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Protein Expr Purif. 1999 Mar;15(2):236-42.
Affinity purification of recombinant interferon-alpha on a mimetic ligand adsorbent.

Swaminathan S, Khanna N.

Recombinant Gene Products Laboratory, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India.

A method for improved refolding and purification of recombinant human interferon-alpha (rh-IFN-alpha) from inclusion bodies is described. The optimal conditions of refolding were obtained by the addition of 0.5 M l-arginine to the refolding buffer. The rh-IFN-alpha was purified to near homogeneity utilizing a single-step chromatography on a mimetic dye-ligand matrix. Improved refolding, coupled to a single-column affinity purification strategy, resulted in a 10-fold increase in the yield of rh-IFN-alpha. This single-step purification protocol yielded approximately 50 mg of purified rh-IFN-alpha from 1 liter of shake flask culture. The rh-IFN-alpha prepared by this protocol was found to be essentially monomeric based on HPLC gel filtration and nonreducing SDS-PAGE. It had a specific activity of approximately 2.8 x 10(8) IU/mg, measured as inhibition of cytopathic effect of encephalomyocarditis virus on A549 human lung carcinoma cells. 1999 Academic Press.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10049681&dopt=Abstract

J Interferon Cytokine Res. 1999 Jan;19(1):41-8.
Dietary omega-3 polyunsaturated fatty acids reduce IFN-gamma receptor expression in mice.

Feng C, Keisler DH, Fritsche KL.

Graduate Nutritional Sciences Program, College of Agriculture, Food & Natural Resources, University of Missouri Columbia 65211, USA.

Enrichment of immune cells in vivo or in vitro with omega-3 polyunsaturated fatty acid (n-3 PUFA) has been reported to diminish their response to interferon-y (IFN-gamma). We hypothesized that the n-3 PUFA-induced hyporesponsiveness to IFN-gamma is mediated, in part, by a reduction in the number of IFN-gamma receptors (IFNGR) expressed on the surface of these cells. To test this hypothesis, we fed mice experimental diets containing low or high amounts of n-3 PUFA. Thioglycollate-elicited peritoneal macrophages (PEC) were collected and tested for binding and internalization of [125I]-labeled recombinant murine IFN-gamma. High n-3 PUFA intake was associated with a significant (n = 2, p < 0.01) reduction in [125I]-IFN-gamma binding without affecting binding affinity (Kd). When studies were performed at 37 degrees C, high n-3 PUFA intake reduced internalization of [125I]-rmIFN-gamma by 20%-30% (n = 2,p < 0.001). Results from flow cytometric analysis of IFNGR-1 expression on the surface of murine splenocytes were in agreement with the binding studies. Further, total cellular IFNGR-1 from PEC and splenocytes was examined via immunoprecipitation and Western blotting. High n-3 PUFA diet was associated with a 50% decline (n = 3-6, p < 0.05) in total IFNGR-1 in both immune cell populations studied. These data suggest that reduced IFNGR expression may be responsible for immune cell hyporesponsiveness to IFN-gamma, which may, in part, explain some of the immunomodulatory and anti-inflammatory effects associated with the consumption of diets high in n-3 PUFA.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10048767&dopt=Abstract

Cancer Res. 1999 Feb 15;59(4):872-9.
Suppression of angiogenesis, tumorigenicity, and metastasis by human prostate cancer cells engineered to produce interferon-beta.

Dong Z, Greene G, Pettaway C, Dinney CP, Eue I, Lu W, Bucana CD, Balbay MD, Bielenberg D, Fidler IJ.

Department of Cancer Biology, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA. zdonotes.mdacc.tmc.edu

We determined whether the IFN-beta gene can be used to suppress angiogenesis, tumor growth, and metastasis of human prostate cancer cells growing in the prostate of nude mice. Highly metastatic PC-3M human prostate cancer cells were engineered to constitutively produce murine IFN-beta subsequent to infection with a retroviral vector containing murine IFN-beta cDNA. Parental (PC-3M-P), control vector-transduced (PC-3M-Neo), and IFN-beta-transduced (PC-3M-IFN-beta) cells were injected into the prostate (orthotopic) or subcutis (ectopic) of nude mice. PC-3M-P and PC-3M-Neo cells produced rapidly growing tumors and regional lymph node metastases, whereas PC-3M-IFN-beta cells did not. PC-3M-IFN-beta cells also suppressed the tumorigenicity of bystander nontransduced prostate cancer cells. PC-3M-IFN-beta cells produced small tumors (3-5 mm in diameter) in nude mice treated with anti-asialo GM1 antibodies and in severe combined immunodeficient/Beige mice. Immunohistochemical staining revealed that PC-3M-IFN-beta tumors were homogeneously infiltrated by macrophages, whereas control tumors contained fewer macrophages at their periphery. Most tumor cells in the control tumors were stained positive by an antibody to proliferative cell nuclear antigen; very few were positively stained by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling. In sharp contrast, PC-3M-IFN-beta tumors contained fewer proliferative cell nuclear antigen-positive cells and many terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling-positive cells. Staining with antibody against CD31 showed that control tumors contained more blood vessels than PC-3M-IFN-beta tumors. PC-3M-IFN-beta cells were more sensitive to lysis mediated by natural killer cells in vitro or to cytostasis mediated by macrophages than control transduced cells. Conditioned medium from PC-3M-IFN-beta cells augmented splenic cell-mediated cytolysis to control tumor cells, which could be neutralized by antibody against IFN-beta. Collectively, the data suggest that the suppression of tumorigenicity and metastasis of PC-3M-IFN-beta cells is due to inhibition of angiogenesis and activation of host effector cells.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10029078&dopt=Abstract

J Autoimmun. 1999 Feb;12(1):35-42.
Differential activities of immunogenic collagen type II peptides in the induction of nasal tolerance to collagen-induced arthritis.

Chu CQ, Londei M.

Kennedy Institute of Rheumatology, London, UK. cchrudeauinstitute.org

Nasal tolerance has recently been used to modulate immune responses in animal models of autoimmunity. We have compared immunogenic collagen type II (CII) peptides for induction of nasal tolerance in DBA/1 mice to collagen-induced arthritis (CIA). Three synthetic peptides corresponding to T cell-stimulating sequences of alpha1(II)-CB11, 260-270, 245-270 and 259-273, one peptide analog 245-270 (A260B261N263) and one myelin basic protein (MBP) peptide 89-101 were administered intranasally to DBA/1 mice respectively (total 300 microg peptide/mouse on three consecutive days) 10 days prior to CII immunization. Forty percent of CII245-270 (P<0.05) and 20% CII260-270 (P>0.05) treated mice did not develop arthritis whilst all of the mice treated with CII245-270 (A260B261N263) or CII259-273 developed arthritis compared to those in control groups (PBS- and MBP89-101-treated). The mice in either the CII245-270- or CII260-270-treated group which developed arthritis had a significantly delayed onset and their disease was less severe both clinically and histologically. All mice in both CII245-270- and CII260-270-treated groups had a reduced serum level of anti-CII antibody (P<0.01), with a marked reduction of IgG2a. Drain lymph node (LN) cells taken 7 days after CII immunization from these mice showed a significant reduction of interferon (IFN)-gammaP<0.01) production uponin vitro stimulation with CII. These results indicate that intranasal administration of synthetic CII peptides can control CIA, which is achieved by down-regulating the Th1 CII-induced responses. In addition, they stress that a fine 'tuning' of the peptide able to induce 'tolerance' is required to achieve the optimal effect. 1999 Academic Press.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10028020&dopt=Abstract

J Exp Med. 1999 Mar 1;189(5):803-10.
T helper cell type 1-associated and cytotoxic T lymphocyte-mediated tumor immunity is impaired in interleukin 4-deficient mice.

Schuler T, Qin Z, Ibe S, Noben-Trauth N, Blankenstein T.

Max-Delbruck-Center for Molecular Medicine, 13122 Berlin, Germany. tschudc-berlin.de

It is widely accepted that cellular immune responses are induced by CD4(+) T helper 1 (Th1) cells secreting interleukin (IL)-2 and interferon (IFN)-gamma. Tumor immunity is often mediated by cytotoxic T lymphocytes (CTLs) whose activation is supported by Th1 cytokines. Since IL-4 directs Th2 development and has been shown to inhibit Th1-dominated responses, we assumed that IL-4-deficient (IL-4(-/-)) mice would develop vigorous CTL-mediated tumor immunity compared with IL-4-competent (IL-4(+/+)) mice. Surprisingly, IL-4(-/-) mice were severely impaired to develop tumor immunity to both a mammary adenocarcinoma line and a colon carcinoma line. The lack of tumor immunity in IL-4(-/-) mice was associated with reduced IFN-gamma production, diminished levels of tumor-reactive serum IgG2a, and undetectable CTL activity, indicating a defective Th1 response in the absence of endogenous IL-4. Anti-IL-4 monoclonal antibody blocked tumor immunity in IL-4(+/+) mice when administered at the time of immunization but not at the time of challenge. Additionally, tumor immunity could be induced in IL-4(-/-) mice, if IL-4 was provided by gene-modified cells together with immunizing tumor cells. These results demonstrate that tumor immunity requires IL-4 in the priming phase for the generation of effector cells rather than for their maintenance and exclude secondary, developmental defects in the "knockout" strain. Together, our results demonstrate a novel and previously unanticipated role of IL-4 for the generation of Th1-associated, CTL-mediated tumor immunity.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10049944&dopt=Abstract

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