Expression of Human Growth Hormone in Silkworm Larvae through RecombinantBombyx moriNuclear Polyhedrosis Virus

We have generated a recombinantBombyx morinuclear polyhedrosis virus, vBmhGH, harboring the full-length human growth hormone gene (2.4-kb genomic DNA, with four introns and the signal peptide sequences) under the control of the polyhedrin promoter. BmN cells in culture infected with the recombinant virus showed the presence of RNA corresponding to the authentic growth hormone mRNA as well as its incompletly processed precusor. Electrophoretic analysis and immunoprecipitation of proteins of recombinant virus-infected BmN cells revealed the presence of the growth hormone protein. Infection of silkworm larvae with vBmhGH led to the synthesis and efficient secretion of the protein into hemolymph. The recombinant human growth hormone was biologically active in a radioreceptor competition binding assay. The secreted protein was isolated and purified to homogeneity by a single step immunoaffinity chromatography, to a specific activity of 2.4 × 104U/mg. The recombinant hGH retained the immunological and biolological properties of the native peptide. We conclude that BmNPV vectors can be used successfully for expressing chromosomal genes harboring multiple introns.

Microbe-induced Innate Immune Responses in Human Dendritic Cells

Two types of antigen-presenting cells (APCs), macrophages and dendritic cells (DCs), function at the interface of innate and adaptive immunity. Through recognition of conserved microbial patterns, they are able to detect the invading pathogens. This leads to activation of signal transduction pathways that in turn induce gene expression of various molecules required for immune responses and eventually pathogen clearance. Cytokines are among the genes induced upon detection of microbes. They play an important role in regulating host immune responses during microbial infection. Chemotactic cytokines, chemokines, are involved in migratory events of immune cells. Cytokines also promote the differentiation of distinct T cell responses. Because of the multiple roles of cytokines in the immune system, the cytokine network needs to be tightly regulated. In this work, the induction of innate immune responses was studied using human primary macrophages or DCs as cell models. Salmonella enterica serovar Typhimurium served as a model for an intracellular bacterium, whereas Sendai virus was used in virus experiments. The starting point of this study was that DCs of mouse origin had recently been characterized as host cells for Salmonella. However, only little was known about the immune responses initiated in Salmonella-infected human DCs. Thus, cellular responses of macrophages and DCs, in particular the pattern of cytokine production, to Salmonella infection were compared. Salmonella-induced macrophages and DCs were found to produce multiple cytokines including interferon (IFN) -gamma, which is conventionally produced by T and natural killer (NK) cells. Both macrophages and DCs also promoted the intracellular survival of the bacterium. Phenotypic maturation of DCs as characterized by upregulation of costimulatory and human leukocyte antigen (HLA) molecules, and production of CCL19 chemokine, were also detected upon infection with Salmonella. Another focus of this PhD work was to unravel the regulatory events controlling the expression of cytokine genes encoding for CCL19 and type III IFNs, which are central to DC biology. We found that the promoters of CCL19 and type III IFNs contain similar regulatory elements that bind nuclear factor kappaB (NF-kappaB) and interferon regulatory factors (IRFs), which could mediate transcriptional activation of the genes. The regulation of type III IFNs in virus infection resembled that of type I IFNs a cytokine class traditionally regarded as antiviral. The induction of type I and type III IFNs was also observed in response to bacterial infection. Taken together, this work identifies new details about the interaction of Salmonella with its phagocytic host cells of human origin. In addition, studies provide information on the regulatory events controlling the expression of CCL19 and the most recently identified IFN family genes, type III IFN genes.

Stability of assembled epitopes on human chorionic gonadotropin to covalent modifications

A measure of stability of a given epitope is an important parameter in the exploration of the utility of a desired MAb. It defines the conditions necessary for using MAbs as an investigative tool in several research methodologies and therapeutic protocols. Despite these obvious interests the lack of simple and rapid assay systems for quantitating MAb-Ag interactions has largely hampered these studies. A single step MAb-Solid Phase Radioimmunoassay (SS-SPRIA), is described which eliminates errors that may arise with multistep sandwich assays. SS-SPRIA has been used to demonstrate the differential stability of the assembled epitopes on gonadotropins. Differential stability towards specific reagents can be exploited to identify aminoacid residues at the epitopic site. Inactivation of an epitopic region is indicative of the presence of the group modified, provided conformational relaxations are not induced due to modifications at distant sites. Here we provide evidence to validate these conclusions.

Distinct subpopulations of gamma delta T cells are present in normal and tumor-bearing human liver

Gamma delta T cells are thought to mediate immune responses at epithelial surfaces. We have quantified and characterized hepatic and peripheral blood gamma delta T cells from 11 normal and 13 unresolved tumor-bearing human liver specimens. gamma delta T cells are enriched in normal liver (6.6% of T cells) relative to matched blood (0.9%; P = 0.008). The majority express CD4(-)CD8(-) phenotypes and many express CD56 and/or CD161. In vitro, hepatic gamma delta T cells can be induced to kill tumor cell lines and release interferon-gamma, tumor necrosis factor-alpha, interleukin-2 and interleukin-4. Analysis of V gamma and V delta chain usage indicated that V delta 3(+) cells are expanded in normal livers (21.2% of gamma delta T cells) compared to blood (0.5%; P = 0.001). Tumor-bearing livers had significant expansions and depletions of gamma delta T cell subsets but normal cytolytic activity. This study identifies novel populations of liver T cells that may play a role in immunity against tumors.