C. Källander - Academia.edu (original) (raw)

Papers by C. Källander

British Journal of Haematology, 1986

... GAREWAL. H., DURIE, BGM, KYLE. RA, FINELY. P.. BOWER, B. & SEKOKMAN, R. (1984) Serum b*-m... more ... GAREWAL. H., DURIE, BGM, KYLE. RA, FINELY. P.. BOWER, B. & SEKOKMAN, R. (1984) Serum b*-microglobulin in the initial staging and subsequent monitoring of multiple myeloma. ... logi~u. 64. 79-86. TEASDALE. C., ~IANDER. AM, FIFIELD, R.. KEYSER. JW, NCW(!OMBB. ...

Antiviral Chemistry and Chemotherapy

A novel reverse transcriptase (RT) assay based on the combined use of macrobead-bound template an... more A novel reverse transcriptase (RT) assay based on the combined use of macrobead-bound template and 125I-iododeoxyuridine-triphosphate (IUTP) was used to determine the IC50 values of various RT inhibitors. The results showed that this assay and the conventional assay gave similar IC50 values. The introduction of carrier bound template-primer, template, or primer also made it possible to design assays revealing the mechanism of action of various RT inhibitors. Unlabelled inhibitor substance could be incubated with carrier bound template-primer in the presence of excess enzyme, after which the inhibitor was removed and the residual template-primer function was analysed by RT assay. By this procedure it was found that chain elongation terminators like 2′,3′-dideoxy-TTP and 3′-azido-TTP destroyed the template-primer at low concentrations which corresponded to the amount of primer. In contrast, 20–200 times higher concentrations were needed for template-primer destruction when using subst...

British Journal of Haematology

ABSTRACT

A one-step procedure which uses enzymes in a crude extract of herpes simplex virus (HSV) type 1-i... more A one-step procedure which uses enzymes in a crude extract of herpes simplex virus (HSV) type 1-infected cells to synthesize 5-[125I]iodo-2'-deoxyuridine triphosphate [( 125I]dUTP) from [125I]dU is described. The design of a one-step procedure for the purification of the product is also presented. The recovery of [125I]dUTP from [125I]dU varied between 50 and 75%, the radiochemical purity of the product was greater than 90%, and both synthesis and purification were completed within 8 h. The sensitivity and specificity of [125I]dUTP as a substrate for both DNA-dependent DNA polymerase (DNAp) and RNA-dependent DNA polymerase (reverse transcriptase, RT) were evaluated and compared to those of [3H]dTTP for the following specimens: purified cloned Klenow fragment, crude extracts of HeLa-, BHK-, and HSV-2-infected BHK cells, purified avian myeloblastosis virus RT, and purified cloned human immunodeficiency virus (HIV) RT. The [125I]dUTP was accepted as a substrate equally as well [3H]dTTP by all of the specimens at all of the concentrations tested. When the same amount of radiolabel was used, [125I]dUTP gave a sensitivity 10- to 25-fold higher than that of [3H]dTTP. The gain in sensitivity was due to the higher specific activity and a higher counting efficiency of the 125I-label compound. The use of [125I]dUTP also offered technical advantages over alternative substrates available, such as product separation without acid precipitation and exclusion of the need for scintillation cocktails. The half-life of the nucleic also gives a reasonable shelf-life for use in routine assays. Activity of less than 0.3 pg of HIV RT could be detected when the new substrate was used, and this made it possible to quantitate HIV RT antibodies (abs) in diluted serum samples without purifying the immunoglobulin. Analysis of 31 HIV-infected individuals showed that all of them had anti-HIV RT ab and that the amount of serum needed for 50% inhibition of the HIV RT activity corresponded to an amount of immunoglobulin 100-fold smaller (i.e., 0.02-31.4 micrograms) than has been previously reported. With the substrate it was also possible to detect DNAp activity in serum from healthy individuals, although a long-duration assay was required. In a long-duration assay the DNAp activity found in sera from healthy individuals was linear with respect to time, whereas the DNAp activity found in many sera from tumor patients was not. [125I]dUTP is judged to be an excellent substrate for detecting and quantifying the activity of various DNA-synthesizing enzymes and their blocking abs.

Antiviral Chemistry and Chemotherapy, 1997

A new sensitive colorimetric reverse transcriptase (RT) activity assay utilizing a 96-well microt... more A new sensitive colorimetric reverse transcriptase (RT) activity assay utilizing a 96-well microtitre plate format, with solid phase-conjugated polyadenylic acid (prA), was investigated for simple analyses of the RT inhibiting capacity and mode of action of various substances. Three different technical procedures using the assay were evaluated: (i) direct lC50 determinations with various substances, using four different combinations of primer and dNTP amounts; (ii) analyses of the capacity of the substances to interfere with the binding of RT to template or template-primer (BIC50); (iii) analyses of the capacity of the substances to destroy the template-primer in presence or absence of RT (TDC50). The assay was found to be useful for all three purposes using small amounts of recombinant RT. In the IC50 analyses, the test substances gave values similar to those reported for soluble RT assays, and the values varied in accordance with their known mode of action in relation to the combi...

Antiviral chemistry & chemotherapy, 1998

Four non-nucleoside reverse transcriptase (RT) inhibitors, 9-CI-TIBO [(+)-S-4,5,6,7-tetrahydro-9-... more Four non-nucleoside reverse transcriptase (RT) inhibitors, 9-CI-TIBO [(+)-S-4,5,6,7-tetrahydro-9- chloro-5-methyl-6-(3-methyl-2-butenyl)imidazo(4,5,1-jk)(1,4)- benzodiazepin-2(1H)-thione)], nevirapine (6,11-dihydro-11-cyclopropyl-4-methyl-dipyrido[2,3-b:2',3'-e]-[1,4]di azepin- 6-one), MSA-300 (N-[cis-2-(2-hydroxy-3-acetyl-6-methoxy-phenyl)-cyclopropyl]-N'- (5-chloropyrid-2-yl)-thiourea) and delavirdine ¿1-(5-methanesulphonamido-1H-indol-2-yl-carbonyl)-4-[3- (1-methylethylamino)pyridinyl]piperazine¿ were analysed for the mode of action of their inhibition of human immunodeficiency virus type 1 (HIV-1) RT in three different assays utilizing a 96-well microtitre plate format, with solid-phase conjugated poly(rA) as template. These were: (i) direct RT assay, for determination of IC50 values of RT inhibitors; (ii) RT template/primer binding inhibition (BIC) assay, for measuring the effect of various substances on the RT activity binding to template/primer; (iii) RT protein E...

Journal of clinical microbiology, 1997

Standardization and calibration of a new colorimetric assay for detection of reverse transcriptas... more Standardization and calibration of a new colorimetric assay for detection of reverse transcriptase (RT) was carried out for optimal detection of RT activity-blocking antibody (RTb-Ab) in serum. A total of 99 of 100 Swedish and 54 of 54 African human immunodeficiency virus type 1 (HIV-1) antibody-positive individuals had RTb-Ab. The one RTb-Ab-negative HIV-1 serum sample from a Swedish individual was obtained early during seroconversion. Five of 615 HIV-1-negative sera from tumor patients, pregnant women, patients undergoing routine viral diagnostics, and blood donors gave false-positive results. In addition, 3 of 126 HIV-1-negative African serum samples and 2 of 91 serum samples selected because of false reactivity in other commercially available HIV antibody assays were positive for RTb-Ab. RT activity and RTb-Ab were measured in sera from newly HIV-1-infected individuals during seroconversion. Peak RT activity was usually detected between days 8 and 13 after the onset of symptoms ...

British journal of cancer, 1983

A recently developed enzyme assay, utilizing [125I]-iododeoxyuridine as substrate, and capable of... more A recently developed enzyme assay, utilizing [125I]-iododeoxyuridine as substrate, and capable of detecting normal levels of serum deoxythymidine kinase (s-dTk), was used in an investigation of sera from 155 untreated patients with non-Hodgkin's lymphoma (NHL). The patients were classified at the discovery of disease, both according to spread (stages I-IV according to the Ann Arbor classification) and to tumour histology (the Kiel classification). The results showed a significant correlation between s-dTk level and the extent of disease, as well as to the malignancy; i.e. the more advanced the disease or the more aggressive the tumour, the higher the s-dTk values. Greater than 100-fold increases in s-dTk levels were found in some patients compared to those reported for healthy individuals. A high pretreatment level of s-dTk for patients in stages III-IV correlated with a poor prognosis for the patient in terms of survival. This was consistent even when only patients in stages II...

Scandinavian journal of haematology, 1984

The prognostic value of different pretreatment laboratory and clinical findings at diagnosis was ... more The prognostic value of different pretreatment laboratory and clinical findings at diagnosis was assessed in a series of 141 patients with generalized non-Hodgkin's lymphoma. Univariate and multivariate survival analysis (Cox's regression model) was performed, using serum analysis of deoxythymidine kinase (S-TK), beta 2-microglobulin, lactic dehydrogenase, alpha 1-acid glycoprotein = orosomucoid (S-alpha 1 AGP), haptoglobin and ferritin. In addition, Hb and the erythrocyte sedimentation rate (ESR) were measured. The clinical variables were age, presence or absence of B-symptoms, histopathology ('low-grade'; 'intermediate grade' and 'high-grade' malignancy) and bone marrow involvement. Of the 8 biochemical markers, all except Hb and the ESR showed a significant relationship to survival. Among the clinical variables, this finding was made for B-symptoms and histopathology. Using a multivariate analysis on all variables, S-TK was found to be the best fac...

Current topics in microbiology and immunology, 1983

Developments in biological standardization, 1982

This report summarizes the essential features of a sensitive deoxythymidine kinase (dTk) assay, w... more This report summarizes the essential features of a sensitive deoxythymidine kinase (dTk) assay, which can be used for the diagnosis of Herpes virus infections. A type specific antibody response towards the different Herpes virus dTk isozymes, the Herpes simplex virus (HSV) type 1, type 2, and Varicella Zoster virus (VZV), was found in human sera. The occurrence of dTk antibody in relation to complement fixing (cf) antibody and the state of infection, was found to differ between HSV and VZV. The VZV dTk antibody was only found in patients with Herpes Zoster, while most HSV cf positive individuals had HSV dTk antibody. Absence of dTk in HSV cf positive individuals was related to a "recent primary infection". The type specificity of the dTk isozymes makes these antigens suitable for typing of isolates, and this in combination with the sensitivity of the assay, made it possible to verify and type HSV infection by direct analysis of blister secrete without previous virus isolat...

Journal of clinical microbiology, 1982

We describe two methods for typing of herpes simplex virus (HSV). One procedure is based on the f... more We describe two methods for typing of herpes simplex virus (HSV). One procedure is based on the finding that the multiplication of HSV type 1 strains in primary rabbit kidney cells is inhibited by 2 x 10(-5) M iododeoxyuridine, whereas growth of HSV type 2 strains is considerably less affected. Forty-nine different HSV isolates were typed according to this method. For all isolates except two the results were found to be in agreement with results obtained by another typing procedure, the counterimmunoelectroosmophoretic method (S. Jeansson, Appl. Microbiol. 24:96-100, 1972). One HSV type 1 isolate behaved as a type 2 strain and was found to be a deoxythymidine kinase-negative mutant strain. The other deviant strain exhibited an intermediate iododeoxyuridine sensitivity, thus being impossible to type with this method. Another, faster typing procedure which is based on the immunological difference between HSV type 1 and 2 deoxythymidine kinase is also presented. This assay, in combinat...

Infection and immunity, 1980

An optimized assay for herpes simplex virus type 1- and 2-induced deoxythymidine kinase (dTk) is ... more An optimized assay for herpes simplex virus type 1- and 2-induced deoxythymidine kinase (dTk) is described which used [125I]iododeoxyuridine (IUdr) as a substrate. Values for Km and Vsat were determined for both viral and cellular dTK, using either deoxythymidine or IUdR as a substrate. A comparison between the two substrates revealed that higher reaction velocities and lower Km values were obtained with IUdR. A standard assay was designed which uses 10(-7) M IUdR as a substrate. This assay can detect herpes simplex-induced dTK from as few as two infected cells and is several orders of magnitude more sensitive than conventional dTK assays which use 10(-5) M dT as a substrate. An easily detectable blocking activity, which was shown to be mainly confined to the immunoglobulin G antibody class, was found in most human sera which were positive for complement-fixing antibody against herpes simplex virus.

Infection and immunity, 1982

The conditions required for the production of varicella-zoster virus (VSV)-induced deoxythymidine... more The conditions required for the production of varicella-zoster virus (VSV)-induced deoxythymidine kinase (dTk) have been studied. Extracts from Vero cells harvested 62 h after VZV infection were found to contain VZV-induced dTk activity, with a minimal contribution from the cellular dTk activity. VZV dTK was shown to have a broad substrate specificity phosphorylating both deoxythymidine, deoxycytidine, and iododeoxyuridine. Deoxythymidine triphosphate inhibition studies revealed an intermediate deoxythymidine triphosphate sensitivity when compared with that of the cellular cytosolar enzyme and the deoxythymidine triphosphate-insensitive herpes simplex virus dTk. An assay for VZV dTk-blocking antibodies was developed, with [125I]iododeoxyuridine as a substrate in the presence of a deoxythymidine triphosphate concentration which selectively blocked the dTK of host cell origin. A total of 79 serum samples were studied; these included serum pairs from patients with varicella or herpes z...

Annals of clinical research, 1986

Twenty patients, cadaveric renal transplant recipients, were retrospectively analysed for serum l... more Twenty patients, cadaveric renal transplant recipients, were retrospectively analysed for serum levels of deoxythymidine kinase. Special reference was made to the thymidine kinase level in relation to rejection, and viral infection. Seven of the patients experienced clinically suspected cytomegalovirus infection. All these patients had elevated levels of serum thymidine kinase during the period of clinical disease. Usually the thymidine kinase level parallelled the severity of the disease. All patients with irreversible rejection had increased levels of serum thymidine kinase, but normally not as high, as seen in patients with severe cytomegalovirus infection. There was also some correlation between clinically suspected rejection, that lead to rejection treatment, and moderate increase in thymidine kinase. However, not all rejection episodes were accompanied by a thymidine kinase increase. Serum thymidine kinase was analysed and compared in patients with self-healing cytomegalovirus...

Transplantation proceedings, 1988

ABSTRACT The development of a colorimetric capture assay for HIV-1 reverse transcriptase (RT) act... more ABSTRACT The development of a colorimetric capture assay for HIV-1 reverse transcriptase (RT) activity is described. This assay consisted of three basic steps: enzyme purification, RT reaction and product detection, which were all performed in the same microtitre plate. Mouse monoclonal anti-RT antibodies of subclass G2a were bound by polyclonal goat anti-(mouse IgG2a) immobilized in the wells of a microtitre plate. The monoclonal antibodies (mAbs) were selected for their ability to bind HIV-1 RT without hampering the polymerase activity. The assay system first involved the RT's adherence to the immobilized mAbs. Non-specific enzymes and other impurities were removed by a simple wash, after which an RT reaction mixture containing BrdUTP as nucleotide substrate was added. After the RT reaction substrate and product had been separated by washing of the plate, the amount of BrdUMP-DNA in the wells was finally detected with alkaline-phosphatase-conjugated mouse anti-BrdU antibodies of subclass IgG1. The background signal in this system was similar to the signals obtained with control wells coated with BSA only. A detection limit of 1.2 micro-units of RT activity, corresponding to 0.3 pg of RT protein, was obtained for the capture assay when applying colorimetric product detection. The assay detected RTs from HIV-1 subtypes A and B and one of the two D type isolates tested. None of the five non-HIV-1 RTs tested was found positive. At least 50 microl of human serum or plasma per sample could be included in the capture assay without adverse effects on the recovery of the RT activity.