Cytoplasm Of Cultured Cells Research Articles

Intracellular Salmonella delivery of an exogenous immunization antigen refocuses CD8 T cells against cancer cells, eliminates pancreatic tumors and forms antitumor immunity.

Immunotherapies have shown great promise, but are not effective for all tumors types and are effective in less than 3% of patients with pancreatic ductal adenocarcinomas (PDAC). To make an immune treatment that is effective for more cancer patients and those with PDAC specifically, we genetically engineered Salmonella to deliver exogenous antigens directly into the cytoplasm of tumor cells. We hypothesized that intracellular delivery of an exogenous immunization antigen would activate antigen-specific CD8 T cells and reduce tumors in immunized mice. To test this hypothesis, we administered intracellular delivering (ID) Salmonella that deliver ovalbumin as a model antigen into tumor-bearing, ovalbumin-vaccinated mice. ID Salmonella delivers antigens by autonomously lysing in cells after the induction of cell invasion. We showed that the delivered ovalbumin disperses throughout the cytoplasm of cells in culture and in tumors. This delivery into the cytoplasm is essential for antigen cross-presentation. We showed that co-culture of ovalbumin-recipient cancer cells with ovalbumin-specific CD8 T cells triggered a cytotoxic T cell response. After the adoptive transfer of OT-I CD8 T cells, intracellular delivery of ovalbumin reduced tumor growth and eliminated tumors. This effect was dependent on the presence of the ovalbumin-specific T cells. Following vaccination with the exogenous antigen in mice, intracellular delivery of the antigen cleared 43% of established KPC pancreatic tumors, increased survival, and prevented tumor re-implantation. This response in the immunosuppressive KPC model demonstrates the potential to treat tumors that do not respond to checkpoint inhibitors, and the response to re-challenge indicates that new immunity was established against intrinsic tumor antigens. In the clinic, ID Salmonella could be used to deliver a protein antigen from a childhood immunization to refocus pre-existing T cell immunity against tumors. As an off-the-shelf immunotherapy, this bacterial system has the potential to be effective in a broad range of cancer patients.

Intracellular delivery of drugs by chitosan-based multi-liposomal complexes

Positively charged linear chitosan molecules were cross-linked with sulfate-anions to form chitosan nanoparticles which were used as a scaffold of negatively charged cardiolipin/egg lecithin liposomes loaded with doxorubicin (DOX). Thus formed multi-liposomal complexes (MLCs) containing 55 liposomes/chitosan and bearing a slight positive net charge effectively transmitted DOX into the cytoplasm of cells in culture. The efficiency of DOX delivery increased 4–5-fold upon drug incorporation in MLCs. Inside the cells, the penetrated MLCs released DOX thus enabling its accumulation in the nuclei and interaction with its intracellular target DNA leading to a decrease in cell survival. The effects were demonstrated on 3T3 line of mouse fibroblasts, drug sensitive tumor MCF-7 cells and drug resistant human ovarian carcinoma OVCAR-8 cells (formerly called MCF-7/ADR). Thus, MLC particles can be used for effective delivery of payloads in cells of different origin.

Towards a novel influenza vaccine: engineering of hemagglutinin on a platform of adenovirus dodecahedron

BackgroundThe production process for the current influenza vaccine takes about 6 months and its antigenic composition must be modified annually. In the attempt towards developing influenza vaccine production that would be faster, safer and cheaper we engineered an influenza vaccine in which multiple copies of hemagglutinin (HA) would be delivered by a vector, adenovirus dodecahedron (Ad Dd). Dd is a virus-like particle, formed by assembly of twelve copies of pentameric penton base (Pb) proteins responsible for virus penetration. In order to attach HA to the vector, an adaptor containing WW domains was used. The WW domain is a linear peptide fragment identified as a partner of proline-proline-x-tyrosine (PPxY) motif present at the N-terminal extremity of the Pb protein, which is a building block of Dd. That tandem of three WW domains in fusion with the protein of interest enables interaction with Dd and efficient translocation to the cytoplasm of cells in culture.ResultsSince HA is an oligomeric protein with complicated processing, we prepared six different constructs of HA (A/swan/Poland/467/2006(H5N1)) in fusion with the WW adaptor. Herein we report baculovirus expression and functional analysis of six HA-WW variants. The best behaving variant was successfully delivered into human cells in vitro.ConclusionsEngineering of a soluble complex of HA with Dd, a virus-like particle that serves as a vector, an adjuvant and as a multivalent presentation platform, is an important step toward a novel influenza vaccine.

Inducible nitric oxide synthase in the epithelial epididymal cells of the rat.

The expression of mRNA for inducible nitric oxide synthase (iNOS) in rat epithelial cells of epididymis was investigated with reverse transcription followed by polymerase chain reaction. Immunocytochemical reaction for iNOS was performed to confirm the enzyme's localization in the epididymal epithelium. Additionally, an indirect spectrophotometric method for nitric oxide (NO) determination was applied for measurement of nitrite production by cultured epididymal epithelial cells. Inducible NOS mRNA was detected in freshly isolated epithelial cells, in cultured cells without stimulation as well as in cultured cells after stimulation by lipopolysaccharide and interferon-gamma. Inducible NOS immunoreactivity was observed in the apical part of epithelial cells of epididymal sections and in the cytoplasm of cells in culture. Release of nitrite was observed in vitro in both the unstimulated and stimulated cells of caput (1.44+/-0.94 v. 4.37+/-2.42 microM) and cauda (0.69+/-1.21 v. 5.21+/-2.76 microM) epididymis (P < 0.001). To the best of our knowledge, this is the first study to demonstrate iNOS in the epididymal epithelial cells of the rat. Nitric oxide released by epididymal epithelial cells may act on cells and tissues located nearby. The results may help explain epididymal function: sperm storage, passage and maturation. Excessive epididymal NO production may also play a role in the inflammatory infertility of the male.

28] Microinjection of antibodies

This chapter describes the use of the microinjection technique as a way to deliver immunopurified antibodies into the cytoplasm of cells in culture. The success in the microinjection of antibodies for neutralization purposes relies mainly on the quality of the antibodies used. Polyclonal antibodies have the advantage of recognizing different epitopes on a same target protein and therefore should have stronger neutralizing activities. By using glass needles, miscellaneous molecules like DNA, RNA, and protein can be injected into the cytoplasm or into the nucleus of cells in culture. This procedure, therefore, allows the investigation of the biological role of different molecules directly in their natural cellular environment. Intracellular microinjection of purified antibodies against different target proteins has been successfully used to inhibit the function of these proteins in vivo. Using this technique, useful information has been obtained about the role of several proteins in different biological situations. This approach can be used to neutralize the regulatory activities of the Fos and Jun proteins in an effort to elucidate the role of these transcription factors in the proliferation of fibroblasts. The requirement of Fos and Jun proteins for cell cycle progression during different growth conditions by monitoring DNA synthesis of the injected cells has been investigated.

Mobile dispersed genetic element MDGI of Drosophila melanogaster: transcription pattern

Virtually the whole region of the mobile dispersed genetic element mdg1 including the direct repeats that frame mdg1 /1/ is transcribed. The end of the transcriptional unit is located within the direct repeat. Although the transcription of mdg1 is symmetric, one direction is transcribed preferentially and is a full-length transcript represented by approximately 29S poly(A)+RNA. Besides it, approximately 15S poly(A)+RNA is present in the cytoplasm of culture cells which corresponds to the 3'-part of 29S RNA and possibly to a small region near the 5'-end of the latter. The major fraction of mdg1 transcripts is present in the fraction of free cytoplasmic RNP particles while only 10-20% are recovered in polysomes. In this respect, mdg1 is similar to mdg3 and quite different from two single-copy genes transcripts of which are almost completely recovered in polysomes. The relationship between mobile dispersed genetic elements and endogeneous viral genomes is discussed.

Uptake of proteins by red blood cells

During hypotonic hemolysis red cells can take up 125I-myoglobin and 125I-immunoglobulin G. Cells which contain these proteins have distinctive cell morphology and are called gray ghosts. The association of protein with gray ghosts is fairly stable: these cells retain half of the proteins after 3 days. Passive diffusion of protein into the internal cell volume is the most plausible mechanism for uptake, and several lines of evidence indicate that the loaded proteins are freely diffusable within the red cells. Bacteriophage T4 is not taken up during hemolysis so uptake through large gaps in the red cell membrane with subsequent resealing seems unlikely. If an efficient procedure for fusing loaded gray ghosts to culture cells can be devised, it will be possible to introduce selected macromolecules into the cytoplasm of culture cells quite easily.