Biomedical subjects
Publications and source records attributed to G Andersson.
Chronic respiratory deficiency is a complication in a number of diseases and results in poor quality of life and increased mortality. Notwithstanding that the long-term prognosis is poor the patient should be offered respiratory support via a nasal mask or a tracheotomy tube; this issue was discussed at an international meeting on ventilation at home held in Lyons in France.
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A reverse modification of ELISPOT assay using nitrocellulose membranes and epitope-specific monoclonal antibodies is described for the detection of single lymphokine-secreting cells. As a model, the production of gamma-interferon by mitogen stimulated human peripheral blood lymphocytes has been examined. The assay can also be modified to permit microscopic examination of spot-forming cells.
Rat liver somatogenic receptors have been characterized by gel permeation chromatography, sucrose density gradients in H2O and D2O, and affinity cross-linking using 125I-bovine growth hormone (bGH) as a specific somatogenic receptor ligand. Cross-linking of 125I-bovine growth hormone to a Triton X-100-treated low density fraction isolated from livers of late pregnant rats followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis under reducing conditions showed three major binders with Mr 95,000, 86,000, and 43,000 and a minor binder of Mr 55,000, after correction for bound ligand assuming a 1:1 binding ratio of ligand-receptor. The Mr 86,000, 55,000, and 43,000 species were recovered in the detergent-soluble supernatant after high-speed centrifugation, whereas the Mr 95,000 species remained Triton X-100 insoluble. Detergent-soluble 125I-bGH-receptor complexes were further analyzed by sedimentation into sucrose density gradients. The sedimentation coefficient was S20,w = 5.2 S and the partial specific volume v = 0.72 ml/g. Gel permeation chromatography on a Sepharose S-400 column indicated a Stokes radius of 61 A for the 125I-bGH-receptor-Triton X-100 complex. Based on these figures, the molecular weight of the complex was calculated as 131,100. The molecular weight of the ligand-free receptor-Triton X-100 complex was calculated as Mr 109,100. Affinity cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the 61 A peak from Sephacryl S-400 chromatography (cf. above) showed two binding entities, one major and one minor with Mr values 86,000 and 43,000, respectively, in the absence of reductant. When electrophoresis was run in the presence of reductant the Mr 43,000 species was the major binding entity. Furthermore, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (first dimension, nonreducing and second dimension, reducing) showed that a disulfide-linked binder at Mr 43,000 is contained within the Mr 86,000 species. As with pregnant rats, female and male rats both showed 125I-bovine growth hormone binders of Mr 95,000, 84,000, 55,000, 43,000, and additionally an Mr 35,000 binder.
1. Climbing fibres were activated by peripheral nerve stimulation at 'high' frequencies (greater than 3 Hz) for 15-25 s and then at 0.9 Hz for about 1 min. The high frequency activation induced a post-conditioning inhibition, lasting up to about 1 min, of climbing fibre responses recorded from the cerebellar surface. 2. Electrolytic lesions were made in the superior cerebellar peduncle (brachium conjunctivum). After the lesion, the post-conditioning inhibition was completely eliminated. 3. Injections of the GABA-receptor blocker bicuculline methiodide into the inferior olive reversibly blocked the post-conditioning inhibition. 4. The results support the hypothesis proposed by Andersson and Hesslow (1987a), that post-conditioning inhibition is mediated by a GABA-ergic interposito-olivary pathway.
In most individuals two HLA-DR beta genes are expressed from each chromosome. One of these genes encodes one of the classical DR specificities, while the other encodes either of the supertypic DRw52/DRw53 specificities. In addition to these genes usually one or two DR beta pseudogenes are present. In contrast, the DRw8 chromosomal region only contains a single DR beta gene. To determine the relationship of this single gene to the multiple DR beta genes of other DR specificities, comparisons of Southern genomic blots were carried out. In this analysis genomic clones for each individual DR beta chain locus were included. The DR beta w8 gene was indistinguishable from the DR beta III gene of DR3 cells (encoding DRw52), suggesting that it is closely related to the latter gene. The functional implications of this finding are discussed.
Several immunodominant B-cell epitopes of the P. falciparum antigen blood stage Pf155/RESA, a major vaccine candidate antigen, are located in the molecular regions containing amino acid repeats. We started to map Pf155/RESA for T cell reactive epitopes. For this purpose, short synthetic peptides corresponding to the 3'- and 5' repeat regions of the molecule as well as to non-repeated sequences outside these regions were prepared. T cells from P. falciparum primed donors from two highly endemic areas of Africa were tested for their responsiveness to the peptides by thymidine incorporation and/or interferon gamma (IFN-gamma) release. There was a considerable variation in the response to the different peptides. However, the strongest and most frequent responses were seen with a few peptides from the 3'- and 5'-repeat regions. Thus, the immunodominant B cell epitope regions of Pf155/RESA, contain several T cell epitopes. Since the repeat regions are known to be conserved in different P. falciparum strains, the T cell epitopes reported here may be suitable constituents of a P. falciparum subunit vaccine.
Receptor-mediated endocytosis of radiolabelled bovine growth hormone (125I-bGH) via somatogenic receptors in the liver was studied following in vivo intraportal injection. At different times after injection, subcellular membrane fractions involved in binding (plasma membranes), endocytosis (endocytic vesicles) and degradation (lysosomes) of peptide hormones were isolated by sucrose density gradient centrifugation. These fractions were evaluated for the time-course accumulation of radiolabelled bGH and for the presence of internalized 125I-bGH-receptor complexes. These uptake studies indicate that after initial plasma membrane association of 125I-bGH, the ligand is transported in two successive endocytic compartments prior to arrival in lysosomes. The molecular weight of the somatogenic binders of male and female rat livers involved in internalization of 125I-bGH was determined to 95,000, 64,000, 55,000, 43,000 and 35,000, assuming a 1:1 binding of the hormone to the binder. These binders were seen in both endosomes and lysosomes, which suggests that growth hormone is transported to the lysosomes in a complex with its receptor. Binding and uptake of 125I-bGH was also compared in male and female rat livers, and endocytosis of 125I-bGH was compared to that of radiolabelled ovine prolactin (125I-oPrl). The specific uptake of 125I-bGH appeared not to be sexually differentiated in contrast to that of 125I-oPrl which showed a 35-fold higher uptake in female rat liver. Degradation of 125I-bGH was studied under in vitro binding assay conditions. A distinct 15,000 Da fragment was generated by plasma membrane, endosomal and lysosomal fractions. Based on protease inhibitor studies, a non-trypsin-like serine protease is suggested to be involved in the degradation of bGH. The 15,000 Da proteolytic fragment of GH can be affinity cross-linked to somatogenic binders of similar molecular weights as those involved in the binding of intact GH.
Immunogens included in a subunit vaccine should contain both B- and T-cell-activating sites to ensure anamnestic responses following reinfection after vaccination as well as antibody-independent cellular immunity. The Plasmodium falciparum antigen Pf155/RESA, a major candidate for a vaccine against the asexual blood stages of this malaria parasite, was investigated for T-cell epitopes in its C-terminal amino acid repeat region, a region known to be conserved in different P. falciparum strains. It was found to contain several related sequences that activated T cells from humans primed to P. falciparum by natural exposure, to proliferation, and/or interferon-gamma release in vitro. T cells from approximately half of the donor group investigated responded to the intact protein, and 65% of these responders also responded to short synthetic peptides, probably representing a small number of partly overlapping T-cell epitopes. Thus, sequences from the C terminus of Pf155 may be suitable constituents of a P. falciparum subunit vaccine and also provide a basis for epitope-specific epidemiological studies relating cellular immune responses in vitro to clinical immunity and P. falciparum endemicity.
Insulin-like growth factor I (IGF-I) and IGF-II are peptides that presumably are required for normal fetal and postpubertal growth. The production of IGFs is developmentally regulated and the liver appears to be a major site of production. By analysing mRNA levels for IGF-I and IGF-II in the rat liver we have attempted to further study the expression of these growth factors during development and regeneration as well as during the course of hepatic carcinogenesis. Fetal livers are characterized by a high level of IGF-II mRNA and a low level of IGF-I mRNA, while in adult livers the opposite situations occur, i.e. a high level of IGF-I mRNA and a non-measurable level of IGF-II mRNA. During the course of experimentally induced hepatic cancer, IGF-I mRNA was consistently reduced and in a majority of cancers analysed (6/9) IGF-II mRNA was increased, i.e. a fetal type of IGF expression can be switched on in some experimentally induced hepatocellular carcinomas. The onset of IGF-II production during hepatic carcinogenesis appears to be a late phenomenon since liver nodules, preceding the development of hepatocellular cancer, were found not to contain IGF-II mRNA. Furthermore, during hepatic regeneration following partial hepatectomy no marked change in IGF-I or IGF-II mRNA levels was noted. The above results suggest that the fetal growth factor IGF-II could have a role in hepatic cancer.
In order to study the natural course of venous flow and temperature reaction in the legs after symptomatic first episode of deep vein thrombosis (DVT), 65 patients (57 with proximal DVT) without further thromboembolic complications during the observation period were followed for 1 year by repeated plethysmography (PG) and thermography (TG). Regarding the non-invasive parameters substantial individual variations was observed during the observation period. After 1 year pathologic PG and TG were still demonstrated in 39% (I mu (95%) = /0.43 +/- 0.05/) and 65% (I mu (95%) = /1.18 +/- 0.11/), respectively, of the patients after proximal DVT. Only a minority of the patients were normalized permanently in both PG and TG during the first year. The high frequency of remaining venous obstruction and especially, persistent thermoactivity is notable and may be of clinical importance.
The binding specificities of three biologically active anti-lymphocyte monoclonal antibodies (MoAb) (K46M, K3G, and 3-19-2) produced against human T-cell surface components reactive with the mitogenic lectin leucoagglutinin from Phaseolus vulgaris (La) were analysed. K46M is a strong T-cell mitogen, while K3G and 3-19-2 inhibited cell-mediated cytotoxicity. Resting peripheral blood lymphocytes (PBL) contained 4-16% K46M+ cells, 8-35% K3G+ cells, and less than 0.3-4% 3-19-2+ cells. After stimulation with T-cell mitogens the proportion of K46M+ and 3-19-2+ cells increased markedly (mean 59 and 30% positive cells, respectively), while the increase in K3G+ cells was less prominent (38%). K46M-reactive structures were expressed on mature T cells and probably also on B cells. K3G reacted with B and T cells while 3-19-2 showed a broader specificity reacting also with erythrocytes. All three MoAb reacted with lipid extracts of resting and activated PBL as well as with purified neutral glycolipids of lymphoid origin. In addition 3-19-2 reacted with lipid extracts of erythrocytes. K46M immuno-precipitated four surface peptides from lectin-stimulated PBL. Their apparent molecular weights were 53,000, 42,000, and 16,000 (doublet). The 53,000 and 42,000 MW peptides were identified as the alpha and beta chains of the T-cell antigen receptor. The identity of the 16,000 MW peptides is presently unknown. K3G and 3-19-2 did not specifically precipitate any lymphocyte surface peptide.
A monoclonal antibody, K46M (IgM kappa), obtained after immunization with leucoagglutinin (La)-reactive T-cell surface components, stimulated human lymphocytes to proliferate. It induced maximal proliferation at greater than 20 micrograms IgM/ml after 3-4 days of culture. Cells stimulated by K46M produced interleukin 2 (IL-2) and gamma interferon (IFN-gamma) and expressed receptors for IL-2 and transferrin. The majority of the activated cells were phenotypically T cells as defined by monoclonal antibodies against CD3 and CD2, and an increase in the K46M-positive cells was also observed during the activation period. K46M-activated cells display major histocompatibility complex (MHC)-unrestricted cytotoxicity against several cultured target cells. The frequencies of the cytotoxic and of the proliferative precursor cells were determined using a limiting dilution assay. K46M seems to activate a larger fraction of cytotoxic precursor cells against Molt 4 than against K562, but the statistical significance of these observations requires further exploration. Both K46M or La activated 40% of PBL to proliferate, whereas 70% of PBL were induced by OKT3. However, the frequency of K46M-activated cells was 40% only when the lymphocytes were plated at low cell densities, i.e. less than 0.5 cells per well. At higher densities an inhibition of proliferation was seen that resulted in a biphasic response curve, indicating that the activation of PBL by K46M was not a single hit event. This was not found with either La or OKT3. Whether K46M, in contrast to OKT3 and La, activates a subpopulation with suppressor activity remains to be established.
The immunohistochemical expression of the trophic peptide insulin-like growth factor I (IGF-I; somatomedin C) was investigated in the post-ischaemic regenerating rat kidney. The part of the nephron most severely injured by the ischaemic insult was the pars rectae or S3 region of the proximal tubules. In this part of the nephron no specific IGF-I immunoreactivity could be demonstrated under control conditions. Three days after injury, however, low regenerating tubular cells expressing IGF-I immunoreactivity could be demonstrated invading the damaged region. Five and 7 days post-ischaemia some of the regenerating cells showed IGF-I immunoreactivity while adjacent and similar-looking cells did not. In parallel with an increasing differentiation of the regenerating cells the IGF-I immunoreactivity vanished. Fourteen days after the injury the S3 cells had regained their normal morphology, and by this time the IGF-I immunoreactivity had returned to a normal undetectable level. It is concluded that there is a transient increase in the expression of IGF-I mainly confined to the early phase of post-ischaemic rat kidney regeneration. It is proposed that IGF-I exerts its trophic role by paracrine mechanisms.
Parenchymal and non-parenchymal cell fractions isolated from rat liver were analysed for the distribution of somatogenic receptors and for insulin-like growth factor-I (IGF-I) mRNA. The effect of hypophysectomy either alone or in combination with a single injection of human GH (hGH) on the levels of IGF-I mRNA was also studied in the two cell fractions. The contamination of parenchymal cells in the non-parenchymal cell fraction was in the range of 2-3%. Somatogenic receptors were found only in the parenchymal cell fraction. IGF-I mRNA was detected in both cell fractions, although the level of this mRNA was about fivefold higher in parenchymal cells. When RNA was studied by Northern gel analysis no major differences were observed in the size distribution of IGF-I transcripts in the two cell fractions. In hypophysectomized animals, the IGF-I mRNA level was decreased to 10 and 30% of control values in the parenchymal and non-parenchymal cell fractions respectively. Treatment of hypophysectomized animals with a single dose of hGH restored IGF-I mRNA in parenchymal and in non-parenchymal cells to the extent found in intact animals. In conclusion, our data indicate that somatogenic receptors are exclusively, and IGF-I mRNA predominantly, expressed in liver parenchymal cells compared with a total non-parenchymal cell fraction. The most marked effect of GH, correlating with the presence of somatogenic receptors, was seen in the parenchymal cells. The minor non-GH dependent expression of IGF-I mRNA suggests that this peptide may elicit effects in addition to the well-established GH-dependent activity.
Nickel sulphate antigen-induced peripheral blood lymphocyte activation in vitro was characterized by lymphokine measurement (IL-2, IFN-gamma) and phenotyping of the IL-2 responsive cells. Mononuclear cells from nickel-sensitive patients synthesized more DNA, produced more IL-2 and had more IL-2 receptor positive cells in response to nickel than did those of the control subjects. On the other hand no IFN-gamma was detectable in the nickel supernatants, while PPD, used as the control antigen, induced pronounced quantities of IFN-gamma with an equal amount of DNA synthesis. The increase in IL-2 receptor positive cells was due to activation of CD4+ (helper/inducer) T cells. T cells with HLA-DR antigen surface markers were more numerous on each day of culture than cells with IL-2 receptors. These two activation markers were co-expressed on the same cells only to a certain extent, thus perhaps reflecting different types or phases of activation. In conclusion, nickel-induced peripheral blood mononuclear cell activation in vitro differs from microbial antigen-induced activation with respect to its modest or non-existent IFN-gamma response.
Peripheral blood mononuclear cells from clinically immune Gambian adults were assayed for in vitro proliferation in response to crude and partially purified Plasmodium falciparum antigens. Lymphoproliferative responses to malaria antigens, lectin mitogens and Candida albicans were compared with those of control donors with no previous exposure to malaria. Cells of malaria-immune individuals were significantly more responsive to conconavalin A, and less responsive to phytohaemagglutinin, than cells from the control donors in both non-immune human serum and autologous serum. Cells from a proportion of immune donors proliferated in response to soluble malaria antigens but a substantial minority did not. Young adults and women were over-represented in the non-responding population. Responses to soluble malaria antigens were depressed in autologous serum compared with normal human serum. Both immune and control cells produced low levels of gamma-IFN when stimulated with crude P. falciparum schizont antigens. Approximately half the immune donors, and none of the controls, produced significant levels of gamma-IFN in response to purified soluble malaria antigen or malaria parasite culture supernatant. There was no direct correlation between lymphoproliferation and gamma-IFN production.
Peripheral blood mononuclear cells from 63 Gambian children with acute Plasmodium falciparum malaria were examined for lymphoproliferation and interferon-gamma (IFN) production in response to stimulation by mitogens, malaria antigens and other soluble antigens. Mitogen or Candida-induced proliferation was not depressed during acute infection but was enhanced 2 to 4 weeks after treatment. Responses to partially purified soluble P. falciparum antigens were minimal or absent in all children in the acute phase but approximately 50% of the children responded by proliferation or IFN-gamma production during the 2 to 8 week convalescent period. These proliferative responses were severely depressed in the presence of the patient's own serum. Nine children with significant convalescent phase proliferative responses were re-examined several months after acute infection. Of these, four remained responsive for at least 8 months in the probable absence of reinfection.