Chimerism analysis in sex-mismatched murine transplantation using quantitative real-time PCR.

Marine experimental stem cell transplantations require the accurate discrimination and quantification of donor cells from host cells. A Y-chromosome-specific, quantitative real-time PCR (kinetic PCR) protocol for blood-derived DNA was developed. The assay sensitivity was extremely high with accurate detection of only 10 pg (six copies of Y target DNA) in a variable background of female DNA background ranging from 2.5 to 50 ng. The dynamic range of the assay provided accurate results ranging from 2.2 x 10(-2)% to 100% of male DNA in female background. The kinetic PCR assay can be used in all mouse strains, and a sample size as low as 2.5 ng total DNA is sufficient for analysis. Therefore, kinetic PCR allows engraftment kinetic studies on repeated blood draws of individual animals with no need for sacrifice. Compared to conventional PCR, the assay is much simplified, as neither the accurate adjustment of sample DNA concentration nor a post-reaction analysis procedure is required. The procedure is simple, free of radioactivity, and permits a throughput of 500-600 reactions per day.

Effect of alcohol consumption on host release of interleukin-17 during pulmonary infection with Klebsiella pneumoniae.

BACKGROUND: A link between alcohol abuse and bacterial pneumonia has been recognized for centuries, but mechanisms to explain this relationship are unclarified. Interleukin-17 (IL-17) is a lymphocyte-derived cytokine that is part of the inflammatory cytokine cascade. Previous studies from our laboratory indicated that IL-17 is released in lung tissue in a murine model of bacterial pneumonia caused by Klebsiella pneumoniae. The effects of alcohol consumption on pulmonary release of IL-17 are unknown. METHODS: Mice were maintained on 20% ethanol in drinking water or on a control diet without alcohol. After 2 weeks, alcohol and control mice were challenged with intratracheal K. pneumoniae. Mice were followed for survival after bacterial challenge, neutrophil recruitment was assayed as myeloperoxidase, and IL-17 was measured in lung lavage fluid by enzyme-linked immunosorbent assay. In additional experiments, splenocytes from control mice were incubated with ethanol in vitro, and release of IL-17 was measured in culture supernatants. Finally, control and alcohol mice received intrapulmonary gene transfer of E-1-deleted adenovirus containing the murine IL-17 gene. These mice were then challenged with K. pneumoniae and followed for survival and neutrophil recruitment. RESULTS: In these studies, we demonstrate that a 2-week history of ethanol consumption in mice suppresses release of IL-17 into lung tissue, decreases neutrophil recruitment, and increases mortality from experimental K. pneumonia. In vitro experiments confirm a direct suppressive effect of ethanol on the release of IL-17 from splenocytes. In vivo administration of the IL-17 gene in an adenoviral vector to alcohol-consuming mice results in release of IL-17 into lavage fluid and normalizes neutrophil recruitment and mortality after bacterial challenge. CONCLUSIONS: The results of these experiments strongly implicate IL-17 as an important pathway for the immunosuppression associated with alcohol abuse and support gene therapeutic approaches to augment immune function in the alcoholic host or to treat infections associated with alcoholism.

Ischemia/reperfusion injury-mediated down-regulation of adenovirus-mediated gene expression in a rat heart transplantation model is inhibited by co-application of a TNFRp55-Ig chimeric construct.

E1-deleted adenoviral vectors are efficient vectors for somatic gene therapy. Recently, we have shown that intratracheal application of an adenoviral reporter construct leads to significant reporter gene expression in rat lungs within 24 h. In contrast, reporter gene expression in syngeneic rat heart transplants after adenovirus-mediated gene transfer was delayed. Since the adenovirus cannot replicate, down-regulation of the hCMV-IE promoter controlled reporter gene expression in initially infected cells by cytokines, which are released as a result of ischemia/reperfusion injury, might be involved. In order to investigate the role of proinflammatory cytokines, eg TNF-alpha in affecting hCMV-IE promoter-driven reporter gene expression, transient blockade of TNF-alpha was achieved by local co-application of an Ad-construct encoding for a soluble TNFRp55-Ig chimeric molecule in a syngeneic rat heart transplantation model. Co-application of the reporter construct together with the TNFRp55-Ig chimeric molecule significantly increased the early reporter gene expression after transplantation. Moreover, infiltration of inflammatory cells (T cells, macrophages, NK cells) and production of TNF-alpha in the transplant was markedly reduced. Our results indicate that: (1) proinflammatory cytokines are involved in down-regulation of reporter gene expression in ischemia/reperfusion injured tissues; and (2) inhibition of TNF-alpha might be a useful tool to increase early gene expression in gene therapy protocols, particularly in transplantation. Gene Therapy (2000) 7, 1238-1243.

Immunomodulation and adenoviral gene transfer to the lungs of nonhuman primates.

Previous data from our laboratory and others have demonstrated a critical role for the CD4+ T lymphocyte in in vivo immune responses to recombinant adenoviral vectors. In rodent models, this subset of T cells is required for T cell proliferation, subsequent cytotoxic T cell generation, and production of anti-adenoviral antibodies by B cells. Both depleting and nondepleting anti-CD4 antibodies can attenuate these immune responses to recombinant adenovirus. On the basis of these data, we hypothesized that a nondepleting CDR-engrafted anti-human CD4 antibody (OKT4A) with cross-reactivity to rhesus macaques would attenuate both T and B cell responses to intrapulmonary administration of recombinant adenovirus and permit prolonged reporter gene expression and permit secondary gene transfer. Juvenile rhesus macaques were treated with PBS or OKT4A antibody (10 mg/kg) daily beginning 1 day prior to and up to 11 days after gene transfer. OKT4A resulted in significant attenuation of lymphocyte recruitment into the lung, lymphocyte-proliferative responses to both adenovirus capsid proteins and transgene protein, and adenovirus-induced interferon-gamma elaboration in whole blood and hilar lymph nodes. However, OKT4A was ineffective in attenuating adenovirus-induced IL-4 production in whole blood or hilar lymph nodes, generating neutralizing anti-adenoviral antibodies, or permitting secondary gene transfer. As all the monkeys in this protocol had baseline-detectable anti-adenoviral antibodies by ELISA that were nonneutralizing, analogous to most patients with cystic fibrosis, we postulate that anti-CD4 did not block the proliferation of memory B cells. Moreover, these data suggest that for transient immunomodulation to be successful, strategies need to focus specifically on B cell activation independent of CD4+ T cell help.

IFN-gamma and CD8+ T cells restore host defenses against Pneumocystis carinii in mice depleted of CD4+ T cells.

Host defenses against infection are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes and defective cell-mediated immunity. Although recent advances in antiretroviral therapy can dramatically lower HIV viral load, blood CD4+ T lymphocytes are not restored to normal levels. Therefore, we investigated mechanisms of host defense other than those involving CD4+ T lymphocytes against a common HIV-related opportunistic infection, Pneumocystis carinii (PC) pneumonia. Using CD4-depleted mice, which are permissive for chronic PC infection, we show that up-regulation of murine IFN-gamma by gene transfer into the lung tissue results in clearance of PC from the lungs in the absence of CD4+ lymphocytes. This resolution of infection was associated with a >4-fold increase in recruited CD8+ T lymphocytes and NK cells into the lungs. The role of CD8+ T cells as effector cells in this model was further confirmed by a lack of an effect of IFN-gamma gene transfer in scid mice or mice depleted of both CD4+ and CD8+ T cells. Cytokine mRNA analysis revealed that recruited, lung-derived CD8+ T cells had greater expression of IFN-gamma message in animals treated with the IFN-gamma gene. These results indicate that CD8+ T cells are capable of clearing PC pneumonia in the absence of CD4+ T cells and that this host defense function of CD8+ T cells, as well as their cytokine repertoire, can be up-regulated through cytokine gene transfer.