Biomedical subjects

I Pettersson

Publications and source records attributed to I Pettersson.

At least 19 recordsLinked to original sources

PubMed2002

Natural (ghrelin) and synthetic (hexarelin) GH secretagogues stimulate H9c2 cardiomyocyte cell proliferation.

Recent experimental data demonstrate cardiovascular effects of the GH secretagogues (GHSs) hexarelin and ghrelin, the proposed natural ligand for the GHS receptor. Moreover, specific cardiac binding sites for GHSs have been suggested. The aim of the present study was to investigate if the natural ligand ghrelin and synthetic GHS peptide hexarelin and analogues have direct effects on the cardiomyocyte cell line, H9c2. Hexarelin stimulated thymidine incorporation in a dose-dependent manner with significant responses at 3 micro M (147+/-3% of control, P<0.01) and elicited maximal effects at concentrations around 30 micro M. This activity was seen already after 12 h of incubation with a maximal effect after 18 h (176+/-9% of control, P<0.01). Ghrelin also had a significant stimulatory effect on thymidine incorporation (129+/-2% of control at 3 micro M and 18 h, P<0.05). The stimulatory effect on thymidine incorporation of hexarelin, Tyr-Ala-hexarelin, EP80317 and ghrelin was specific and no stimulatory effect was observed with the truncated GH-releasing peptide EP51389 or the non-peptidyl GHS MK-0677. In competitive binding studies, (125)I-labeled Tyr-Ala-hexarelin was used as radioligand and competition curves showed displacement with hexarelin, Tyr-Ala-hexarelin, EP80317 and ghrelin, whereas MK-0677 and EP51389 produced very little displacement at 1 micro M concentration, adding further support for an alternative subtype binding site in the heart compared with the pituitary. In conclusion, we have demonstrated a dose-dependent and specific stimulation of cardiomyocyte thymidine incorporation by natural and synthetic GHS analogues, suggesting increased cell proliferation and binding of GHS to H9c2 cardiomyocyte cell membranes. These findings support potential peripheral effects of GHS on the cardiovascular system independent of an increased GH secretion.

Binding, Competitive↗

PubMed2001

"Tolerosomes" are produced by intestinal epithelial cells.

The development of immunological tolerance to orally fed antigens depends on the sampling, processing and transportation events followed in the intestinal epithelium. We present here a description of a "tolerosome": a supra-molecular, exosome-like structure assembled in and released from the small intestinal epithelial cell. The tolerosome is a approximately 40 nm large vesicular structure that carries MHC class II (MHC II) with bound antigenic peptides sampled from the gut lumen. Tolerosomes isolated from serum shortly after antigen feeding or from an in vitro pulsed intestinal epithelial cell line are fully capable of inducing antigen specific tolerance in naive recipient animals. Purified tolerosomes represent a structure by which fed antigens can be efficiently presented to the immune system. Removal of the tolerosomes from serum by ultracentrifugation or absorption of MHC II results in abrogated tolerance development.

Animals↗

PubMed2001

An improved nicotinic pharmacophore and a stereoselective CoMFA-model for nicotinic agonists acting at the central nicotinic acetylcholine receptors labelled by.

A study of a series of compounds with agonistic effect at the alpha4beta2 nicotinic acetylcholine receptors resulted in an improved pharmacophore model as well as a CoMFA model. The pharmacophore was composed of three pharmacophoric elements: (1) a site point (a) corresponding to a protonated nitrogen atom, (2) a site point (b) corresponding to an electronegative atom capable of forming a hydrogen bond, and (3) the centre of a heteroaromatic ring or a C=O bond (c). The pharmacophoric elements were related by the following parameters: (a-b) 7.3-8.0 A, (a-c) 6.5-7.4 A, and the angle between the two distance vectors (delta bac) 30.4-35.8 degrees. In addition to this, a stereoselective CoMFA model was developed, which showed good predictability even for compound classes not present in the training set.

Animals↗

PubMed2000

Ro 52kD autoantibodies are detected in a subset of ANA-negative sera.

To define Ro 52kD, Ro 60kD, and La specificities of autoantibodies within ANA-negative sera, samples from 64 ANA-negative but SSA positive patients undergoing investigation due to suspected CTD were analysed, using recombinant antigens and synthetic peptides by immunoblotting and ELISA. The sera were selected from 4025 sera submitted for routine ANA analysis. Antibodies to Ro or La were detected in 42/64 sera (65%). Anti-Ro 52kD antibodies occurred most frequently and were present in 42/64 sera (65%). This was the only specificity of autoantibody detected in 18 sera. No patient had only anti-La or anti-Ro 60 antibodies. In total 18.64 patients (28%) had Ro 60 antibodies and 14/64 had anti-La antibodies (21%). Eight patients had antibodies reacting with all three antigens. We used the same set of sera to test the antigenicity of different regions of Ro 52kD represented by deletion clones and peptides derived from the Ro 52kD sequence. Out of 30 sera reacting with a recombinant deletion clone encompassing as residues 136-227, 12 sera reacted with a peptide corresponding to a 200-239. Some sera gave a low positive OD value with a peptide of a 176-196. Based on the results of this study in which we demonstrate Ro 52kD autoantibodies in 65% of selected ANA negative sera and define an autocephitope within the Ro 52kD protein composed of the leucine zipper domain, we suggest that testing for Ro 52kD antibodies could be included in an extended investigation of ANA negative patients with suspected connective tissue disease.

Antibodies, Antinuclear↗

PubMed1999

Key residues revealed in a major conformational epitope of the U1-70K protein.

Epitopes depending on three-dimensional folding of proteins have during recent years been acknowledged to be main targets for many autoantibodies. However, a detailed resolution of conformation-dependent epitopes has to date not been achieved in spite of its importance for understanding the complex interaction between an autoantigen and the immune system. In analysis of immunodominant epitopes of the U1-70K protein, the major autoantigen recognized by human ribonucleoprotein (RNP)-positive sera, we have used diversely mutated recombinant Drosophila melanogaster 70K proteins as antigens in assays for human anti-RNP antibodies. Thus, the contribution of individual amino acids to antigenicity could be assayed with the overall structure of the major antigenic domain preserved, and analysis of how antigenicity can be reconstituted rather than obliterated was enabled. Our results reveal that amino acid residue 125 is situated at a crucial position for recognition by human anti-RNP autoantibodies and that flanking residues at positions 119-126 also appear to be of utmost importance for recognition. These results are discussed in relation to structural models of RNA-binding domains, and tertiary structure modeling indicates that the residues 119-126 are situated at easily accessible positions in the end of an alpha-helix in the RNA binding region. This study identifies a major conformation-dependent epitope of the U1-70K protein and demonstrates the significance of individual amino acids in conformational epitopes. Using this model, we believe it will be possible to analyze other immunodominant regions in which protein conformation has a strong impact.

Amino Acid Sequence↗

PubMed1999

Improving the nicotinic pharmacophore with a series of (Isoxazole)methylene-1-azacyclic compounds: synthesis, structure-activity relationship, and molecular modeling.

A series of (isoxazole)methylene-1-azacyclic compounds was prepared. The compounds were tested for affinity to central nicotinic acetylcholine receptors (nAChRs) and central muscarinic receptors. The compounds covered a broad range of affinities for the nAChRs (IC(50) = 0.32 to >1000 nM), with selectivities for the nAChRs over the muscarinic receptors in the range of 3-183. The high-affinity compound (Z)-26 (3-(4-methyl-5-isoxazolyl)methylene-1-azabicyclo[2.2. 2]octane, IC(50) = 3.2 nM) having only one energy minimum was used as the reference structure in a computational study. This ligand has enabled definition of an important distance parameter, and the existence of this parameter was supported by showing that other potent nicotinic ligands (for example, nicotine and epibatidine) fit the model.

Animals↗

PubMed1999

Antibodies given orally in the neonatal period can affect the immune response for two generations: evidence for active maternal influence on the newborn's immune system.

Two day old Wistar rats were tube fed with 1 or 10 micrograms of a mouse IgG1 monoclonal anti-idiotypic (a-Id) antibody that was directed against an anti-Escherichia coli-K13 capsular polysaccharide antibody. A control group was given 10 micrograms of an unrelated control antibody. Six weeks after the administration of antibodies, the rats were intestinally colonised with an ovalbumin (OVA)-producing E. coli O6K13 strain. At 8 weeks of age, the male rats (first generation) and the offsprings of the female rats (second generation), were parenterally immunised with OVA and dead wild type E. coli O6K13, and the immune response was followed. In the rats of the first generation, there were no major differences between the groups in the immune response to the bacterium. However, the offspring of the neonatally a-Id administered rats had a profoundly affected immune response to the idiotypically connected antigen K13, but also to other antigens on the bacteria. Thus, a-Id treatment in the first generation gave, in the second generation, a greatly enhanced serum antibody response to the spatially related antigens OVA and O6 LPS, as well as to the idiotypically connected antigen K13. Concurrently, the in vitro spleen cell proliferative response to both OVA and the wild type bacterium was lowered. Overall, greater effects were seen with the higher dose of a-Id. In conclusion, our results demonstrate that by giving monoclonal antibodies idiotypically connected to a single bacterial component to neonatal rats, one profoundly influence the immune response also to other-spatially related-bacterial antigens in their offsprings.

Administration, Oral↗

PubMed1998

Autoantibody profiles in canine ANA-positive sera investigated by immunoblot and ELISA.

Canine systemic lupus erythematosus (SLE) has a similar disease expression as human SLE, but the serological characterisation of the canine disease is as yet incomplete. In the present study, we examined the specificity of antinuclear antibodies (ANA) in indirect immunofluorescence (IIF) positive canine sera. Sixty-four canine IIF ANA positive sera were characterised using HeLa cell nuclear extract immunoblots and recombinant U1-70K ELISA. We compared these results with a previously shown concordance between indirect immunofluorescence and immunodiffusion in canine SLE serological diagnosis. One canine serum reacting with Sm proteins was observed, and five canine sera presented anti-RNP autoantibodies against the antigens 70K, A, C, and/or B/B'. The autoantigen most frequently recognised was a 43 kDa nuclear protein, previously described as hnRNP G. This prominent canine autoantigen was missing in the commercially available extract designed for immunodiffusion testing of human sera. Other prominent canine autoantigens were found not to be identical with the principal human ones, thus making present human test systems deficient for the use in canine systemic connective disease diagnosis. The development of antigenic extract designed for canine autoimmune autoantigens is necessary in order to make immunodiffusion a useful method in canine diagnosis. The anti-RNP positive canine sera were examined in more detail and we found that the human major antigenic region of the most prominent RNP antigen, the U1-70K protein, also is targeted by canine autoantibodies. Thus, the response against the RNP antigen seems to be conserved between man and dog.

Animals↗

PubMed1998

The Zn2+ binding domain of the human Ro 52 kDa protein is a target for conformation-dependent autoantibodies.

The Ro 52 kDa protein was originally identified using autoimmune sera directed against the Ro/SS-A antigen, also known as Ro ribonucleoprotein particles (Ro RNPs). In most human cells these consist of one of four small human cytoplasmic (hY) RNA molecules complexed with the Ro 60 kDa protein. This protein interacts directly with the hY RNAs, whereas the Ro 52 kDa protein is thought to associate via protein-protein interactions. The Ro RNPs are present in all mammalian cells, but their intracellular location and function remain unclear. The primary structure shows that the Ro 52 kDa protein is a member of a small family of proteins sharing two features, a leucine zipper region and an aminoterminal cysteine-histidine rich region with two different putative zinc finger motifs. To study if the Ro 52 kDa protein actually binds Zn2+, both the full-length Ro 52 kDa clone and various subclones were expressed as recombinant proteins and assayed for Zn2+ binding in vitro. A fragment containing the first cysteine-histidine cluster at residues 14-54 and other larger overlapping fragments were found to bind Zn2+. In this report we also demonstrate that the Zn2+ binding domain is a target for conformation-dependent anti-Ro 52 kDa autoantibodies. The antigenicity is dramatically increased by the same reducing conditions that promote Zn2+ binding. This is in contrast to the previously described immunodominant Ro 52 kDa domain.

Autoantibodies↗

PubMed1998

Ro/SS-A and La/SS-B antibody level variation in patients with Sjögren's syndrome and systemic lupus erythematosus.

To examine both possible correlations between anti-Ro/SS-A and anti-La/SS-B levels and their correlation with clinical disease activity in patients with Sjögren's syndrome (SS) or systemic lupus erythematosus (SLE), an ELISA was developed using purified recombinant Ro 60 kDa, Ro 52 kDa and La antigens. The ELISA was used for testing sequential serum samples from 16 patients with either SS or SLE. The patients were followed for periods between 15 and 128 months, and 3-15 serum samples per patient were analysed and compared with clinically apparent disease activity at the time of sampling in 14 patients. A temporal correlation of antibody levels to Ro and La antigens was found, and antibodies to different epitopes of the Ro 60 kDa protein showed parallel variation in seven of eight patients tested. Co-variation of autoantibody levels and disease activity was found in 11 of 14 patients. In seven of these 11 patients the anti-Ro and anti-La levels were stable and changes in disease activity were minimal during the observation period. In the other four of these 11, changes in disease activity were noted, with an associated change in autoantibody levels. The results suggest that the serological response to Ro and La antigens, as well as to different epitopes of the Ro 60 kDa protein, is antigen driven and regulated by common mechanisms, and indicate a correlation of Ro and La antibodies with pathogenic events.

Adult↗

PubMed1998

Intracellular localisation of the Ro 52kD auto-antigen in HeLa cells visualised with green fluorescent protein chimeras.

Autoantibodies to the Ro/SSA and La/SSB antigens are found in patients with Sjogren's syndrome and systemic lupus erythematosus. The Ro/SSA autoantigen consists of a 52 kD and a 60 kD protein, complexed with one of four small RNA molecules. The La protein can associate with the complex. The Ro/SSA autoantigens are present in all mammalian cells, but their intracellular location is subject of controversy and their function remains unclear. To study the intracellular sorting and targeting of Ro 52 kD we have constructed expression plasmids encoding fusion proteins between the full-length Ro 52 kD protein as well as Ro 52 kD fragments and the green fluorescent protein (GFP) from the jelly fish, Aequorea Victoria. The subcellular distribution of the GFP-Ro 52 kD fusion proteins was investigated in transient expression experiments using transfected HeLa cells. The GFP-full-length Ro 52 kD fusion protein was accumulated in the cytoplasm and excluded from the nucleus. When GFP was fused with the La protein, the fluorescence was located in the nucleus. Clones coding for Ro 52 kD fragments containing the hydrophilic central part of the Ro 52 kD protein gave the same intracellular location and type of cytoplasmic speckles as the full-length Ro 52 kD protein. In contrast, both amino terminal and carboxy terminal fragments were uniformly distributed throughout the cell just like the GFP protein itself. These observations indicated a cytoplasmic location of the Ro 52 kD protein and demonstrated the crucial role of the hydrophilic domain in restricting the Ro 52 kD protein to this intracellular compartment.

Animals↗

PubMed1997

Thrombomucin, a novel cell surface protein that defines thrombocytes and multipotent hematopoietic progenitors.

MEP21 is an avian antigen specifically expressed on the surface of Myb-Ets-transformed multipotent hematopoietic precursors (MEPs) and of normal thrombocytes. Using nanoelectrospray tandem mass spectrometry, we have sequenced and subsequently cloned the MEP21 cDNA and named the gene thrombomucin as it encodes a 571-amino acid protein with an extracellular domain typical of the mucin family of proteoglycans. Thrombomucin is distantly related to CD34, the best characterized and most used human hematopoietic stem cell marker. It is also highly homologous in its transmembrane/intracellular domain to podocalyxinlike protein-1, a rabbit cell surface glycoprotein of kidney podocytes. Single cell analysis of yolk sac cells from 3-d-old chick embryos revealed that thrombomucin is expressed on the surface of both lineage-restricted and multipotent progenitors. In the bone marrow, thrombomucin is also expressed on mono- and multipotent progenitors, showing an overlapping but distinct expression pattern from that of the receptor-type stem cell marker c-kit. These observations strengthen the notion that the Myb-Ets oncoprotein can induce the proliferation of thrombomucin-positive hematopoietic progenitors that have retained the capacity to differentiate along multiple lineages. They also suggest that thrombomucin and CD34 form a family of stem cell-specific proteins with possibly overlapping functions in early hematopoietic progenitors.

Animals↗

PubMed1997

Autoepitope-mapping of the U1-70K protein with human-Drosophila chimeric proteins.

The 70K protein is the major autoantigen for anti-RNP autoantibodies directed against the U1 small nuclear ribonucleoprotein complex particle. The U1-70K protein has been epitope-mapped by various groups, and a major antigenic region of about 70 amino acids has been found which overlaps with the RNA binding motif. Attempts to map the major antigenic region further with smaller cloned fragments or with peptides have been hampered by total loss of, or strongly reduced, antigenicity. Thus the major antigenic region is composed of conformational epitopes and a detailed analysis of particular epitopes has not been possible. In the present work, we examine the antigenicity of chimeric proteins assembled from the highly conserved Drosophila melanogaster 70K proteins grafted with human 70K segments. With this approach, the effects on antigenicity of exchanging particular segments can be assayed with the overall structure of the major antigenic domain kept relatively constant. Our results, supported by depletion experiments, show that residues 99-128 from the human protein are essential for recognition by both human and canine anti-RNP autoantibodies. These residues have to be presented in a manner that allows correct conformational interaction between the different protein domains.

Amino Acid Sequence↗

PubMed1996

Autoantibody repertoire to Ro/SSA and La/SSB antigens in patients with primary and secondary Sjögren's syndrome.

Autoimmune diseases are characterized serologically by the presence of antibodies to specific autoantigens. Antibodies to the two antigens Ro/SSA and La/SSB are found in patients with primary (pSS) and secondary Sjögren's syndrome (sSS). To explore if differences in the fine specificity of these autoantibodies could be distinguished in sera from patients with primary (n = 17) and secondary (n = 20) Sjögren's syndrome, sera were analysed by immunoblotting and ELISA using recombinant antigens and synthetic peptides. Minor differences were detected when the frequencies of the Ro 60 kD, Ro 52 kD and La autoantibody specificities to full-length proteins in the pSS and sSS groups were compared. However, when reactivity to different parts of the Ro 60 kD antigen was analysed, including recombinant fragments encompassing amino acid (aa) 1-134, aa 181-320 and aa 397-525, only two sera, both from pSS patients, reacted to the aminoterminal fragment aa 1-134, and 3/4 sera that reacted with the carboxyterminal aa 397-525 fragment derived from sSS patients. Of all the anti-Ro 60 kD positive sera, 80% reacted with the middle fragment encompassing aa residues 181-320. The fine specificity of the autoantibodies reacting with this 181-320 aa region was further mapped with synthetic peptides, and a peptide (VSLVCEKLCNEKLLKKARIH) recognized by 8 out of 16 sera from both pSS and sSS patients was identified.

Adult↗

PubMed1996

Human anti-RNP sera contain both human-specific and cross-reactive anti-70K autoantibodies.

The U1 snRNP (small nuclear ribonucleoprotein complex) associated 70K protein is the main autoantigen for the anti-RNP autoantibodies which are directed against the U1 snRNP particle. The major antigenic region of the 70K protein has by various laboratories been mapped to an RNA binding domain required for the 70K-U1 snRNA interaction. We have used recombinant proteins comprising this region from the human and the Drosophila melanogaster 70K proteins to examine the species specificity of the human anti-70K autoantibodies found in 42 patient sera. Most, but not all, anti-70K positive sera in this cross-sectional sample contained both human 70K specific anti-bodies and Drosophila 70K reactive antibodies. Results of a longitudinal follow-up of 14 patients indicated that the cross-reactive anti-70K antibodies developed secondarily to the establishment of a species-specific anti-70K reaction. In a fraction of the patient sera this broadening of the response never occurred. Taken together, the data in this study support the hypothesis that the endogenous human 70K protein is the immunogen driving the production of anti-70K autoantibodies.

Amino Acid Sequence↗

PubMed1996

Nuclear colocalization of the Ro 60 kDa autoantigen and a subset of U snRNP domains.

The Ro 60 kDa protein is an RNA binding molecule present both in the cytoplasm and in the nucleus. Cytoplasmic Ro 60 kDa is complexed to other proteins and to certain RNAs denoted hYRNAs. This RNA-protein complex is also known as the Ro/SSA antigen recognized by sera from patients with certain autoimmune disorders. Components interacting with the nuclear Ro 60 kDa protein fraction in mammalian cells have not been identified. To look for an association with previously known nuclear structures, rabbit antisera to the amino- and carboxy-terminal parts of the Ro 60 kDa protein were used in immunomorphological studies on HeLa cells. A strong speckled nuclear pattern and a weak cytoplasmic staining were detected. Double immunofluorescence staining with affinity purified anti-Ro 60 kDa antibodies and monoclonal antibodies recognizing the Sm and RNP antigens of the U snRNPs, displayed colocalization. Another U snRNP containing nuclear compartment, the coiled bodies, did not contain any Ro 60 kDa protein. Cells infected with a toga virus demonstrated redistribution of both U snRNP antigens and the Ro 60 kDa protein with retained colocalization. These results indicate a role for the nuclear fraction of the Ro 60 kDa protein in RNA processing.

Animals↗

PubMed1995

U2-snRNP B" protein gene is an early growth-inducible gene.

In this work we isolated mouse U2-snRNP-specific b" clones and analysed the expression of the mouse U2-snRNP-specific b" and U1-snRNP-specific 70K genes in NIH-3T3 fibroblasts. Stimulation of growth-arrested NIH-3T3 cells with serum was found to evoke a rapid increase in the amount of cytoplasmic b" and 70K mRNAs. These increases in mRNA did not require de novo protein synthesis. Moreover, the inhibition of protein synthesis by cycloheximide caused a superinduction in the amounts of the U1-snRNP-specific 70K transcripts. We also found that c-Ha-rasVal12 oncogene-transformed NIH-3T3 cells have higher levels of the b" and 70K mRNAs than the normal 3T3 cells. These data imply that the b" and 70K are early growth response genes, and their enhanced expression might be of significance in the processing of pre-mRNAs into mature mRNAs.

3T3 Cells↗

PubMed1995

Single-view screening mammography: psychological, endocrine and immunological effects of recalling for a complete three-view examination.

To investigate influences of a recall due to inconclusive findings on screening mammography, 45 women were examined with psychological ('mood' and 'coping'), endocrine and immunological tests immediately after complete mammography (first interview), 2-3 days after the initial screening mammography, and 3 weeks after the women had been informed of normal findings (second interview). The mood score in the first interview was significantly lower than in the second. No differences were found in the endocrine and immunological tests. The recall for complete mammography provoked a significant short-term emotional reaction not reflected in changes in the endocrine and immune functions.

Affect↗