene in a high-throughput screening strategy to identify successful transformed mouse embryonic stem cells; (vi) demonstration of pluripotency of IL-12 p40 expressing ES cells in vitro and in vivo; and (vii) germline transmission of the IL-12 p40 reporter gene. CONCLUSION: IL-12 p40 showed several advantages as a reporter gene in terms of sensitivity and ease of the detection procedure. The IL-12 p40 assay was rapid and simple, in as much as the reporter protein secreted from the transfected cells was accurately measured by ELISA using a small aliquot of the culture medium. Remarkably, expression of Il-12 p40 does not affect the pluripotency of mouse ES cells. To our knowledge, human IL-12 p40 is the first secreted reporter protein suitable for high-throughput screening of mouse ES cells. In comparison to other secreted reporters, such as the widely used alkaline phosphatase (SEAP) reporter, the IL-12 p40 reporter system offers other real advantages.

Efficacy of topical immunoglobulins against experimental adenoviral ocular infection.

PURPOSE: Presently, there is no U.S. Federal Drug Administration (FDA)-approved antiviral therapy for the treatment of adenoviral (Ad) ocular infections. The goal of the present study was to determine the antiviral efficacy of human immunoglobulin (Ig), a preparation of highly purified and concentrated immunoglobulin (IgG) antibodies isolated from a large pool of human plasma donors, in vitro and on acute Ad replication in the Ad5 New Zealand White (NZW) rabbit ocular model. METHODS: The antiviral activity of human Ig against multiple wild-type and human ocular isolates of adenovirus serotypes was investigated in vitro by using neutralizing assays in different human epithelial cell lines. In vivo bilateral topical ocular toxicity and antiviral efficacy were evaluated with established Ad5/NZW rabbit ocular models. In vivo Ig antiviral results were compared with those obtained with topical 0.5% cidofovir and saline. RESULTS: In three different epithelial cell lines,

Effects of a SARS-associated coronavirus vaccine in monkeys.

The causative agent of severe acute respiratory syndrome (SARS) has been identified as a new type of coronavirus. Here, we have investigated the ability of adenoviral delivery of codon-optimised SARS-CoV strain Urbani structural antigens spike protein S1 fragment, membrane protein, and nucleocapsid protein to induce virus-specific broad immunity in rhesus macaques. We immunised rhesus macaques intramuscularly with a combination of the three Ad5-SARS-CoV vectors or a control vector and gave a booster vaccination on day 28. The vaccinated animals all had antibody responses against spike protein S1 fragment and T-cell responses against the nucleocapsid protein. All vaccinated animals showed strong neutralising antibody responses to SARS-CoV infection in vitro. These results show that an adenoviral-based vaccine can induce strong SARS-CoV-specific immune responses in the monkey, and hold promise for development of a protective vaccine against the SARS causal agent.

Hemozoin inhibition and control of clinical malaria.

Malaria has a negative impact on health and social and economic life of residents of endemic countries. The ultimate goals of designing new treatment for malaria are to prevent clinical infection, reduce morbidity, and decrease mortality. There are great advances in the understanding of the parasite-host interaction through studies by various scientists. In some of these studies, attempts were made to evaluate the roles of malaria pigment or toxins in the pathogenesis of malaria. Hemozoin is a key metabolite associated with severe malaria anemia (SMA), immunosuppression, and cytokine dysfunction. Targeting of this pigment may be necessary in the design of new therapeutic products against malaria. In this review, the roles of hemozoin in the morbidity and mortality of malaria are highlighted as an essential target in the quest for effective control of clinical malaria.

Evaluation of rubella immunity in a community prenatal clinic.

Since the introduction of the Rubella vaccine in 1969, prevalence of congenital Rubella syndrome (CRS) has greatly declined in the United States. However, reports of sporadic adult cases of the disease and frequent identification of non-Rubella immune (NRI) women in prenatal units may result in outbreak of CRS in susceptible communities. Identifying populations with high rates of NRI will assist in evidence-based public health intervention that may prevent epidemic of CRS in the United States. Method. This is a retrospective, cross-sectional study involving chart audit of Rubella screening results of 642 women who attended a high-risk prenatal care at a northwestern Iowa clinic between January 1 and December 31, 2007. Results. NRI was found in 6.9% of the study population. The highest prevalence rate of 10.2% was found among adolescents. NRI was highest among Native American women at 17.3%, compared to Whites 7.3%, African Americans 5.9%, and Hispanics 4.6%. Multivariate analysis demonstrated that Native Americans were 2.5 times more likely to be NRI compared to Whites (OR 2.7; 95% CI: 1.1, 6.6). Conclusion. This study demonstrated higher rate of non-Rubella immunity among adolescent pregnant women and supports Rubella booster immunization for all non-pregnant teenage women. The observed high rate of NRI among Native Americans may require further studies and evaluation of Rubella vaccination programs in tribal communities.

Broad cellular immunity with robust memory responses to simian immunodeficiency virus following serial vaccination with adenovirus 5- and 35-based vectors.

Adenovirus serotype 35 (Ad35) is a promising vaccine platform for human immunodeficiency virus (HIV) infection and emerging infectious diseases as it is uncommon in humans worldwide and is distinct from Ad5, the major vaccine serotype for which many individuals have pre-existing immunity. The immunogenicity of a first-generation, replication-competent Ad35-based vaccine was tested in the simian immunodeficiency virus (SIV) rhesus macaque model by evaluating its capacity to boost immunity generated by Ad5-based vectors. A series of four immunizations with replication-defective Ad5 vectors expressing SIVmac239 gag induced high-frequency responses mediated by both CD8(+) and CD4(+) T cells directed against several epitopes. Ad5-specific neutralizing antibody responses that did not neutralize Ad35 were rapidly induced but waned over time. Subsequent immunization with Ad5-based vectors was minimally effective, whereas immunization with Ad35-based vectors generated a strong increase in the frequency of Gag-specific T cells with specificities that were unchanged. While this boosting response was relatively transient, challenge with the distinct pathogenic isolate SIV/DeltaB670 generated robust and selective recall responses to Gag with similar specificities as induced by vaccination that were elevated for 25 weeks relative to controls. Vaccination had measurable albeit minor effects on virus load. Unexpectedly, regional hypervariability within the Gag sequence of SIV/DeltaB670 was associated with mutation of the conserved CD8(+) T-cell epitope CM9 without concurrent flanking mutations and in the absence of immune pressure. These findings support the further development of Ad35 as a vaccine vector, and promote vaccine regimens that utilize serial administration of heterologous adenoviruses.

Prevalence of neutralizing antibodies to adenoviral serotypes 5 and 35 in the adult populations of The Gambia, South Africa, and the United States.

One of the major limitations of the use of adenoviruses as gene therapy vectors is the existence of preformed immunity in various populations. Recent studies have linked failure of adenoviral gene therapy trials to the presence of antiadenoviral neutralizing antibodies (NAb). Understanding the distribution and specificity of such antibodies will assist in the design of successful recombinant adenoviral gene therapies and vaccines. To assess the prevalence of NAb to adenovirus serotypes 5 and 35 (Ad5 and Ad35), we analyzed serum samples from adult immunocompetent individuals living in The Gambia, South Africa, and the United States by using a neutralization assay. Serum samples were incubated with A549 lung carcinoma cells and adenoviruses encoding enhanced green or yellow fluorescent proteins; results were analyzed by fluorescence microscopy and flow cytometry. Using this technique, we found a high prevalence of NAb against Ad5 in Gambian, South African, and U.S. subjects at both low and high titers. Conversely, all subjects displayed a low prevalence of NAb to Ad35; when present, anti-Ad35 NAb were seen at low titers. Because of the ability of adenoviruses to elicit systemic and mucosal immune responses, Ad35 with its low NAb prevalence appears to be an attractive candidate vector for gene therapy applications.