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Biomedical subjects

A Salama

Publications and source records attributed to A Salama.

At least 73 records · Page 4Linked to original sources
PubMed1998

Detection of tumor cells in peripheral blood samples from patients with germ cell tumors using immunocytochemical and reverse transcriptase-polymerase chain reaction techniques.

The aim of this study was to establish sensitive techniques for the detection of residual germ cell tumor cells in peripheral blood and progenitor cell harvests. For this purpose, we used immunocytochemical staining for cytokeratin filaments and reverse transcriptase-polymerase chain reaction (RT-PCR) for epidermal growth factor receptor (EGF-R) and germ cell alkaline phosphatase (GCAP). Germ cell tumor lines Tera-1 and Tera-2 were titrated into normal peripheral blood. Immunocytochemical staining allowed detection of one cytokeratin-positive tumor cell in 10(5) cells. RT-PCR for EGF-R revealed one tumor cell in 10 cells for Tera-1 and one tumor cell in 10(3) cells for Tera-2, while RT-PCR for GCAP displayed a sensitivity of one tumor cell in 10(6) cells for Tera-1, one tumor cell in 10(4) cells for Tera-2, and no positivity in normal mononuclear cells (n = 20) and progenitor cell harvests (n = 5). The analysis of peripheral blood and progenitor cell harvests from 20 patients with germ cell tumors revealed tumor cell contamination in three patients using immunocytochemical staining and in seven patients by RT-PCR for GCAP. We conclude that RT-PCR for GCAP appears to be suitable for the sensitive detection of residual germ cell tumor cells in peripheral blood and progenitor cell harvests.

Alkaline Phosphatase↗
PubMed1998

Use of the gel agglutination technique for determination of fetomaternal hemorrhage.

BACKGROUND: Adequate administration of Rh immune globulin requires an accurate determination of the number of D-positive cells in the circulation of D-negative women. Although several tests have been described for the detection of fetomaternal hemorrhage, there is still a need for a rapid, simple, and clinically relevant screening test. STUDY DESIGN AND METHODS: Serial dilutions of a monoclonal anti-D were incubated with stock solutions (0.2 mL) of adult D-negative red cells in the absence or presence of various amounts of fetal D-positive cells (0.1, 0.2, 0.3, 0.4, and 0.5%). After incubation, the supernatants were tested against D-positive red cells by using the new, gel agglutination technique (GAT). After the GAT was adapted to detect D-positive cells at concentrations of > or = 0.2 percent, unselected postpartum samples from D-negative women (n = 420) who delivered D-positive infants were analyzed by both the new test and the Kleihauer-Betke test (KBT). RESULTS: Three of a total of 420 postpartum samples were positive (> or = 0.4% fetal cells), and 406 were negative in both tests. One had 0.5-percent fetal cells in the KBT and gave negative results in the GAT. The latter test was, however, performed after administration of Rh immune globulin. The KBT gave false-positive results in two cases, because of hereditary persistence of hemoglobin F, and the GAT gave a false-positive reaction in one case because of a maternal weak D variant. In the remaining seven cases, the KBT results were only weakly positive (0.2%) and could not be attributed solely to D positive red cells. CONCLUSION: The GAT is suited for routine screening. It provides rapid and specific detection of D-positive red cells at clinically relevant concentrations. The test may (rarely) yield false-negative results due to insufficient administration of Rh immune globulin before testing.

Adult↗
PubMed1998

Epidemiological and clinical aspects of hepatitis G virus infection in blood donors and immunocompromised recipients of HGV-contaminated blood.

BACKGROUND AND OBJECTIVES: The infectiousness and clinical relevance of the newly discovered blood-borne Flaviviridae-like agent, termed hepatitis G virus (HGV), are not well understood. MATERIALS AND METHODS: Twenty-three transfusion recipients of two HGV-affected long-term blood donors were studied for HGV genome and antibodies to the putative envelope 2 glycoprotein (anti-E2) of HGV. Nine recipients had nonhematological disorders and 14 suffered from severe hematological diseases and 7 of them received allogeneic bone marrow or blood stem cell transplantation. The molecular epidemiology of the observed HGV infection was studied by direct sequencing of parts of the 5'-noncoding region, NS3, and NS5 region of HGV in the 2 long-term donors and in their 6 recipients who became HGV RNA positive. Additionally, 549 individuals-homologous (n = 254) and autologous blood donors (n = 202), and medical staff (n = 89)--were investigated for the presence of HGV RNA. RESULTS: HGV RNA in serum was found in 15 of the 23 (65%) transfusion recipients with known exposure of HGV-contaminated blood. Seven of the remaining 8 recipients showed only an anti-E2 response, indicating previous HGV infection with spontaneous clearance of the virus. In one recipient neither HGV RNA nor anti-E2 could be detected. Molecular evidence for HGV transmission by the 2 donors was found in 3 of the 6 recipients studied. The alanine aminotransferase levels were not significantly different in the HGV RNA positive and negative recipients, and none of the 23 recipients developed posttransfusion hepatitis. Persistent HGV infection was observed especially in recipients with severe hematological disorders or in those in whom intensive immunosuppressive treatment was necessary. Of the 549 individuals studied, 10 (1.8%) were healthy carriers of HGV RNA. CONCLUSION: The persistence of transfusion-acquired HGV infection is not associated with acute or chronic hepatitis, but may be influenced by the recipient's underlying disease.

Adult↗
PubMed1998

Regulation of ion transport across lamprey (Lampetra fluviatilis) erythrocyte membrane by oxygen tension.

We have measured the effects of oxygen tension on the transport of Na+, K+ and Cl- across the erythrocyte membrane of the lamprey Lampetra fluviatilis. The transport of each ion was affected by the oxygen tension of the medium. Hypoxic conditions (PO2 2 kPa) caused an increase in the acidification-induced influx of Na+ via Na+/H+ exchange. The influx of K+ was only slightly affected by the oxygenation of the medium. In contrast, the basal K+ efflux, measured using the radioactive isotope 43K, was markedly reduced by decreasing the oxygen tension of the medium, whereas the K+ flux in hypotonic medium was not affected. Only minor effects of hypoxic conditions on the influx of Cl- were observed in either isotonic or hypotonic conditions (there was a tendency for the isotonic influx to increase) or on the efflux in isotonic conditions. However, deoxygenation caused a marked reduction in the Cl- efflux in hypotonic conditions. The results show that oxygen tension has a marked effect on the pH and volume regulatory transport pathways of lamprey erythrocytes. For K+ and Cl-, the regulation appears to be asymmetric, i.e. influx and efflux are affected differently.

Animals↗
PubMed1997

Pulsed high-dose dexamethasone in chronic autoimmune haemolytic anaemia of warm type.

The management of patients with autoimmune haemolytic anaemia of warm type (AIHA) is often problematic. Recently, pulsed high-dose dexamethasone (HDD) has been shown to be effective in the treatment of autoimmune thrombocytopenic purpura (AITP). In this study we treated seven patients with AIHA with HDD. The regimen recommended for treatment of refractory AITP (40 mg dexamethasone for 4 d at the beginning of each 28 d cycle) was employed in almost all cases. Prior to dexamethasone administration, haemolysis was decompensated in all seven patients. HDD was well tolerated and led to an improvement of haemolysis in all cases.

Adult↗
PubMed1997

The diversity of the HLA class I introns reflects the serological relationship of the coding regions.

The introduction of PCR-based HLA typing techniques has uncovered that the HLA system is much more variable than it has been expected from the conventional typing methods. More and more new alleles are detected which are not characterized by new sequence motifs, but by new combinations of already existing sequence motifs. This variability, reflecting the immunological necessity to have the greatest capacity for presenting antigenic peptides, is increasingly complicating DNA typing techniques based on the diversity of the coding region. We have determined the sequence of the 1st through 3rd intron of the majority of HLA-A and HLA-B alleles in 48 well-defined cell lines and 195 PCR-typed clinical samples. The few published sequences emerged to contain substantial errors. The introns turned out to be highly polymorphic. Besides extensive homologies, numerous locus- and group-specific sites could be identified. The most intriguing finding was that most of the polymorphic motifs were related to serological families. These sequence motifs were extremely beneficial for setting up PCR-based typing systems. In particular, sequencing-based typing strategies benefited from intron-restricted priming for amplification and sequencing by enabling complete analysis of the polymorphic exons. The determination of cis/trans linkages of sequence motifs was substantially facilitated. Apart from these advantages, the intron sequences were useful for evolutionary studies, delivering more insights into the genetic relationship between different alleles and the mechanisms involved in the development of the diversity of HLA. Moreover, the variability of the introns may provide a structural basis for the identification of regulatory elements acting on the level of transcription.

Cell Line↗
PubMed1996

Discrimination of HLA-B27 alleles by group-specific amplification followed by solid-phase sequencing.

HLA-B27 is known to be highly associated with ankylosing spondylitis. Until now, nine B27 subtypes have been sequenced and may contribute in different fashions to ankylosing spondylitis. Additionally, the divergent subtypes may be of clinical importance in bone marrow transplantation with alternative donors. The purpose of this study was to determine the different subtypes of HLA-B27 by a direct sequencing approach. The typing strategy is based on a group-specific amplification of the second and third exon followed by automated fluorescence sequencing of the polymorphic regions. The extensive sharing of sequence motifs between the different B alleles made it impossible to specifically amplify the B27 group under the precondition of including all sequence variations necessary for a postamplification specificity step. Therefore, for setting up a direct sequencing approach of B27, co-amplified B alleles had to be taken into account. In order to get unambiguous sequencing chromatograms without any heterozygous positions, nested sequencing primers were used which selectively matched sequence motifs only present in the second and third exon of the amplified B27 alleles. This strategy allowed in all cases investigated a clear separation of the haplotypes, revealing unequivocal sequencing results. Using this method, we have investigated 93 B27-positive individuals. Sequencing identified the alleles B*2702, 2703, 2704, 2705, and 2707. B*2701, 2706, 2708, and 2709 were not represented in the population studied.

Alleles↗
PubMed1996

Diclofenac-induced immune haemolytic anaemia: simultaneous occurrence of red blood cell autoantibodies and drug-dependent antibodies.

During the last 5 years we have identified a total of 17 patients (nine females and eight males aged between 53 and 85 years) with immune haemolytic anaemia related to diclofenac (a nonsteroidal anti-inflammatory drug). All patients developed acute intravascular haemolysis. Two patients died, and eight patients developed temporary renal failure that required haemodialysis. The direct antiglobulin test was positive with anti-IgG and anti-C3d in all cases, with anti-IgA in 4/10 cases tested, and negative with anti-IgM. The indirect antiglobulin test was moderately or weakly positive in 11 cases, and IgG autoantibodies could be eluted from the red blood cells (RBCs) of all patients. Initially, the diagnosis of autoimmune haemolytic anaemia of warm type was suggested in all cases. All patients had simultaneously developed autoantibodies and drug-dependent antibodies. The majority of drug-dependent antibodies (n = 13) reacted with urine containing the drug and its metabolites (ex vivo antigen), the native drug, and diclofenac-treated RBCs. The antibodies in the remaining four cases were detectable only in the presence of ex vivo antigen. Diclofenac appears to bind only weakly to RBCs in the absence of the drug-dependent antibodies. We conclude that diclofenac forms neoantigens with RBCs that may stimulate the production of autoantibodies and drug-dependent antibodies. The resulting haemolytic syndrome is very similar to autoimmune haemolytic anaemia of warm type.

Aged↗
PubMed1996

Sequence analysis of the 2nd intron revealed common sequence motifs providing the means for a unique sequencing based typing protocol of the HLA-A locus.

We here present a sequencing strategy for the HLA-A locus which is generally applicable for all HLA class I genes. The typing strategy is based on a group-specific amplification according to the serologically defined antigens. The PCR products carry the typing-relevant polymorphic regions of the 2nd and 3rd exon including the 2nd intron. The sequencing primers were designed to match conserved sequence motifs in the 2nd intron allowing a nested sequencing approach in 3' and 5' direction. These conserved regions were identified after sequence compilation of the 2nd intron of 143 clinical samples and 48 cell lines mostly from the 9th and 10th IHWC representing all serologically defined groups of alleles. This strategy allowed the use of only one 5' and one 3' sequencing primer regardless of the amplified allele. Therefore, it was possible to use dye terminator as well as dye primer sequencing chemistry. The amplification strategy allowed the separation of the haplotypes in almost all cases. Thus, an assignment of heterozygous positions requiring high sequencing quality was not necessary, allowing the application of Sequenase as well as TaqPolymerase as sequencing enzyme. Concerning the resolution of heterozygosity it is obvious that this approach is superior to a typing system using a single pair of generic primers followed by direct sequencing, since the latter technique is not capable of defining the cis/trans linkage of polymorphic sequences and, hence, cannot exclude the presence of unknown alleles.

Alleles↗
PubMed1996

The effects of repeated physical stress on the b-adrenergic response of the rainbow trout red blood cell

The effects of a 7-day period of daily physical stress (chasing until exhaustion) on the beta-adrenergic response of the rainbow trout (Oncorhynchus mykiss) red blood cell (rbc) were examined in vitro. Physical stress was associated with pronounced increases in the circulating levels of the catecholamine hormones (adrenaline and noradrenaline) measured on days 1, 3 and 7 of the stress regime. After 7 days, the numbers of high-affinity cell surface beta-adrenoceptors were reduced in the physically stressed fish when measured in vitro under conditions of normoxia (20 % reduction) or hypoxia (30 % reduction). Under hypoxic conditions, the binding affinity of the rbc beta-adrenoceptor was significantly higher in the stressed fish. Although the stressed fish had fewer beta-adrenoceptors, rbc adrenergic responsiveness was enhanced after 7 days of physical stress as determined from dose-response curves relating noradrenaline concentration to water and Na+ accumulation (indices of rbc adrenergic Na+/H+ exchange activity). The EC50 values (concentrations yielding half-maximal responses) for noradrenaline were lowered significantly by 1.7- to 3.9-fold in the blood from physically stressed fish. The enhanced adrenergic responsiveness of the rbcs appeared to be unrelated to changes in the initial steps of the beta-adrenergic signal transduction pathway leading to cyclic AMP production because physical stress was without effect on the magnitude or the dose-dependency of rbc cyclic AMP accumulation. To determine whether post-cyclic-AMP events were affected by physical stress, water and Na+ accumulation were measured in rbcs that had been incubated with the permeable cyclic AMP analogue 8-bromo cyclic AMP. The EC50 values for 8-bromo cyclic AMP were lowered by 1.6- to 1.7-fold in the blood from stressed fish. These experiments demonstrate that repeated physical stress significantly enhances the adrenergic responsiveness of the rainbow trout rbc, presumably by modifying the sensitivity of the Na+/H+ exchanger (or the steps immediately preceding exchanger activation) to cyclic AMP. The results are discussed with respect to the interrelationships between chronic and acute stress responses in fish.

Journal Article↗
PubMed1996

Vesicouterine fistula--variable clinical presentation.

Vesicouterine and vesicocervical fistula are mostly caused by trauma during lower segment caesarian section. The clinical presentation and management of nine cases are described. Presentation was typical, i.e. cyclic haematuria (menouria) without urinary leakage, in three of the nine patients, and atypical--vaginal leakage of urine, urethral passage of lochia and/or regular vaginal menes--in six patients. The diagnosis was made clinically, radiologically and endoscopically. Surgical repair was successfully performed in eight cases and one was managed conservatively with satisfactory outcome. Atypical presentation of such fistulas may considerably delay the diagnosis. In contrast to vesicovaginal fistula, conservative management may be tried in selected cases.

Adult↗
PubMed1995

A novel HLA-DR13 allele (DRB1*1314) identified by single-strand conformation polymorphism analysis and confirmed by direct sequencing.

A novel variant of the HLA-DR13 group is described. The new allele was found in a DR10, 13 heterozygous patient of Turkish origin, two HLA genotypically identical children of the patient typed as DR11,13, and one child typed as DR13,13. DR13 subtyping of the patient was initially performed by SSCP analysis of PCR-amplified DNA, using the 11th OHWS primers DRBAMP-3 and DRBAMP-B. Due to an unusual SSCP banding pattern, the PCR product was subjected to solid-phase sequencing. The sequence of the new allele, DRB1*1314, is different from that of DRB1*1307 by a single nucleotide substitution in codon 47, with T replacing consensus A. This results in a single amino acid change of tryptophan to phenylalanine in the first domain of the DR beta chain.

Anemia, Sickle Cell↗
PubMed1995

Complete subtyping of the HLA-A locus by sequence-specific amplification followed by direct sequencing or single-strand conformation polymorphism analysis.

A variety of reasons related to the HLA class I system has complicated the application of molecular approaches to HLA class I typing. Here we present a PCR-based HLA-A typing strategy considering the sequence variations of the two most polymorphic exons which allows complete subtyping of the HLA-A locus. The method is based on a sequence-specific amplification identifying the serologically defined HLA-A specificities. The PCR products generated by these group-specific primers bear the sequence information necessary for a postamplification specificity step. The primer pairs are located within one exon, either exon 2 or exon 3, which avoids amplification of polymorphic intron sequences allowing subsequent single-strand conformation polymorphism analysis and facilitating direct sequencing. Using this method we investigated 48 cell lines and 153 clinical samples. 23 PCR reactions are performed per individual for the assignment of the serological specificities A1-A80. The reproducibility was 100% in all cell lines and 85 clinical samples typed on two separate occasions. With the exception of 13 out of 231 possible serological combinations all homozygous and heterozygous combinations of A1-A80 can be distinguished by specific amplification patterns. Comparing the PCR based typing results with those of serology in 12% a discrepancy was found. Solid-phase sequencing or SSCP analysis of the group-specific PCR fragments allowed complete subtyping of the HLA-A locus. This strategy can identify all 48 HLA-A alleles based on the sequence variations of the 2nd and 3rd exon. 1128 homozygous and heterozygous allele combinations are possible for the HLA-A locus. Only 4 out of these 1128 allele combinations remained unresolved.

Alleles↗
PubMed1994

Uptake of fluorine-18-fluorodeoxyglucose in sarcoidosis.

Whole-body PET scanning was performed using 18F-fluorodeoxyglucose (FDG) in two patients with hilar lymphadenopathy in whom the clinical differential diagnosis was between sarcoidosis and lymphoma. Both patients were later proven to have sarcoidosis. Uptake of 18FDG was seen in both intra- and extrathoracic lesions as well as in associated erythema nodosum. One patient underwent a repeat scan after steroid therapy where a marked decrease in hilar uptake was seen. Fluorine-18-fluorodeoxyglucose uptake is observed in lymph nodes with sarcoid involvement. Further investigation is necessary to assess if quantitative differences exist between sarcoid and malignant lymphadenopathy.

Adult↗