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Biomedical subjects

L Rydberg

Publications and source records attributed to L Rydberg.

At least 37 records · Page 2Linked to original sources
PubMed1999

Typing for the human lewis blood group system by quantitative fluorescence-activated flow cytometry: large differences in antigen presentation on erythrocytes between A(1), A(2), B, O phenotypes.

BACKGROUND: Lewis phenotyping by hemagglutination is an unreliable routine method for Lewis antigen designation. Now genomic typing of the Lewis gene is available. Additionally, flow cytometry has been used for typing. We wanted to compare the results of Lewis typing in healthy individuals using the three methods. MATERIALS AND METHODS: Ninety-three randomly selected plasma donors were genotyped for inactivating Secretor (FUT2) G428A and Lewis (FUT3) T59G, T202C, C314T, G508A and T1067A point mutations. All Le(a+b-) individuals (nonsecretors) were homozygous for the FUT2 G428A mutation and all Le(a-b-) individuals had inactivating mutations on both FUT3 alleles. Fixed erythrocytes were analyzed by fluorescence-activated flow cytometry and the results were compared with hem- agglutination and genotypic data. Antigen availability was expressed as median fluorescence intensity and as percentage positive cells with fluorescence intensities > or =10(2). RESULTS: Using an anti-Le(a) reagent a mean of 99% of erythrocytes from Le(a+b-) individuals and 1% of erythrocytes from Le(a-b-) or Le(a-b+) individuals were stained positive. Using an anti-Le(b) reagent, a mean of 71% of erythrocytes from A(1), 95% from B and 99% from O and A(2) Le(a-b+) individuals and less than 10% of erythrocytes from Le(a-b-) or Le(a+b-) individuals were stained positive. After papain treatment 100% of the erythrocytes from A(1) and A(1)B Le(a-b+) individuals stained positive without increase in background staining. The flow cytometric technique revealed large differences in staining intensities, within each ABO Le(a-b+) subgroup which was not directly correlated to plasma donation frequencies nor to Secretor or Lewis genotypes. CONCLUSION: Flow cytometry may prove valuable as a Lewis blood group typing technique but also as a research tool when investigating Lewis phenotypes of human erythrocytes.

ABO Blood-Group System↗
PubMed1998

No evidence of pig DNA or retroviral infection in patients with short-term extracorporeal connection to pig kidneys.

BACKGROUND: The xenotransplantation of organs and tissues, in particular those from pigs, is viewed as a means to alleviate the shortage of human donor organs and cells available for transplantation and also as a therapy for other diseases. The potential microbiological hazards of xenotransplantation have recently attracted much attention. One concern is over pig endogenous retroviruses (PERV). Until the possible consequences of infection by PERV are better understood it is unlikely that a significant number of porcine xenotransplants will proceed. However, a small number of patients have already been treated with or exposed to living porcine cells or tissue, and investigation of these patients may provide valuable information. METHODS: We took serial blood samples from two renal dialysis patients whose circulation had been linked extracorporeally to pig kidneys and tested them for pig DNA and PERV DNA by nested PCR. The patients' plasma was also tested for neutralising antibodies to two anthropotropic PERV strains. FINDINGS: Having established that the nested PCRs could detect single molecules of target sequence, we analysed DNA isolated from patients' peripheral blood mononuclear cells. We found no evidence of pig or PERV DNA in either patient, even in samples taken as early as 6 h after the perfusion. Furthermore, we found no evidence of seroconversion for PERV-specific antibodies. INTERPRETATION: The absence of porcine cells in the circulation of both patients, even in the samples taken soon after the perfusion experiment, suggests that any porcine cells dislodged from the kidney became rapidly sequestered from the circulation. Since cell-to-cell contact increases the efficiency of infection of PERV this removal of porcine cells may increase the risk of transmission of PERV to the xenograft recipient. We did not, however, detect indications of infection by PERV by PCR or neutralisation assay. The genetic and serological methods described here will be useful for detection of possible PERV infection in other patients.

Adult↗
PubMed1998

An ELISA technique for quantitation of human xenoantibodies binding to pig cells: application in patients with pig kidneys extracorporeally connected to the circulation.

A quantitative ELISA technique for determination of human anti-pig xenoantibody number in serum samples has been established using pig lymphocytes and pig/rabbit erythrocytes as target cells and a pool of serum from human blood group AB donors. The number of low affinity antibodies binding to the cells was determined by quantitation following the use of aqueous washing of the cells and separation of bound and unbound antibodies with the phthalate oil method. The efficiency of different soluble Gal(alpha)1-3Gal-terminating di- and tri-saccharides to inhibit antibody binding was tested and found to vary between 70-90% at a saccharide concentration of 10 mg/ml. The assay was used to evaluate the antibody changes in two patients who, after plasmapheresis treatments, had pig kidneys extracorporeally connected to their blood circulation. The number of anti-pig IgM/IgG antibodies bound to each pig lymphocyte were reduced from 5,600/13,200 to 1,300/3,100 in patient 1 and from 1,200/6,500 to 500/2,100 in patient 2 by three consecutive daily plasmapheresis treatments. Although the lymphocytotoxic titers were reduced to very low levels, the antibody numbers still present in the blood of patient 1 caused a hyperacute rejection of the pig kidney. However, the antibody levels in patient 2 did not cause rejection of this kidney during 15 min perfusion time. A strong anti-pig antibody response 3 weeks after the perfusion experiment was found in patient 1 as shown by 27,600/245,300 IgM/IgG molecules bound to pig lymphocytes corresponding to an increase of lymphocytotoxic titer from 8 to 512. The second patient showed a much weaker immune response with 1,400/19,800 IgM/IgG antibodies corresponding to a lymphocytotoxic titer increase from 8 to 32. The use of this quantitation technique enables more accurate investigation of antibody binding to xenogenic target cells than conventional titration techniques.

Animals↗
PubMed1998

Characterisation of human natural anti-sheep xenoantibodies.

Currently, the pig species is regarded as the most likely organ donor for human xenotransplantation in the future. However, it cannot be granted that the pig will be the optimal species of choice. We have studied human anti-sheep antibodies in comparison with anti-pig antibodies. The anti-sheep lymphocytotoxic and hemagglutination titers were in the range 8 to 128 and 2 to 32, respectively, in single individuals, which were considerably lower than the anti-pig titers of these individuals. Perfusion of sheep kidneys with human blood reduced the anti-sheep xenoantibody titers to zero as measured by lymphocytotoxic, hemagglutination, and sheep aortic endothelial cell antibody binding assays. The perfused kidneys showed generalised depositions of human IgM and C3c in the vascular tree and focal depositions of C1q and fibrin. Obliteration of capillaries by human platelets and polymorphonuclear cells were observed. Total neutral glycolipid fractions were isolated from sheep intestinal, pancreatic, and kidney tissues. By using a chromatogram binding assay, a monoclonal anti-Forssman antibody identified a single compound with five sugar residues in all organs. Several glycolipid bands were stained in all organs by the Gal(alpha)1-specific lectin I-B4 from Griffonia (Bandeiraea) Simplicifolia. A human AB serum pool showed staining by both IgG and IgM antibodies of the Forssman and Gal(alpha)1-terminating components as well as some other, not structurally identified, components. The Forssman and Gal(alpha)1-reactivity in human sera could be eliminated by immunoadsorption using Forssman and Gal(alpha)1-3Gal-immunoadsorbent columns, respectively. Immunostaining of sheep kidney tissue sections showed the presence of Gal(alpha)1-terminating epitopes by immunoperoxidase and immunogold silver staining techniques. Proximal convoluted tubules showed a strong staining, while thin loops of Henle, collecting ducts, urothelium, and vessels showed a weaker staining. Distal convoluted tubules and thick loops of Henle were completely negative. In summary, human serum contains anti-sheep xenoantibodies reacting mainly with the Forssman and Gal(alpha)1-determinants in sheep tissues and the anti-sheep antibody titers are lower than the corresponding anti-pig titers.

Animals↗
PubMed1998

Extracorporeal ("ex vivo") connection of pig kidneys to humans. III. Studies of plasma complement activation and complement deposition in the kidney tissue.

The complement system is one of the important factors involved in the hyperacute rejection of xenografts. This report deals with the activation of the complement system in a clinical trial where pig kidneys were extracorporeally connected to two volunteer dialysis patients who were pretreated with plasmapheresis in order to substantially reduce anti-pig xenoantibodies. The clinical data of the perfusion experiments and the patients humoral immune response to pig xenoantigens have been reported in detail (Xenotransplantation 1996; 3:328-339, 340-353). Three consecutive daily plasmapheresis treatments of the patients reduced the plasma complement protein (C3, C4, and C5) concentrations to 8-27% of the baseline values. The perfusion of the pig kidney connected to patient 1 was terminated at 65 min due to graft rejection and this patient was not hemodynamically affected by the experiment. The second experiment was terminated at 15 min due to an anaphylactic like reaction of the patient. In patient 1 a slight reduction of plasma C3, C4, and C5 and an increase of C5a and SC5b-9 occurred, while C3a decreased during the perfusion. Patient 2 had an increase of all complement parameters, most prominent for C4d and SC5b-9, which occurred concomitant with the appearance of the anaphylactic like side effects. In general, plasma levels of PMN elastase, IL6 and IL8 increased in both patients during the perfusion. Immunohistochemical investigation of the kidney tissues revealed deposition of human complement factors C1q, C4c, and C3c in a congruent pattern with the vasculature of the kidney in patient 1. In kidney 2 only trace amounts of C1q and C3c were found. Both kidneys were negative for properdin. Therefore, in this experimental set up with extracorporeal connection of pig kidneys to the human circulation the human complement cascade is activated mainly through the classical pathway.

Adult↗
PubMed1997

Blood spillage during salvage.

Recovery of blood components by salvage was evaluated in a randomized laboratory investigation using donor whole blood. Three methods of salvage were studied; conventional red cell salvage, red cell salvage with continuous processing, and salvage by haemofiltration using recirculation through a 30,000-Da filter. Less than 10% of the haemoglobin was wasted during processing by all three methods. About half of the leukocytes were recovered with no significant difference between the techniques. The recovery of platelets was 15, 4 and 41% during salvage with conventional red cell centrifugation, continuous centrifugation processing and haemofiltration, respectively. In contrast with the other methods, haemofiltration gave a major recovery of albumin.

Blood Cell Count↗
PubMed1997

Complement activation during storage of whole blood, red cells, plasma, and buffy coat.

BACKGROUND: The process of separating whole blood into components and the storage of blood components may cause the release of toxic metabolites from the complement cascade. The aim of this study was to determine whether the storage of blood components leads to the activation of the complement cascade and the release of anaphylatoxins. STUDY DESIGN AND METHODS: Blood from 12 healthy volunteers was collected and stored either as whole blood or as components: red cells in saline-adenine-glucose-mannitol solution, plasma, and buffy coat. The concentrations of anaphylatoxins and other complement proteins in the various blood components were intermittently analyzed during a 5-week storage period. RESULTS: Increasing levels of anaphylatoxins were demonstrated during the storage of whole blood and plasma. Elevated concentrations of the anaphylatoxins C3a and C5a were observed during the storage of whole blood. Increased C5a levels were observed after 7 days of storage. High concentrations of C3a were found in plasma after 14 days of storage. Low or non-detectable levels of C3a; C5a, and other complement components were found in red cells stores in saline-adenine-glucose-mannitol solution. CONCLUSION: The study demonstrated activation of complement during the storage of whole blood and plasma but not in red cells in storage solution. The transfusion of larger volumes of stored whole blood or plasma may contribute to the risk of development of organ dysfunction. Therefore, it is advisable to use red cells in storage solution.

Anaphylatoxins↗
PubMed1997

Report from the HLA class II typing by PCR-SSP Multicentre Study.

Results from 360 HLA-DR and -DQ 'low-resolution' typings with polymerase chain reaction sequence-specific primers (PCR-SSP), performed by nine laboratories, were analysed for their overall utility in routinely defining the HLA-DR1-DR18, DR51-DR53 and DQ1-DQ9 specificities in less than 2.5 h. Thirty EDTA blood samples and 10 DNA samples were distributed and analysed by each laboratory. DNA was extracted using a rapid bromide salt extraction protocol. Complete HLA-DR and -DQ typings were performed, three by three, on pre-aliquoted 96-tube PCR trays. When compared with reference typing, 351/360 (98%) correct DR typings were obtained, whereas 320/360 (89%) of the DQ phenotypes were correctly assigned. The time for three complete HLA-DR and -DQ 'low-resolution' typings, including DNA extraction, ranged from 2.0 h to 2.3 h. Unfortunately, an unusually high level of PCR amplification failures was observed (3%), probably due to diffusion and a significant volume loss from some of the pre-aliquoted primer mixes. Consequently, only 52% of the typings were without any amplification failure, and 0-2 amplification failures where found in 88% of the PCR-SSP typings performed. The number of HLA-DR-DQ retypings needed was 7 and 8%, respectively, reflecting the low number of typings where allelic identification was directly affected by the relatively high level of amplification failures in this study. Thus, a 91-98% success rate of correctly identified HLA-DR and -DQ alleles could be maintained, even under suboptimal typing conditions.

Alleles↗
PubMed1997

Influence of infant feeding and gluten intake on coeliac disease.

OBJECTIVES: To study the impact of infant feeding habits and actual gluten intake on gluten induced enteropathy. METHODS: A case-referent design, controlling for the HLA alleles conferring increased genetic risk, was used. All 164 siblings of 97 probands were investigated. Eighty five of the siblings, carrying the genes DQA1*0501-DQB1*02 conferring susceptibility for the disease, were investigated by interview, food recording, and taking a small intestinal biopsy sample. Eight cases of silent coeliac disease were found and these were compared with the 73 siblings in whom the diagnosis was excluded. RESULTS: No statistically significant differences were found between cases and referents in terms of duration of breast feeding, age at introduction of cows' milk products, frequency of breast feeding after gluten introduction, and gluten consumption. CONCLUSIONS: The studies factors may be of less importance for the development of gluten induced enteropathy.

Adolescent↗
PubMed1997

Does oesophageal motor function improve with time after successful antireflux surgery? Results of a prospective, randomised clinical study.

MAIN OBJECTIVE: There is a continuing debate whether motor abnormalities associated with gastro-oesophageal reflux disease (GORD) are primary phenomena or occur as a consequence of repeated injury caused by inflammation. To get new insights into mechanisms involved, patients were studied before and three years after effective and durable reflux control induced by two types of fundoplications. PATIENTS AND METHODS: Thirty three consecutive patients with chronic GORD entered the trial. All patients had a clinical history of GORD assessed symptomatically, endoscopically, and by use of 24 hour pH-metry. Eighteen were randomised to a 180 degrees semifundoplication (Toupet) and 15 to a total fundic wrap (Nissen-Rossetti). Manometry was done preoperatively, six months, and three years after the operation assessing motor function in defined areas of the tubular oesophagus and lower oesophageal sphincter. RESULTS: All patients had a proper control of GORD both when objectively and clinically assessed. Postoperatively, the resting tone of the lower oesophageal sphincter was significantly higher in the Nissen-Rossetti group (p < 0.05), and the intra-abdominal portion of the lower oesophageal sphincter was of identical length in the two groups. A significant increase in peristaltic amplitude in the middle and distal third of the oesophagus was recorded at long term follow up compared with the preoperative findings (p < 0.05), but there was no corresponding effect on propagation speed and duration of contraction. However, an increase in peristaltic amplitude and, as a tentative consequence, a significant decrease (p < 0.05) in the frequency of primary peristalsis was found only in patients operated on with a total fundic wrap. CONCLUSION: Despite adequate and durable reflux control after fundoplication in patients with chronic GORD, no change was found in oesophageal motor function with time. The higher contraction amplitude and decreased frequency of failed primary peristalsis seen in patients having a total fundic wrap were thus most likely due to a mechanical outflow obstruction in the gastro-oesophageal junction. These results could therefore be interpreted in favour of the hypothesis that GORD is pathogenetically linked to a primary defect in oesophageal motor function.

Esophagus↗
PubMed1996

Long-term results of a prospective randomized comparison of total fundic wrap (Nissen-Rossetti) or semifundoplication (Toupet) for gastro-oesophageal reflux.

The importance of the extent of the fundic wrap that encircles the distal oesophagus for the establishment of long-term control of gastro-oesophageal reflux disease (GORD) and for the risk of symptoms after fundoplication was evaluated in a prospective, randomized clinical trial. Of 137 consecutive patients with GORD, 72 were allocated to a semifundoplication (180-200 degrees, Toupet) and 65 to a total fundoplication (360 degrees, Nissen-Rossetti). Dysphagia was more common in the early postoperative period after a total fundic wrap, a difference which disappeared with time. This corresponded to a higher resting tone in the lower oesophageal sphincter area. Seven patients (5 per cent) experienced relapse of GORD during follow-up of more than 3 years. Although no difference in the cumulative relapse rate (5 per cent for Nissen-Rossetti versus 6 per cent for Toupet) was found between the two study groups, the total failure rate was higher (P < 0.05) among patients who had a Nissen-Rossetti procedure because of a procedure-specific complication: intrathoracic herniation of the fundoplication in five patients caused obstructive symptoms without reflux (four had no posterior crural repair). In addition, symptoms in the form of flatulence were more frequently seen after Nissen-Rossetti fundoplication (P < 0.05 at 2 years and P < 0.01 at 3 years). Both Nissen-Rossetti and Toupet fundoplication equally well and durably controlled GORD. Fewer symptoms occurred in those having a semifundoplication, both in the early and late postoperative period.

Adult↗
PubMed1996

Blood group A antigen expression in platelets is prominently associated with glycoprotein Ib and IIb. Evidence for an A1/A2 difference.

Blood group ABH antigens are associated with platelets as intrinsic determinants and extrinsically adsorbed antigens, and exist both on glycosphingolipids and on glycoproteins (GPs). We now provide direct evidence that the blood group ABH antigens are prominently associated with platelet GPIb and GPIIb. By immunoprecipitation, a murine monoclonal anti-A antibody precipitated surface-biotin-labelled blood group A1 platelet membrane proteins with electrophoretic characteristics identical to those of GPIb/IX and GPIIb/IIIa. By immunoblotting of SDS-PAGE separated blood group A1 platelet proteins the monoclonal anti-A antibody bound to proteins with electrophoretic characteristics identical to those of GPIb and GPIIb. When immunoaffinity purified GPIb/IX and GPIIb/IIIa, derived from blood group O, A1 and A2 platelets, were employed for immunoblotting, GPIb and GPIIb only from A1 platelets bound the monoclonal anti-A antibody. By ELISA, wherein monoclonal antibodies specific for GPIb (APl) and the GPIIb/IIIa complex (AP2) were used to capture and hold antigens from platelet lysate, human anti-A antibodies reacted with these proteins derived from blood group A1 platelets; proteins from blood group A2, O and B platelets showed no reactivity. These results indicate that blood group A antigen is associated with GPIb and GPIIb derived from blood group A1 but not A2 platelets.

ABO Blood-Group System↗
PubMed1996

Evidence for an expression of blood group A antigen on platelet glycoproteins IV and V.

Blood group ABO antigens are known to be carried by several platelet glycoproteins (GP), e.g. GPIb, GPIIa, GPIIb, GPIIa and PECAM. Beside these proteins, we recently observed that blood group A antigen was also expressed on some other uncharacterized platelet proteins (70-90 kDa) having electrophoretic mobilities closely resembling those of GPIV and GPV. These findings prompted us further to characterize these latter ABO-expressing platelet proteins. By antigen capture ELISA, wherein the monoclonal antibodies (mAbs) CLB-IVC7 and CLB-SWI6 were used to hold the corresponding antigens GPIV and GPV, human anti-A specifically bound to these proteins derived from A1-platelets; neither GPIV nor GPV derived from A2-, B- or O-platelets bound anti-A. In a Western blot assay using immunoprecipitated GPIV and GPV as antigens, mAb anti-A immunostained GPIV and GPV precipitated from A1, but not from A2 and O platelets. These results conclusively demonstrate that blood group A antigen is expressed on platelet GPIV and GPV.

ABO Blood-Group System↗
PubMed1996

DNA sequencing and screening for point mutations in the human Lewis (FUT3) gene enables molecular genotyping of the human Lewis blood group system.

The human Lewis gene encodes an alpha(1,3/1,4)-fucosyltransferase responsible for synthesis of the Le(a) and a Le(b) antigens. To define the molecular background for non-functional Lewis genes we have sequenced PCR-amplified DNA fragments from two Le(a-b-) individuals. One was homozygously mutated at nucleotides 202(T --> C) and 314 (C --> T), altering Trp68 to Arg and Thr105 to Met, and the other was homozygously mutated at nucleotides 59 (T --> G) and 1067 (T --> A), altering Leu20 to Arg and Ile356 to Lys. Using PCR we screened for these and additionally one other mutation at nucleotide 508 (G --> A) among 40 Caucasians. Of 15 Le(a-b-) individuals, 7 typed as le59/1067le202/314, 4 as le202/314le202/314 and 1 as le59/1067le59/1067. Of 21 Le(a-b+) and 4 Le(a+b-), 17 typed as LeLe and 7 as Lele202/314. A pedigree study of 8 Lewis-positive individuals showed that the mutations at nucleotides 202 and 314 were located on the same allele.

Alleles↗
PubMed1996

Genotyping of the platelet-specific alloantigen HPA-5 (Br(a)/Br(b)) using polymerase chain reaction with sequencespecific primers (PCR-SSP).

A DNA-based one-stage technique, polymerase chain reaction with sequence-specific primers (PCR-SSP) was developed for genotyping of the platelet specific alloantigen HPA-5 (Bra/Brb). Sequence-specific primers, matching the wild type and the point mutation responsible for the HPA-5 (Bra/Brb) phenotype, were constructed. Conjointly a fragment of the gene coding for glycoprotein (GP) IIIa was amplified as an internal control of the enzyme reaction. Using these HPA-5 (Bra/Brb) sequence-specific primers the correct fragment of the GPIa gene was amplified, as evidenced by the PCR product size, the restriction map and by the nucleotide sequence. This assay was applied on 187 Swedish blood donors; 157 individuals were found to have a homozygous HPA-5a (Bra/Brb) genotype and 30 individuals a heterozygous HPA-5a,b (Bra/Brb) genotype. None of the donors was found to display a homozygous HPA-5b (Bra/Brb) genotype. Thus, the (HPA-5b) Bra antigen frequency in this population will be approximately 16.0% with a gene frequency of 8.0%. It is concluded that this assay is an attractive technique for genotyping of the HPA-5 (Bra/Brb) alloantigens on genomic DNA. The technique can replace serological alloantigen typing, especially in cases where platelets and rare human alloantisera are not available.

Adult↗