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1.
Immunohematology ; 39(1): 1-10, 2023 Apr 01.
Article in English | MEDLINE | ID: mdl-37017600

ABSTRACT

The high-prevalence blood group antigen, Sda, had been puzzling blood bankers and transfusionists for at least a decade when it was reported in 1967. The characteristic mix of agglutinates and free red blood cells (RBCs), caused by anti-Sda, is seen with the RBCs from 90 percent of individuals of European descent. However, only 2-4 percent of individuals are truly Sd(a-) and may produce anti-Sda. The antibodies, generally considered insignificant, may cause hemolytic transfusion reactions with high-expressing Sd(a+) RBCs (e.g., the unusual Cad phenotype, which can also be polyagglutinable). The Sda glycan, GalNAcß1-4(NeuAcα2-3)Gal-R, is produced in the gastrointestinal and urinary systems, while its origin on RBCs is more controversial. According to current theory, Sda is likely to be passively adsorbed in low amounts, except in Cad individuals, where it has been found on erythroid proteins and at higher levels. The long-standing hypothesis that B4GALNT2 encodes the Sda synthase was confirmed in 2019, since homozygosity for a variant allele with rs7224888:C produces a non-functional enzyme associated with most cases of the Sd(a-) phenotype. Thereby, the SID blood group system was acknowledged as number 038 by the International Society of Blood Transfusion. Although the genetic background of Sd(a-) was settled, questions remain. The genetic background of the Cad phenotype has not yet been determined, and the source of the RBC-carried Sda is unknown. Furthermore, the interest of Sda stretches beyond transfusion medicine. Some tantalizing examples are lowered antigen levels in malignant tissue compared with normal tissue and interference with infectious agents like Escherichia coli, influenza virus, and malaria parasites.


Subject(s)
Blood Group Antigens , Humans , Erythrocytes , Blood Transfusion , Antibodies , Carbohydrates
2.
Vox Sang ; 113(2): 185-197, 2018 Feb.
Article in English | MEDLINE | ID: mdl-29214632

ABSTRACT

BACKGROUND AND OBJECTIVES: ABO remains the clinically most important blood group system, but despite earlier extensive research, significant findings are still being made. The vast majority of catalogued ABO null alleles are based on the c.261delG polymorphism. Apart from c.802G>A, other mechanisms for O alleles are rare. While analysing the data set from the 1000 Genomes (1000G) project, we encountered two previously uncharacterized deletions, which needed further exploration. MATERIALS AND METHODS: The Erythrogene database, complemented with bioinformatics software, was used to analyse ABO in 2504 individuals from 1000G. DNA samples from selected 1000G donors and African blood donors were examined by allele-specific PCR and Sanger sequencing to characterize predicted deletions. RESULTS: A 5821-bp deletion encompassing exons 5-7 was called in twenty 1000G individuals, predominantly Africans. This allele was confirmed and its exact deletion point defined by bioinformatic analyses and in vitro experiments. A PCR assay was developed, and screening of African samples revealed three donors heterozygous for this deletion, which was thereby phenotypically established as an O allele. Analysis of upstream genetic markers indicated an ancestral origin from ABO*O.01.02. We estimate this deletion as the 3rd most common mechanism behind O alleles. A 24-bp deletion was called in nine individuals and showed greater diversity regarding ethnic distribution and allelic background. It could neither be confirmed by in silico nor in vitro experiments. CONCLUSION: A previously uncharacterized ABO deletion among Africans was comprehensively mapped and a genotyping strategy devised. The false prediction of another deletion emphasizes the need for cautious interpretation of NGS data and calls for strict validation routines.


Subject(s)
ABO Blood-Group System/genetics , Gene Deletion , Genome, Human , Genotype , Humans , Phenotype , Polymorphism, Genetic
3.
Immunohematology ; 29(1): 19-24, 2013.
Article in English | MEDLINE | ID: mdl-24046919

ABSTRACT

The P blood group antigen of the GLOB system is a glycolipid structure, also known as globoside, on the red blood cells (RBCs) of almost all individuals worldwide. The P antigen is intimately related to the Pk and NOR antigens discussed in the review about the P1PK blood group system. Naturally occurring anti-P is present in the serum of individuals with the rare globoside-deficient phenotypes p, P1k, and P2k and has been implicated in hemolytic transfusion reactions as well as unfavorable outcomes of pregnancy. The molecular genetic basis of globoside deficiency is absence of functional P synthase as a result of mutations at the B3GALNT1 locus. Other related glycolipid structures, the LKE and PX2 antigens, remain in the GLOB blood group collection pending further evidence about the genes and gene products responsible for their synthesis.


Subject(s)
Erythrocytes/chemistry , Erythrocytes/immunology , P Blood-Group System/chemistry , P Blood-Group System/immunology , Animals , Humans , Molecular Diagnostic Techniques
4.
Immunohematology ; 29(1): 25-33, 2013.
Article in English | MEDLINE | ID: mdl-24046920

ABSTRACT

The antigens in the P1PK blood group system are carried on glycosphingolipids. The system currently includes three different antigens, P1, Pk, and NOR. The P1 antigen was disovered in 1927 by Landsteiner and Levine, and Pk and NOR were described in 1951 and 1982, respectively. As in the ABO system, naturally occurring antibodies of the immunoglobulin (Ig) M or IgG class, against the missing carbohydrate structures, can be present in the sera of people lacking the corresponding antigen. Anti-P1 is generally a weak and cold-reactive antibody not implicated in hemolytic transfusion reaction (HTR) or hemolytic disease of the fetus and newborn while Pk antibodies can cause HTR, and anti-NOR is regarded as a polyagglutinin. A higher frequency of miscarriage is seen in women with the rare phenotypes p, P1k, and P2k. Furthermore, the Pk and P1 antigens have wide tissue distributions and can act as host receptors for various pathogens and toxins. Why p individuals lack not only Pk and P expression but also P1 has been a longstanding enigma. Recently, it was shown that the same A4GALT-encoded galactosyltransferase synthesizes both the P1 and Pk antigens and that a polymorphism in a new exon in this gene predicts the P1 and P2 phenotypes.


Subject(s)
Globosides/immunology , P Blood-Group System/immunology , Humans
5.
Thorac Cardiovasc Surg ; 58(1): 11-6, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20072970

ABSTRACT

OBJECTIVE: Transmyocardial laser revascularization for angina relief and intramyocardial autologous endothelial progenitor cell injection for neoangiogenesis may offer a new treatment strategy for patients with intractable ischemic heart disease. METHODS: Transmyocardial laser revascularization and intramyocardial injection of bone marrow-derived CD133+ cells was performed in six highly symptomatic patients. Transmyocardial laser channels were created and isolated CD133+ cells were injected intramyocardially. All patients were followed up for a minimum of 6 months postoperatively. RESULTS: One patient died shortly after the operation due to refractory heart failure. In the five survivors, CCS class improved as well as left ventricular ejection fraction. Left ventricular end-diastolic volume and myocardial perfusion varied between the patients. All patients described a considerable improvement in quality of life postoperatively. Repeated 24-hour Holter monitoring revealed no significant arrhythmias. CONCLUSIONS: In this small patient cohort, intramyocardial CD 133+ cell injection combined with transmyocardial laser revascularization led to an improvement in clinical symptomatology in all patients and in left ventricular function in 4 out of 5 patients, with an unclear effect on myocardial perfusion. Caution is advised when employing this therapy in patients with severely depressed left ventricular function.


Subject(s)
Endothelial Cells/transplantation , Laser Therapy , Myocardial Ischemia/surgery , Myocardial Revascularization/methods , Stem Cell Transplantation , Aged , Combined Modality Therapy , Female , Humans , Male , Middle Aged , Stem Cells , Treatment Outcome
6.
Immunohematology ; 25(4): 165-9, 2009.
Article in English | MEDLINE | ID: mdl-20406024

ABSTRACT

The Jk(a-b-) phenotype results from alterations in the JK gene and is characterized by absence of the RBC urea transporter in the cell membrane. The frequency of Jk(a-b-) varies among populations,but this phenotype is most commonly found in people of Polynesian and Finnish descent. Although rare, Jk(a-b-) individuals present a clinical challenge because anti-Jk3 is produced readily in response to transfusion and pregnancy, and Jk(a-b-) blood is not routinely available. Identification of Jk(a-b-) patients and donors is most often performed serologically. However, ten JK*0 alleles have been identified, and this information can be used in DNA-based typing. We selected five JK*0 alleles that had been encountered by our reference laboratory in two or more samples from unrelated individuals and designed an allele-specific primer PCR assay for use as an initial screening tool. After in-house validation,we tested genomic DNA from a family: a mother and her two sons referred to us for genetic investigation of their Jk(a-b-)phenotypes. Two different nucleotide substitutions, -1g>a in intron 5 (IVS5) and 956C>T in exon 10, originally associated with Polynesian and Indian/African populations respectively, were identified in the family. The mother and one son were compound heterozygotes, and the second son was homozygous for IVS5-1g>a. We conclude that the effort to design and validate such a screening assay was cost-efficient when compared with DNA sequencing costs. Furthermore, selection of the more common JK*0 mutations was a practical approach that resulted in rapid identification of the genetic bases behind the Jk(a-b-) phenotypes in this unusual family. Although an obvious target for eventual inclusion into high-throughput genotyping platforms for clinical diagnostic services, current systems are very limited. Our approach provides a simple and inexpensive method for the identification of these rare alleles.


Subject(s)
Blood Group Incompatibility/diagnosis , Genetic Testing , Heterozygote , Neoplasm Proteins/genetics , Antibodies/blood , Blood Group Incompatibility/genetics , Blood Group Incompatibility/immunology , Female , Gene Frequency , Guam , Humans , Intracellular Signaling Peptides and Proteins , Male , Membrane Proteins , Membrane Transport Proteins/deficiency , Membrane Transport Proteins/immunology , Mutation/genetics , Neoplasm Proteins/metabolism , Pedigree , Polymorphism, Genetic , Urea Transporters
7.
Transfus Med ; 15(5): 435-42, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16202060

ABSTRACT

Apparent deviation from Mendelian rules of blood group inheritance is rarely observed. Blood group O parents with children expressing weak A subgroups have occasionally been described but not explained. A detailed serological investigation of such a family is described here. The ABO locus was analysed by PCR-ASP/restriction fragment length polymorphism genotyping and DNA sequencing. The propositus' RBCs were very weakly agglutinated with monoclonal anti-A but distinctly with polyclonal anti-A,B, i.e. typical for Ax. Serum anti-A1 (titre 4) and -B were present. Her parents' blood groups were both clearly O, with titres of serum anti-A1, and -A at 16 and 4, respectively. Adsorption/ elution studies demonstrated A antigen on the daughter's cells only. The ABO genotypes were: mother, AxO1; father, O1vO2; and propositus, AxO2. The Ax allele was an A1-O1v hybrid allele with a crossing-over breakpoint between positions 235 and 446 in intron 6 (Ax-4). Compared to the A1 glycosyltransferase, this allele predicts a protein with two amino acid substitutions (Phe216Ile and Met277Val) known to yield either weakly expressed or no A antigen on RBCs. This study suggests that the nature of the ABO allele in trans can influence A antigen expression, a phenomenon previously described as allelic enhancement (or reinforcement). Potential mechanisms for this are discussed.


Subject(s)
ABO Blood-Group System/genetics , Alleles , Crossing Over, Genetic , Gene Expression , Polymorphism, Restriction Fragment Length , Quantitative Trait Loci , Family , Female , Genotype , Humans , Male
8.
Vox Sang ; 89(1): 27-38, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15938737

ABSTRACT

BACKGROUND AND OBJECTIVES: The chemical basis of the subgroups of A is largely unknown. We used thin-layer chromatography immunochemical staining techniques together with a range of characterized monoclonal reagents to analyse glycolipids isolated from a variety of weak subgroups. MATERIALS AND METHODS: Glycolipids isolated from red cells collected from nine genetically defined individuals of the rare subgroups of A, including a novel A(3) allele (A(2) 539G>A) not described previously, were subjected to a highly sensitive thin-layer chromatographic immunochemical analysis. RESULTS: Semicharacterized monoclonal antibodies revealed that, in addition to the expected quantitative differences between common phenotypes and the weak subgroups, qualitative glycolipid differences (or at least an apparent qualitative basis), caused by major changes in the ratios of different structures exist. Specifically it was found that the weakest A-expressing samples (A(el) phenotype) appeared to express an unusual A structure in the 8-12 sugar region. Variable expression of several structures in one of the A weak samples were suggestive of novel blood group A structures. CONCLUSIONS: Although no structural characterization could be undertaken, the results are clearly indicative that the variant glycosyltransferases of the rare ABO subgroups are not only inefficient, but they may potentially synthesize novel ABO structures.


Subject(s)
ABO Blood-Group System/chemistry , Glycolipids/analysis , Immunoenzyme Techniques , ABO Blood-Group System/genetics , ABO Blood-Group System/immunology , Antibodies, Monoclonal , Chromatography, Thin Layer , Genotype , Glycolipids/chemistry , Glycosyltransferases , Humans , Lewis Blood Group Antigens , Phenotype
9.
Vox Sang ; 88(4): 265-70, 2005 May.
Article in English | MEDLINE | ID: mdl-15877648

ABSTRACT

BACKGROUND AND OBJECTIVES: Reagent red blood cells (RBCs) for antibody detection should express certain important antigens as a double dose, that is, the donors must be homozygous for the corresponding alleles. Traditionally, dose is determined by serological typing and known allele frequencies. However, RHD zygosity cannot be predicted serologically owing to the absence of an antithetical antigen, and FY zygosity is confounded by two variant haplotypes, FY*0 and FY*X. Furthermore, lack of reagents hampers our ability to type for some clinically important antigen pairs such as Do(a)/Do(b). MATERIALS AND METHODS: Genomic DNA was isolated from reagent RBC samples. Established, validated methods were used to determine the RHD, FY, and DO genotypes. RESULTS: Three of 52 D+ samples gave results that differed from the predicted genotype: two presumed R(1)R(1) samples and an R(2)R(2) sample were shown to be R(1)r' and R(2)r'', respectively. Five of 59 samples that were from presumed homozygotes for either FY*A or FY*B were heterozygous, together with either FY*X (three samples) or FY*0 (two samples). Seventy-five samples tested for DO were DO*A/A (n = 14), DO*A/B (n = 39), or DO*B/B (n = 22). CONCLUSIONS: The results show that serologically determined RhD and Duffy phenotypes of reagent RBCs are unreliable and that antigens we thought were represented as a double dose were single dose. The addition of Dombrock genotyping provides information which is useful in antibody identification. We conclude that selected genotype analyses are a valuable quality assurance measure to ensure that reagent RBCs comply with national and international recommendations for test sensitivity.


Subject(s)
Antibodies/analysis , Blood Group Antigens/genetics , Blood Grouping and Crossmatching/methods , Erythrocytes/immunology , ADP Ribose Transferases/genetics , Blood Group Antigens/immunology , Blood Grouping and Crossmatching/standards , Duffy Blood-Group System/genetics , Gene Dosage , Genotype , Humans , Membrane Proteins/genetics , Quality Control , Receptors, Cell Surface/genetics , Rh-Hr Blood-Group System/genetics , Serologic Tests/standards
10.
Br J Haematol ; 125(4): 528-36, 2004 May.
Article in English | MEDLINE | ID: mdl-15142124

ABSTRACT

The aim of this study was to further explore the molecular genetic bases of the clinically important but rare blood group phenotypes p, P(1) (k) and P(2) (k) by analysis of the 4-alpha-galactosyltransferase (P(k)) and 3-beta-N-acetylgalactosaminyltransferase (P) genes responsible for synthesis of the related P(k) (Gb(3)) and P (Gb(4)) antigens respectively. Lack of these glycolipid moieties is associated with severe transfusion reactions and recurrent spontaneous abortions but also offers immunity against certain infectious agents. Blood samples from 20 p and 11 P(1) (k) or P(2) (k) individuals of different geographic and ethnic origin were investigated. DNA sequencing by capillary electrophoresis was performed following amplification of the coding regions in the P(k) or P genes. In the P(k) gene, nine novel and five previously described mutations were detected. One of the newly found mutations introduced an immediate stop, five shifted the reading frame introducing premature stop codons and three were missense mutations causing amino acid substitutions in conserved regions of the transferase. Four new and two previously described mutations in the P gene were found. Three of the novel alleles reported here carried nonsense mutations whilst the fourth allele had a missense mutation. The finding of 13 novel mutations in 14 alleles emphasizes further the genetic heterogeneity at the glycosyltransferase loci underlying the GLOB blood group system and collection.


Subject(s)
Glycosyltransferases/genetics , Mutation , P Blood-Group System , Amino Acid Sequence , Base Sequence , Female , Genetic Heterogeneity , Humans , Male , Molecular Sequence Data , Phenotype , Sequence Analysis, DNA
11.
Surg Endosc ; 18(10): 1509-13, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15791379

ABSTRACT

BACKGROUND: Suspected appendicitis is one of the most common indications for acute laparotomy or laparoscopy. The negative laparotomy and laparoscopy rates are high, often in the range of 15-30%, and especially high in some groups of patients such as women of child-bearing age and young patients. Different scoring systems have been introduced in order to improve diagnostic accuracy. The aim of the present study was to analyse the outcome of the Fenyö-Lindberg scoring system in a prospectively randomized multicenter trial and to analyze how well the score performed in stratified subgroups. METHODS: The variables of the Fenyö-Lindberg scoring system were collected in a prospective study comparing laparoscopic and open surgery in suspected appendicitis and with four participating centers. None of the hospitals had used the scoring system previously. Since surgeons were unfamiliar with the score, they could not use it as a diagnostic aid. When comparing the score with the clinical outcome, retrospectively, the investigators interpreting the score were blinded regarding the surgical outcome. RESULTS: Positive predictive value (PPV) of the Fenyö-Lindberg score was higher than that of the surgeon's clinical diagnosis in the patient cohort [0.90 vs 0.79 (p < 0.001)]. The score demonstrated an improvement of PPV in women [0.83 vs 0.70 (p < 0.01)]. PPV was increased in women between 15 and 50 years of age. In women aged 15-30 years and 31-50 years PPV increased from 0.69 to 0.82 and 0.68 to 0.86, respectively (p < 0.01). Both the sensitivity (0.77) and the specificity (0.69) of the score were, however, low. CONCLUSION: The Fenyö-Lindberg score is an inexpensive clinical tool that may improve the diagnostic accuracy for acute appendicitis in women of childbearing age, which is a group of patients where the diagnostic accuracy usually is low and where the arsenal of diagnostic tools such as computed tomography is limited because of radiation. The low specificity of the score in women of childbearing age must, however, be kept in mind.


Subject(s)
Appendectomy/methods , Appendicitis/diagnosis , Appendicitis/surgery , Laparoscopy , Adolescent , Adult , Aged , Diagnostic Techniques, Digestive System , Female , Humans , Male , Middle Aged , Predictive Value of Tests , Prospective Studies , Single-Blind Method
12.
Acta Radiol ; 43(4): 391-5, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12225481

ABSTRACT

AIM: To evaluate the role of duplex scanning in the selection of patients with infrainguinal arterial occlusive disease for percutaneous transluminal angioplasty (PTA). MATERIAL AND METHODS: From January 1995 through May 2000, 702 patients (952 limbs), with chronic lower extremity ischemia due to infrainguinal atherosclerotic disease diagnosed by duplex scanning, were retrospectively studied. Diagnostic angiography (130 limbs) or infrainguinal PTA (108 limbs) was performed in 238 limbs. Two investigators retrospectively analyzed the duplex examinations and angiographies in a blinded manner and used similar criteria for the interpretation of lesions suitable or not suitable for PTA. RESULTS: The superficial femoral, popliteal and crural artery lesions were correctly selected for PTA in 85%, 66% and 32%, respectively. The accuracy, sensitivity, specificity, negative predictive value and positive predictive value of duplex scanning to appropriately categorize femoropopliteal lesions as suitable or unsuitable for PTA were 89%, 83%, 92%, 94% and 78%, respectively. The accuracy of duplex scanning for predicting the performance of infrainguinal PTA was 83%. CONCLUSION: Duplex scanning has an important impact on the selection of treatment modalities in limbs with infrainguinal arterial occlusive disease. Femoropopliteal lesions can be reliably selected to PTA according to duplex scan findings.


Subject(s)
Angioplasty, Balloon , Arteriosclerosis/diagnostic imaging , Arteriosclerosis/therapy , Peripheral Vascular Diseases/diagnostic imaging , Peripheral Vascular Diseases/therapy , Aged , Female , Humans , Leg/blood supply , Male , Predictive Value of Tests , Retrospective Studies , Sensitivity and Specificity , Ultrasonography, Doppler, Duplex
13.
Vox Sang ; 83(1): 55-62, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12100390

ABSTRACT

BACKGROUND AND OBJECTIVES: During the past 10 years several DNA-typing methods have been developed to complement routine serological typing for determination of polymorphisms in the ABO, RH, KEL, JK and FY blood group genes. However, the molecular basis of blood groups can differ widely between ethnic groups. The purpose of this study was to evaluate selected DNA-based methods for phenotype prediction in a population not previously investigated. MATERIALS AND METHODS: Blood samples from a random sample of Jordanian blood donors were collected and red cells isolated from these blood samples were phenotyped for common ABO (n = 150) and KEL/FY/JK (n = 90) antigens. RHD-negative and -positive donors were selected for RH typing (n = 120 and 30, respectively). DNA was prepared and blood group genotyping performed according to selected methods in current use. Discordant samples required further investigation by extended serology and DNA sequencing. RESULTS: The degree of concordance between phenotype and genotype was high, but some exceptions were noted. Two of 14 A2/A2B samples lacked all mutations associated with known A2 alleles of the ABO system. RH typing revealed four samples with the c(cyt48) marker, causing false-positive RHC typing. A single D-negative sample was positive for D-specific exon 10 markers. The RHD pseudogene was not found in the 150 donors tested. Nine samples revealed discrepancies that were associated with unknown silent or weakly expressing Fyb-like alleles. CONCLUSIONS: With the exception of the FY system, we conclude that the molecular background of the clinically important blood group antigens studied here is similar to that reported for Caucasoids.


Subject(s)
Blood Grouping and Crossmatching/methods , Immunophenotyping , Polymerase Chain Reaction/methods , Alleles , Blood Donors , Blood Group Antigens/analysis , Blood Group Antigens/genetics , Blood Group Antigens/immunology , Blood Grouping and Crossmatching/standards , DNA Primers , Gene Frequency , Genotype , Humans , Jordan , Polymerase Chain Reaction/standards , Sequence Analysis, DNA
14.
Eur J Vasc Endovasc Surg ; 23(2): 140-5, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11863331

ABSTRACT

OBJECTIVE: to evaluate preoperative duplex as the sole investigation prior to lower limb reconstruction. Design retrospective analysis. MATERIALS AND METHODS: between January 1995 and December 1999, 157 of 329 surgical interventions for chronic infrainguinal arterial or aneurysmal disease were performed without preoperative angiography. RESULTS: in patients undergoing femoral artery endarterectomy, the extent of the stenosis and the status of the distal deep femoral artery were correctly diagnosed with duplex scanning in all but one patient. Duplex scan findings in patients undergoing infrainguinal bypass procedures were in agreement with the findings obtained from on-table angiography in regard to the selection of optimal outflow anastomotic sites in 123 (98%). Duplex scanning correctly evaluated the status of runoff in 113 (90%). There were no significant differences in 30-day occlusion rate and patency at 12 months between reconstructions performed with and without preoperative angiography. CONCLUSION: in patients with conclusive duplex scan findings there is no need to perform angiography prior to lower limb reconstruction.


Subject(s)
Arterial Occlusive Diseases/diagnostic imaging , Arterial Occlusive Diseases/surgery , Leg/blood supply , Leg/surgery , Preoperative Care , Ultrasonography, Doppler, Duplex , Adolescent , Adult , Aged , Aged, 80 and over , Arterial Occlusive Diseases/complications , Female , Femoral Artery/diagnostic imaging , Femoral Artery/surgery , Follow-Up Studies , Graft Occlusion, Vascular/etiology , Humans , Image Processing, Computer-Assisted , Male , Middle Aged , Popliteal Artery/diagnostic imaging , Popliteal Artery/surgery , Radiography , Reoperation , Time Factors , Treatment Outcome , Vascular Patency/physiology
15.
Protein Expr Purif ; 24(1): 131-7, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11812234

ABSTRACT

Cysteine proteinases and in particular cysteine proteinase 5 (EhCP5) of Entamoeba histolytica are considered important for ameba pathogenicity. To study EhCP5 in more detail a protocol was elaborated to produce considerable amounts of the enzyme in its active form. The protein was expressed in Escherichia coli as a histidine-tagged pro-enzyme and purified to homogeneity under denaturing conditions in the presence of guanidine-HCl using nickel affinity chromatography. Renaturation was performed by 100-fold dilution in a buffer containing reduced and oxidized thiols, which led to soluble but enzymatically inactive pro-enzyme. Further processing and activation was achieved in the presence of 10 mM DTT and 0.04% SDS at 37 degrees C. Recombinant enzyme (rEhCP5) was indistinguishable from native EhCP5 purified from E. histolytica lysates. Both runs in SDS-PAGE under reducing and nonreducing conditions at positions corresponding to 27 and 29 kDa, respectively, had the same pH optima and displayed similar specific activity against azocasein. Moreover, both enzymes were active against a broad spectrum of biological and synthetic substrates such as mucin, fibrinogen, collagen, human hemoglobin, bovine serum albumin, gelatin, human IgG, Z-Arg-Arg-pNA, and Z-Ala-Arg-Arg-pNA, but not against Z-Phe-Arg-pNA. The identity of rEhCP5 as a cysteine proteinase was confirmed by inhibition with specific cysteine proteinase inhibitors. In contrast, various compounds known to specifically inhibit aspartic, metallo, or serine proteinases had no effect on rEhCP5 activity.


Subject(s)
Cysteine Endopeptidases/genetics , Entamoeba histolytica/enzymology , Animals , Cloning, Molecular , Cysteine Endopeptidases/isolation & purification , Cysteine Endopeptidases/metabolism , Enzyme Activation , Escherichia coli , Gene Expression , Protein Folding , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism
16.
FEBS Lett ; 508(1): 36-8, 2001 Nov 09.
Article in English | MEDLINE | ID: mdl-11707263

ABSTRACT

Plasma and urinary levels of 8-iso-PGF(2alpha) and 15-keto-dihydro-PGF(2alpha) were analysed at baseline and during the ischemia-reperfusion period in experimental spinal cord ischemia. A significant and immediate increase of 8-iso-PGF(2alpha) in plasma at the start and up to 60 min, and in the urine at 90-150 min following ischemia indicate an association of oxidative injury. The inflammatory response indicator 15-keto-dihydro-PGF(2alpha) in plasma increased significantly at the start and up to 60 min after ischemia. No such increase was seen in animals with no spinal cord ischemia. Thus, free radical mediated and cyclooxygenase catalysed products of arachidonic acid are increased during spinal cord ischemia as a consequence of oxidative injury and inflammation.


Subject(s)
Dinoprost/analogs & derivatives , Dinoprost/blood , F2-Isoprostanes/metabolism , Reperfusion Injury/physiopathology , Spinal Cord Ischemia/physiopathology , Spinal Cord/physiopathology , Animals , Aorta, Thoracic/surgery , Biomarkers , Cerebrospinal Fluid/chemistry , F2-Isoprostanes/blood , F2-Isoprostanes/urine , Free Radicals/metabolism , Inflammation/physiopathology , Oxidation-Reduction , Reperfusion Injury/metabolism , Spinal Cord Ischemia/metabolism , Swine , Time Factors
17.
Vasc Surg ; 35(1): 31-5, 2001.
Article in English | MEDLINE | ID: mdl-11668366

ABSTRACT

This retrospective study was conducted to analyze a new concept of evaluation of the effect of distal runoff on patency in infrainguinal bypass surgery for arterial insufficiency. Distal runoff was evaluated on postreconstruction angiograms in 191 limbs undergoing femoropopliteal and femorodistal reconstruction. Runoff was characterized as good, fair, or poor. Determination of graft patency was made by clinical examination, ankle-brachial index measurement, or duplex scanning at 1 month and thereafter at 6-month intervals. Cumulative patency rates were calculated according to the actuarial life table method. Patency rates in limbs with good runoff were better than in limbs with fair and poor runoff; at 6 months, patency rates were 88.2%, 70.9%, and 21.8%, respectively (p < 0.01). Similar patency rates were found for good runoff in femoropopliteal and femorodistal reconstructions (84.7% in femoropopliteal and 75% in femorodistal reconstructions) at 6 months. The authors conclude that this method of angiographic evaluation accurately predicts patency in infrainguinal bypass reconstructions.


Subject(s)
Femoral Vein/surgery , Graft Occlusion, Vascular/surgery , Inguinal Canal/blood supply , Leg/blood supply , Popliteal Vein/surgery , Vascular Patency/physiology , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Plastic Surgery Procedures/mortality , Retrospective Studies , Time Factors , Veins/transplantation
18.
J Vasc Surg ; 34(1): 114-21, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11436083

ABSTRACT

PURPOSE: The aim of the study was to evaluate the results of percutaneous transluminal angioplasty (PTA) of femoropopliteal arteries in patients with subcritical or critical lower limb ischemia. MATERIALS AND METHODS: Ninety-two patients underwent 121 PTA procedures, 68 were of the superficial femoral artery (SFA), 13 of the popliteal and 40 of both arteries. Fifty-seven procedures were performed for treatment of occlusions. Eighty-four patients (94 procedures) were monitored with duplex scanning. RESULTS: Technical success rate was 88%. Primary success rates at 12 and 60 months in the whole series were 40% and 27%, respectively. The primary success rate in limbs with SFA occlusion of longer than 5 cm was only 12% after 5 years compared with 32% if the occlusion was

Subject(s)
Angioplasty, Balloon , Femoral Artery , Ischemia/therapy , Leg/blood supply , Popliteal Artery , Adult , Aged , Chronic Disease , Female , Humans , Ischemia/diagnostic imaging , Life Tables , Male , Middle Aged , Radiography
19.
Scand Cardiovasc J ; 35(2): 147-50, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11405492

ABSTRACT

OBJECTIVES: To investigate the effect of 100% oxygen ventilation on cerebrospinal fluid (CSF) oxygenation in 11 pigs during thoracic aortic cross-clamping. DESIGN: An aorto-aortic shunt was used for control of central hemodynamics and study of hypoperfusion by exsanguination. CSF PO2, PCO2 and pH were continuously monitored before and during clamping. The changes in hemodynamic parameters and intrathecal gas tensions in response to variations in proximal mean aortic pressure and fraction of inspired oxygen (FiO2) were recorded. RESULTS: Baseline CSF PO2 decreased from 4.8 +/- 1.9 to 2.6 +/- 2.2 kPa following aortic occlusion. Increasing FiO2 to 1.0 resulted in a significant increase in CSF PO2 to 4.1 +/- 3.0 with a return to 2.7 +/- 2.1 kPa after reducing FiO2 to 0.4 again. The same variations in FiO2 did not induce any significant changes in CSF PO2 during hypotension. CONCLUSION: Increased FiO2 during experimental thoracic aortic cross-clamping with stable proximal arterial pressure helps to maintain CSF PO2, whereas severe hypotension could not be compensated for by hyperoxemia.


Subject(s)
Oxygen/cerebrospinal fluid , Vascular Surgical Procedures/methods , Animals , Aorta, Thoracic , Constriction , Disease Models, Animal , Female , Hemodynamics , Male , Swine , Vascular Surgical Procedures/instrumentation
20.
Surg Endosc ; 15(4): 387-92, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11395821

ABSTRACT

BACKGROUND: Laparoscopic appendectomy (LA) has been associated with a faster recovery and less postoperative pain than the open technique. However, few data are available on the clinical outcome of LA in overweight patients. METHODS: A group of 106 patients with a body mass index (BMI) > 26.4, representing the upper quintile of 500 prospectively randomized patients, were included in the study. They were randomized to undergo either laparoscopic or open appendectomy (OA). Operating and anesthesia times, postoperative pain, complications, hospital stay, functional index (1 week postoperatively), sick leave, and time to full recovery were documented. RESULTS: In OA, the operating time for overweight patients was significantly longer than that for patients in the normal weight range (40 vs 35 min, p < 0.05). In LA, there was no difference in operating time between the normal and overweight patients. Overweight patients who underwent LA had longer operating and anesthesia times than their OA counterparts (55 vs 40 min, p < 0.001; and 125 vs 100 min, p < 0.001, respectively). Postoperative pain was significantly greater in overweight patients who underwent OA than in those treated with the laparoscopic technique. Postoperative pain was also significantly greater in overweight patients subjected to OA than in patients of normal weight after 4 weeks; the clinical significance may, however, be of less importance since the values are low (0.26 vs 0.09, p < 0.05). There were no significant differences between the two operating techniques in terms of complications. Hospital stay was longer for overweight patients than for normal-weight patients undergoing OA (3.0 vs 2.0, p < 0.01). The functional index did not differ between any group of patients. Sick leave was longer for overweight patients who underwent OA than for normal-weight patients treated with the same technique (17 vs 13 days, p < 0.01). In the laparoscopic group, however, there were no differences between the overweight and normal-weight patients. Time to full recovery was greater in overweight patients subjected to OA than in the overweight patients in the LA group (22 vs 15 days, p < 0.001). CONCLUSION: In this study, overweight patients who were submitted to LA had less postoperative pain and a faster postoperative recovery than overweight patients who had OA. LA also abolished some of the negative effects that overweight had on operating time, hospital stay, and sick leave with the open technique. However, anesthesia and operating times were significantly longer in LA for both overweight patients and those with a normal BMI.


Subject(s)
Appendectomy/methods , Appendicitis/surgery , Body Weight , Laparoscopy/methods , Adolescent , Adult , Aged , Appendectomy/statistics & numerical data , Appendicitis/epidemiology , Body Mass Index , Comorbidity , Female , Humans , Laparoscopy/statistics & numerical data , Male , Middle Aged , Obesity/epidemiology , Pain, Postoperative/epidemiology , Postoperative Complications/epidemiology , Prospective Studies , Time Factors , Treatment Outcome
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