Selective modification of fluciclovine (18F) transport in prostate carcinoma xenografts.

We investigated if previously demonstrated inhibition of fluciclovine (18F) in vitro could be replicated in a PC3-Luc xenograft mouse model. Following intratumoral injection of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), alpha-(methylamino)isobutyric acid (MeAIB) or saline, fluciclovine PET tumor-to-background activity was 43.6 (± 5.4)% and 25.3 (± 5.2)% lower in BCH (n = 6) and MeAIB (n = 5) injected PC3 Luc xenografts, respectively, compared to saline-injected controls (n = 2). Partial inhibition of fluciclovine uptake by BCH and MeAIB can be demonstrated in vivo similar to previous in vitro modeling.

A pair of concentric capillaries as an interface for gas chromatography and supersonic jet/multiphoton ionization/mass spectrometry.

A pair of concentric capillaries was developed to mix helium, which was used as the carrier gas for gas chromatography, with argon for efficient molecular cooling by supersonic jet expansion. A simple instrument was constructed for the evaluation of nozzle diameter using the Hagen-Poiseuille equation. The effects of nozzle diameter, type of expansion gas, flow rate, and the distance from the nozzle to the observation region were investigated. Mixing argon gas with the carrier gas helium resulted in efficient molecular cooling from 30 to 10 K and the complete disappearance of the background signal from the multiphoton ionization spectrum. Consequently, the spectral selectivity was significantly improved and the nozzle was successfully applied to isomer-selective analysis of dichlorotoluenes. Since the dead volume in the nozzle was negligible, it was suitable as an interface for gas chromatography and supersonic jet/multiphoton ionization/mass spectrometry.

Genetic contribution of the tumour necrosis factor (TNF) B + 252*2/2 genotype, but not the TNFa,b microsatellite alleles, to systemic lupus erythematosus in Japanese patients.

The contribution of the tumour necrosis factor (TNF) B + 252 (TNFB) dimorphism and microsatellite polymorphisms of TNFa and TNFb to the pathogenesis of systemic lupus erythematosus (SLE) was studied in Japanese patients. The TNFB dimorphism was determined using the restriction fragment length polymorphism (RFLP) method with NcoI digestion followed by specific polymerase chain reaction (PCR) amplification. TNFa and TNFb microsatellite polymorphisms were determined using the DNA sequencer and GeneScan program (Applera Corporation, Foster City, CA) followed by specific PCR amplification. HLA-DRB1*15 typing was carried out by the PCR-sequence specific conformational polymorphism (SSCP) method. In SLE, the allele frequency of TNFB*2 significantly increased (68.9%, P < 0.05) and the genotype frequency of TNFB*2/2 also increased (52.8%, P < 0.05). TNFB*2 showed no significant linkage disequilibrium with HLA-DRB1*1501. The prevalence of TNFa13 and TNFb4 showed very slight increases, but these increases were not significant. An association analysis indicated that TNFB*2/2 conferred greater, or at least equal, susceptibility to SLE in Japanese patients in comparison with HLA-DRB1*1501. The TNFB*2/2 genotype may contribute additively with DRB1*1501 to SLE in Japanese patients. No association was observed between auto-antibodies and TNF. TNFB*2 is a genetic marker for SLE in Japanese patients, while TNFa and TNFb microsatellites are not associated with SLE.

Cloning and expression of cDNA encoding the complete prepro-form of an isoform of Der f 1, the major group 1 allergen from house dust mite Dermatophagoides farinae.

cDNA clones encoding a major house dust mite allergen, Der f 1, were isolated from a Dermatophagoides farinae cDNA library by plaque immunoscreening using rabbit anti-Der f 1 serum. The sequences cover the complete open reading frame encoding the prepro-form. The sequence is different from previously reported cDNA of Der f 1 in six bases and the encoded amino acid sequence is different in two residues. Pro-forms of Der f 1 and its mutant, in which the N-glycosylation motif was disrupted, expressed in Pichia pastoris were converted to the mature forms by an in vitro activation process and they showed significant IgE-binding. The biologically active rDer f 1 molecules would be useful for diagnostic testing and allergen-specific immunotherapy. In contrast, Der f 1 directly expressed in Escherichia coli without the prosequence had very low IgE binding. The hypoallergenic Der f 1 polypeptide could be useful for safer and more effective immunotherapy.

Biologically active recombinant forms of a major house dust mite group 1 allergen Der f 1 with full activities of both cysteine protease and IgE binding.

BACKGROUND: Group 1 allergens from mite faeces, Der f 1 and Der p 1, are the most significant in-door allergens. Therefore, they are the most important component in the standardization of house dust mite extract for diagnosis and allergen-specific immunotherapy (AIT). Although their cDNAs have been cloned, efforts to prepare biologically active recombinant forms in expression systems using bacteria or yeast have failed. OBJECTIVE: Our purpose is to establish an efficient system to prepare recombinant Der f 1(rDer f 1), identical in quality to native Der f 1. METHODS: The preproforms of Der f 1 and a mutant N53Q, whose consensus motif for N-glycosylation was disrupted, were expressed in yeast Pichia pastoris. Cysteine protease activity and IgE reactivity were analysed using synthetic substrates and by RAST-EIA, respectively. RESULTS: The proforms of the two rDer f 1 molecules were efficiently secreted into culture medium. Their prosequences were removed autocatalytically by dialysis against acidic buffer. Although the wild-type rDer f 1 was more highly glycosylated than native Der f 1, N53Q had almost the same apparent molecular weight as native Der f 1 on SDS-PAGE. Both the protease and IgE binding activities of the mature rDer f 1 molecules were the same as those of native Der f 1, whereas the proforms had no or markedly reduced activities. CONCLUSION: The efficient system to prepare active rDer f 1s established in this study is useful for diagnosis and standardized AIT for house dust mite allergy. Furthermore, the system would be a tool for analysis of IgE epitopes, determination of tertiary structure, allergen engineering for safer and more effective AIT, resolving the relation between the enzymatic activity and pathogenesis, and the development of therapeutic inhibitors.

Concepts of the pathogenesis of allergic disease: possible roles of Epstein-Barr virus infection and interleukin-2 production.

The prevalence of atopic diseases in so-called developed countries has risen over the past several decades, which is too short a period for genetic changes to have taken place. Furthermore, the increase has occurred irrespective of race or geographical differences in the prevalence of atopic diseases. These facts strongly suggest that the increase is due not to genetic factor(s) but to environmental factor(s). In this article, previous investigations into the pathogenesis of allergic disease are reviewed. The roles of immune cells, cytokines, oncoproteins and viral infections are examined.

[Invasive amebiasis at an institution for the mentally retarded in Hyogo Prefecture].

An outbreak of amebiasis caused by Entamoeba histolytica occurred at an institution for mentally retarded persons in Hyogo Prefecture. Twelve out of a total of 49 admitted persons exhibited E. histolytica cysts in their stool, and 13 including persons in whom no cysts had been detected showed positive serological reactions for E. histolytica infection. However, neither the cyst nor the antibody against the organism was detected in the staff members of the institution. Indirect fluorescence antibody test and sandwich enzyme-linked immunosorbent assay with a monoclonal antibody specific for pathogenic strains of E. histolytica revealed that all trophozoite strains grown from cysts in stool samples from five patients were pathogenic. Epidemiological analysis strongly suggested that a patient in the institution had been infected with an organism from a patient outside the institution, and that infection may have spread among the admitted persons due to abnormal behavior. Administration of metronidazole resulted in effective elimination of the cysts from the stool of the cyst-carriers.

Cyclic AMP suppresses interleukin-5 synthesis by human helper T cells via the downregulation of the calcium mobilization pathway.

1. To delineate the mechanism by which cyclic AMP (cAMP) suppresses interleukin (IL)-5 synthesis, the effects of prostaglandin (PG) E2, forskolin, dibutyryl (db)-cAMP and the Ca2+ ionophore, ionomycin on cytokine synthesis, proliferation and CD25 expression of human T cells were investigated. Further studies were performed by measurement of the intracellular concentrations of cyclic AMP ([cAMP]i) and Ca2+ ([Ca2+]i) and by electrophoretic mobility shift analysis (EMSA). 2. PGE2, forskolin and db-cAMP suppressed IL-5 production by human T cell line following T cell receptor (TCR)-stimulation. PGE2 suppressed TCR-induced messenger RNA (mRNA) expression of IL-2, IL-4 and IL-5, as well as proliferation and CD25 expression. 3. Cyclic AMP-mediated suppression of cytokine synthesis, proliferation and CD25 expression in human T cells were attenuated by ionomycin. 4. [cAMP]i was increased by PGE2 and forskolin. PGE2 suppressed the TCR-induced biphasic increase in [Ca2+]i. EMSA revealed that four specific protein-DNA binding complexes related to NF-AT were detected at the IL-5 promoter sequence located from -119 to -90 relative to the transcription initiation site. The slowest migrating complex induced by TCR stimulation was enhanced by PGE2 and further upregulated by ionomycin. Another binding which did not compete with cold AP-1 oligonucleotides, was constitutively present and was unaffected by PGE2 but enhanced by ionomycin. 5. The suppressive effect of cyclic AMP on human IL-5 synthesis is mediated by interference with intracellular Ca2+ mobilization but distinct from the NF-AT-related pathway.

Transcriptional control of the IL-5 gene by human helper T cells: IL-5 synthesis is regulated independently from IL-2 or IL-4 synthesis.

BACKGROUND: IL-5 is fundamentally involved in eosinophilic inflammation. Control of IL-5 production may be effective for the management of allergic diseases. OBJECTIVE: We aimed to find the transcriptional mechanisms that regulate the IL-5 gene to selectively control IL-5 synthesis. METHODS: Allergen-specific T-cell clones and T-cell hybridomas were established from the peripheral blood lymphocytes of patients with asthma, and the transcriptional regulation of the IL-5 gene was investigated with transient transfection and electrophoretic mobility shift analysis. RESULTS: A human IL-5 promoter/enhancer-luciferase gene construct, pIL-5(-511)Luc, was transcribed on activation of IL-5-producing T-cell clones, but not IL-5-nonproducing clones. pIL-5(-511)Luc was transcribed by T-cell hybridomas derived from fusion between IL-5-producing T-cell clones and an IL-5 gene-nonexpressing T-cell line, but not by hybridomas derived from IL-5-nonproducing T-cell clones. IL-5 synthesis was not only induced by T-cell receptor stimulation but also by IL-2 receptor stimulation. Binding of NF-AT, NF-kappaB, and AP-1 was induced by T-cell receptor (TcR) stimulation, although there was no significant upregulation of binding by IL-2 stimulation. CONCLUSION: IL-5 synthesis by human helper T cells is regulated at the transcriptional level. A unique transcriptional mechanism distinct from those regulating the IL-2 or IL-4 genes seems to control the IL-5 gene. Selective regulation of IL-5 gene transcription may be useful for treating eosinophlic inflammation.

IL-6 synthesis by rheumatoid synoviocytes is autonomously upregulated at the transcriptional level.

BACKGROUND: Involvement of IL-6 in the pathogenesis of rheumatoid arthritis has recently been demonstrated, but the mechanism of its production by rheumatoid synoviocytes is still poorly defined. OBJECTIVE: The purpose of this study was to clarify the cellular and molecular mechanisms involved in the spontaneous production of IL-6 by fibroblast-like synoviocytes obtained from patients with rheumatoid arthritis. METHODS: Cloned synoviocytes were established by the limiting dilution method. IL-6 synthesis was evaluated by ELISA and Northern blot analysis. IL-6 gene transcription and transcription factors were analyzed by the transient transfection of luciferase reporter plasmids and the electrophoretic mobility shift assay, respectively. RESULTS: IL-6 synthesis by cloned rheumatoid synoviocytes was spontaneously upregulated at the transcriptional level. Enhanced IL-6 production by high-producing clones was independent of cytokines from other cell populations or autocrine production of tumor necrosis factor-alpha and IL-1. Deletion analysis showed that the IL-6 promoter was regulated by 2 positive elements (-159 to -142 base pair and -77 to -59 base pair). The transcriptional activity of the latter element was upregulated in clones showing high IL-6 production. The binding activity of NF-kappaB p50/p65 heterodimer and RBP-Jkappa was enhanced in these clones. CONCLUSION: IL-6 production by rheumatoid synoviocytes is autonomously upregulated at the transcriptional level and spontaneous activation of NF-kappaB and RBP-Jkappa seems to be involved.

Non-anaphylactic combination of partially deleted fragments of the major house dust mite allergen Der f 2 for allergen-specific immunotherapy.

Allergen-specific immunotherapy, in which repeated injections of allergens over prolonged periods are used to induce tolerance, has proven an effective treatment of allergy. A major side effect of allergen-specific immunotherapy is anaphylactic reaction. House dust mite allergens are major causative factors associated with various allergic diseases. Der f 2 is the major house dust mite allergen composed of 129 amino acid residues. Analysis using deletion mutants of Der f 2 suggested that T-cell epitopes of Der f 2 were multiple in mite-allergic patients. We found that some IgE epitopes were renatured by dialysis of a mixture of two denatured C- and N-terminal deletion mutants, 1-112 and 85-129 in 13 patients out of 14. On the other hand, IgE binding activity was negative in the separately dialyzed fragments and their mixture in each patient tested. Furthermore, we demonstrated that neither of the two separately prepared polypeptides induced in vivo skin prick test reactivity. These findings are important for improvement of T-cell targeting allergen-specific immunotherapy and development of monovalent IgE haptens. The use of combinations of overlapping non-anaphylactic fragments of allergen covering all of the T-cell epitopes achieves the removal of IgE reactivity, the cause of harmful anaphylactic reactions, without affecting the T-cell reactivity essential for immunotherapy, offering potentially safer and more effective treatment for allergic disease.

Regulation of interleukin-1beta-induced interleukin-6 gene expression in human fibroblast-like synoviocytes by glucocorticoids.

Involvement of interleukin-6 (IL-6) in the pathogenesis of rheumatoid arthritis (RA) has recently been demonstrated. In the present study, the regulation of IL-6 gene expression by glucocorticoids and IL-1beta in fibroblast-like synoviocytes (FLSs) was investigated. Both synthetic and natural glucocorticoids, i.e., dexamethasone (DEX) and hydrocortisone (HC), respectively, concentration-dependently inhibited protein production and gene expression of IL-6 by human FLSs. The effect of DEX was dependent on de novo protein synthesis. DEX significantly reduced the rate of IL-6 gene transcription without affecting the stability of IL-6 mRNA. The IkappaBalpha pathway seemed not to be involved in DEX-mediated inhibition of IL-6 gene expression in IL-1beta-stimulated human FLSs. These findings suggest that glucocorticoids suppress IL-6 gene transcription by an as yet undefined mechanism.

Establishment and characterization of a novel human rheumatoid fibroblast-like synoviocyte line, MH7A, immortalized with SV40 T antigen.

A novel immortalized rheumatoid fibroblast-like synoviocyte (FLS) line, MH7A, was established by stably transfecting FLS cells with SV40 T antigen gene. MH7A cells expressed SV40-specific small t and large T antigens as well as an elevated level of p53 protein. They have already reached over 150 population doublings through culture crisis, and have been growing rapidly compared with the parental FLSs. Constitutive activation of p42/p44 mitogen-activated protein (MAP) kinase was detected in MH7A cells. Serum requirements for the growth of MH7A were markedly decreased compared with those for the parental FLSs. MH7A cells were stained positively for interleukin (IL)-1R, intercellular adhesion molecule-1 (ICAM-1), CD16, CD40, CD80, and CD95. IL-1beta enhanced the production of IL-6 and stromelysin-1, and the surface expression of ICAM-1, in a manner similar to that in the parental FLSs. SB203580, a specific inhibitor of p38 MAP kinase, significantly inhibited IL-1beta-induced IL-6 and stromelysin-1 production by both parental FLSs and MH7A cells; although PD098059, an inhibitor of the p42/p44 MAP kinase pathway, did not affect it. Our results clearly indicate the usefulness of MH7A cells for investigating the regulation of rheumatoid FLSs and the IL-1 signal transduction pathway to develop future RA therapy.

Regulation of interleukin-1beta-induced interleukin-6 gene expression in human fibroblast-like synoviocytes by p38 mitogen-activated protein kinase.

Involvement of p38 mitogen-activated protein (MAP) kinase in interleukin (IL)-6 gene expression of human fibroblast-like synoviocytes (FLSs) was assessed. p38 MAP kinase was constitutively expressed in human FLSs and activated in response to IL-1beta. A pyridinylimidazole compound, SB203580, inhibited p38 MAP kinase activity in vivo, since the activity of MAPKAP kinase-2 (a substrate of p38 MAP kinase) in IL-1beta-stimulated FLSs was totally suppressed by it. SB203580 concentration-dependently inhibited protein production and gene expression of IL-6 by human FLSs. The effect of SB203580 was dependent on de novo protein synthesis. SB203580 significantly reduced the stability of IL-6 mRNA without affecting the rate of IL-6 gene transcription. Here, we provide evidence that p38 MAP kinase is activated in response to IL-1beta in human FLSs and is involved in IL-6 synthesis by stabilizing IL-6 mRNA.

Hyposensitization to allergic reaction in rDer f 2-sensitized mice by the intranasal administration of a mutant of rDer f 2, C8/119S.

C8/119S is a mutant of recombinant Der f 2 (rDer f 2), and lacks a disulphide bond possessed by wild-type rDer f 2. In humans and mice, C8/119S has a very weak IgE-binding capacity compared with the wild-type, but possesses a T cell reactivity comparable to that of the wild-type. C8/119S may thus be a safe immunotherapeutic agent for house dust mite allergy. The aim of the present study was to evaluate whether the intranasal administration of C8/119S could suppress an immediate allergic reaction in mice sensitized with wild-type rDer f 2, possessing an allergic activity comparable to native counterparts purified from mite extract. Seven-week-old male A/J mice were immunized with wild-type rDer f 2 four times, and then intranasally administered 0.2-2 microg of wild-type, 0.2-20 microg of C8/119S, or PBS alone, three times a week for 4 weeks. Seven days after the last administration, the mice were examined for an immediate allergic reaction. The animals administered 2 microg of C8/119S (C2.0 group) showed significantly reduced immediate bronchoconstriction provoked by the i.v. injection of 1 and 10 microg of wild-type rDer f 2, compared with the PBS-treated mice. Similar results were obtained when we examined mice 10 weeks after the last administration. The reactions in the other groups given wild-type or C8/119S also tended to decrease in severity in comparison with the animals of the PBS group. The allergic phenotypes of the T cells, B cells, and basophils in the C2.0 group were shifted to that of naive mice without immunization. We conclude that C8/119S has hyposensitizing activities in mice sensitized with wild-type rDer f 2. C8/119S may be useful for immunotherapy of house dust mite allergy.

Transcriptional roles of CCAAT/enhancer binding protein-beta, nuclear factor-kappaB, and C-promoter binding factor 1 in interleukin (IL)-1beta-induced IL-6 synthesis by human rheumatoid fibroblast-like synoviocytes.

The involvement of interleukin (IL)-6 in the pathogenesis of rheumatoid arthritis (RA) has been recently demonstrated. IL-1beta stimulated rheumatoid fibroblast-like synoviocytes (FLSs) to produce IL-6 in a concentration- and time-dependent manner. In the present study we investigated how the IL-6 promoter is transcriptionally regulated in rheumatoid FLSs in response to a physiologically relevant mediator of inflammation, IL-1beta. Deletion analysis showed that the IL-6 promoter is regulated by two positive elements (located at -159 to -142 base pairs (bp) and -77 to -59 bp). Electrophoretic mobility shift assays revealed that CCAAT/enhancer binding protein-beta (C/EBPbeta) binding to nucleotides -159 to -142 bp was constitutively present. The probe corresponding to nucleotides -77 to -59 bp gave three positive bands. The two slower migrating bands were induced by IL-1beta and comprised an nuclear factor (NF)-kappaB p50/p65 heterodimer and a p65/p65 homodimer. The faster migrating band was constitutively expressed and identified as Epstein-Barr virus C-promoter binding factor 1, CBF1. Site-specific mutagenesis analysis demonstrated that the NF-kappaB and CBF1 binding elements regulated inducible activity of the IL-6 promoter in response to IL-1beta stimulation, whereas the C/EBPbeta binding element mainly regulated basal activity. We also provide the first evidence that CBF1 functions as a positive regulator of human IL-6 gene transcription.

Constitutive transcription of the human interleukin-6 gene by rheumatoid synoviocytes: spontaneous activation of NF-kappaB and CBF1.

The involvement of IL-6 in the pathogenesis of rheumatoid arthritis (RA) has been recently demonstrated. In the present study, we investigated the cellular and molecular mechanisms involved in the spontaneous IL-6 production by the fibroblast-like synoviocytes (FLSs) obtained from patients with RA. Cloned FLSs were established from the bulk cultures of FLSs by the limiting dilution method. Some FLS clones spontaneously produced large amounts of IL-6, whereas others produced low amounts of it. Neither anti-human TNF-alpha nor IL-1 antibody affected spontaneous IL-6 production of these FLS clones, suggesting that IL-6 production of the FLSs was endogenously up-regulated. A luciferase reporter plasmid containing the human IL-6 promoter region was significantly transcribed when transfected into the IL-6 high-producing clones, indicating that the rheumatoid FLSs retained constitutive transcriptional activity of the IL-6 gene. Electrophoretic mobility shift assays revealed that the binding activity of p50 and p65 NF-kappaB subunits and CBF1 was significantly enhanced in the IL-6 high-producing clones compared with that of IL-6 low-producing clones and cultured sarcoma cells, suggesting that spontaneous activation of NF-kappaB and CBF1 may lead to the constitutive transcription of the IL-6 gene by rheumatoid FLSs.

A selective type 4 phosphodiesterase inhibitor, T-440, modulates intracellular cyclic AMP level and interleukin-2 production of Jurkat cells.

Effect of a selective type 4 phosphodiesterase (PDE4) inhibitor, T-440, on intracellular cyclic AMP (cAMP) level and interleukin (IL)-2 production of Jurkat cells was investigated. T-440 suppressed both cAMP-PDE activities in cytosolic and membrane fractions of Jurkat cells. Intracellular cAMP level in Jurkat cells was elevated by PGE2 and forskolin but not by T-440. T-440, however, significantly enhanced the increase of cAMP by PGE2. PGE2 and forskolin inhibited IL-2 production of Jurkat cells stimulated with concanavalin A. T-440 by itself did not affect IL-2 production, but significantly enhanced the effect of PGE2 on IL-2 production. The increase of intracellular cAMP by T-440, PGE2, forskolin and T-440 plus PGE2 was well correlated with the inhibition of IL-2 production. These results indicate that IL-2 production of T cells is regulated by cAMP-PDE activity. Immunomodulatory effects of PDE4 inhibitors like T-440 should further be explored in vivo.

Development of lung eosinophilic inflammation by the infusion of IL-5-producing T cell clones.

To delineate the critical role of T cells on asthma, we tested whether eosinophilic inflammation of the bronchial mucosa is induced by transfer of IL-5-producing T cell clones, in the absence of antigen-specific immunoglobulins (IgE, A and G). Ovalbumin-specific T cell clone, FI5, that produced IL-5 upon challenge with relevant antigen was established. Eosinophilic inflammation of the lung occurred when unprimed mice were transferred with FI5 and challenged by the inhaled antigen. Eosinophil infiltration was completely suppressed by the administration of anti-IL-5 neutralizing antibody, indicating the essential role of IL-5. We concluded that the existence of IL-5-producing helper T cells is sufficient for the development of airway eosinophilic inflammation.