Enhancement by Recombinant Human Interleukin 2 of Host Resistance to Toxoplasma gondii Infection in Pregnant Mice

The lymphokine production by pregnant mice infected with a lethal dose of Toxoplasma gondii was evaluated in comparison with that by virgin mice infected with a sublethal dose of this protozoan parasite. Splenocytes taken from mice before and on the day after infection produced considerable amounts of IL-2 in response to concanavalin A (Con A) stimulation, but the titers rapidly declined in both pregnant and virgin mice as infection progressed. A trace amount or undetectable level of IL-2 was produced by splenocytes from acutely infected mice when stimulated with Toxoplasma lysate antigen (TLA). In contrast to the kinetics of IL-2 production, the levels of IFN-γ produced by splenocytes cultured with Con A or TLA increased steadily in the later stage of infection in both pregnant and virgin mice. Thus, the response to Con A or TLA of splenocytes to produce IL-2 and IFN-γ differed strikingly in acute toxoplasmosis in mice. The administration of rHuIL-2 resulted in a significant decrease in the mortality of pregnant mice infected with a lethal dose of Toxoplasma. The combination of rHuIL-2 and rMuIFN-γ increased the survival rate slightly but not significantly compared with pregnant mice receiving either rHuIL-2 or rMuIFN-γ. Moreover, exogenously administered rHuIL-2 enhanced the production of both IFN-α and IFN-γ in the bloodstreams of pregnant mice, in accordance with the decreased mortality. These results indicate that IL-2 may play a significant role in modulating the host defense against Toxoplasma infection in pregnant mice.     

Expression and characterization of human glycosylated interleukin-1 receptor antagonist in Pichia pastoris

Interleukin-1 receptor antagonist is an inhibitor of the pro-inflammatory action of interleukin-1. The gene encoding for interleukin-1 receptor antagonist (IL-1ra) was cloned into a Pichia pastoris expression vector pPICzalphaA (Invitrogen, USA) and transformed into P. pastoris strain SMD1168H. Multi-copy selection of the gene produced a high expressing strain of IL-1ra that produced 17mg/L of total secreted purified protein. The IL-1ra produced in P. pastoris was a mixture of glycosylated and non-glycosylated IL-1ra where 70% of the total protein was glycosylated. SP-Sepharose purification allowed for separation of the two expressed forms of IL-1ra, which permits biochemical investigation of glycosylated and non-glycosylated IL-1ra using one expression system. Mass spectrometric analysis revealed the expression of the full-length protein and that the glycosylated IL-1ra contained high mannose glycoforms that ranged from Man(9)GlcNAc(2) to Man(14)GlcNAc(2).     

Immunopharmacology of recombinant human interleukin-18 in non-human primates

Recombinant human interleukin (IL)-18 (rHuIL-18) has a potential as a therapeutic agent in cancer and is currently in drug development. Since human IL-18 displays 96% and 100% amino acid sequence homology with cynomolgus monkey and chimpanzee IL-18, respectively, the biological responses to rHuIL-18 were evaluated in these species. A single intravenous dose of rHuIL-18 at 1 or 10mg/kg in cymonolgus monkeys caused a transient reduction in lymphocyte counts, induction of IL-1alpha and tumour necrosis factor alpha (TNF-alpha) mRNA in whole blood cells and a marked increase in plasma neopterin. rHuIL-18 administered to cynomolgus monkeys at doses of 0.3 or 3mg/kg for two 5-day cycles (Days 1-5 and 15-19) resulted in increased monocyte counts, induction of NK cells and concomitant increases in plasma IL-12 and neopterin. Administration of repeat doses of rHuIL-18 at 10mg/kg to chimpanzees was associated with increased monocyte counts, upregulation of FcgammaRI surface expression on monocytes, and increased IL-8, IL-12 and neopterin in plasma. These studies demonstrate, for the first time, the immunostimulatory activity of rHuIL-18 in vivo. The described pharmacological profile of rHuIL-18 in both cynomolgus monkeys and chimpanzees is indicative of the immunotherapeutic potential of rHuIL-18 in the treatment of cancer.     

Human recombinant interleukin-1 receptor antagonist inhibits lymphocyte blastogenesis induced by concanavalin A. Restorative effect of hrIL-1.

Interleukin-1 (IL-1), mainly produced by monocyte-macrophages, is a polypeptide cytokine with pleiotropic biological effects. IL-1 plays an important role in mediating immune response and inflammation. Recently a natural inhibitor to IL-1 has been discovered, interleukin-1 receptor antagonist (IL-1ra), produced by human monocytes cultured on adherent IgG which binds to the IL-1 receptors. In our study we found that the pretreatment of cells with serial dilutions of IL-1ra (250 ng/ml-2.5 pg/ml) inhibits, in a dose-dependent manner, lymphocyte DNA synthesis stimulated with Con A (10 micrograms/ml). IL-1ra did not have any effect on resting peripheral blood mononuclear cells (PBMC). Time course experiments show that IL-1ra at 250 ng/ml has its maximum inhibitory effect on lymphocyte blastogenesis when cells are pretreated 2 h before Con A. No effect was found when hrIL-1ra was added after Con A. Moreover, hrIL-1ra also inhibits the enhancing effects of exogenous hrIL-1 (400, 200, 100 and 50 ng/ml) on lymphocytes stimulated with Con A; while when hrIL-1ra was used on cells treated with only Con A, the inhibition was more pronounced. When PBMC were removed from monocytes, by adherence, the Con A-treated lymphocytes were not influenced by 2 h pretreatment of hrIL-1ra; while a strong inhibition was found when exogenous hrIL-1 was added at different concentrations. In addition, hrIL-1ra also inhibits the enhancing effect of hrIL-2 on lymphocyte DNA synthesis. In another set of experiments PBMC were pretreated with hrIL-1ra (250 ng/ml) for 2 h and then added LPs (10 ng/ml) and IL-1 alpha generation was determined using ELISA. In these experiments IL-1ra completely abolished the generation of IL-1 alpha. These data suggest that hrIL-1ra exhibits a dose-response inhibition of lymphocyte blastogenesis induced by Con A, probably through the down-regulation of IL-1 synthesis necessary as an early signal for T-cell activation and IL-2 production.     

Subcutaneous injection of interleukin 12 induces systemic inflammatory responses in humans: implications for the use of IL-12 as vaccine adjuvant

Interleukin 12 (IL-12) is a cytokine with important regulatory functions bridging innate and adaptive immunity. It has been proposed as an immune adjuvant for vaccination therapy of infectious diseases and malignancies. The inflammatory properties of IL-12 play an important role in the adjuvant effect. We studied the effect of s.c. injections of recombinant human IL-12 (rHuIL-12) in 26 patients with renal cell cancer and demonstrated dose-dependent systemic activation of multiple inflammatory mediator systems in humans. rHuIL-12 at a dose of 0.5 g/kg induced degranulation of neutrophils with a significant increase in the plasma levels of elastase (p<0.05) and lactoferrin (p=0.01) at 24 h. Additionally, rHuIL-12 injection mediated the release of lipid mediators, as demonstrated by a sharp increase in the plasma secretory phospholipase A₂ (sPLA₂) level (p=0.003). rHuIL-12, when administered at a dose of 0.1 g/kg, showed minimal systemic effects. In conclusion, when IL-12 is used as an adjuvant, doses should not exceed 0.1 g/kg, in order to avoid severe systemic inflammatory responses.     

Correlation of neutrophil and monocyte derived interleukin-1 receptor antagonist and interleukin-8 with colitis severity in the rabbit

Activated neutrophils and monocytes produce interleukin (IL)-8, a pro-inflammatory chemokine, but also IL-1 receptor antagonist (IL-1ra), which is an anti-inflammatory cytokine. We were interested to see the profiles of IL-8 and IL-1ra in the colonic tissue and in the peripheral blood leukocytes (PBL) during the development of immune complex induced colitis in rabbits. IL-1ra and IL-8 in PBL were measured in 26 rabbits at time 0 h, 24 h, and 48 h after induction of colitis. The colons were removed at 48 h for measuring myeloperoxidase (MPO), ulcer area, IL-1ra and IL-8. Epithelial damage, crypt abscess formation and leukocyte infiltration of the colonic tissue were major features of this colitis model. During the development of colitis, there was an increase in circulating neutrophils and monocytes (P < 0.0001), but not lymphocytes. Likewise, elevated amounts of IL-1ra (P = 0.0001) and IL-8 (P = 0.0219) production by PBL were observed following induction of colitis. Flow cytometry revealed major source of IL-1ra was monocytes, while the main sources of IL-8 were neutrophils and monocytes. There was correlation between MPO and ulcer area (R_s = 0.6327, P < 0.0001). At 24 h, PBL from MPO^High group (n = 11) showed increased IL-1ra (P = 0.027) and IL-8 (P = 0.0128) levels vs MPO^Low group (n = 15). IL-8 production by PBL showed correlation with tissue MPO (R_s = 0.4273, P = 0.0295). The colitis in this model was associated with an increase in circulating monocytes and neutrophils, which released increased amounts of IL-8 and IL-1ra. Further, IL-8 and IL-1ra showed correlation with the severity of colitis. These observations should significantly further understandings on the role of neutrophils and monocytes in the immunopathogenesis of ulcerative colitis.     

Interaction of recombinant monocyte-derived interleukin 1 receptor antagonist with rheumatoid synovial cells.

Interleukin 1 receptor antagonist (IL-1ra) is a newly described cytokine that is produced by human monocytes cultured on adherent immunoglobulin G (IgG). These studies have characterized the binding of IL-1ra to receptors on human rheumatoid synovial cells in comparison to binding of IL-1 alpha. The human synovial cells bound 35S-IL-1ra with a Kd of 213 pM and a Ki of 134 pM. 125I-IL-1 alpha bound to the synovial cells with similar values, showing a Kd of 205 pM and a Ki of 58 pM. Cross-inhibition studies were performed to examine whether IL-1ra and IL-1 alpha interacted with the same receptors and in an identical fashion. At the highest concentrations of inhibitory proteins, the binding of each ligand was inhibited 100% by the same or opposite ligand. This result indicated that IL-1ra and IL-1 alpha bound to the same receptors and not to overlapping subsets of receptors. In addition, the binding of 35S-IL-1ra was inhibited in an identical fashion by equimolar amounts of IL-1ra or IL-1 alpha. However, twofold or greater amounts of IL-1ra in comparison to IL-1 alpha were required to offer comparable inhibition of binding of 125I-IL-1 alpha. These results suggest that IL-1ra and IL-1 alpha bind with equal avidity to IL-1 receptors but may not bind identically. Additional experiments are necessary to establish whether these two ligands may bind to different regions of the extracellular portion of the IL-1 receptor.     

In vivo anti inflammatory effects of the M20 IL-1 Inhibitor: I. Effects on acute inflammatory parameters

Previous studies have described an IL-1 Inhibitor produced by a myelomonocytic line developed in our laboratory (Eur J Immunol 1986; 16: 1449). This IL-1 Inhibitor was secreted by the M20 line constitutively in addition to IL-1, from which it could be separated. We have recently shown that the M20 IL-1 Inhibitor is distinct from the IL-1ra.In vitro this factor inhibited IL-1 induced proliferative responses as well as PGE₂ secretion by IL-1 induced fibroblasts. We also showed for the first time (Lymphokine Research 1988; 7(3): 268) that an IL-1 inhibitor can reduce IL-1 induced inflammatory effects. This study describes the specific effect of the M20 IL-1 Inhibitor on IL-1 induced parameters of inflammation: fever, leukocytosis and local foot pad swelling or lymph node enlargement. Purified preparations of the IL-1 Inhibitor, when injected together with IL-1, or before the IL-1, reduced fever, leukocytosis, foot pad swelling and lymph node enlargement caused by IL-1. Similar responses were obtained by injection of IL-6 or TNF, but were unaffected by the IL-1 Inhibitor, when injected together.These results indicate that the M20 IL-1 Inhibitor acts specifically on IL-1 induced responsesin vivo. The potential importance of this factor as an anti-inflammatory and immune regulatory factor, is supported by the findings of this study.Abbreviations IL-1 Interleukin 1 - IL-6 Interleukin 6 - IL-1ra Interleukin 1 receptor antagonist - TNF tumor necrosis factor     

<Emphasis Type="Italic">Paenibacillus</Emphasis> strain MP-1: a new source of mutanase

A mutan-degrading bacterium, closely related to Paenibacillus curdlanolyticus, was isolated from soil. It produced 0.4 U mutanase ml⁻¹ in 2 days in shake-flask cultures when bacterial mutan was the sole carbon source. Mutanase activity was optimal at pH 6.2 and 45°C over 1 h and was stable between pH 5.8 and 12 at 4°C for 24 h and up to 40°C for 1 h. Mutan produced by Streptococcus mutans was rapidly hydrolyzed by this enzyme. The hydrolysis of mutan (1 g l⁻¹) resulted in 17% saccharification over 2 h and, at the same time, glucan was entirely solubilized.     

Modulation of the acute phase response by altered expression of the IL-1 type 1 receptor or IL-1ra

A complete understanding of the role for endogenously produced interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and IL-1 receptor antagonist (IL-1ra) in the acute phase response to inflammation remains unknown. In the present studies, knockout mice lacking either a functional IL-1 type I receptor (IL-1RI(-/-)), a TNF type I receptor (TNFR-I(-/-)), or both IL-1 type I and TNF type I receptors (IL-1RI(-/-)/TNFR-I(-/-)) received a turpentine abscess. Additional mice deficient in IL-1ra protein (IL-1ra(-/-)) or overexpressing IL-1ra protein (IL-1ra(tg)) were similarly treated. After a turpentine abscess, IL-1 receptor knockout mice exhibited an attenuated inflammatory response compared with wild-type or animals lacking a functional TNFR-I. Mice overexpressing IL-1ra also had an attenuated hepatic acute phase protein response, whereas IL-1ra knockout mice had a significantly greater hepatic acute phase response. We conclude that the inflammatory response to a turpentine abscess is the result of a balance between IL-1ra expression and IL-1 binding to its type I receptor. Endogenously produced IL-1ra plays a central role in mitigating the magnitude of the IL-1-mediated inflammatory response and, ultimately, the outcome to a turpentine abscess.     

The autocrine production of transforming growth factor-beta 1 during lymphocyte activation. A study with a monoclonal antibody-based ELISA

We describe the production and characterization of three mAb to transforming growth factor-beta (TGF-beta) and the use of two of them for the development of a TGF-beta 1-specific ELISA and for the study of the regulation of immune function in vitro. All three mAb bound recombinant human TGF-beta 1 (rHuTGF-beta 1) with high affinity and recognized the dimer form of this molecule in immunoblots. mAb 2G7 immunoprecipitated rHuTGF-beta 1, TGF-beta 2, and rHuTGF-beta 3 and neutralized the growth inhibitory activity of all three molecules in vitro on mink lung epithelial-like cells, Mv1Lu, indicating a shared neutralization epitope. mAb 4A11 neutralized and immunoprecipitated only rHuTGF-beta 1, and mAb 12H5 immunoprecipitated rHuTGF-beta 1 but had no effect on the bioactivity of either rHuTGF-beta 1, TGF-beta 2, or rHuTGF-beta 3. These results suggest that a second neutralization epitope may be unique to TGF-beta 1. The ELISA was developed with mAb 4A11 and 12H5, with a range of 0.63 to 40 ng/ml, i.e., a sensitivity of 0.63 ng/ml or 63 pg/sample. The assay is accurate, precise, and specific for the active but not the inactive or latent TGF-beta 1 complex and fails to react with TGF-beta 2, rHuTGF-beta 3, inhibin A, and activin A. Supernatants obtained from serum-free cultures of human PBMC from multiple donors contained significant quantities of TGF-beta 1 (3 to 15 ng/ml), which was detected in the ELISA only after pH 2 treatment to convert latent TGF-beta to the active form. Treatment of the PBMC with either recombinant human IL-2 (rHuIL-2) or PHA-P/PMA enhanced the production of latent TGF-beta 1. mAb 4A11 and 2G7, but not mAb 12H5 enhanced both the proliferative response of PBMC to rHuIL-2/rHuTNF-alpha and PHA-P and the development of the rHuIL-2/rHuTNF-alpha treated PBMC into LAK cells with cytotoxic activity against COLO target cells. These findings suggest that although PBMC secrete latent TGF-beta 1, mechanisms that convert the latent TGF-beta complex into an active form exist in vitro and that the endogenously produced TGF-beta can regulate immune functions in an autocrine fashion.     

A Combined Hydroxylation of 3‐Cyanopyridine to 3‐Cyano‐6‐hydroxypyridine and 6‐Hydroxynicotinic Acid by Resting Cells of Comamonas testosteroni JA1 Grown on Nicotinic Acid

A strain of Comamonas testosteroni JA1 known for its capacity to hydroxylate 3‐cyanopyridine to 3‐cyano‐6‐hydroxypyridine was found to be also capable to hydroxylate nicotinic acid at a higher rate. In the course of the induced cultivation the forming 6‐hydroxynicotinic acid was degraded either slightly, in the presence of nicotinic acid in the medium, or faster, in the absence of nicotinic acid. In a combined process of hydroxylation of nicotinic acid by growing culture and hydroxylation of 3‐cyanopyridine by resting cells of Comamonas testosteroni JA1, not only an additional amount of 50.38 g of solid 6‐hydroxynicotinic acid was produced from 1 L of cultivation broth with a 99.97 % molar conversion yield, but also the yield of 3‐cyano‐6‐hydroxypyridine produced was more than doubled. This can be compared to that of the resting cells from the induced cultivation broth where within 8 h an amount of 5.77 g of solid 3‐cyano‐6‐hydroxypyridine was produced by resting cells from 1 L of the cultivation broth. This also was superior to 4.39 g/L of cultivation broth of resting cells reported in the literature.     

Features of bacterial cellulose synthesis in a mutant generated by disruption of the diguanylate cyclase 1 gene of <Emphasis Type="Italic">Acetobacter xylinum</Emphasis> BPR 2001

The diguanylate cyclase 1 (DGC1) (dgc1) gene in Acetobacter xylinum BPR 2001—a bacterial cellulose (BC) producer—was cloned and sequenced, and a DGC1 gene-disrupted mutant, strain DD, was constructed. The production and structural characteristics of the BC formed by DD were compared with those of the parental strain BPR 2001. BC production by DD was almost the same as that by BPR 2001 in static cultivation and in shake flask cultivation. However, in a jar fermentor DD produced about 36% more BC than the parental strain. DD produced suspended particle materials that cannot aggregate owing to their random structural characteristics in static cultivation; more uniformly dispersed BC pellicles and smaller BC pellets are produced on average in a jar fermentor, as reflected by the higher BC production by DD than by the parental strain in a jar fermentor. Micrographs of BC produced by DD revealed that the width of cellulose ribbons assemblies decreased as a result of differences in the ultrastructure and mechanism of formation of BC between the two strains. These results reveal that disruption of the dgc1 gene, which catalyzes synthesis of c-di-GMP (an effector of BC synthase), is not fatal for BC synthesis, although it affects BC structure.     

Enzymatic reduction of the α, β-unsaturated carbon bond in citral

Bacteria, yeasts and filamentous fungi were screened for enantio-specific reduction of the α, β-unsaturated carbon bond in citral to produce citronellal. While a traditional aqueous screening system revealed only Zymomonas mobilis as positive, citronellal was produced in an aqueous/organic two liquid phase system by 11 of the 46 tested strains, which demonstrates the relevance of applying two-phase systems to screening strategies. Z. mobilis and Citrobacter freundii formed 1 mM citronellal in 3 h in the presence of a NADPH regenerating system and 20% (v/v) toluene. In comparison to these bacteria, the eukaryotic strains showed at least five-fold lower citral reductase activities. The bacterial strains produced preferentially the (S)-enantiomer of citronellal with e.e. values of >99% for Z. mobilis and 75% for Citrobacter freundii. In contrast the yeasts produced preferentially (R)-citronellal, i.e. Candida rugosa with an enantiomeric excess value of more than 98%. Many strains formed alcoholic by-products, viz. geraniol, nerol and citronellol. For Z. mobilis the production of these alcohols was suppressed in the presence of various organic solvents, e.g. toluene, and further decreased after EDTA addition.     

Interleukin (IL)-1 gene polymorphisms: relevance of disease severity associated alleles with IL-1beta and IL-1ra production in multiple sclerosis

BACKGROUND: Multiple sclerosis (MS) is an autoimmune disorder, with a considerable genetic influence on susceptibility and disease course. Cytokines play an important role in MS pathophysiology, and genes encoding various cytokines are logical candidates to assess possible associations with MS susceptibility and disease course. We previously reported an association of a combination of polymorphisms in the interleukin (IL)-1B and IL-1 receptor antagonist (IL-1RN) genes (i.e. IL-1RN allele 2+/IL-1B(+3959)allele 2-) with disease severity in MS. Extending this observation, we investigated whether IL-1beta and IL-1ra production differed depending on carriership of this gene combination. METHODS: Twenty MS patients and 20 controls were selected based upon carriership of the specific combination. In whole blood, in vitro IL-1beta and IL-1ra production was determined by enzyme-linked immunosorbent-assay after 6 and 24 h of stimulation with lipopolysaccharide. RESULTS: Carriers of the specific combination produced more IL-1ra, especially in MS patients, although not significantly. IL-1ra production was significantly higher in individuals homozygous for IL-1RN allele 2. In patients, Il-1ra production was higher and IL-1beta production lower compared with controls. In primary progressive patients, the IL-1beta /IL-1ra ratio was significantly lower than in relapsing-remitting patients. CONCLUSION: Our results suggest higher in vitro IL-1ra production in carriers of IL-1RN allele 2, with an indication of an allelic dose-effect relationship.     

Dmrt1 polymorphism covaries with sex‐determination patterns in Rana temporaria

Patterns of sex‐chromosome differentiation and gonadal development have been shown to vary among populations of Rana temporaria along a latitudinal transect in Sweden. Frogs from the northern‐boreal population of Ammarnäs displayed well‐differentiated X and Y haplotypes, early gonadal differentiation, and a perfect match between phenotypic and genotypic sex. In contrast, no differentiated Y haplotypes could be detected in the southern population of Tvedöra, where juveniles furthermore showed delayed gonadal differentiation. Here, we show that Dmrt1, a gene that plays a key role in sex determination and sexual development across all metazoans, displays significant sex differentiation in Tvedöra, with a Y‐specific haplotype distinct from Ammarnäs. The differential segment is not only much shorter in Tvedöra than in Ammarnäs, it is also less differentiated and associates with both delayed gonadal differentiation and imperfect match between phenotypic and genotypic sex. Whereas Tvedöra juveniles with a local Y haplotype tend to ultimately develop as males, those without it may nevertheless become functional XX males, but with strongly female‐biased progeny. Our findings suggest that the variance in patterns of sex determination documented in common frogs might result from a genetic polymorphism within a small genomic region that contains Dmrt1. They also substantiate the view that recurrent convergences of sex determination toward a limited set of chromosome pairs may result from the co‐option of small genomic regions that harbor key genes from the sex‐determination pathway.     

Chemical synthesis of polysaccharides. 7. Enzymatic hydrolysis of (1 → 6)-α-DL-glucopyranan (DL-dextran)

Hydrolysis of (1 → 6)-α-DL -glucopyranan (synthetic DL -dextran) by an endo-dextranase from a Penicillium species was examined in an acetate buffer solution (pH 5.3) at 37°C. Three samples of different tacticities (isotactic dyad content, 55, 63, and 72%) were employed with a clinical dextran for comparison. Colorimetric determination of the reducing end units of the saccharides produced during hydrolysis showed that the maximum degrees of hydrolysis based on the D-glucose units, (D.H.)_D, for the DL -dextrans were 21.4, 27.8, and 33.0% in the order of increasing isotacitic dyad content, whereas the (D.H.)_D value for the clinical dextran was 51.9%. A statistical treatment of the enzymatic hydrolysis is proposed to interpret the experimental results.