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.

Transcriptional regulation of the IL-5 gene in peripheral T cells of asthmatic patients.

Mechanisms that underlie the regulation of IL-5 gene expression in human peripheral T cells remain incompletely defined because of the low efficiency of transfection of plasmid constructs into non-transformed T cells. To elucidate the cellular and molecular mechanisms of IL-5 production, concanavalin A (ConA)-stimulated blastocytes derived from peripheral blood lymphocytes of asthmatic patients were employed in this study. Transcriptional activity of the synthetic human IL-5 promoter in ConA-stimulated blastocytes correlated with the production of IL-5. Deletion analysis of the reporter gene showed that the cis-regulatory element located at - 119 to - 80 is critical for inducible IL-5 promoter activity. Electrophoretic mobility shift assay revealed that an oligonucleotide probe corresponding to the element (- 119 to - 90) gave two specific bands. The slower migrating band was absolutely dependent on stimulation and was composed of a co-operative complex of the transcription factors, nuclear factor of activated T cells (NFAT) and activating protein-1 (AP-1). The faster migrating band was also inducible and was identified as AP-1-less NFAT. Mutation of either the NFAT or AP-1 element abrogated the slower migrating band and at the same time abolished transcriptional activity of the human IL-5 promoter/enhancer gene. Cyclosporin A equivalently suppressed DNA-binding activity of the composite NFAT/AP-1 site, promoter activity and protein production of IL-5. In conclusion, these data suggests that the composite NFAT/AP-1 binding element (- 115 to - 100) plays a crucial role in IL-5 synthesis by peripheral T cells of asthmatic patients.

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.

Selective regulation of T cell IL-5 synthesis by OM-01, JTE-711 and p38 MAP kinase inhibitor: independent control of Th2 cytokines, IL-4 and IL-5.

BACKGROUND: Helper T cells are involved in chronic eosinophilic inflammation. Control of cytokine production seems to be an effective management. METHODS: The effect of nonactin, SB203580, a p38 MAP kinase inhibitor, and JTE-711 on the cytokine production of allergen-specific T cell clones was determined. The effect of nonactin on the airway eosinophilia was investigated using murine asthma model. RESULTS: Nonactin suppressed IL-5 synthesis by human Th cells in a dose-dependent manner without affecting IL-2 or IL-4 synthesis, and still significantly suppressed murine airway eosinophilia induced by the antigen inhalation. SB203580 and JTE-711 also selectively inhibited IL-5 synthesis in vitro. CONCLUSION: Synthesis of IL-5 by human Th cells can be differentially regulated from that of other major T cell cytokines. The in vivo effects of selective IL-5 synthesis inhibitors suggest that IL-5 is the reasonable target for the regulation of allergic disorders accompanied by eosinophilic inflammation.

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.

Transcriptional regulation of IL-5 gene by nontransformed human T cells through the proximal promoter element.

OBJECTIVE: IL-5 is strongly involved in the eosinophilic inflammation in bronchial asthma and atopic dermatitis. We have previously reported that IL-5 synthesis in atopic and nonatopic asthmatics is significantly enhanced compared to control subjects. T cell IL-5 synthesis is regulated through several transcriptional elements, one of which is the proximal human IL-5 promoter (-62 to -46). The present study was undertaken to delineate the transcriptional regulation through this element using nontransformed human T cells. METHODS: Con A blast lymphocytes which tolerate electroporation were derived from peripheral blood lymphocytes. Luciferase reporter analysis and gel shift analysis were performed. RESULTS: The proximal promoter element is the overlapping binding site for the constitutive binding factor, Oct-1, and the inducible one, AP-1. The transcriptional induction was ascribed to the inducible binding, while the constitutive binding was rather inhibitory. A mutant element which lost the constitutive binding but retained the inducible binding exerted 3 times more transcriptional activity compared to the wild type element. In contrast, another mutant element which lost the inducible binding and retained the constitutive binding exhibited no transcriptional induction. Gel shift analysis clarified that the inducible binding was more prominent and the constitutive binding was less in IL-5-producing T cells derived from asthma patients compared to IL-5-nonproducing cells derived from control subjects. CONCLUSIONS: The ratio of the inducible/constitutive binding to the proximal promoter element may determine the capacity of human Th cells to transcribe IL-5 gene, and its regulation may control eosinophilic inflammation.

Control of IL-5 production by human helper T cells as a treatment for eosinophilic inflammation: comparison of in vitro and in vivo effects between selective and nonselective cytokine synthesis inhibitors.

BACKGROUND: Helper T cells are involved in the pathophysiologic condition of asthma, so modulation of cytokine production may be effective therapy. OBJECTIVE: We aimed to selectively control the synthesis of IL-5 by helper T cells and tested in vivo effects using a murine asthma model. METHODS: The effect of dexamethasone, FK506, cyclosporin A, and nonactin (a macrolide compound produced by Streptomyces griseus) on cytokine production by allergen-specific T-cell clones was determined. The effect of these agents and an anti-IL-5 neutralizing antibody on airway eosinophilic inflammation was investigated in a murine asthma model. RESULTS: Dexamethasone, FK506, and cyclosporin A suppressed the production of IL-2, IL-4, and IL-5 by human helper T cells, which shows a similar concentration-response relationship in each case. Cyclosporin A and dexamethasone inhibited airway eosinophilia in vivo. Nonactin suppressed IL-5 synthesis but not IL-2 or IL-4 synthesis, and it also significantly suppressed airway eosinophilia. CONCLUSION: Nonactin only suppressed IL-5 synthesis and was as effective against eosinophilia as cyclosporin A and dexa-methasone, which indicates that IL-5 is a reasonable therapeutic target in allergic disorders that are accompanied by eosinophilic inflammation.

Pathogenesis of allergic diseases: interactions between pollutants and pollens really important?

Nowadays, the prevalence of atopic diseases in so-called developed countries is estimated to exceed 30%. Furthermore, it may reach over 50% in two generations. Based on such facts, the so-called "atopic predisposition" can not be defined as an abnormal genotype consisting of certain "atopic gene(s)" possessed by a minority of unfortunate people. Rather, the gene(s) that cause atopic diseases reside in the common human gene repertoire, and several environmental factors would cause the overexpression of some constitutive gene(s), leading to the development of atopic diseases. The author considers that overexpression and production of Th2 cytokines, especially IL-5, may be a key event. The recent prevalence of atopic diseases in developed countries may be mediated by a change of Th2 polarization due to modern retrogression of environmental factors such as bacterial and viral infections that favor Th1 polarization. The "atopic trait" might be actually an "atopic phenotype" rather than an "atopic genotype". In other words, the atopic trait seems not to be a genotype that decides the development of an atopic disease on an all or nothing basis, but a phenotype that determines the susceptibility to the disease. Of course, the familial nature of atopic diseases is undeniable, but this does not necessarily indicate the genetic nature of atopic diseases. For example, if a parent suffers from tuberculosis, the possibility that the children will develop tuberculosis markedly increased. However, tuberculosis is a truly infectious disease. In this article, several environmental factors those may affect the recent sharp increase of atopic diseases are discussed.

Comparison of motor reactivity of the human colon cold-stored in different preservative solutions to carbachol and noradrenaline in vitro.

We have compared the reactivity to carbachol and noradrenaline of circular smooth muscle isolated from the human colon which was cold-stored at 4 degrees C in three different preservative solutions: Krebs bicarbonate solution (KBS), phosphate buffer solution (PBS) or minimum essential medium (MEM). Concentration-dependent contractions in response to carbachol were reduced in terms of both their sensitivity (pEC50) and reactivity (Emax), depending on the period of cold storage. The reduction was more marked when the tissue was cold-stored in either MEM or KBS than in PBS. Similar reduction of the relaxation response to noradrenaline was also observed after cold storage. It is concluded that the cold storage of surgically resected human colon in PBS for two to three days best preserved smooth muscle functions for pharmacological examinations.

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.

p38 mitogen-activated protein kinase regulates human T cell IL-5 synthesis.

Involvement of p38 mitogen-activated protein (MAP) kinase in human T cell cytokine synthesis was investigated. p38 MAP kinase was clearly induced in human Th cells activated through the TCR. SB203580, a highly selective inhibitor of p38 MAP kinase, inhibited the induction of p38 MAP kinase in human Th cells. Major T cell cytokines, IL-2, IL-4, IL-5, and IFN-gamma, were produced by Der f 2-specific Th clones upon stimulation through the TCR. IL-5 synthesis alone was significantly inhibited by SB203580 in a dose-dependent manner, whereas the production of IL-2, IL-4, and IFN-gamma was not affected. The proliferation of activated T cells was not affected. IL-5 synthesis of human Th clones induced upon stimulation with rIL-2, phorbol ester plus anti-CD28 mAb, and immobilized anti-CD3 mAb plus soluble anti-CD28 mAb was also suppressed by SB203580 in the same concentration response relationship. The results clearly indicated that IL-5 synthesis by human Th cells is dependent on p38 MAP kinase activity, and is regulated distinctly from IL-2, IL-4, and IFN-gamma synthesis. Selective control of IL-5 synthesis will provide a novel treatment devoid of generalized immune suppression for bronchial asthma and atopic dermatitis that are characterized by eosinophilic inflammation.

Primary role of CD4+ T cells and supplemental role of mast cells in allergic pulmonary eosinophilia.

BACKGROUND: We have recently demonstrated that allergic eosinophilic inflammation is transferred to unprimed mice by infusing IL-5-producing CD4+ T cells. The contribution of mast cells to the development of eosinophilic inflammation is controversial. METHODS: To clarify the possible different roles of CD4+ T cells and mast cells in eosinophilic inflammation, we compared antigen-induced airway eosinophilia between mast-cell-deficient mice (WBB6F1-W/W(v)) and their congenic normal littermates (WBB6F1-+/+). RESULTS: The time course study indicated that equivalent numbers of eosinophils were recruited into the airway of both +/+ and W/W(v) mice 6, 24, 96, and 216 h after antigen challenge, whereas the number of eosinophils 48 h after antigen challenge was significantly lower in W/W(v) compared to +/+ mice. Administration of either anti-CD4 or anti-IL-5 monoclonal antibody almost completely inhibited antigen-induced eosinophil recruitment in W/W(v) mice 48 h after antigen challenge. In contrast, the inhibitory effect of these antibodies in +/+ mice were partial (approximately 50% inhibition). Anti-CD4 and anti-IL-5 antibodies equally suppressed airway eosinophilia in both +/+ and W/W(v) mice 96 h after antigen challenge. CONCLUSION: Our study indicates that CD4+ T cells are crucially involved in the development of allergic eosinophilic inflammation, while mast cells may play a supplemental role depending on the kinetics of the response.

[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.

Cloned Th cells confer eosinophilic inflammation and bronchial hyperresponsiveness.

BACKGROUND: The essential role of Th cells and T cell cytokines in eosinophilic inflammation has been established. METHODS: To determine whether Th cells are sufficient for the development of airway eosinophilic inflammation, ovalbumin-reactive murine Th clones were established and infused into unprimed mice. RESULTS: Eosinophilic infiltration into the lung was induced upon antigen inhalation in parallel with the rise in bronchoalveolar lavage fluid (BALF) eosinophil peroxidase activity. Neither IgG, IgA, nor IgE antibodies were present in this model. Pathological examination showed swelling and desquamation of epithelial cells, mucous plugs, and goblet cell hyperplasia, all of which well resemble human asthma. Fluorescent probe labeled Th clones migrated into the lung prior to the eosinophil accumulation. Bronchial hyperresponsiveness (BHR) was clearly induced upon antigen inhalation. Anti-IL-5 monoclonal antibody abrogated the responses. Dexamethasone and cyclosporin A suppressed cytokine production by Th cells both in vitro and in vivo, BALF eosinophilia, and BHR. The number of eosinophils recovered in the BALF correlated with the intensity of BHR. CONCLUSION: The results clearly indicated that monoclonal Th cells are sufficient for the development of both airway eosinophilia and BHR. Agents capable of downregulating IL-5 production seem promising for the treatment of bronchial asthma.