Murine Peritoneal Exudate Cells Research Articles

Inactivated vaccine with glycyrrhizic acid adjuvant elicits potent innate and adaptive immune responses against foot-and-mouth disease.

Foot-and-mouth disease (FMD) is an extremely contagious viral disease that is fatal to young animals and is a major threat to the agricultural economy by reducing production and limiting the movement of livestock. The currently commercially-available FMD vaccine is prepared using an inactivated viral antigen in an oil emulsion, with aluminum hydroxide [Al(OH)3] as an adjuvant. However, oil emulsion-based options possess limitations including slow increases in antibody titers (up to levels adequate for defense against viral infection) and risks of local reactions at the vaccination site. Further, Al(OH)3 only induces a T helper 2 (Th2) cell response. Therefore, novel adjuvants that can address these limitations are urgently needed. Glycyrrhizic acid (extracted from licorice roots) is a triterpenoid saponin and has great advantages in terms of price and availability. To address the limitations of the currently used commercial FMD vaccine, we added glycyrrhizic acid as an adjuvant (immunostimulant) to the FMD bivalent (O PA2 + A YC) vaccine. We then evaluated its efficacy in promoting both innate and adaptive (cellular and humoral) immune reactions in vitro [using murine peritoneal exudate cells (PECs) and porcine peripheral blood mononuclear cells (PBMCs)] and in vivo (using mice and pigs). Glycyrrhizic acid has been revealed to induce an innate immune response and enhance early, mid-, and long-term immunity. The studied bivalent vaccine with glycyrrhizic acid increased the expression of immunoregulatory genes such as pattern-recognition receptors (PRRs), cytokines, transcription factors, and co-stimulatory molecules. Collectively, glycyrrhizic acid could have utility as a novel vaccine adjuvant that can address the limitations of commercialized FMD vaccines by inducing potent innate and adaptive immune responses.

The role of zinc sulfate in enhancing cellular and humoral immune responses to foot-and-mouth disease vaccine

Foot-and-mouth disease (FMD) is a rapidly propagating infectious disease of cloven-hoofed animals, especially cattle and pigs, affecting the productivity and profitability of the livestock industry. Presently, FMD is controlled and prevented using vaccines; however, conventional FMD vaccines have several disadvantages, including short vaccine efficacy, low antibody titers, and safety issues in pigs, indicating the need for further studies. Here, we evaluated the efficacy of a novel bivalent vaccine containing zinc sulfate as an immunostimulant and FMD type O and A antigens (O PA2 and A YC, respectively) against FMD virus in mice and pigs. Zinc sulfate induced cellular immunity in murine peritoneal exudate cells (PECs) and porcine peripheral blood mononuclear cells (PBMCs) by increasing IFNγ secretion. Additionally, FMD vaccine containing O PA2 and A YC antigens and zinc sulfate induced early, mid-, and long-term immune responses in mice and pigs, and enhanced cellular and humoral immunity by regulating the expression of pathogen recognition receptors (PRRs), transcription factors, co-stimulatory molecules, and cytokines in porcine PBMCs from vaccinated pigs. Overall, these results indicated that the novel immunostimulant zinc sulfate induced potent cellular and humoral immune responses by stimulating antigen-presenting cells (APCs) and T and B cells, and enhanced long-term immunity by promoting the expression of co-stimulatory molecules. These outcomes suggest that zinc sulfate could be used as a novel vaccine immunostimulant for difficult-to-control viral diseases, such as African swine fever (ASF) or COVID-19.

Differential mobility separation of leukotrienes and protectins.

Differential mobility spectrometry (DMS) is capable of separating stereoisomeric molecular ions based on their mobility in an oscillating electrical field with an asymmetric waveform. Thus, it is an "orthogonal" technique to chromatography and (tandem) mass spectrometry. Bioactive lipids, particularly of the eicosanoid and docosanoid class feature numerous stereoisomers, which exhibit a highly specific structure-activity relationship. Moreover, the geometry of these compounds also reflects their biochemical origin. Therefore, the unambiguous characterization of related isomers of the eicosanoid and docosanoid classes is of fundamental importance to the understanding of their origin and function in many biological processes. Here we show, that SelexION DMS technology coupled to μLC-MS/MS is capable of differentiating at least five closely related leukotrienes partially coeluting and (almost) unresolvable using LC-MS/MS only. We applied the developed method to the separation of LTB4 and its coeluting isomer 5S,12S-diHETE in murine peritoneal exudate cells, showing that LTB4 is present only after zymosan A injection while its isomer 5S,12S-diHETE is produced after saline (PBS) administration. Additionally, we show that the SelexION technology can also be applied to the separation of PD1 and PDX (10S,17S-diHDHA), two isomeric protectins.

Tyrosine Phosphorylation of CD13 Regulates Inflammatory Cell–Cell Adhesion and Monocyte Trafficking

CD13 is a large cell surface peptidase expressed on the monocytes and activated endothelial cells that is important for homing to and resolving the damaged tissue at sites of injury. We showed previously that cross-linking of human monocytic CD13 with activating Abs induces strong adhesion to endothelial cells in a tyrosine kinase- and microtubule-dependent manner. In the current study, we examined the molecular mechanisms underlying these observations in vitro and in vivo. We found that cross-linking of CD13 on U937 monocytic cells induced phosphorylation of a number of proteins, including Src, FAK, and ERK, and inhibition of these abrogated CD13-dependent adhesion. We found that CD13 itself was phosphorylated in a Src-dependent manner, which was an unexpected finding because its 7-aa cytoplasmic tail was assumed to be inert. Furthermore, CD13 was constitutively associated with the scaffolding protein IQGAP1, and CD13 cross-linking induced complex formation with the actin-binding protein α-actinin, linking membrane-bound CD13 to the cytoskeleton, further supporting CD13 as an inflammatory adhesion molecule. Mechanistically, mutation of the conserved CD13 cytoplasmic tyrosine to phenylalanine abrogated adhesion; Src, FAK, and ERK phosphorylation; and cytoskeletal alterations upon Ab cross-linking. Finally, CD13 was phosphorylated in isolated murine inflammatory peritoneal exudate cells, and adoptive transfer of monocytic cell lines engineered to express the mutant CD13 were severely impaired in their ability to migrate into the inflamed peritoneum, confirming that CD13 phosphorylation is relevant to inflammatory cell trafficking in vivo. Therefore, this study identifies CD13 as a novel, direct activator of intracellular signaling pathways in pathophysiological conditions.

Neutralization escape variant of West Nile virus associated with altered peripheral pathogenicity and differential cytokine profile

In order to understand the factors influencing pathogenicity of a virus, two neutralization escape (NE) variants were selected from wild type lineage 1 West Nile virus (WNV) 68856 strain pathogenic by intra-peritoneal (i.p.) route using monoclonal antibodies (MAbs) against envelope (E) protein. Both NE IF1A7 1.1 and NE IVC3F10 1.2 were resistant to neutralization and were neurovirulent by intra-cranial (i.c.) inoculation. Growth kinetics in porcine stable (PS) kidney and baby hamster kidney (BHK) cells was unchanged. In contrast to parent WNV only NE IF1A7 1.1 failed to cause lethal encephalitis on i.p. inoculation and was non pathogenic. NE IF1A7 1.1 variant showed delayed replication kinetics in murine peritoneal exudate cells (PEC) and Neuro 346 cells in vitro. In comparison with parent WNV and NE IVC3F10 1.2 variant, non pathogenic variant exhibited significantly reduced tumour necrosis factor α (TNF-α) induction in infected animals and PEC. Other cytokines like Interleukin (IL)-10, IL-6 and Interferon (IFN)-β remained unchanged. However, IL-1β did not follow the pattern and was higher only in parent WNV-infected PEC. The E gene sequences of these NE variants showed three common amino acid substitutions at residues E50, E89 and E242. A unique E156 (ser → pro) substitution in NE IF1A7 1.1, was absent in NE IVC3F10 1.2 variant suggested probable virulence marker. Our data indicates possible role of WNV E protein in induction of TNF-α and IL-1β and its association with WNV pathogenesis.

Isolation, structural elucidation and immunomodulatory activity of fructans from aged garlic extract

Traditionally, garlic ( Allium sativum) is known to be a significant immune booster. Aged garlic extract (AGE) possesses superior immunomodulatory effects than raw garlic; these effects are attributed to the transformed organosulfur compounds. AGE is also known to contain fructans; the amount of fructans in AGE represents a small fraction (0.22%) of the total fructans in raw garlic. In order to evaluate the biological activity of fructans present in AGE, both high molecular weight (>3.5 kDa; HF) and low molecular weight (<3 kDa; LF) fructans were isolated. The structures of purified HF and LF from AGE determined by 1H NMR and 13C NMR spectroscopy revealed that both have (2 → 1) β- d-fructofuranosyl bonds linked to a terminal glucose at the non-reducing end and β- d-fructofuranosyl branching on its backbone. Biological activity of fructans was assessed by immunostimulatory activity using murine lymphocytes and peritoneal exudate cells (source of macrophages). Both HF and LF displayed mitogenic activity and activation of macrophages including phagocytosis. These activities were comparable to that of known polysaccharide immunomodulators such as zymosan and mannan. This study clearly demonstrates that garlic fructans also contribute to the immunomodulatory properties of AGE, and is the first such study on the biological effects of garlic fructans.

Chemerin Contributes to Inflammation by Promoting Macrophage Adhesion to VCAM-1 and Fibronectin through Clustering of VLA-4 and VLA-5

Chemerin is a potent macrophage chemoattractant protein. We used murine peritoneal exudate cells (PECs) in adhesion, flow cytometry, and confocal microscopy assays to test the hypothesis that chemerin can also contribute to inflammation by promoting macrophage adhesion. Chemerin stimulated the adhesion of PECs to the extracellular matrix protein fibronectin and to the adhesion molecule VCAM-1 within a minute, with an EC(50) of 322 and 196 pM, respectively. Experiments using pertussis toxin and PECs from ChemR23(-/-) mice demonstrated that chemerin stimulated the adhesion of macrophages via the Gi protein-coupled receptor ChemR23. Blocking Abs against integrin subunits revealed that 89% of chemerin-stimulated adhesion to fibronectin was dependent on increased avidity of the integrin VLA-5 (alpha(5)beta(1)) and that 88% of adhesion to VCAM-1 was dependent on increased avidity of VLA-4 (alpha(4)beta(1)). Although chemerin was unable to induce an increase in integrin affinity as judged by the binding of soluble ligand, experiments using confocal microscopy revealed an increase in valency resulting from integrin clustering as the mechanism responsible for chemerin-stimulated macrophage adhesion. PI3K, Akt, and p38 were identified as key signaling mediators in chemerin-stimulated adhesion. The finding that chemerin can rapidly stimulate macrophage adhesion to extracellular matrix proteins and adhesion molecules, taken together with its ability to promote chemotaxis, suggests a novel role for chemerin in the recruitment and retention of macrophages at sites of inflammation.

Binding of the Staphylococcus aureus leucotoxin LukM to its leucocyte targets

The range of leucocytes susceptible to the leucotoxin LukM/F′, a two-component pore-forming toxin of Staphylococcus aureus causing mastitis in ruminants, had not been defined. We used fluorescent-labeled LukM to investigate the binding of this toxin to bovine cells and to identify its cellular targets among bovine, human and murine leucocytes. LukM bound to bovine blood neutrophils from all the individuals tested with similar affinity, with an apparent dissociation constant (Kd) of 1.81 ± 0.14 nM and 13 3100 ± 506 binding sites. The amount of LukM bound to bovine neutrophils did not depend on the presence of the complementary component LukF’, suggesting that the binding of LukM to its ligand does not depend on the formation of pore-forming oligomers, and that the number of bound LukM molecules corresponds to the number of available cell membrane ligands. Other staphylococcal class S components of bipartite leucotoxins (HlgA, HlgC, LukE, LukS-PV) were inefficient competitors of LukM for the binding to bovine neutrophils, indicating that LukM has a distinct ligand on target cells. Bovine blood neutrophils bound slightly more LukM than did milk neutrophils, and much more than did ovine and caprine blood neutrophils. Bovine monocytes and milk macrophages readily bound LukM, whereas blood lymphocytes did not. Human neutrophils bound little LukM and were resistant to LukM/F′ at the highest tested concentration (40 nM). Murine neutrophils bound LukM and were susceptible to the toxicity of LukM/F′, exhibiting flattening and nucleus alteration beginning at 0.3 nM concentration. Among murine peritoneal exudate cells, T lymphocytes (CD3+) and monocytes/macrophages (F4/80+) bound LukM, whereas binding to B lymphocytes (CD19+) was not detected. These results indicate that cells of the myeloid lineage are the main targets of LukM/F′ in dairy ruminants, and that resident or inflammatory migrated phagocytes are susceptible to this toxin.

3,5-Dimethyl-1-thiocarbamoylpyrazole and its Pd(II) complexes: Synthesis, spectral studies and antitumor activity

Complexes of the type [PdX 2(tdmPz)] { X = Cl −( 1), Br −( 2); I −( 3); SCN −( 4); tdmPz = 1-thiocarbamoyl-3,5-dimethylpyrazole} have been synthesized and characterized. Compound 1 was formed from the reaction between [PdCl 2(CH 3CN) 2] and 1-thiocarbamoyl-3,5-dimethylpyrazole. Complexes 2, 3 and 4 were obtained by metathesis of the chloro groups from 1 by bromide, iodide and thiocyanate ions, respectively. All the compounds and cisplatin have been tested in vitro by MTT assay for their cytotoxicity against three murine cancer cell lines: mammary adenocarcinoma (LM3 and LMM3) and lung adenocarcinoma (LP07) as well towards normal murine peritoneal exudate cells (PEC). Promising cytotoxic effect against LM3 has been found for 3 showing IC 50 equal to 24.5 μM which is comparable to the value obtained for cisplatin (30.3 μM).

Potential of Selected Strains of Lactic Acid Bacteria to Induce a Th1 Immune Profile

Lactic acid bacteria (LAB) might switch the Th2 biased immune response in allergic patients towards a balanced Th1/Th2 immune profile, leading to amelioration of allergy. To select strains of LAB that could be of potential application for foods in controlling allergy, 35 bacterial strains were screened in vitro using murine splenocytes and peritoneal exudate cells (PECs). Streptococcus thermophilus AHU1838 (FERM AP-21009), and Lactobacillus paracasei subsp. casei AHU1839 (FERM AP-21010) enhanced the secretion of Th1 cytokines such as interferon-gamma (IFN-gamma) and interleukin-12 (IL-12). The two strains of LAB also up-regulated the expression of CD40, and CD86 in dendritic cells (DCs), and activated cytotoxic T lymphocytes (CTL). These two strains could therefore be used in producing fermented food products that can enhance the Th1 immune profile which is important in ameliorating allergy.

Alcohol extracts of Echinacea inhibit production of nitric oxide and tumor necrosis factor-alpha by macrophages in vitro

It has been suggested that Echinacea has anti-inflammatory activity in vivo. Nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), and interleukin-1beta are important mediators in the inflammatory response. The effect of alcohol extracts of E. angustifolia (EA), E. pallida (EPA) and E. purpurea (EP) on the production of these inflammatory mediators in both LPS-stimulated RAW 264.7 macrophages in vitro and murine peritoneal exudate cells (PECs) in vivo were investigated. As macrophages produce these inflammatory mediators in response to pathogenic infection, parallel cultures of macrophages were studied for phagocytosis and intracellular killing of Salmonella enterica. EPA and EP in vitro inhibited NO production and TNF-α release in a dose-dependent manner. RAW 264.7 cells treated with EA or EP showed decreased killing over 24 h, although EA enhanced bacterial phagocytosis. Upon bacterial infection, RAW 264.7 cells produce high levels of NO; however, an Echinacea-mediated decrease in NO production was observed. Echinacea alcohol extracts administered orally at 130 mg/kg per day for seven days had a weak effect on NO production and phagocytosis by LPS-stimulated PECs. The results indicated that all Echinacea species significantly decreased inflammatory mediators in vitro, however, only EA and EP reduced bacterial killing. Oral administration of Echinacea alcohol extracts did not adversely affect the development and anti-bacterial function of inflammatory PECs in vivo, however, NO production was decreased during bacterial infection of PECs.

3,4-Dichloropropionaniline Suppresses Normal Macrophage Function

Macrophages are a critical part of the innate immune response and natural surveillance mechanisms. As such, proper macrophage function is crucial for engulfing bacterial pathogens through phagocytosis and destroying them by generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). The production of a number of cytokines by macrophages, such as tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6, plays an important role in the initiation of the acquired immune response creating an inflammatory environment favorable for fighting a bacterial infection. 3,4-Dichloropropionaniline (DCPA) suppresses several inflammatory parameters, including TNF-alpha production through a mechanism where nuclear factor-kappaB (NF-kappaB)-DNA binding is inhibited but not entirely abrogated. The goal of the present study was to evaluate the effects of DCPA on the inflammatory mediators of macrophages, including ROS and RNS in both murine peritoneal exudate cells and the human monocytic cell line, THP-1. The ability to perform phagocytosis and directly kill Listeria monocytogenes was also assessed. The results indicate that DCPA decreases the ability of both types of macrophages to phagocytize beads and generate both types of reactive species, which was correlated with a decrement in listericidal activity. These results demonstrate that DCPA has profound effects on macrophage function and provide insight into the potential mechanisms of immunosuppression by DCPA.

Resistance ofFrancisella tularensisStrains against Reactive Nitrogen and Oxygen Species with Special Reference to the Role of KatG

Francisella tularensis is a facultative intracellular bacterial pathogen capable of proliferating within host macrophages. The mechanisms that explain the differences in virulence between various strains of the species are not well characterized. In the present study, we show that both attenuated (strain LVS) and virulent (strains FSC200 and SCHU S4) strains of the pathogen replicate at similar rates in resting murine peritoneal exudate cells (PEC). However, when PEC were activated by exposure to gamma interferon (IFN-gamma), they killed LVS more rapidly than virulent strains of the pathogen. Addition of N(G)-monomethyl-l-arginine, an inhibitor of inducible nitric oxide synthase, to IFN-gamma-treated PEC, completely inhibited killing of the virulent strains, whereas it only partially blocked the killing of LVS. Similarly, in a cell-free system, SCHU S4 and FSC200 were more resistant to killing by H(2)O(2) and ONOO(-) than F. tularensis LVS. Catalase encoded by katG is a bacterial factor that can detoxify bactericidal compounds such as H(2)O(2) and ONOO(-). To investigate its contribution to the virulence of F. tularensis, katG deletion-containing mutants of SCHU S4 and LVS were generated. Both mutants demonstrated enhanced susceptibility to H(2)O(2) in vitro but replicated as effectively as the parental strains in unstimulated PEC. In mice, LVS-DeltakatG was significantly attenuated compared to LVS whereas SCHU S4-DeltakatG, despite slower replication, killed mice as quickly as SCHU S4. This implies that clinical strains of the pathogen have katG-independent mechanisms to combat the antimicrobial effects exerted by H(2)O(2) and ONOO(-), the loss of which could have contributed to the attenuation of LVS.

Effects of Ni2+ Ions on Cell Viability and NO Production of Murine Peritoneal Exudate Cells (Macrophages) with and without Lipopolysaccharide Stimulation

The purpose of this study was to clarify the cytotoxicity of Ni2+ ions against murine peritoneal exudate cells (PEC) (macrophages). First, we examined the cell viability of PEC with and without lipopolysaccharide (LPS) stimulation in culture media containing Ni2+ ions up to 1000 micromol/L. Results showed that the cytotoxicity of Ni2+ ions against PEC was dose-dependent and accelerated by LPS stimulation, especially in media with Ni2+ ions exceeding 100 micromol/L. Second, we measured the production of nitric oxide (NO) from PEC and found that LPS caused the PEC to produce abundant NO. However, high dose of Ni2+ ions at concentration more than 200 micromol/L hindered and inhibited NO production. These results pointed out that the cytotoxicity of Ni2+ ions against macrophages depended on both the Ni2+ ion concentration and the presence of bacteria with LPS. Further, NO--a killer of bacteria--was lost when LPS-stimulated macrophages were exposed to high dose of Ni2+ ions.

Effect of various catechins on the IL-12p40 production by murine peritoneal macrophages and a macrophage cell line, J774.1.

Interleukin-12 (IL-12) is a heterodimeric cytokine comprising p40 and p35 subunits produced mainly by monocytes and macrophages, and plays an essential role in the regulation of the differentiation of Th1 cells. Green tea polyphenols exhibit potent anti-oxidative activities and anti-inflammatory effects by modulating cytokine production. We investigated the effect of catechins on IL-12p40 production in murine macrophages induced by bacterial lipopolysaccharide (LPS). Pretreatment with several catechins at doses of 0.3-30 microM suppressed IL-12 p40 production by murine peritoneal exudate cells (PEC) and J774.1 cells in a dose-dependent manner. Decreases in protein production were primarily due to down-regulation of the transcription of IL-12p40 mRNA. Of the various catechins, (-)-epigallocatechin gallate (EGCG) was the most potent inhibitor, followed by (-)-gallocatechin gallate (GCG) and (-)-epicatechin gallate (ECG). EGCG inhibited LPS-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), but not Jun N-terminal kinase (JNK), while EGCG augmented LPS-induced phosphorylation of p44/p42 extracellular signal-related kinase (ERK). In addition, both EGCG and GCG inhibited LPS-induced degradation of IkappaBalpha with concomitant inhibition of nuclear protein binding to NF-kappaB site and synthesis of IRF-1. These results suggest that gallate-containing catechins, particularly EGCG, inhibits LPS-induced IL-12p40 production in murine macrophages by inhibiting p38 MAPK while enhancing p44/p42 ERK, leading to the inhibition of IkappaBalpha degradation and NF-kappaB activation.

Purification and characterization of a rhamnose-binding lectin with immunoenhancing activity from grass carp ( Ctenopharyngodon idellus) ovaries

A rhamnose-specific lectin was isolated from ovaries of the grass carp ( Ctenopharyngodon idellus). The grass carp lectin possesses a molecular mass of 205 kDa. It is composed of six subunits each with a molecular mass of 35 kDa. The N-terminal amino acid sequence of the grass carp shows similarity to those of other fish species with 26–35% amino acid identity. It is mitogenic toward murine splenocytes and peritoneal exudate cells.

Inhibition Of Natural, Interleukin-2 Stimulated and Bacillus Calmette-Guerin Enhanced Cytotoxicity With Anti-CD16 Antibodies

We studied the cytolytic mechanism of nonstimulated, bacillus Calmette-Guerin (BCG) stimulated and interleukin (IL)-2 (Chiron Corp., Amsterdam, The Netherlands) stimulated peripheral blood mononuclear cells. We inhibited the cytotoxicity of nonstimulated, BCG stimulated and IL-2 stimulated peripheral blood mononuclear cells against various target cells using 3 monoclonal antibodies directed against the CD16 receptor of natural killer cells or alternatively monoclonal antibodies against the alpha and beta subunits of the IL-2 receptor complex (IL-2R). The main target cell was the poorly differentiated transitional cell line T24. Of the 3 anti-CD16 antibodies tested only CLB FcR-gran/1 effectively inhibited natural, IL-2 stimulated and BCG enhanced cytotoxicity. Cytotoxicity was also markedly diminished after depletion of CD16+CD56+/- cells with CLB FcR-gran/1. An hour of pretreatment with CLB FcR-gran/1 was enough to reduce significantly the level of cytotoxicity evoked by overnight stimulation with BCG or IL-2. Simultaneous administration of anti-IL-2Ralpha and anti-IL-2Rbeta significantly decreased the killing of target cells by BCG stimulated and IL-2 stimulated peripheral blood mononuclear cells. Within the stimulation times chosen the same killing mechanisms seemed to explain the nonstimulated, BCG stimulated and IL-2 stimulated cytotoxicity with CD16 positive cells as central effectors. Anti-CD16 antibodies may deliver a target cell independent down-regulatory signal to natural killer cells or alternatively mimic a nonIg ligand and block the detection of the target cell.

Prostaglandin E2 down-regulates viable Bacille Calmette-Guérin-induced macrophage cytotoxicity against murine bladder cancer cell MBT-2 in vitro.

The regulatory effect of prostaglandin (PG) E2 and a cyclooxygenase (COX) inhibitor on Bacille Calmette-Guérin (BCG)-induced macrophage cytotoxicity in a bladder cancer cell, MBT-2, was studied in vitro. BCG stimulated thioglycollate-elicited murine peritoneal exudate cells (PEC) to induce cytotoxic activity and to produce cytokines such as interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and PGE2. NS398, a specific COX-2 inhibitor, and indomethacin (IM), a COX-1 and COX-2 inhibitor, enhanced viable BCG-induced cytotoxic activity and IFN-gamma and TNF-alpha production of PEC. However, NS398 and IM did not enhance these activities induced by killed BCG. Enhanced cytotoxicity was mediated by increased amounts of IFN-gamma and TNF-alpha. Exogenous PGE2 reduced cytotoxic activity and IFN-gamma and TNF-alpha production of PEC. These results suggest that PGE2 produced by BCG-activated macrophages has a negative regulatory effect on the cytotoxic activity of macrophages. Accordingly, a PG synthesis inhibitor may be a useful agent to enhance BCG-induced antitumour activity of macrophages.

Antigenic characterisation of a novel Streptococcus anginosus antigen that induces nitric oxide synthesis by murine peritoneal exudate cells.

A novel antigen that induces nitric oxide (NO) synthesis by murine peritoneal exudate cells (PEC) was prepared from a culture supernate of Streptococcus anginosus NCTC 10713 in dialysed medium by column chromatography with DEAE-Sephacel followed by size-exclusion high performance liquid chromatography (HPLC). A chemical analysis of the S. anginosus antigen (SAA) revealed that it mainly consisted of carbohydrates (rhamnose, N-acetylglucosamine, glucose and galactose), smaller quantities of protein and a trace amount of phosphorus. The SAA stimulated PEC from C57BL/6N mice to produce NO and accumulate induced NO synthetase (iNOS) mRNA in a dose-dependent manner, reaching a plateau with 10-30 microg/ml. Furthermore, a reverse transcription-PCR assay revealed that SAA 10 microg/ml could induce mRNA accumulation of tumour necrosis factor-alpha, interleukin (IL)-1beta and IL-6 as well as iNOS. In contrast, Rantz-Randall antigen (RRA), a carbohydrate antigen prepared from the organisms, could not induce NO synthesis or cause the accumulation of iNOS mRNA, although cytokine production was observed after stimulation. The SAA-induced NO synthesis, but not the cytokine production, was sensitive to heat. Furthermore, an immunoblot analysis of SAA indicated that the 43-kDa protein band reacted with anti-SAA but not anti-RRA antibodies. In immunodiffusion, SAA reacted with both anti-SAA and anti-RRA antibodies, and the precipitin bands formed crossing lines, suggesting that SAA could possess two different antigenic components--one that reacts specificially with anti-SAA antibodies and another that has an identity similar to that of RRA. Taken together, SAA, a novel antigen of S. anginosus, was found to induce NO synthesis as well as produce inflammatory cytokines in murine PEC. It is suggested that the protein molecule of SAA may exclusively induce NO synthesis, and its carbohydrate component(s) could have a relationship to cytokine production.

MURINE IL-2 SECRETING RECOMBINANT BACILLUS CALMETTE-GUÉRIN AUGMENTS MACROPHAGE-MEDIATED CYTOTOXICITY AGAINST MURINE BLADDER CANCER MBT-2

This study was to establish a more effective anti-cancer immunomodulating agent by constructing recombinant (r) Bacillus Calmette-Guérin (BCG) secreting alpha-antigen (alpha-Ag) fused murine (m) interleukin (IL)-2, and to study its biological activity on cell-mediated cytotoxicity against murine bladder cancer cell, MBT-2, in vitro. pSO246 plasmid vector ligated with mIL-2 gene was introduced into BCG by electroporation. Thioglycollate-elicited murine peritoneal exudate cells (PEC) were stimulated in vitro with parental BCG or rBCG and their cytotoxic activity and the cytokine production was studied. Cytokines were assayed by an enzyme-linked immunosorbent assay (ELISA) and L929 bioassay. Cytotoxicity was measured by 51Cr releasing assay. rBCG (alpha-Ag-IL-2) secreted functional IL-2 and augmented more efficient cytotoxicity to MBT-2 and cytokines such as IL-12, tumor necrosis factor and interferon (IFN)-gamma in PEC than parental BCG did. rBCG (alpha-Ag) had the same activity as BCG. Anti-IL-2 antibody reduced rBCG (alpha-Ag-IL-2)-mediated cytotoxicity and IFN-gamma production. Exogenous IL-2 also enhanced BCG-mediated cytotoxicity, but 100 times more IL-2 was required to express the same activity as rBCG (alpha-Ag-IL-2). Anti-IL-12 neutralizing antibody and the depletion of T cells and NK cells reduced IFN-gamma production by PEC stimulated with rBCG (alpha-Ag-IL-2), suggesting that T cells, NK cells and IL-12 participate in the enhancement of IFN-gamma production. rBCG secreting IL-2 showed significant antitumor activity and cytokine production and this will be a promising agent for bladder cancer patient to reduce both clinical dose and side effects of BCG for immunotherapy.