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At least 73 records · Page 4Linked to original sources
PubMed2002

The ability of fish oil to suppress tumor necrosis factor alpha production by peripheral blood mononuclear cells in healthy men is associated with polymorphisms in genes that influence tumor necrosis factor alpha production.

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) mediates inflammation. High TNF-alpha production has adverse effects during disease. Polymorphisms in the TNF-alpha and lymphotoxin alpha genes influence TNF-alpha production. Fish oil suppresses TNF-alpha production and has variable antiinflammatory effects on disease. OBJECTIVE: We examined the relation between TNF-alpha and lymphotoxin alpha genotypes and the ability of dietary fish oil to suppress TNF-alpha production by peripheral blood mononuclear cells (PBMCs) in healthy men. DESIGN: Polymorphisms in the TNF-alpha (TNF*1 and TNF*2) and lymphotoxin alpha (TNFB*1 and TNFB*2) genes were determined in 111 healthy young men. TNF-alpha production by endotoxin-stimulated PBMCs was measured before and 12 wk after dietary supplementation with fish oil (6 g/d). RESULTS: Homozygosity for TNFB*2 was 2.5 times more frequent in the highest than in the lowest tertile of inherent TNF-alpha production. The percentage of subjects in whom fish oil suppressed TNF-alpha production was lowest (22%) in the lowest tertile and doubled with each ascending tertile. In the highest and lowest tertiles, mean TNF-alpha production decreased by 43% (P < 0.05) and increased by 160% (P < 0.05), respectively. In the lowest tertile of TNF-alpha production, only TNFB*1/TNFB*2 heterozygous subjects were responsive to the suppressive effect of fish oil. In the middle tertile, this genotype was 6 times more frequent than the other lymphotoxin alpha genotypes among responsive individuals. In the highest tertile, responsiveness to fish oil appeared unrelated to lymphotoxin alpha genotype. CONCLUSION: The ability of fish oil to decrease TNF-alpha production is influenced by inherent TNF-alpha production and by polymorphisms in the TNF-alpha and lymphotoxin alpha genes.

Adult↗
PubMed2002

Mechanism of TCDD-induced suppression of antibody production: effect on T cell-derived cytokine production in the primary immune reaction of mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to suppress antigen-specific antibody production in humoral immune reactions, but the precise mechanism remains unclear. Since T cell activation and subsequent production of type 2 helper T (Th2) cell-derived cytokines are required for antigen-specific antibody production in humoral immunity, we examined the effects of TCDD on splenic T cell numbers, T cell growth factor IL-2 production, and Th2 cell-derived cytokine production. C57BL/6N mice were orally given TCDD (20 microg/kg) or vehicle, and immediately intraperitoneally immunized with ovalbumin (OVA) adsorbed to alum, and cellular changes in the spleen and cytokine production by spleen cells were investigated from Day 1 to Day 14. In vehicle-control mice the numbers of splenic CD3(+) T cells increased from Day 7 onward, but no increase was observed in the TCDD-exposed mice. When spleen cells from control mice were cultured and restimulated with OVA ex vivo, a significant amount of IL-2 was found from Day 1, but it decreased on Day 7, whereas TCDD exposure promptly suppressed the increase on Day 4. TCDD exposure significantly suppressed the production of Th2 cell-derived cytokines IL-4, IL-5, and IL-6, which were prominently increased from Day 4 onward in control mice. The dose-dependent study showed that IL-5 production was significantly suppressed in a dose-dependent manner starting at 1 microg/kg TCDD. Moreover, separation and reconstitution studies showed that the TCDD-induced suppression of IL-5 production was due to the impaired function of T cells rather than that of antigen-presenting cells. The results of this study suggest that TCDD-induced suppression of T cell activation and Th2-type cytokine production is involved in the impairment of antigen-specific antibody production.

Administration, Oral↗
PubMed1997

The decrease in nonsplenic interleukin-6 (IL-6) production after splenectomy indicates the existence of a positive feedback loop of IL-6 production during endotoxemia in dogs.

The spleen is involved in endotoxin-induced interleukin-6 (IL-6) production. To quantitate the relative contribution of the spleen to endotoxin-induced IL-6 production, we studied the effect of endotoxin (1.0 microg/kg of body weight) in control dogs (n = 7) and splenectomized dogs (n = 7). Blood for analysis of tumor necrosis factor (TNF) and IL-6 was sampled from the femoral artery and the portal, hepatic, and splenic (only in controls) veins. Arterial plasma endotoxin and cortisol levels were also measured. Whole-body IL-6 production was calculated by a deconvolution technique. Splenic IL-6 production in control dogs was measured from splenic blood flow and arteriovenous concentration differences. Endotoxin levels were higher in splenectomized dogs (P < 0.05) because of a decreased distribution volume (P < 0.05) and decreased clearance of endotoxin (P < 0.05). Endotoxin-induced plasma IL-6 levels were decreased by approximately 75% in splenectomized dogs (P < 0.01), and whole-body IL-6 production rates were severalfold lower (median of 8.7 mg/4 h and range of 3.9 to 11.4 mg/4 h versus a median of 32.3 mg/4 h and a range of 22.7 to 70.2 mg/4 h) (P < 0.05). However, in control dogs splenic IL-6 production (0.6 +/- 0.2 mg/4 h) was only approximately 2% of whole-body IL-6 production. Plasma TNF levels increased in both groups (P < 0.01) but were not different between the groups. Plasma cortisol levels were slightly higher in splenectomized dogs than in control dogs (P < 0.05). In conclusion, splenectomy decreases the distribution volume and clearance rate of endotoxin. Splenectomy results in decreased endotoxin-induced IL-6 production, which is caused not by the absence of splenic IL-6 production, but by a decrease in nonsplenic IL-6 production. Therefore, the spleen is an important mediator in the complete activation of nonsplenic IL-6 production by endotoxin.

Animals↗
PubMed2006

The chrondoprotective actions of a natural product are associated with the activation of IGF-1 production by human chondrocytes despite the presence of IL-1beta.

BACKGROUND: Cartilage loss is a hallmark of arthritis and follows activation of catabolic processes concomitant with a disruption of anabolic pathways like insulin-like growth factor 1 (IGF-1). We hypothesized that two natural products of South American origin, would limit cartilage degradation by respectively suppressing catabolism and activating local IGF-1 anabolic pathways. One extract, derived from cat's claw (Uncaria guianensis, vincaria), is a well-described inhibitor of NF-kappaB. The other extract, derived from the vegetable Lepidium meyenii (RNI 249), possessed an uncertain mechanism of action but with defined ethnomedical applications for fertility and vitality. METHODS: Human cartilage samples were procured from surgical specimens with consent, and were evaluated either as explants or as primary chondrocytes prepared after enzymatic digestion of cartilage matrix. Assessments included IGF-1 gene expression, IGF-1 production (ELISA), cartilage matrix degradation and nitric oxide (NO) production, under basal conditions and in the presence of IL-1beta. RESULTS: RNI 249 enhanced basal IGF-1 mRNA levels in human chondrocytes by 2.7 fold, an effect that was further enhanced to 3.8 fold by co-administration with vincaria. Enhanced basal IGF-1 production by RNI 249 alone and together with vincaria, was confirmed in both explants and in primary chondrocytes (P < 0.05). As expected, IL-1beta exposure completely silenced IGF-1 production by chondrocytes. However, in the presence of IL-1beta both RNI 249 and vincaria protected IGF-1 production in an additive manner (P < 0.01) with the combination restoring chondrocyte IGF-1 production to normal levels. Cartilage NO production was dramatically enhanced by IL-1beta. Both vincaria and RNI 249 partially attenuated NO production in an additive manner (p < 0.05). IL-1beta - induced degradation of cartilage matrix was quantified as glycosaminoglycan release. Individually RNI 249 or vincaria, prevented this catabolic action of IL-1beta. CONCLUSION: The identification of agents that activate the autocrine production of IGF-1 in cartilage, even in the face of suppressive pro-inflammatory, catabolic cytokines like IL-1beta, represents a novel therapeutic approach to cartilage biology. Chondroprotection associated with prevention of the catabolic events and the potential for sustained anabolic activity with this natural product suggests that it holds significant promise in the treatment of debilitating joint diseases.

Cells, Cultured↗
PubMedDate not supplied

Pharmacy placement of nonprescription nicotine replacement therapy products and community pharmacists' counseling for product use.

OBJECTIVE: To investigate the relationship between nonprescription nicotine replacement therapy (NRT) product placement within community pharmacies and the number of patients whom pharmacists report counseling for use of these products. DESIGN: Cross-sectional study. SETTING: Four Northern California counties. PARTICIPANTS: 327 community pharmacists. INTERVENTIONS: Survey mailed in 1999-2000. MAIN OUTCOME MEASURES: Estimated number of NRT purchasers who received counseling in their pharmacy and pharmacists' level of control over NRT purchases and counseling as functions of the location of the NRT products within the pharmacy setting. RESULTS: Of 327 community pharmacists surveyed, 26 (8.0%) reported that all nonprescription NRT products were stocked behind the pharmacy counter (full control over NRT purchases and counseling), 46 (14.1%) reported that all NRT products were stocked outside of the pharmacy department in an area that could not be seen easily by the pharmacist (no control), and the remainder (n = 255; 78.0%) indicated that all NRT products were located within their visual field but not behind the counter and/or that NRT products were located in more than one of the three previously described store locations (i.e., behind counter, not behind counter but within visual field, and outside visual field) (partial control). While adjusting for known confounders (pharmacists' self-efficacy for counseling, practice setting, and perceived pros of counseling), NRT product location was significantly associated with counseling of four or more patients a month for use of these products (P = .005). Specifically, compared with pharmacists with no control over NRT purchases and counseling, pharmacists with full control were 4.73 (95% CI, 1.46-15.37) times as likely to counsel four or more patients per month, and pharmacists with partial control were 2.95 (95% CI, 1.47-5.94) times as likely. Compared with pharmacists with partial control, pharmacists with full control were 1.60 (95% CI, 0.58-4.41) times as likely to counsel at least four patients monthly (P = .361). CONCLUSION: The location of nonprescription NRT products within the community pharmacy setting is associated with the number of patients whom pharmacists report counseling for use of these products.

Administration, Cutaneous↗
PubMed2005

Fas ligand induces cell-autonomous IL-23 production in dendritic cells, a mechanism for Fas ligand-induced IL-17 production.

Fas ligand (FasL) has the potential to induce inflammation accompanied by massive neutrophil infiltration. We previously reported that FasL rapidly induces the production of various inflammatory cytokines including IL-1beta and IL-17. In this study, we investigated the mechanism of the FasL-induced IL-17 production. We found that the culture supernatant of mouse resident peritoneal exudate cells (PEC) cocultured with FasL-expressing tumor (FFL) cells induced IL-17 production in freshly isolated resident PEC. Anti-IL-1beta Ab strongly inhibited the IL-17-inducing activity. However, rIL-1beta by itself induced only weak IL-17 production. Intriguingly, anti-IL-12 Ab but not an IL-15-neutralizing agent, IL15R-Fc, strongly inhibited the FasL-induced IL-17-inducing activity. IL-23, which shares the p40 subunit with IL-12, but not IL-12 itself, induced IL-17 production synergistically with IL-1beta in resident PEC. FasL induced the production of IL-23 in PEC in vivo and in vitro, and IL-17 production following the i.p. injection of FFL cells was severely impaired in p40-/- mice, indicating that IL-23 plays an important role in the FasL-induced IL-17 production. FFL also induced the production of IL-23 in bone marrow- or PEC-derived dendritic cells (DCs). Finally, FasL induced only weak p40 production in a mixture of p40-/- and Fas-/- DC, indicating that FasL induces IL-23 production in DC mainly in a cell-autonomous manner.

Animals↗
PubMed2006

The classification of antiseptic products to be administered to wounds--another borderline case between medicinal products and medical devices?

The importance of a correct demarcation between a Medicinal Product (MP) and a Medical Device (MD) is undisputedly one of the major topics related to the development and launch of a new healthcare product. However, for some products the correct demarcation between MPs and MDs can turn out to be somewhat complicated. This article aims to provide an overview on the existing legislation and its adequate application based on a suitable example at hand. Article 2 (2) of the European Directive 2001/83/EC as amended by Directive 2004/27/EC on the Community code relating to medicinal products for human use stipulates that the respective Medicinal Products Legislation must be applied whenever a product can be covered by both the definitions for MPs and for products regulated by other legal provisions enacted by the European Community, e.g. Cosmetic Products (CPs) or MDs. This basic principle implies that the decision to base the risk-benefit assessment of the product in question on the Medical Device Directive (MDD) would contradict the aforementioned constitutional principle, pursuant to which the stricter of the regulatory procedures theoretically possible is to apply in cases of doubt. In contrast to the approval procedure established for MPs, the MDD requires a Conformity Assessment Procedure to be performed by the manufacturer himself and a "Notified Body". Thus, in the majority of cases the responsibility for the risk assessment of MDs lies solely with the manufacturer and is prior to launch not subject to further scrutiny by regulators. Only in specific cases, i.e. for the Conformity Assessment Procedure of Class III MDs which contain an Active Pharmaceutical Ingredient one of the Member States competent authorities designated in accordance with Directive 65/ 65/EEC has to be involved before taking a decision. It is therefore important that the classification of the product is carried out carefully in full compliance with existing legal provisions, also taking into account the related guidance documents issued by the European Commission. The adequate application of these rules is explained using the example of the antiseptic compound polihexanide, which is used both in approved medicinal products (wound antiseptics) and wound irrigation solutions labelled as medical devices.

Animals↗
PubMed1991

Disodium cromoglycate (DSCG) selectively inhibits IgE production and enhances IgG4 production by human B cell in vitro.

The effect of DSCG on human IgE production in vitro was studied. DSCG selectively inhibited interleukin-4 (IL-4) induced IgE production by mononuclear cells (MNC) from normal donors without affecting IgM, IgA, IgG1, IgG2 or IgG3 production. In contrast, DSCG enhanced IgG4 production. To achieve this effect, DSCG must be added to the culture at the initiation and be present throughout the entire culture period. Interferon-gamma (IFN-gamma) also inhibited IL-4-induced IgE production, but IgG4 production was not affected by IFN-gamma. Monoclonal anti-IFN-gamma antibody blocked the inhibition of IgE production by IFN-gamma, but did not block the inhibition of IgE production by DSCG. DSCG also selectively inhibited spontaneous IgE production and enhanced IgG4 production by B cells from atopic patients in the presence of T cells and monocytes. These results indicate that there is a mechanism of IgE production inhibition which is not mediated by IFN-gamma. We also found that DSCG is an excellent reagent for the study of IgE and IgG4 regulation in vitro.

B-Lymphocytes↗
PubMed1997

Interleukin-12 (IL-12) production in whole blood cultures from human immunodeficiency virus-infected individuals studied in relation to IL-10 and prostaglandin E2 production.

The role of interleukin-12 (IL-12) in Th1 cell differentiation is well established. The heterodimer p70, composed of a p40 and a p35 chain, is the biologically active form. IL-12 production by human monocytes is enhanced by interferon-gamma (IFN-gamma) and inhibited by IL-10 and prostaglandin E2 (PGE2). Peripheral blood mononuclear cells from human immunodeficiency virus (HIV)-infected individuals reportedly have impaired IL-12 p40 and p70 production on stimulation with Staphylococcus aureus Cowan I (SAC) in vitro. Both PGE2 and IL-10 previously were proposed to be instrumental in this defect in IL-12 production. Here, we studied IL-12 p40 and p70 production in relation to IL-10 and PGE2 production in whole blood cultures from HIV-infected individuals. On stimulation with lipopolysaccharide, IL-12 production was normal. However, on stimulation with SAC, IL-12 p40 and p70 production was decreased in HIV-infected individuals and correlated significantly with decreased peripheral blood CD4+ T-cell number and T-cell reactivity to CD3 monoclonal antibody in vitro. However, IL-10 and PGE2 production in cultures from HIV-infected individuals was normal and did not relate to IL-12 production. In conclusion, IL-12 production by cells from HIV-infected individuals is impaired under certain conditions in vitro and this decrease is independent of IL-10 or PGE2 production.

Blood Cells↗
PubMed1996

Control of the dehydration process in production of intermediate-moisture meat products: a review.

IM meat products are produced by lowering the aw to 0.90 to 0.60. Such products are stable at ambient temperature and humidity and are produced in nearly every country in the world, especially in developing areas where refrigeration is limited or unavailable. Traditionally IM meats use low cost sources of energy for drying, such as sun drying, addition of salt, or fermentation. Products produced by different processes are of interest since they do not require refrigeration during distribution and storage. Many different IM meat products can be produced by utilizing modern processing equipment and methods. Production can be achieved in a relatively short period of time and their advantages during marketing and distribution can be utilized. Nevertheless, a better understanding of the principles involved in heat transfer and efficiency of production are still needed to increase efficiency of processing. A basic understanding of the influence of water vapor pressure and sorption phenomena on water activity can materially improve the efficiency of drying of IM meats. Predrying treatments, such as fermentation and humidity control, can also be taken advantage of during the dehydration process. Such information can lead to process optimization and reduction of energy costs during production of IM meats. The development of sound science-based methods to assure the production of high-quality and nutritious IM meats is needed. Finally, such products also must be free of pathogenic microorganisms to assure their success in production and marketing.

Absorption↗
PubMed2001

In vitro-prepared advanced glycation end-products and the modulating potential of their low-molecular weight degradation products in IRPTC-A rat proximal-tubular derived kidney epithelial cell line.

Low-molecular advanced glycation end-products (AGEs)-degradation products resulting from a proteolysis of tissue or circulating AGEs represent up to 80% of AGE plasma immunoreactivity. These AGE peptides contribute to the dramatic increase in AGE levels in end-stage renal disease even in the absence of diabetes. Because glomerular filtered AGE-degradation products may accumulate within intracellular compartments of proximal tubular epithelial cells, we investigated whether there is a pathway potentially mediating damaging effects of AGE-degradation products by perturbation of the function of the tubuloepithelium. Proximal tubular-derived rat kidney cells (IRPTC) were incubated with high-molecular AGEs highly modified by incubation of bovine serum albumin (BSA) with glucose for 50 days in vitro, and with low-molecular AGE-degradation products derived from proteolytic cleavage and isolated in the molecular range between 1 and 30 kDa. The proliferation of IRPTC (3H-thymidine incorporation) was reduced to 89+/-1% and 69+/-2% after 24 hr of incubation with BSA-AGE and BSA-AGE-degradation products, respectively. The cell viability of IRPTC was reduced significantly to 59+/-15% and 31+/-13% after 144 hr of incubation with BSA-AGE and BSA-AGE-degradation products, respectively. Conditioned media obtained from IRPTC incubated for 72 hr with BSA-AGE and its degradation products increased the proliferation rate of renal fibroblasts (RFb) to 222+/-24% and 449+/-40%, respectively. Incubation of IRPTC with BSA-AGE-degradation products increased the expression of endothelin-1 (ET-1) mRNA to 210% after 1 hr; the expression of platelet-derived growth factor-B (PDGF-B) mRNA reached 184% after 2 hr. Regarding the toxicity of AGEs to the kidney, low-molecular weight AGE-degradation products possibly form an individual fraction with a comparatively higher toxic potential.

Animals↗
PubMed1991

The detection of an early advanced glycation product which co-elutes with the Amadori product on aminophenylboronate affinity chromatography.

The production of an antiserum recognizing an early advanced glycation product of glycated human serum albumin (HSA) is reported. The antiserum was produced with the intention of recognizing the Amadori product, i.e. the monofructosamine derivative, of any glycated protein. In retrospect, however, the immunogen appears to have been transformed in vivo which led to the production of antibodies to an early advanced glycation product. Two-site immunometric and competitive ELISAs showed that the affinity-purified antibodies recognized glycated HSA only after it had been stored for several months. This recognition, by the antibody, was more specific for the transformed product than for the original hapten (1-amino-1-deoxy-D-fructose-6-aminohexanoic acid) used for immunization by a factor of more than 1,000. These antibodies also detected immunoreactive material present in the elution fraction after in vivo glycated HSA had been chromatographed on an aminophenylboronate affinity column, indicating that an early advanced glycation product can co-elute with the Amadori product of glycated HSA on aminophenylboronate affinity chromatography. This suggests that the antiserum recognized an early advanced glycation product that also contained cis-diols as does the Amadori product, and may prove useful in the early detection of clinical complications in diabetic patients.

Aminocaproic Acid↗
PubMed1996

Interferon-gamma-inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin-12 for interferon-gamma production.

The novel cytokine interferon-gamma-inducing factor (IGIF) augments natural killer (NK) cell activity in cultures of human peripheral blood mononuclear cells (PBMC), similarly to the structurally unrelated cytokine interleukin (IL)-12. IGIF has been found to enhance the production of interferon-gamma (IFN-gamma) and granulocyte/macrophage colony-stimulating factor (GM-CSF) while inhibiting the production of IL-10 in concanavalin A (Con A)-stimulated PBMC. In this study, when anti-CD3 monoclonal antibody (mAb)-stimulated human enriched T cells were exposed to IGIF, the cytokine dose-dependently enhanced the proliferation of the cells and this could be completely inhibited by a neutralizing antibody against IL-2 at lower concentrations of IGIF. Neutralizing antibody against IFN-gamma had only insignificant inhibitory effects on T cell proliferation at higher concentrations of IGIF. Enzyme-linked immunosorbent assays (ELISA) revealed that, like PBMC, T cells exposed to IGIF produced large amounts of IFN-gamma; however, changes in the production of IL-4 and IL-10 were minimal. IGIF, but not IL-12, significantly enhanced IL-2 and GM-CSF production in T cell cultures, as determined by CTLL-2 bioassay and ELISA, respectively; however, both IGIF and IL-12 enhanced IFN-gamma production by the T cells. When T cells were exposed to a combination of IGIF and IL-12, a synergistic effect was observed on the production of IFN-gamma, but not on production of IL-2 and GM-CSF. In conclusion, IGIF enhances T cell proliferation apparently through an IL-2-dependent pathway and enhances Th1 cytokine production in vitro and exhibits synergism when combined with IL-12 in terms of enhanced IFN-gamma production but not IL-2 and GM-CSF production. Based on structural and functional differences from any known cytokines, it was recently proposed that this cytokine be designated interleukin-18.

Adult↗
PubMed2006

Recent advances in polyhydroxyalkanoate production by mixed aerobic cultures: from the substrate to the final product.

Numerous bacteria have been found to exhibit the capacity for intracellular polyhydroxyalkanoates (PHA) accumulation. Current methods for PHA production at the industrial scale are based on their synthesis from microbial isolates in either their wild form or by recombinant strains. High production costs are associated with these methods; thus, attempts have been made to develop more cost-effective processes. Reducing the cost of the carbon substrates (e.g., through feeding renewable wastes) and increasing the efficiency of production technologies (including both fermentation and downstream extraction and recovery) are two such examples of these attempts. PHA production processes based on mixed microbial cultures are being investigated as a possible technology to decrease production costs, since no sterilization is required and bacteria can adapt quite well to the complex substrates that may be present in waste material. PHA accumulation by mixed cultures has been found under various operational conditions and configurations at both bench-scale and full-scale production. The process known as "feast and famine" or as "aerobic dynamic feeding" seems to have a high potential for PHA production by mixed cultures. Enriched cultures submitted to a transient carbon supply can synthesize PHA at levels comparable to those of pure cultures. Indeed, the intracellular PHA content can reach around 70% of the cell dry weight, suggesting that this process could be competitive with pure culture PHA production when fully developed. Basic and applied research of the PHA production process by mixed cultures has been carried out in the past decade, focusing on areas such as microbial characterization, process configuration, reactor operational strategies, process modeling and control, and polymer characterization. This paper presents a review of the PHA production process with mixed cultures, encompassing the findings reported in the literature as well as our own experimental results in relation to each of these areas.

Bacteria, Aerobic↗
PubMed1983

Cyclosporin A inhibits the production of gamma interferon (IFN gamma), but does not inhibit production of virus-induced IFN alpha/beta.

The effect of cyclosporin A (CsA) on the production of gamma interferon (IFN gamma) versus IFN alpha/beta was studied using mouse and human lymphocytes and fibroblasts. Spleen cells from C57Bl/6 mice produced low but significant levels (40-60 U/ml) of IFN gamma after 2 to 3 days of culture with irradiated DBA spleen cells. The addition of CsA at concentrations as low as 0.1 microgram/ml completely inhibited (less than 10 U/ml) IFN gamma production in these cultures. High levels of IFN gamma (170-1200 U/ml) were produced when either C57Bl/6 spleen cells or Ficoll-Hypaque-purified human peripheral blood lymphocytes (PBL) were cultured with the T-cell mitogen staphylococcal enterotoxin A (SEA). The addition of CsA (0.1 microgram/ml) to these cultures also completely inhibited (less than 10 U/ml) IFN gamma production. This inhibition was shown not to be due to a change in the kinetics of IFN gamma production or to a change in the amount of SEA required for stimulation. IFN gamma production in SEA-stimulated mouse spleen cells was inhibited at 3 days of culture even when CsA was added at 24 or 48 hr postculture initiation. Thus, CsA inhibits IFN gamma production even when early events associated with lymphocyte activation have been allowed to take place. In contrast to IFN gamma production, IFN alpha/beta production by Newcastle disease virus (NDV)-infected mouse and human lymphocytes or fibroblasts was not inhibited by the addition of CsA (1 microgram/ml). CsA also did not block the action of IFN gamma or IFN alpha/beta since addition of CsA (1 microgram/ml) to reference IFN standards had no effect on their antiviral activity. Thus, CsA inhibits the production of IFN gamma by T cells but appears to have no effect on the production of IFN alpha/beta by virus-infected cells or on the antiviral action of already produced IFN gamma and IFN alpha/beta.

Animals↗
PubMed2005

Development of PLEX, a plasmid-based expression system for production of heterologous gene products by the gram-positive bacteria Streptococcus gordonii.

While Escherichia coli expression systems have been widely utilized for the production of heterologous proteins, these systems have limitations with regard to the production of particular protein products, including poor expression, expression of insoluble proteins into inclusion bodies, and/or expression of a truncated product. Using the surface protein expression (SPEX) system, chromosomally integrated heterologous genes are expressed and secreted into media by the naturally competent gram-positive organism Streptococcus gordonii. After E. coli turned out to be an inappropriate expression system to produce sufficient quantities of intact product, we successfully utilized SPEX to produce the heterologous antigen BH4XCRR that is designed from sequences homologous to the S. pyogenes M-protein C-repeat region. To further enhance production of this product by S. gordonii, we sought to develop a novel system for the production and secretion of heterologous proteins. We observed that under various growth conditions, S. gordonii secreted high levels of a 172 kDa protein, which was identified by N-terminal sequence analysis as the glucosyltransferase GTF. Here we report on the development of a plasmid-based expression system, designated as PLEX, which we used to enhance production of BH4XCRR by S. gordonii. A region from the S. gordonii chromosome that contains the positive regulatory gene rgg, putative gtfG promoter, and gtfG secretion-signal sequence was cloned into the E. coli/Streptococcus shuttle plasmid pVA838. Additionally, the bh4xcrr structural gene was cloned into the same plasmid downstream and in-frame with rgg and gtfG. This plasmid construct was transformed into S. gordonii and BH4XCRR was detected in culture supernatants from transformants at greater concentrations than in supernatants from a SPEX strain expressing the same product. BH4XCRR was easily purified from culture supernatant using a scalable two-step purification process involving hydrophobic-interaction and gel-filtration chromatography.

Antigens, Bacterial↗
PubMed2001

Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress gene expressions and production of tumor necrosis factor-alpha in murine macrophages stimulated with lipopolysaccharide or lipoteichoic acid.

We previously reported that the ES products from the plerocercoids of Spirometra erinaceieuropaei reduce nitric oxide synthase and chemokine gene expression in macrophages. In this study, we show that ES products suppressed tumor necrosis factor-alpha mRNA expression and tumor necrosis factor-alpha production in murine peritoneal macrophages stimulated with lipopolysaccharide or lipoteichoic acid in vitro. When macrophages from ES product-injected mice were stimulated with lipopolysaccharide in vitro, these cells produced smaller amounts of tumor necrosis factor-alpha compared with those taken from control mice. The suppressive effects of ES products were not restored by the treatment of indomethacin or anti-IL-10 antibody, and the ES products did not induce mRNA expression of secretory leukocyte protease inhibitor. Macrophages from C3H/HeJ mice, which have a single point mutation in the Toll-like receptor 4 gene, expressed tumor necrosis factor-alpha and IL-1alpha mRNA in the presence of lipopolysaccharide, but these expressions were less than those of macrophages from C3H/HeN. ES products significantly suppressed tumor necrosis factor-alpha gene expression and tumor necrosis factor-alpha production in macrophages from C3H/HeN and C3H/HeJ mice stimulated with lipopolysaccharide. However, ES products had no effect on IL-1 mRNA expression. Our data suggest that the plerocercoids secrete the tumor necrosis factor-alpha inhibitory products to evade the host's immune system, and that tumor necrosis factor-alpha mRNA expression might be inhibited downstream from Toll-like receptor 4 in the lipopolysaccharide signaling pathway.

Animals↗
PubMed1999

Isolation and identification of cyclic imide and deamidation products in heat stressed pramlintide injection drug product.

This report summarizes the identification of six cyclic imide [Asu] and two deamidation products from a sample of pramlintide final drug product that had been stressed at 40 degrees C for 45 days. The pramlintide degradation products were isolated by cation exchange high-performance liquid chromatography (HPLC) followed by reversed-phase HPLC. The isolated components were characterized by mass spectrometry (MS), tandem MS (MS/MS) and when necessary, by enzymatic (thermolysin) digestion followed by liquid chromatography/mass spectrometry (LC/MS) and sequence analysis. The isolated products were identified as [Asu14]-pramlintide, [Asu21]-pramlintide, [Asu22]-pramlintide, [Asu35]-pramlintide, [1-21]-succinimide-pramlintide, and [1-22]-succinimide-pramlintide. Also identified were [Asp35]-pramlintide, the deamidation product of pramlintide at Asn35, and [Tyr37-OH]-pramlintide, the deamidation product of the pramlintide amidated C-terminal Tyr. Together these data support those presented earlier (C. Hekman et al., Isolation and identification of peptide degradation products of heat stressed pramlintide injection drug product. Pharm Res 1998;15:650-9) indicating that the primary mechanism of degradation for pramlintide in this pH 4.0 formulation is deamidation, with six of the eight possible deamidation sites observed to undergo deamidation. Gln-10 and Asn-31 are the only two residues subject to deamidation for which none is observed. The data indicate that the cyclic imide products account for approximately 20% of the total thermal degradation while the deamidation products account for 64%. The remaining degradation is due to peptide backbone hydrolysis.

Amino Acid Sequence↗