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  • 1
    ISSN: 1432-0428
    Keywords: Interleukin 1 ; pancreatic islets ; preproinsulin mRNA ; proinsulin biosynthesis ; Type 1 (insulin-dependent) diabetes mellitus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Human crude and recombinant interleukin 1 (IL-1) was found to dose- and time-dependently affect the biosynthesis of (pro)insulin in isolated rat islets of Langerhans. Incubation of rat islets with either 0.5 U/ml or 5 U/ml of crude IL-1 for 1 h had no detectable effect on (pro)insulin biosynthesis. After 24 hours of exposure 0.5 U/ml of crude or 0.6 ng/ml of recombinant IL-1 (beta) increased the (pro)insulin biosynthesis by 42% and 58%, respectively, whereas a 10-fold greater concentration of IL-1 decreased the (pro)insulin biosynthesis by 74% and 89%, respectively. The increase in (pro)insulin biosynthesis was accompanied by an increase in total protein biosynthesis indicating a nonspecific stimulatory action of low IL-1 concentrations. In contrast, high IL-1 concentrations caused a more selective decrease of the (pro)insulin biosynthesis when compared to the total protein biosynthesis. In addition, low IL-1 concentrations were found to increase and high concentrations to decrease the relative levels of pre-proinsulin mRNA suggesting that IL-1 may act both at a pre- and post-translational level of insulin biosynthesis.
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  • 2
    ISSN: 1365-3083
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The effects of dietary supplementation with ω-3-polyunsaturated fatty acids (ω-3-PUFA) on the proliferative response of PBMC and on the secretion of monokines and arachidonic acid metabolites from PBMC and monocytes (Mo) from healthy subjects and patients with recent-onset insulin-dependent diabetes mellitus (IDDM) were examined. Three groups of eight to nine healthy individuals were randomized to either 2.0 g/day or 4.0 g/day of ω-3-PUFA devoid of vitamins A and D, or an isocaloric amount of placebo. Furthermore, eight patients with recent-onset IDDM received 4.0 g/day of ω-3-PUFA. IL-lβ production and TNF-α secretion was determined before and after 7 weeks of treatment, and 10 weeks after withdrawal of treatment. Significant increases in platelet and PBMC membrane eicosapentaenoic acid was found in ω-3-PUFA-treated individuals. ω-3-PUFA treatment significantly reduced the content of IL-Ib in lysates of PBMC, but did not affect PBMC or Mo secretion of IL-1β, TNF-α or prostaglandin E2 (PGE2) or PBMC leukotriene B4 (LTB4) secretion in healthy subjects or in IDDM patients. A significant inhibition of the PHA-stimulated. but not the spontaneous or PPD-stimulated, proliferative response of PBMC was observed in healthy and diabetic subjects treated with (o-3-PUFA. No correlation was found between PHA-stimulated PBMC proliferation and PBMC secretion of TNF-α and IL-1β. There were no significant differences in the spontaneous or the PPD-or PHA-stimulated proliferative responses of PBMC between diabetic and healthy individuals at entry. We conclude that although dietary supplementation with 4.0 g/day of ω-3-PUFA inhibits the proliferation of PBMC and reduces IL-I immunoreactivity in PBMC and Mo, it does not alter monokine, PGE2 or LTB4, secretion in healthy or IDDM subjects.
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  • 3
    ISSN: 1365-3083
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: HLA-class III region genes may be associated with susceptibility to insulin-dependent diabetes mellitus (IDDM). In this study an Ncol polymorphism of the tumour necrosis factor beta (TNF-β) gene, which is positioned next lo the tumour necrosis factor alpha (TNF-α) gene in the HLA class 111 region, was detected by restriction fragment length polymorphism (RFLP). This polymorphism has previously been reported lo be located in the TNF-α gene. Caucasian HLA-DR3.4 heterozygous IDDM patients (n=-26) and DR-matched healthy controls (n=19). as well as randomly selected IDDM patients (n = 27) and controls (n = 25) were studied. In addition four multiplex families (49 individuals) and eight HLA-non-identical sibpairs concordant for IDDM were analysed.The TNF-β gene RFLP analysis showed fragments of 5.5 kb and 10.5 kb, which behaved as alleles. In all groups there was a haplotype assignment of the TNF-β 5.5-kb allele to BS, DR3 haplotypes, and of the TNF-β 10.5-kb allele to B15.DR4-positive haplotypes. The allelic and genotypic frequencies differed between DR3.4 IDDM patients and DR3,4 controls, and the DR3.4 control group differed significantly from the randomly selected control group (P 〈 0.0079), In HLA-DR3,4-atid DQw8-positive persons, the DR3 haplotypes carried the 10.5-kb allele ihrcL- times more frequently in IDDM patients than in controls, suggesting that the 10.5-kb allele when present on DR3 haplotyes may contribute lo susceplibility to IDDM in DR3.4 heterozygous individuals, A contributory role of the Hl.5-kballele in genetic IDDM susceptibility was supported by the sibpair analysis, in which all were TNF-/1 identical. Five were 10,5 kb homozygous. and the remaining three pairs were 5.5.10,5 kb heterozygous.Twenty-five healthy and eight newly diagnosed IDDM patients were randomly selected to study the Escherichia coli lipopolysaccharides (LPS)-purified protein derivate (tuberculin) (PPD)-, and phytohaemagglutinin (PHA)-stimulated monocyte (Mo) secretions of interleukin 1 bela (IL-1/J)and TNb-α in relation lo the Ncol TNF-/f gene polymorphism. The LPS- and PHA stimulated Mo IL-l/f and TNF-a: secretions were significantly lower for the TNF-β 5.5.10,5 kb heterozygous individuals than for TNF-β 10.5 kb homozygous individuals. Furthermore, the Mo IL-1β and TNF-a secretions of IDDM patients were significantly higher than the Mo secretions of TNF-β genolype-matched healthy controls.This study suggests an association between the 10.5 kb TNF-β allele and IDDM, and demonstrates an association between monokine responses and TNF-β genotypes. These observations may have implications for understanding the pathogenesis of HLA-associated autoimmune diseases including IDDM.
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  • 4
    ISSN: 1365-3083
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The particular susceptibility to insulin-dependent diabetes mellitus (IDDM) conferred by HLA-DR3,4 heterozygosity has been suggested to be an effect of transcomplementation of HLA class II molecules. To test this hypothesis of special IDDM-specific hybrid determinants and to evaluate the T-cell repertoire towards a specific antigen in IDDM patients we generated a total of 352 PPD-specific T-cell lines by the soft-agar cloning technique and studied their restriction by HLA class II molecules. Of these lines, 227 were from nine IDDM patients, of whom six were DR3,4 heterozygotes, and 125 from 10 healthy controls Forty-six T-cell lines elicited specific responses in at least two experiments and in addition to T-cell lines demonstration class-II-restricted PPD specificity, lines with an alloreactivity occurred. HLA-DQ-restricted PPD-specific T-cell line were not identified and a possible DP restriction (DPw2) was only observed with one line. These data indicate that PPD is preferentially presented to T cells in the context of HLA-DR/Dw, Presentation of PPD by hybrid molecules in IDDM patients or by IDDM-specific class II epitopes recognized by the T-cell lines was not demonstrated. By restriction fragment length polymorphism analysis using a probe for the joining region of the T-cell receptor gamma gene, T-cell lines generated by the soft-agar cloning technique were found to be oligoclonal. It is concluded that soft-agar cloning should be followed by subsequent limiting dilution in order to assure monoclonality. Different preparations of antigen-presenting cells (APC) were tested. In several cases the T-cell lines were not able to respond to PPD presenting by Epstein -Barr-virus transformed lymphoblastoid cell lines (LCL). It was demonstrated that lipopolysaccharides (LPS) of E. coli potently reduce the proliferative response of antigen-specific and alloreactive T cells when T-cell-depleted peripheral blood mononuclear cells (E− cells) were used as APC, whereas only limited inhibition was observed when LCL were used as APC in the presence of LPS. This effect of LPS is suggested to be mediated by increased prostaglandin secretion by monocytes among the E− cells since indomethacin abolished the effect of LPS. This observation may have implications for T cell cloning procedures since we have found that most commercially available culture media are heavily contaminated with endotoxin.
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  • 5
    ISSN: 1365-3083
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Recombinant human interleukin 1β (rhIL-1β) and supernatants of Escherichia coli lipopolysaccharides-stimulated human monocyte (Mo) cultures, containing native human IL-lβ (nhIL-1β), demonstrate significant differences when tested in the mouse co-stimulatory thymocyte (lymphocyte activating factor [LAF]) assay. The aims of the present study were to investigate this characteristic difference between rhIL-1β and Mo culture supernatants (Mo supernatants), and to compare the biological and the immunological activity of preparations of rhlL-lβ and nhlL-1β during each step of an identical purification procedure. The biological activity of rhIL-1β/nhIL-lβ preparations was characterized by the use of the LAF assay and the rat islet insulin release assay. An IL-1β enzyme-linked immunosorbent assay (ELISA) was established in order to compare the biological and immunological responses of the IL-lβ preparations.We report that the significant difference between rhIL-lβ and supernatants of Mo cultures, which was only demonstrable in the LAF assay, is due to the presence of interleukin 6 (IL-6) in the Mo supernatants. We describe a simple cation exchange chromatography separating nhlL-lβ and lL-6 of Mo supernatants. The highly purified rhIL-β possessing the correct amino-terminal sequence and nhIL-lβ have identical biological and immunological activities demonstrating a specific biological activity (SBA) of 3x102 U/ng IL-lβ, Thus, we have no indications of secondary or tertiary structural differences between rhIL-1β and purified nhIL-lβ.In contrast, both in the LAF assay and in the rat islet insulin release assay the SBA of anaminoextended rhIL-lβ form, Met-Glu-Ala-Glu-rhIL-lβ, was only 1-2% of the SBA of rhIL-lβ, suggesting that structural changes were introduced into the molecule by the amino-terminal extension. In the present study we have demonstrated that systematic combined testing of IL-lβ preparations in two different biological assays and an immunological assay is useful for the characterization and comparison of the activity of recombinant and native IL-1β preparations purified by the use of exactly the same procedures.
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  • 6
    ISSN: 1365-3083
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The secretions of interleukin 1 (IL-1), tumour necrosis factor α (TNF), and prostaglandin E2 (PGE2) of low-dose E. coli lipopolysaccharide (LPSJ-stimulated human monocytes (Mø) were investigated in an endotoxin (ET)-free milieu (〈1.6 pg LPS/ml). Human Mø cultures from nine healthy men were stimulated with 0, 12.5–500, and 250,000 pg LPS/ml as measured by a very sensitive Limulus test. The IL-1 activity was tested by the mouse costimulatory thymocyte (LAF) assay, which was thoroughly standardized and characterized (interassay variation 22–24%, intra-assay variation 3–7%). Spontaneous Mø secretions of IL-1, TNF, and PGE2 were negligible, but 12.5 pg LPS/ml significantly stimulated the secretions of these Mø products and the monokine responses to 500 and 250,000 pg LPS/ml were almost in the same range. It was demonstrated that the secretions of IL-1-TNF and TNF-PGE2 were strongly correlated. Pronounced interindividual differences in LPS responsiveness were demonstrated, and two low-responders, one of whom was HLA-DR 1,2-positive, were identified. Three first-degree relatives of the DR1.2-positive low-responder had similar low responses. Furthermore. Mø cultures were prepared weekly for 4 weeks from four HLA-DR different men and the only DR2.2 homozygous individual had low monokine responses. In conclusion, stable interindividual differences in in vitro monokine and PGE2 secretions of LPS-stimulated Mø were demonstrated. It is suggested that HLA-DR2-positive individuals may be low responders.
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  • 7
    ISSN: 1365-3083
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Interleukin 1β(IL-1β) and tumour necrosis factor alpha (TNF-α) may be pathogenetically important in insulin-dependent diabetes mellitus (IDDM), which is associated with genes of the HLA region. Since a regulatory role of HLA region genes on monokine production may exist, we looked for an association between the monokine and prostaglandin E2 (PGE2) responses of monocytes (Mo) from 20 healthy males (18–50 years) with HLA-DR types relevant for IDDM susceptibility and resistance (DR 1,2, DR 1,3, DR 1,4, DR 3,4). Monokine assays were established and evaluated and the secretions of IL-1β, TNF-α, and PGE2 measured in Mo cultures (2 h, 6 h, 20 h) prepared by endotoxin-free techniques and stimulated by low-dose E. coli lipopolysaccharides (LPS). There were no significant associations between Mo responses and HLA-DR phenotype. Likewise, Mo from DR2 (n=5) and DR4 (n= 5) homozygous healthy males demonstrated no significant differences in monokine and PGE2 responses of Mo.In the HLA class III region a diallelic TNF-β gene Ncol polymorphism consisting of alleles of 5.5 kb and 10.5 kb was recently described and associated with susceptibility to autoimmune diseases including IDDM. We report that IL-1β and TNF-α responses of Mo from TNF-β 10.5 kb homozygous healthy individuals were significantly higher than for TNF-β 5.5/10.5 kb heterozygotes.IL-1β and TNF-α responses of Mo from males (18–35 years) with newly diagnosed (n= 10) and long-standing IDDM (n= 10) and from age- and HLA-DR-matched healthy males (n= 10) were studied. LPS, gamma interferon (IFN), and TNF-α-stimulated Mo cultures were investigated. No significant differences were found between Mo responses of IDDM patients and controls. IFN (1000 U/ml) in the presence of LPS significantly potentiated LPS-stimulated Mo TNF-α secretion and reduced the levels of IL-β immunoreactivity in Mo lysates. IFN and TNF-α did not have any effects on LPS-stimulated Mo secretion of IL-1 β immunoreactivity.We conclude that Mo IL-1β and TNF-α production is normal in patients with recent-onset and long-standing IDDM. The interindividual differences in monokine responses may be accounted for by the diallelic human TNF-β gene polymorphism rather than by HLA class II genes. This observation may be important for understanding the association of certain H LA haplotypes with autoimmune phenomena and disease.
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  • 8
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    ISSN: 1365-3083
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The in vitro study of monocytes (Mo) poses several problems. Minor contamination with endotoxin (ET) of media ami utensils as well as adherence to glass or plastic surfaces may activate the cells and cause pronounced production of monokines. Many commercially liquid culture media were found to contain ET in concentrations above 25x10−12 g/ml. A simple system for the removal of ET from media and solutions was established by use of a comniercially available Polymyxin B Septiarose gel, To measure the lipopolysaccharide (LFS) binding capacity of the gel. known concentrations of LPS were added to culture media, which were passed through a column consisting or the Polymyxin B Sepharose gel. The content of ET and added LPS in media was measured hy the Limulus amoehocyte lysatc (LAL) test before and after passage of the column. The LPS-binding capacity of the gel was approximately 2.4 × 10 g/l0 ml. The biological activity of contaminating ET and added LPS in media, before and alter passage of the column, was also characterized by the capacity of the media to induce interleukin l (IL-1) secretion in human Mo cultures. The content of IL-l in Mo culture supernalants was determined by the mouse thymocyte costimulatory(LAF) assay. By comparison of the activity of ET in these different biological systems, it was demonstrated that 15-20 × 10−12 g/ml of ET stimulate human Mo cultures lo IL-1 secretion.
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