Sivadasan Kanangat

Enhancement of immune response to naked DNA vaccine by immunization with transfected dendritic cells.

Immunization with plasmid DNA encoding various proteins promises to be a valuable vaccine approach especially if its immunogenicity could be optimized. In this study we show that the intramuscular delivery in dendritic cells (DC) of naked plasmid DNA encoding two proteins of herpes simplex virus (HSV) leads to the induction of significantly enhanced levels of resistance to viral challenge. Whereas DC transfected in vitro with DNA induced enhanced immunity, similarly transfected macrophage (Mɸ) populations lacked immunogenicity even though plasmid expression occurred in vitro. The enhanced immunity induced by DC‐delivered DNA appeared to be associated mainly with an increased Thl CD4+ T cell response. Our results add evidence that DC are the essential antigen‐presenting cell types involved in immune responses to intramuscularly administered DNA vaccines. J. Leukoc. Biol. 61: 125–132; 1997.

Limitations and modifications of quantitative polymerase chain reaction. Application to measurement of multiple mRNAs present in small amounts of sample RNA.

Quantification of mRNA by polymerase chain reaction (PCR) is performed by using a competitor DNA standard in the PCR or by employing an internal standard RNA following simultaneous reverse transcription (RT) with the sample RNA. The latter approach is more reliable since it accounts for variations in both the RT and PCR steps. However, we describe in this report that at times the internal standard RNA competes with the target mRNA in the PCR when both are present in disproportionate concentrations in the initial simultaneous RT reaction. To overcome the competition in the PCR, multiple simultaneous RT reactions with the sample RTA and the internal standard RNA are required. Such procedures make the approach time consuming and restrict the use of internal standard RNA for quantification of multiple mRNAs present in small amounts of sample RNA. These limitations are circumvented by the competitor DNA standard approach and mRNA levels can be quantified by calculating the RT efficiency. We illustrate the situations by quantifying the levels of IL-2, IL-4, IL-5, IFN-gamma, and beta-actin mRNAs in mitogen stimulated murine T cells using a multiple mRNA specific internal standard.

Expression of cytokine mRNA in murine splenic dendritic cells and better induction of T cell-derived cytokines by dendritic cells than by macrophages during in vitro costimulation assay using specific antigens.

Among antigen‐presenting cells dendritic cells (DC) have the unique ability to generate primary T cell response. The reasons for the superior inductive property of DC still remain obscure. The explanations offered include higher expression of CD80, MHCII, and ICAMI on DC surface which allows effective cluster formation with T cells. It is also possible that additional cellular characteristics of DC, i.e., their ability to release critical mediators involved in the induction of effective immune response, are important. We examined the ability of DC to express IL‐1, IL‐6, and IL‐12 using the highly sensitive reverse transcription‐quantitative polymerase chain reaction. Our data show that highly purified (97–99% pure) murine splenic DC were capable of expressing IL‐1, IL‐6 and IL‐12 mRNA upon stimulation with lipopolysaccharide. We also compared the ability of DC and macrophages to induce T cell‐derived cytokines IL‐2 and IFN‐γ in an in vitro antigen‐specific costimulation assay. In naive T cells stimulated with antigen presented via DC or macrophages, the levels of mRNA for IL‐2 and IFN‐γ were 2‐ to 4‐fold higher when cells were stimulated with DC. Overall, our data add additional support to the description of DC as superior antigen‐presenting cells for the activation of naive T cells. J. Leukoc. Biol. 57: 310–316; 1995.

Cytokine expression in the gut associated lymphoid tissue after oral administration of attenuated Salmonella vaccine strains

The use of attenuated Salmonella vaccine vectors as delivery vehicles for heterologous antigens has been extensive. The majority of work has analyzed the specific immune responses to the recombinant antigen in question. In addition, most analysis has been performed on animals maintained with sterile food, water, and bedding. This report defines the Salmonella specific cytokine responses in the gut associated lymphoid tissue after oral immunization with two highly publicized attenuated Salmonella carrier strains in animals maintained with nonsterile food, water, and bedding. Increases in expression of both IL-4 and IFN-gamma occur following immunization with either Salmonella construct. In addition, other cytokines (IL-6, IL-7, IL-12) increase at similar levels in either BRD509 or KR1 dosed animals. Proinflammatory cytokines IL-1 and TNF-alpha are also present but unchanged at early time points (6, 24, and 72 hours), increasing only after 7 days postimmunization. These data have implications in the generation of immunity to recombinant antigens since the response to Salmonella will dictate the direction of responses in terms of CD4 T cell activation.