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Biomedical subjects

J Wahlberg

Publications and source records attributed to J Wahlberg.

At least 19 recordsLinked to original sources
PubMed1992

Automated magnetic preparation of DNA templates for solid phase sequencing.

An integrated protocol for solid-phase DNA sequencing using a robotic work station is described involving magnetic separation of DNA and analysis of the sequencing product by electrophoresis with automated detection of the fluorescently labeled fragments. The method, which is based on magnetic beads in combination with streptavidin-biotin technology, can be used for sequencing both genomic and plasmid DNA. The DNA template is obtained by the polymerase chain reaction (PCR). Protocols to prepare five and ten immobilized samples is described, giving 10 and 20 single-stranded templates, respectively. The magnetic purification steps are performed in a microtiter plate and this allows for an integrated scheme involving a subsequent procedure for automated primer annealing and sequencing reactions. Here, the procedure is examplified by direct genomic sequencing of DNA in blood sample from a human immunodeficiency virus (HIV)-infected patient and a cloned human antibody DNA fragment using fluorescently labeled sequencing primers.

Automation↗
PubMed1992

Improvement of renal preservation by adding lidoflazine to University of Wisconsin solution. An experimental study in the rat.

The purpose of this study was to investigate the possibility of improving the organ preservation properties of the University of Wisconsin (UW) solution by adding the calcium entry blocker lidoflazine. We also investigated the possibility of decreasing the cold ischemia and reperfusion damage by pretreatment with lidoflazine of the donor and/or recipient. The protective effects of lidoflazine treatment were estimated by measuring the amount of trapped erythrocytes in the rat renal medulla after 48 h of cold storage, subsequent transplantation, and 20 min of reperfusion. Lidoflazine (20 mg/liter) added to the UW solution decreased the amount of erythrocyte trapping from 14.8 +/- 3.1% in controls to 8.6 +/- 1.7% (P less than 0.01). The flow rate of the flush-out solution during the harvesting procedure was also significantly (P less than 0.01) increased when lidoflazine was included in the UW solution (1.10 +/- 0.21 ml/min vs 0.75 +/- 0.22 ml/min). Administration of lidoflazine (0.28 mg/kg body wt) to the donor and/or the recipient did not further reduce the postischemia/reperfusion damage as estimated by the degree of erythrocyte trapping. In conclusion, the results indicate that the preservation properties of the UW solution can be significantly improved by adding lidoflazine to the solution.

Adenosine↗
PubMed1992

Colorimetric detection of Plasmodium falciparum and direct sequencing of amplified gene fragments using a solid phase method.

A rapid colorimetric assay for the detection of DNA from Plasmodium falciparum malaria is described, allowing direct sequencing of amplified fragments in the positive samples. The method is based on amplification by the polymerase chain reaction (PCR), with incorporation of biotin and a lac operator sequence in the amplified target DNA. The PCR product was immobilized on streptavidin-coupled magnetic beads, and detected by the specific binding of an Escherichia coli lac repressor beta-galactosidase fusion protein. Positive samples were subsequently treated with alkali to generate single stranded templates, which were used for solid phase genomic sequencing. As targets for amplification and sequencing we selected a region of the gene for the antigen Pf155/RESA and a region of the parasite dihydrofolate reductase gene (PfDHFR/TS). We show here that both of these gene targets can be used for specific detection of P. falciparum in patient blood samples. Genomic sequencing of five patient isolates revealed no variation in the Pf155/RESA gene fragment. In a comparison of this sequence with conserved protein domains, a marked similarity to the src homology region 3 was detected. A point mutation was found in the PfDHFR/TS gene fragment of one of the clinical samples, replacing Ser108 with Asn. This mutation has earlier been described in pyrimethamine and cycloguanile-resistant strains of P. falciparum.

Amino Acid Sequence↗
PubMed1992

Loss of synergistic response to combinations containing AZT in AZT-resistant HIV-1.

The use of combination chemotherapy for the treatment of HIV-1 infection offers promise for overcoming the problems of toxicity and development of resistance. Primary HIV-1 isolates from three patients before and after treatment with azidothymidine (AZT) were examined for sensitivity to AZT and synergistic response to three different combinations of drugs: AZT+fluorothymidine (FLT), AZT+dideoxyinosine (ddI), and FLT+ddI. All three patients initially harbored AZT-sensitive virus, but posttherapy isolates were resistant to AZT. The pretreatment, AZT-sensitive virus from each patient showed synergistic inhibition by the combinations of AZT+FLT, AZT+ddI, and FLT+ddI. In contrast, the posttreatment, AZT-resistant virus showed only addition or antagonism by the combinations containing AZT, whereas the synergistic response to the combination of FLT+ddI was preserved. Our study argues for early intervention with combination chemotherapy, since after development of resistance, AZT no longer showed synergy but addition or antagonism in combination with other drugs. After resistance to AZT has developed, combination chemotherapy not involving AZT may offer advantages over continued mono- or combination therapy involving AZT.

Antiviral Agents↗
PubMed1992

Dynamic changes in HIV-1 quasispecies from azidothymidine (AZT)-treated patients.

The emergence of azidothymidine (AZT)-resistant human immunodeficiency virus (HIV) variants in clinical samples was studied by a direct genomic sequencing method. Sequential lymphocyte samples from four patients, who had been treated with AZT for up to 27 months, were shown to gradually accumulate multiple nucleotide changes, some of which are known to be associated with AZT resistance. Several samples were shown to contain mixtures of wild-type and mutated genomes, indicating gradual rather than sudden changes in the HIV-1 quasispecies. These results demonstrate for the first time that automated solid-phase DNA sequencing is a rapid and useful tool for investigation of antiviral drug resistance and suggest that DNA sequencing may be important in routine clinical diagnostics in the future.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Pancreatic islet blood flow after syngeneic pancreaticoduodenal transplantation in rats. Differences between the blood perfusion of the native and the transplanted gland.

Inbred male Sprague-Dawley rats were transplanted with syngeneic pancreaticoduodenal grafts. Diabetes was induced in some of the recipient animals by an i.v. injection of streptozotocin one week before transplantation, while the remaining control rats were untreated, and thus had both an intact native and a transplanted pancreas. The left kidney of the recipient was removed and its blood vessels were used for the graft vascular anastomosis. The graft duodenum was sutured end-to-side to the ileum of the recipient. Two weeks after transplantation the rates of blood flow through both the native and transplanted pancreas and duodenum were measured. The blood perfusion of the whole pancreas and the islets was higher in the tx than in the native gland, although no difference was seen with regard to the duodenum. Administration of glucose to control animals had no effect on either native or tx whole pancreatic or intestinal blood flow. However, the islet blood flow through the native pancreas was increased in response to glucose, while that of the tx gland remained unchanged. The combined data show that the blood flow of a tx pancreas is slightly higher than that of the native organ, and that the response of the islet blood perfusion to glucose administration differs between the native and tx pancreas. This suggests that islets in the tx pancreas 14 days after transplantation are not subject to the same blood flow regulatory mechanisms as native islets.

Animals↗
PubMed1992

Variations in the cytomegalovirus major immediate-early gene found by direct genomic sequencing.

An assay to detect and sequence DNA from human cytomegalovirus (HCMV) immediate-early gene region 1 has been developed; it involves in vitro amplification by the polymerase chain reaction and direct solid-phase sequencing of the amplified material. Urine samples from 16 patients tested positive for HCMV DNA in both a colorimetric assay for the detection of immobilized amplified nucleic acids and a standard polymerase chain reaction assay with agarose gel electrophoresis. Ten urine samples from healthy people tested negative in the same assays. Analysis of 106-bp fragments from seven patients and two laboratory HCMV strains (Ad 169 and Towne) demonstrated that the viral sequences were conserved in samples collected at different times from the same patient and in tissue-cultured samples. Two of the patient strains had variations in the amplified region, with a total of seven nucleotide substitutions yielding five amino acid alterations in the coding sequence.

Amino Acid Sequence↗
PubMed1992

Persistence of azidothymidine-resistant human immunodeficiency virus type 1 RNA genotypes in posttreatment sera.

The rate of reversion from azidothymidine (zidovudine; AZT) resistance was studied by direct sequencing of human immunodeficiency virus type 1 (HIV-1) virion RNA in sera from four patients who discontinued long-term treatment. Before cessation of treatment, all four patients harbored HIV-1 with multiple mutations reported to confer AZT resistance. In three patients, slow reversions of these mutations starting after 9, 9, and 18 months were detected. The slow reversions indicate that AZT-resistant HIV-1 variants are likely to have an unaltered replicative capacity and pathogenic potential. Furthermore, there were discrepancies between the in vivo RNA sequences and the sequences of virus isolates, indicating that the isolation procedure may select for nonrepresentative virus variants.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Safety aspects and diagnostic findings of serial renal allograft biopsies, obtained by an automatic technique with a midsize needle.

Percutaneous biopsy is an important diagnostic procedure in evaluating the renal allograft with compromised function. Graft losses and haemorrhagic complication are major risks. To minimize these problems we used a midsize TruCut needle, controlled by an automatic firing device (Biopty-Cut), fixed to an ultrasound guidance system. Core biopsies of 1.2 x 20 mm were obtained from 1,421 kidney grafts. On 5 occasions a haemorrhagic complication that required prolonged hospitalization or intervention occurred. No grafts were lost as a consequence of the biopsy procedure. Typical histological morphological parameters found during allograft rejection has earlier been established. Using a protocol with 27 histological parameters this study confirms that recognized criteria for rejection can be relied upon even with this smaller needle. The results showed that the degree of oedema and lymphocytic infiltration of the interstitium and in the arterial wall discriminated best between rejecting grafts and non-rejection grafts.

Adult↗
PubMed1992

Outcome of renal transplantation in patients treated with erythropoietin.

The introduction of treatment with recombinant human erythropoietin (rhEPO) has raised the possibility of deleterious effects on early kidney graft function. Due to renal anemia, the great majority of patients waiting for kidney transplantation until now have had a low hematocrit. It has been suggested that a low hematocrit is beneficial for early kidney graft function by protecting the transplanted kidney from so-called reperfusion damage, which results in delayed onset of renal function. We have retrospectively examined the early function of 26 kidney grafts transplanted to uremic patients with rhEPO corrected anemia. Compared with a randomized control group no significant differences were seen in the rate of immediate onset of graft function, graft survival or serum levels of creatinine one year after transplantation. We conclude that the reversing of anemia by rhEPO in recipients of cadaver kidneys does not impair early graft function.

Anemia↗
PubMed1991

Analysis of the V3 loop in neutralization-resistant human immunodeficiency virus type 1 variants by direct solid-phase DNA sequencing.

To study the molecular basis for the emergence of human immunodeficiency virus type 1 (HIV-1) variants with reduced sensitivity to neutralization by autologous sera, the DNA sequence of the envelope V3 loop was determined in HIV-1 isolates derived from four patients with primary HIV-1 infection and sequentially thereafter. The gene fragment encoding the V3 loop of gp120 was amplified by a nested polymerase chain reaction (PCR) and subsequently sequenced by a novel solid phase DNA sequencing approach allowing direct sequencing of the viral genome without the need for previous cloning. The results show that all patients have HIV-1 with unique primary sequence of the V3 loop and antibodies to this structure are produced at seroconversion. The structural analysis also demonstrates that the mechanism for virus escape from neutralization in vivo is complex. Thus, in one patient the emergence of neutralization-resistant viruses coincided with the introduction of several amino acid changes in the V3 loop, while in two other patients the V3 loop remained completely unchanged. These findings suggest that an understanding of the interaction between the humoral immune response and HIV-1 may require detailed structural studies of the entire envelope.

Amino Acid Sequence↗