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Gene Ther. 2003 May;10(9):717-24.
In vivo plasmid DNA electroporation generates exceptionally high levels of epitope-specific CD8+ T-cell responses.

Paster W, Zehetner M, Kalat M, Schuller S, Schweighoffer T.

Department of NBE Discovery, Boehringer Ingelheim Austria, Vienna, Austria. wolfgang.pastenivie.ac.at

Based on observations that DBA/2 mice develop a highly specific response towards an HLA-Cw3-derived epitope, consisting entirely of CD8+CD62L-Vbeta10+ cells, we have established an in vivo mouse model for screening a variety of immunization approaches. Responder cells were readily detectable in small samples of the peripheral blood using three-color FACS analysis. This permitted multiple, sequential determination of CD8+ T-cell responses in living animals at a very high degree of precision. In vivo electroporation delivery of expression construct plasmids, outclassed the other approaches tested. Dominant, specific responses were induced already upon a single administration. Both the peak and the longevity of the response resembled those that are generated by the most active viral infections. The induced CTLs rejected epitope-bearing tumor cells in vivo and released interferon-gamma upon stimulation with the correct MHC::peptide combination in vitro. The potent in vivo response was not influenced by known modulators of the innate immune system, such as CpG DNA and LPS content. In vivo electroporation thus deserves consideration in the future in antitumor and antiviral immunization approaches, where CD8+ T cells play a predominant role.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12704410&dopt=Abstract

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We constructed a live recombinant vaccinia virus vaccine candidate containing a synthesised hybrid gene termed 'HGFSP' encoding circumsporozoite protein (CSP), major merozoite surface antigen-1(MSA1), major merozoite surface antigen-2 (MSA2), and ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum, interleukin-1 (IL-1) and tetanus toxin (TT) epitopes. Anti-recombinant vaccinia virus rabbit sera and IgG were tested in inhibition experiments in vitro. Results showed that the recombinant vaccinia virus had some capability to inhibit the growth of P. falciparum in vitro. The sera of rabbits, rats, and mice immunised with recombinant virus showed obvious IL-2 activity 4-6 weeks after immunisation. The interferon (IFN) level of sera from these animals 6 weeks after immunisation was significantly higher than before immunisation. These results indicate that the recombinant vaccinia virus can stimulate cell mediated responses (Th1 cell response) in immunised animals, and has the capability to inhibit multiplication of in vitro cultured P. falciparum. Thus this recombinant vaccinia virus is an appropriate vaccine candidate for further evaluation in Aotus monkey or human clinical trails.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11165271&dopt=Abstract

Neurosci Lett. 2001 Feb 23;299(3):205-8.
Identification of protein kinase C isoforms involved in interferon-gamma-induced expression of inducible nitric oxide synthase in murine BV2 microglia.

Kang J, Yang M, Jou I, Joe E.

Department of Pharmacology, Ajou University School of Medicine, 442-721, Suwon, South Korea.

Microglia are major inflammatory cells of the brain. It has been known that interferon-gamma (IFN-gamma) induces nitric oxide (NO)/inducible nitric oxide synthase (iNOS) in microglia, and that protein kinase C (PKC) mediates the action of IFN-gamma. In this study, we investigated isoforms of PKC that are involved in IFN-gamma-induced activation of microglia using BV2 murine microglial cells. NO release/iNOS expression in IFN-gamma -treated BV2 cells was reduced in the presence of PKC inhibitors (Go 6976 and BIM), and by long-term pre-treatment (48 h) of cells with phorbol-12-myristate-13-acetate (PMA) or thymeleatoxin. PMA depleted alpha, beta, delta, and epsilon isoforms, and thymeleatoxin depleted alpha, beta, and epsilon isoforms although gamma, eta, iota, lambda, theta, mu, and zeta were also detected in these cells. Furthermore, IFN-gamma phosphorylated alpha and epsilon on their tyrosine residues. These results suggested that alpha and epsilon could be the major PKC isoforms involved in signaling pathways of IFN-gamma to induce NO/iNOS expression in BV2 microglia.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11165771&dopt=Abstract

Free Radic Biol Med. 2001 Feb 1;30(3):268-76.
Comparison of control of Listeria by nitric oxide redox chemistry from murine macrophages and NO donors: insights into listeriocidal activity of oxidative and nitrosative stress.

Ogawa R, Pacelli R, Espey MG, Miranda KM, Friedman N, Kim SM, Cox G, Mitchell JB, Wink DA, Russo A.

Radiation Biology Branch, NIH/National Cancer Institute, Building 10, Room B3-B69, Bethesda, MD 20892, USA.

The physiological function of nitric oxide (NO) in the defense against pathogens is multifaceted. The exact chemistry by which NO combats intracellular pathogens such as Listeria monocytogenes is yet unresolved. We examined the effects of NO exposure, either delivered by NO donors or generated in situ within ANA-1 murine macrophages, on L. monocytogenes growth. Production of NO by the two NONOate compounds PAPA/NO (NH2(C3H6)(N[N(O)NO]C3H7) and DEA/NO (Na(C2H5)2N[N(O)NO]) resulted in L. monocytogenes cytostasis with minimal cytotoxicity. Reactive oxygen species generated from xanthine oxidase/hypoxanthine were neither bactericidal nor cytostatic and did not alter the action of NO. L. monocytogenes growth was also suppressed upon internalization into ANA-1 murine macrophages primed with interferon-gamma (INF-gamma) + tumor necrosis factor-alpha (TNF-alpha or INF-gamma + lipid polysaccharide (LPS). Growth suppression correlated with nitrite formation and nitrosation of 2,3-diaminonaphthalene elicited by stimulated murine macrophages. This nitrosative chemistry was not dependent upon nor mediated by interaction with reactive oxygen species (ROS), but resulted solely from NO and intermediates related to nitrosative stress. The role of nitrosation in controlling L. monocytogenes was further examined by monitoring the effects of exposure to NO on an important virulence factor, Listeriolysin O, which was inhibited under nitrosative conditions. These results suggest that nitrosative stress mediated by macrophages is an important component of the immunological arsenal in controlling L. monocytogenes infections.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11165873&dopt=Abstract

Nutrition. 2001 Jan;17(1):41-5.
The influence of restricted calorie intake on peritoneal macrophage function.

Stapleton PP, Fujita J, Murphy EM, Naama HA, Daly JM.

Department of Surgery, New York-Presbyterian Hospital, Weill Medical College of Cornell University, 525 East 68th Street, Room F-739, New York, NY 10021, USA.

Malnutrition leads to immune dysfunction with greatly increased morbidity. However, restrictive dietary regimens are also known to preserve immune function in autoimmune-susceptible mice. The macrophage (Mo) is central to both immune effector and autoregulatory functions and is critical to host-defense mechanisms. The aim of this study was to investigate the effect of calorie restriction on Mo functions in mice. Female, 6- to 8-wk-old, Swiss Webster mice were randomized to ad libitum feeding for 7 or 21 d (n = 10 mice/group), restricted feeding (13.5 to 14.0 g/cage/d; n = 10) for 7 d, or restricted feeding (16.5 to 17.0 g/cage/d; n = 10) for 21 d. These restrictions were equivalent to a decrease in calorie intake of 21.9% and 5.1%, respectively, over 7 and 21 d. All mice were allowed free access to water. On days 8 and 22, respectively, the mice were killed, and peritoneal Mos were isolated by lavage and adhered to 96-well polystyrene tissue-culture-treated plates. After stimulation with lipopolysaccharide, supernatant prostaglandin E2 and interleukin-6 levels were measured by enzyme-linked immunosorbent assay. Supernatant NO2- in response to stimulation with lipopolysaccharide and interferon-gamma was determined by the Greiss reaction. Prostaglandin E2 production was significantly elevated in peritoneal Mos from the calorie-restricted mice compared with the ad-libitum-fed mice after 7 d. After 21 d, production of both prostaglandin E2 and nitric oxide was significantly increased (P < 0.05) in peritoneal Mos from the restricted mice compared with the ad-libitum-fed mice. These results indicate that calorie restriction influences immune function by altering prostaglandin E2 and nitric oxide generation by Mos.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11165887&dopt=Abstract

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