CD90 Expression on human primary cells and elimination of contaminating fibroblasts from cell cultures.

Cluster Differentiation 90 (CD90) is a cell surface glycoprotein originally identified on mouse thymocytes. Although CD90 has been identified on a variety of stem cells and at varying levels in non-lymphoid tissues such as on fibroblasts, brain cells, and activated endothelial cells, the knowledge about the levels of CD90 expression on different cell types, including human primary cells, is limited. The goal of this study was to identify CD90 as a human primary cell biomarker and to develop an efficient and reliable method for eliminating unwanted or contaminating fibroblasts from human primary cell cultures suitable for research pursuant to cell based therapy technologies.

Mosquito feeding patterns and natural infection of vertebrates with Ross River and Barmah Forest viruses in Brisbane, Australia.

Host feeding patterns of mosquitoes were assessed through the identification of 865 blood meals collected from Brisbane during 2000-2001. Under natural conditions, mosquito feeding (including that of Culex annulirostris, Aedes vigilax, and Aedes notoscriptus) was primarily on dogs (37.4%), but also on birds (18.4%), horses (16.8%), brushtail possums (13.3%), humans (11.6%), and cats, flying foxes, and macropods, depending on site. From 1997 to 1999, sera (N=1706) were collected from dogs, cats, horses, flying foxes, and brushtail possums in the Brisbane area and were analyzed by microneutralization assay for antibodies to Ross River virus (RRV) and Barmah Forest virus (BFV). For RRV, all vertebrate species tested had been naturally infected, and seroprevalence varied from 10.5% to 25.5%, whereas for BFV, rates varied between 0% and 11.3%. Brushtail possums were often infected in the field, with 17.6% and 10.7% of wild individuals having antibodies to RRV and BFV, respectively. Horses and flying foxes also had a relatively high prevalence of antibodies to RRV. This study, therefore, provides data to indicate that brushtail possums play a role in the urban transmission of RRV in Brisbane and that horses, when they occur, also fill the same role.

Activation-induced cell death of aggressive histology lymphomas by CD40 stimulation: induction of bax.

CD40 is present on both normal and neoplastic B-lineage cells. CD40 stimulation of normal B cells has been shown to promote normal growth and differentiation, whereas aggressive histology B lymphomas are growth inhibited. The inhibition of neoplastic B-cell growth is believed to occur via activation-induced cell death in which stimuli that typically promote the growth of normal cells prevent the growth of their neoplastic counterparts. We show here that CD40 stimulation using either a soluble recombinant human CD40 ligand (srhCD40L) or anti-CD40 monoclonal antibody resulted in apoptosis of human Burkitt lymphoma cell lines. Additional studies examining the mechanism of CD40-mediated death revealed an increase in bax messenger RNA with a subsequent increase in Bax protein in the mitochondria of the treated cells. In vitro exposure of the cells to bax antisense oligonucleotides resulted in a significant decline in Bax protein levels and partial protection from CD40-mediated death, indicating that induction of Bax was at least one mechanism underlying this inhibitory effect of CD40 stimulation on lymphomas. When immunodeficient mice bearing Burkitt lymphoma were treated with srhCD40L, significant increases in survival were observed indicating a direct antitumor effect as a result of CD40 stimulation in vivo. Overall, these results demonstrate that CD40 ligation of aggressive histology B-lymphoma cells results in inhibition both in vitro and in vivo and thus may be of potential clinical use in their treatment.

Exogenous DNA expression in eukaryotic cells following microinjection.

Microinjection of nucleic acids, DNA, RNA, proteins, and any soluble material into living eukaryotic cells makes it possible to design experiments focused on single cells. In contrast facilitated transfer protocols requires hundreds of thousands of cells from which the expressed gene or intracellular effect must be detected within the culture. In addition to the immediate observable nature of the expressed product and intracellular reaction, microinjection bypasses the uptake toxicity associated with facilitated transfer of foreign material into cultured cells. The direct injection of material into the nucleus or cytoplasm allows the number of treated cells to be monitored and expression efficiencies to be observed directly. Microinjection of a hundred cells grown on small glass coverslips and subsequently counted for expression of the foreign material determines expression efficiency as a percentage of cells injected. The efficiency is based on detection of the foreign inserted gene product and does not control for relative promoter efficiency between constructs. The purpose is not to compare two constructs to each other but to monitor dual expression. The creation of marker fluorescent proteins, such as the green fluorescent protein (GFP) in the same expression plasmid with a test gene allows the immediate observation of the GFP injected cells and within the same cells the positive or negative expression of the test gene. Expression of a foreign gene, such as SV40 T antigen cloned into an expression vector can be detected four hours after microinjection of the DNA. Fusing GFP into the same expression region of the T coding sequence labels T-GFP as a fusion protein with characteristic T immunological staining nuclear patterns but allows the cells to be studied without fixation through sequential periods of observation. The direct nature of microinjection allows comparison of gene expression in a variety of cells and the determination of the number of cells expressing the exogenous material in relationship to the number of cells injected.