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J Carlsson

Publications and source records attributed to J Carlsson.

At least 253 records · Page 14Linked to original sources
PubMed1986

A competitive enzyme immunoassay of human beta 2-microglobulin based on separation by filtration.

A competitive enzyme immunoassay has been developed for the determination of beta 2-microglobulin in undiluted human serum. In the assay a beta 2-microglobulin-beta-galactosidase conjugate competes with beta 2-microglobulin from the sample for the binding to anti-beta 2-microglobulin antibodies bound to small size agarose particles (micro-Sepharose) via a double antibody. The conjugate is made by providing beta 2-microglobulin with 'reactive disulphide structures.' This is achieved by the heterobifunctional reagent SPDP and the subsequent linkage of the formed derivative to beta-galactosidase by a thioldisulphide exchange reaction. This procedure gives conjugates with high immunoreactivity and enzyme activity. The assay is fast and simple. The reagents are incubated together for 60 min in the wells of a Millititer plate and separation is performed by filtration. Bound enzyme-labelled beta 2-microglobulin is measured by incubation with substrate for 15 min. Both incubations are performed at room temperature without agitation. Due to the high capacity of the micro-Sepharose no sample predilution is needed. The use of the Millititer filtration system gives a rapid and efficient separation and the advantages of access to equipment for dispensing and reading adapted to the microtitre format.

Filtration↗
PubMed1986

Human monoclonal antibodies to Pf 155, a major antigen of malaria parasite Plasmodium falciparum.

Pf 155, a protein of the human malaria parasite Plasmodium falciparum, is strongly immunogenic in humans and is believed to be a prime candidate for the preparation of a vaccine. Human monoclonal antibodies to Pf 155 were obtained by cloning B cells that had been prepared from an immune donor and transformed with Epstein-Barr virus. When examined by indirect immunofluorescence, these antibodies stained the surface of infected erythrocytes, free merozoites, segmented schizonts, and gametocytes. They bound to a major polypeptide with a relative molecular weight of 155K and to two minor ones (135K and 120K), all having high affinity for human glycophorin. The antibodies strongly inhibited merozoite reinvasion in vitro, suggesting that they might be appropriate reagents for therapeutic administration in vivo.

Animals↗
PubMed1986

Rabbit and human antibodies to a repeated amino acid sequence of a Plasmodium falciparum antigen, Pf 155, react with the native protein and inhibit merozoite invasion.

The Plasmodium falciparum-derived antigen of Mr 155,000 designated Pf 155, deposited in the membrane of infected erythrocytes, contains at least two blocks of tandemly repeated amino acid sequences. The peptide Glu-Glu-Asn-Val-Glu-His-Asp-Ala, which corresponds to a subunit of a C-terminally located repeat, was synthesized. Rabbits immunized with the octapeptide conjugated with either keyhole limpet hemocyanine or tetanus toxoid formed antibodies against the octapeptide. These antibodies reacted with Pf 155 as detected by immunoblotting or a modified immunofluorescence assay. Sera from humans exposed to P. falciparum also contained antibodies binding to the octapeptide in a dot-blot immunoassay. Their anti-octapeptide titers were correlated with their immunofluorescence titers in the assay detecting Pf 155 and other parasite antigens in the membrane of infected erythrocytes. Human octapeptide-reactive antibodies were isolated on an affinity column with the octapeptide conjugated to bovine serum albumin as ligand. These human antibodies reacted with Pf 155 in immunoblotting and strongly stained the surface of infected erythrocytes in the modified immunofluorescence assay. Approximately 20% of this immunofluorescence activity in a high-titered human serum could be recovered from the octapeptide column, indicating that a significant fraction of these anti-parasite antibodies react with epitopes associated with the octapeptide. Furthermore, the human octapeptide-reactive antibodies very efficiently inhibited merozoite reinvasion into erythrocytes in vitro. Similarly purified rabbit antibodies also significantly inhibited reinvasion. Our results suggest that the C-terminal segment of repeated peptides in Pf 155 is a major antigenic region of the molecule and may contain target sites for protective immunity in P. falciparum malaria.

Amino Acid Sequence↗
PubMed1985

Influence of the oxygen pressure in the culture medium on the oxygenation of different types of multicellular spheroids.

Six different types of spheroids of both human and rodent origin were cultured, using the liquid-overlay technique. Oxygen gradients were measured with micro-electrodes, when the spheroids were attached to thin cover-glasses. The gradients were measured from the upper surface towards the center of the spheroids. Stable and reproducible gradients were obtained. Large variations were seen in the steepness of the gradients, depending both on the type and on the size of the spheroids. An interesting phenomenon was discovered. When some types of spheroids were cultured in a medium with a low oxygen pressure (medium equilibrated with gas containing 4-5% O2), the gradients continuously changed and became flatter. Detailed studies showed that most of the changes occurred within 2 days after the transfer to the low oxygen pressure. After 2 days, no further dramatic changes took place. This phenomenon was seen in two types of human glioma (U-118 MG and U-178 MG) and two types of embryonic, hamster lung-cell (V-79-379A and CHEL) spheroids. In the cases of human-osteosarcoma (U-393 OS) and human thyroid-cancer (HTh7) spheroids, no such changes could be seen. The low oxygen tension in the culture medium was chosen to mimic the environmental conditions in solid tumors.

Animals↗
PubMed1985

Degradation of human immunoglobulins G and M and complement factors C3 and C5 by black-pigmented Bacteroides.

Strains of Bacteroides, Capnocytophaga and Fusobacterium were examined by immunological methods for their ability to degrade the human serum proteins IgG, IgM, C3 and C5. The proteolytic activity of the strains was measured in terms of the breakdown of serum into trichloroacetic acid-soluble material. Only black-pigmented Bacteroides strains showed proteolytic activity. Strains of B. gingivalis degraded IgG, IgM, C3 and C5, strains of B. intermedius IgG and C3, strains of B. endodontalis C3 and IgG and a strain of B. loeschei degraded only IgG. These findings are discussed in relation to the pathogenicity of the black-pigmented Bacteroides.

Bacteroides↗
PubMed1985

Inactivation of guinea-pig serum proteinase inhibitors by Bacteroides gingivalis.

The activity of proteinase inhibitors of guinea-pig serum was evaluated by using trypsin as test enzyme. Strains of black-pigmented Bacteroides species were incubated with serum under anaerobic conditions. Strains of Bacteroides gingivalis inactivated the trypsin inhibitors of guinea-pig serum within 1 h, while the other strains reduced the activity of the trypsin inhibitors by less than 50% within 48 h. The inactivation of proteinase inhibitors of guinea-pig serum by B. gingivalis may be an important pathogenic mechanism in the spreading necrotic infections induced in guinea pigs by pure cultures of this species.

Animals↗
PubMed1985

Potentiation by sulfide of hydrogen peroxide-induced killing of Escherichia coli.

L-Cysteine potentiates 100-fold the hydrogen peroxide-induced killing of a growing culture of Escherichia coli K-12 (Berglin et al., J. Bacteriol. 152:81-88). In the present study it is shown that hydrogen sulfide is formed from L-cysteine and that sodium sulfide could substitute for L-cysteine in the potentiation of hydrogen peroxide-induced killing of E. coli K-12. Addition of an amino acid, L-leucine, L-valine, or L-alanine, to an L-cysteine-containing medium with a growing culture of E. coli K-12 inhibited hydrogen sulfide formation and the potentiation of hydrogen peroxide-induced killing. These amino acids did not inhibit hydrogen sulfide formation from L-cysteine by a cell extract, and they did not inhibit the potentiation by sulfide of hydrogen peroxide-induced killing. This indicated that the amino acids protected the culture from L-cysteine-potentiated, hydrogen peroxide-induced killing by inhibiting the transport of L-cysteine into the cell. The potentiation by sodium sulfide of hydrogen peroxide-induced killing was abolished by the metal ion chelator 2,2'-bipyridyl. This indicated that metal ions, in addition to sulfide, were involved in the killing. Toxic effects of hydrogen peroxide are often presumed to be mediated by hydroxyl radicals formed in iron-catalyzed reactions. It was demonstrated that iron sulfide was more efficient than ferrous iron in catalyzing the formation of hydroxyl radicals from hydrogen peroxide. It was suggested that hydrogen sulfide formed in polymicrobial infections may play an important role in the host defense by potentiating the antimicrobial effect of hydrogen peroxide produced by phagocytic cells.

Cysteine↗
PubMed1985

Pyruvate dehydrogenase activity in Streptococcus mutans.

Streptococcus mutans NCTC 10449 and Escherichia coli K-12 strain 37 were grown under aerobic and anaerobic conditions. In cell extracts of both strains, pyruvate dehydrogenase activity dependent on thiamine pyrophosphate, coenzyme A, and NAD was shown. The enzyme was induced by pyruvate in the growth medium, and there was higher activity in aerobically grown cells than in anaerobically grown cells. Acetyl phosphate was a potent inhibitor of the activity. This inhibition was partly overcome by inorganic phosphate.

Ferricyanides↗
PubMed1985

Inactivation of key factors of the plasma proteinase cascade systems by Bacteroides gingivalis.

The effect of Bacteroides gingivalis W83 on various key components of the human plasma proteinase cascade systems was studied. When purified C1-inhibitor was incubated with the bacterium, the inhibitor was rapidly inactivated by limited proteolytic cleavage. In citrated whole plasma, C1-inhibitor, antithrombin, plasminogen, prekallikrein, prothrombinase complex, the clotting factor X, and most of the alpha 2-antiplasmin were functionally eliminated after 30 min of incubation with the bacterium. Fibrinogen disappeared from the plasma almost immediately upon mixing with the bacterial suspension. In contrast, there was no appreciable decrease in the bulk of other plasma proteins, such as various transport proteins (albumin, prealbumin, transferrin) and immunoglobulins, during 4 h of incubation with the bacterium. Most of the observed effects can be assigned to the proteolytic activity of the bacterium itself, since there was little evidence for generation of intrinsic plasma proteinase activity, despite the loss of proteinase inhibitory activities. B. gingivalis W83 thus seems to be equipped with proteolytic enzyme systems which selectively recognize and rapidly inactivate the most important proteinase inhibitors and proenzymes present in human plasma. This bacterium therefore seems to be able to efficiently paralyze the host's various defenses against invading microorganisms.

Bacteroides↗
PubMed1985

Abnormal redox status of membrane-protein thiols in sickle erythrocytes.

Although sickle erythrocytes (RBC) undergo excessive autooxidation, investigators have not found evidence for abnormal oxidation of protein thiols in sickle RBC membranes (e.g., protein aggregates linked by intermolecular disulfide bonds). However, the conventional techniques heretofore used cannot detect more subtle changes in thiol status such as abnormal intramolecular disulfide bonds. We examined RBC membranes using thiol-disulfide exchange chromatography which partitions sodium dodecyl sulfate-solubilized proteins on the basis of reactive thiols, yielding gel-bound (reduced-thiol) and filtrate (oxidized/blocked-thiol) fractions. Membranes from normal RBC partition so that only 13.6 +/- 1.4% of all membrane protein is found in the filtrate fraction. An abnormally increased amount of membrane protein from sickle RBC (21.5 +/- 4.3%) partitions into the filtrate fraction (P less than 0.001). Since sickle RBC do not have high molecular weight aggregates of membrane protein, this indicates abnormal intramolecular thiol oxidation in sickle RBC membranes. Treatment of normal RBC with thiol blockers and oxidants simulates this shift of membrane protein into the filtrate fraction. Analysis using polyacrylamide gel electrophoresis reveals that the filtrate fraction derived from normal RBC consists mostly of band 7 and glycophorins, with only trace amounts of other membrane proteins. Superimposed upon this normal background, sickle RBC filtrates are enriched with all proteins (including cytoskeletal protein bands 1, 2, 2.1, and 4.1), suggesting a generalized oxidative perturbation of sickle RBC membranes. These observations support the concept that excessive RBC autooxidation may play a role in sickle disease pathophysiology, and they perhaps help explain the development of those membrane abnormalities that may reflect cytoskeletal dysfunction in sickle erythrocytes.

2,2'-Dipyridyl↗
PubMed1985

Pf 155, a candidate for a blood stage vaccine in Plasmodium falciparum malaria.

Pf 155 is a Mr 155,000 P. falciparum antigen, which is deposited in the erythrocyte membrane at merozoite invasion. The antigen is detected by a modified immunofluorescence assay giving staining of the surface of ring stage infected erythrocytes. Cell lines producing human monoclonal antibodies to Pf 155 were established and two different antibodies of IgM and IgG class, respectively, were characterized further. Both antibodies gave a strong surface immunofluorescence and in immunoblotting they react strongly with Pf 155, but also with some lower molecular weight material, including polypeptides of Mr 135,000 and 120,000. Both antibodies were efficient inhibitors of P. falciparum reinvasion in vitro. Pf 155 as well as the Mr 135,000 and 120,000 polypeptides were shown to bind with high affinity to human erythrocyte glycophorin. An octapeptide (GluGluAsnValGluHisAspAla) corresponding to a repeated sequence in Pf 155 was synthesized. Rabbit antibodies to the octapeptide gave a distinct surface immunofluorescence and reacted with Pf 155 and the Mr 135,000 and 120,000 polypeptides in immunoblotting. Human antibodies reacting with the octapeptide were isolated by affinity chromatography from the serum of a P. falciparum immune individual. These antibodies showed a strong reaction with Pf 155 as determined by immunoblotting and surface immunofluorescence. Pf 155 reactive antibodies affinity purified on monolayers of P. falciparum infected erythrocytes are very efficient inhibitors of parasite reinvasion. Such antibody preparations depleted of octapeptide reactive antibodies showed a markedly decreased reinvasion inhibitory capacity, while a high inhibitory activity was recovered in the octapeptide reactive antibodies. Pf 155 fulfills several criteria thought to be proper for antigens involved in anti-malarial protective immunity.

Amino Acid Sequence↗
PubMed1984

Antibodies in malarial sera to parasite antigens in the membrane of erythrocytes infected with early asexual stages of Plasmodium falciparum.

Monolayers of human erythrocytes (E) infected with Plasmodium falciparum were briefly fixed with 1% glutaraldehyde and air dried. They were then exposed to sera from patients with P. falciparum malaria or from donors immune to this parasite and tested in an indirect immunofluorescence assay (IFA). Parasites in infected E were made visible by counterstaining with ethidium bromide. Immunofluorescence (IF) was restricted to the surface of infected E. No antibody binding was detected unless the E were dried, suggesting that the relevant antigens were not available on the outer layers of the E surface. Staining over large parts of the E surface was seen already when the merozoite penetrated noninfected cells and was strong in E containing early stages of the parasite (rings, trophozoites). It was weak or absent from E containing schizonts. Antibodies in sera from different parts of Africa, Colombia, or Sweden reacted similarly with E infected with a Tanzanian P. falciparum strain kept in culture for many years and with parasitized E freshly drawn from African, Swedish, or Colombian patients. All sera from residents of a holoendemic area (Liberia) were IFA positive. In contrast, some sera from Colombian or Swedish patients with primary infection gave negative results. The results of the IFA and of an enzyme-linked immunosorbent assay in which fixed and dried E were the targets were well-correlated, suggesting that the same antibodies were detected by these assays. The antigens involved in the IFA were susceptible to pronase but not to trypsin or neuraminidase. E surface IF was inhibited by lysates of infected E, merozoite extracts, or soluble antigens present in P. falciparum culture supernatants but not by lysates of normal E or ghost extracts. The inhibitory antigens were heat stable (100 degrees C, 5 min). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting of either antigen-enriched preparations from culture supernatants or merozoite extracts showed that antibodies eluted from monolayers of infected E reacted consistently with a predominant polypeptide of Mr 155,000 and two to four minor polypeptides of lower molecular weights. Metabolic labeling of the parasites with 75Se-methionine indicated that these antigens were parasite derived. We conclude that the antigens involved in these reactions are released from bursting schizonts or merozoites and are deposited in the E membrane in the course of invasion.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies↗
PubMed1984

Liquid-overlay culture of cellular spheroids.

The capacity of different types of human cells to form and grow as spheroids was tested by using suspension culture over a bottom surface, which prevented cell attachment (liquid-overlay culture). The best and most convenient bottom surface so far tested was the bottom of a normal culture dish covered with a thin film of agarose. The agarose film was allowed to dry prior to the addition of medium. Several different human cell lines were tested, and it was found that many types of tumor cells formed and grew as spheroids, Large variations in growth rate, cell morphology, thickness of the viable cell layer and PO2 profiles were found among the tested spheroids. This variability parallels the variability seen in solid tumors. The morphology and the growth rate of solid tumors vary a great deal, depending on the type and position of the tumors (Ackerman and Rosai 1975; Steel 1977). Some tumors have a slow growth rate, with a few proliferating cells, while others grow faster and have higher proportions of proliferative cells (Charbit et al. 1971; Malaise et al. 1973; Friedman 1974; Lightdale and Lipkin 1975; Steel 1977). Local variations in both morphology and proliferation are usually found even within single tumors (Denekamp and Kallman 1973; Ackerman and Rosai 1974). Considering the variability in morphology, growth rate, and radiosensitivity between different types of spheroids, it is not difficult to imagine that wide variations may exist in these variables between different types of tumors or even between different regions within single tumors. The use of a group of different spheroids, showing a spectrum of growth rates, radiosensitivities, etc., will, it is hoped, be helpful in elucidating factors of importance in cancer therapy.

Animals↗
PubMed1984

Penetration and binding of vinblastine and 5-fluorouracil in cellular spheroids.

The kinetics of the penetration and binding of the two commonly used antitumour drugs vinblastine and 5-fluorouracil in nonvascularized tumour tissue were studied. Multicellular human tumour spheroids (glioma U-118 MG and thyroid cancer HTh-7) were used as a model system. Radiolabelled drugs were used in all studies. To avoid disturbances in the distribution of unbound drugs a dry histological technique was used in combination with contact autoradiography. In addition, quantitative measurements of the accumulation and binding of the drugs were made. The results showed that vinblastine penetrated the spheroids less efficiently than 5-fluorouracil. Vinblastine required about 2 h to be isotropically distributed within the studied spheroids, while only a few minutes were required for 5-fluorouracil. Vinblastine seemed to be accumulated in the peripheral parts of the spheroids within 15 min. High concentrations of 5-fluorouracil, isotropically distributed in the spheroids, were observed after 2 h of incubation. Significant amounts (about half) of the accumulated drugs resisted gentle washing for 3 X 20 s plus 15 min in fresh medium. The limited penetration of vinblastine correlated well with a previously observed high resistance of spheroids to treatments of short duration with this drug.

Cell Line↗
PubMed1984

Immunoelectrophoretic analysis of erythrocytic stages of Plasmodium yoelii and P. chabaudi.

Triton X-100 extracts of erythrocytes infected with Plasmodium chabaudi and P. yoelii were analysed in crossed immunoelectrophoresis with rabbit antisera. The parasite origin of the antigens detected was assessed by metabolic radiolabelling of the parasites with 35S-methionine. About 12 immunoprecipitates were obtained with both extracts and their homologous antiserum. Cross-tests showed that the two parasite strains were very similar antigenically. Species-specific antigens could, however, also be demonstrated. Two antigens, present on both P. yoelii- and P. chabaudi-infected erythrocytes, were located on the surface of the host cell membrane as judged from 125I-labellings with lactoperoxidase. Experiments with phenyl-Sepharose showed that most of the antigens detected were hydrophilic and none of them reacted with concanavalin A.

Animals↗
PubMed1984

Rapidly progressing rheumatoid arthritis: an example of polyclonal B-cell activation?

We report the case of a 76-year old woman who presented with a palindromic onset of seropositive rheumatoid arthritis. After suffering from a fulminant pulmonary infection she developed serologic tests for ornitosis, mycoplasma, pneumocystis carinii and low titer yersinia. Also the rheumatoid factor titer increased and the antinuclear antibody test became positive. Clinically, the course of a rather benign polyarthritis changed into a rapidly progressive deforming disease. The course was also complicated by cutaneous necrotizing vasculitis. Further deterioration occurred after a relapse of pulmonary infection, and she died following acute abdominal surgery for perforated sigmoiditis. It is speculated that the clinical course was in part caused by polyclonal B-cell activation.

Aged↗