Biomedical subjects

K Nielsen

Publications and source records attributed to K Nielsen.

At least 37 records · Page 2Linked to original sources

PubMed2004

Comparison of serological tests for the detection of ovine and caprine antibody to Brucella melitensis.

The indirect enzyme-linked immunosorbent assay (IELISA), the competitive enzyme-linked immunosorbent assay (CELISA) and the fluorescence polarisation assay (FPA) were evaluated with sera from sheep experimentally infected with Brucella melitensis and negative Canadian sheep. The sensitivity and specificity of the assays were as follows: IELISA: 91.7% and 97.6%, CELISA: 75.0% and 99.8% and FPA: 91.7% and 89.5%. Sera from the same experimental population were divided according to serological reaction in the rose bengal agglutination test (RBT) and the complement fixation test (CFT). Reactivity relative to the RBT positive and CFT positive sera were as follows: IELISA: 99.7%, CELISA: 93.2% and FPA: 99.1%. Since sera from goats with proven B. melitensis infection were not available, 699 sera from goats judged positive in the buffered antigen plate agglutination test (BPAT) and CFT and 982 BPAT/CFT negative Canadian goats were used. The sensitivity and specificity of the assays relative to the BPAT and CFT positive sera were: IELISA: 99.4% and 98.0%, CELISA: 95.4% and 97.1% and FPA: 92.7% and 99.8%.

Agglutination Tests↗

PubMed2004

Serological diagnosis of bovine brucellosis: a review of test performance and cost comparison.

The authors reviewed over 50 publications in which the sensitivity and specificity values of assays used for the detection of exposure to Brucella abortus had been examined. The sum of the sensitivity and specificity values for each test was averaged to give a performance index (PI) and allow for a comparison between the different methodologies. A score of 200 was perfect. Based on the PI, the buffered antigen plate agglutination test (BPAT) rated highest (PI = 193.1) among the conventional tests. This indicates better accuracy than the other conventional tests including the Rose Bengal test (PI = 167.6) and the complement fixation test (PI = 172.5). Overall, the primary binding assays, including the fluorescence polarisation assay (PI = 196.4), the indirect enzyme-linked immunosorbent assay (PI = 189.8) and the competitive enzyme-linked immunosorbent assay (PI = 188.2), were more accurate than the conventional tests, except for the BPAT. In addition, a fee comparison suggested that the primary binding tests were price competitive with conventional tests for the diagnosis of brucellosis and, therefore, had a better combined cost/efficiency rating.

Agglutination Tests↗

PubMed2003

Protein expression changes in a cell system of beta-cell maturation reflect an acquired sensitivity to IL-1beta.

AIM/HYPOTHESIS: Type 1 diabetes mellitus (T1DM) is caused by specific destruction of the pancreatic beta cells in the islets of Langerhans. Increased sensitivity to cytokines, in particular to interleukin-1beta (IL-1beta) seems to be an acquired trait during beta-cell maturation. In response to cytokines both protective and deleterious mechanisms are induced in beta cells, and when the deleterious prevail, T1DM develops. The aims of this study were to identify perturbation in protein patterns (PiPP) associated with beta-cell maturation, and compare these changes to previous analyses of IL-1beta exposed rat islets. For this purpose, proteome analyses were carried out using a cell-line, which matures from a glucagon-producing pre-beta-cell phenotype (NHI-glu) to an insulin-producing beta-cell phenotype (NHI-ins). We have previously shown that this maturation is accompanied by acquired sensitivity to the toxic effects of IL-1beta. METHODS: 2D-gel electrophoresis was used to separate the proteins and MALDI-MS and database searches were performed to identify the proteins. RESULTS: During beta-cell maturation 135 protein spots out of 2239 detectable changed expression levels. Of these, 74 were down-regulated, 44 up-regulated, 16 were suppressed and 1 was expressed de novo. Using MALDI-MS, positive identification was obtained for 93 out of the 135 protein-spots revealing 97 different proteins. Of these, 22 proteins were in common with changes identified in previous proteome analysis of perturbation in protein pattern in IL-1beta exposed rat islets. Several of the proteins were present in more than one spot suggesting post-translational modification. CONCLUSION/INTERPRETATION: Several proteins and protein modifications were identified that could be critically involved in beta-cell maturation, insulin-gene expression and the acquired IL-1beta sensitivity.

Animals↗

PubMed2003

[Comparative evaluation of the indirect enzyme-linked immunosorbant assay in milk for the detection of cattle infected with Brucella abortus, in herds located in the province of Cundinamarca, Colombia].

To perform a comparative evaluation of the indirect enzyme-linked immunosorbent assay (iELISA) in milk, for the detection of Brucella abortus in cattle, 1,523 milk samples were taken from individual animals and bulk milk belonging to 200 herds in the province of Cundinamarca, Colombia. All these herds were part of the official programme of monitoring free herds and determining herd prevalence in the province. The samples were submitted simultaneously to the milk ring test and the iELISA. A total of 584 individual and 497 bulk milk samples taken from free herds were considered negative, and served to determine the initial ELISA cut-off point. An optimised determination of the cut-off point involved an operational analysis, using a statistical programme. In the analysis, the total population was considered: 756 individual positive and negative samples, as defined by herd history and previously established serologies. The cut-off point was set at > 0.20 optical density units, which, expressed in percentage positivity, corresponded to 20.5% against the controls, with a sensitivity of 95.3%, a specificity of 95.1%, and a confidence interval of 95%. Indirect confirmation of the state of infection was made with competitive ELISA in the serum of the individual animals considered positive, and an attempt was made to determine bacterial presence by isolation in culture and by reverse transcription-polymerase chain reaction. The results confirm that the indirect ELISA is highly sensitive and specific, provides confirmed results in strict quality-control conditions, and may be used to test a large number of herd or individual samples, thus enhancing the efficiency of surveillance programmes and control campaigns.

Agglutination Tests↗

PubMed2002

Failure to demonstrate involvement of antibodies to Acinetobacter calcoaceticus in transmissible spongiform encephalopathies of animals.

Acinetobacter calcoaceticus, a soil microbe, contains molecular sequences which resemble those found in neurofilaments of the brain tissue. It was hypothesized that if cattle ingest large amounts of feedstuff containing A. calcoaceticus, they may develop an autoimmune reaction, with consequences of pathological changes associated with transmissible spongiform encephalopathies (TSEs). The hypothesis was tested using a small number of serum samples collected from cattle and it was found that affected individuals had elevated serum antibody levels to this organism. If this finding was substantiated, it would provide a possible means of diagnosing TSEs in vivo. In the present communication, a larger number of cattle, elk and sheep with or without TSEs were tested using A. calcoaceticus whole cell and lipopolysaccharide antigens as well as myelin basic protein (MBP). It was found that antibody levels in normal and affected animals overlapped considerably, thus casting doubt on the usefulness of these antigens as diagnostic tools for TSEs and on the hypothesis of A. calcoaceticus being a cause of TSEs.

Acinetobacter calcoaceticus↗

PubMed2002

Crystal structure and spectroscopic properties of Na(2)K(6)(VO)(2)(SO(4))(7).

Red-brown crystals of a new mixed alkali oxo sulfato vanadium(V) compound Na(2)K(6)(VO)(2)(SO(4))(7), suitable for X-ray determination, have been obtained from the catalytically important binary molten salt system M(2)S(2)O(7)-V(2)O(5) (M = 80% K and 20% Na). By slow cooling of a mixture with the mole fraction X(V(2)O(5)) = 0.24 from 325 degrees C, i.e., just below the liquidus temperature, to the solidus temperature of around 300 degrees C, a dark reddish amorphous phase was obtained containing crystals of the earlier described V(V)-V(IV) mixed valence compound K(6)(VO)(4)(SO(4))(8) and Na(2)K(6)(VO)(2)(SO(4))(7) described here. This compound crystallizes in the tetragonal space group P4(3)2(1)2 (No. 96) with a = 9.540(3) A, c = 29.551(5) A at 20 degrees C and Z = 4. It contains a distorted VO(6) octahedron with a short V-O bond of 1.552(6) A, a long one of 2.276(5) A trans to this, and four equatorial V-O bonds in the range 1.881(6)-1.960(6) A. The deformation of the VO(6) octahedron is less pronounced compared to that of the known oxo sulfato V(V) compounds. Each VO(3+) group is coordinated to five sulfate groups of which two are unidentately coordinated and three are bidentate bridging to neighboring VO(3+) groups. The length of the S-O bonds in the S-O-V bridges of the two unidentately coordinated sulfato groups are 1.551(6) A and 1.568(6) A, respectively, which are unusually long compared to our earlier measurements of sulfate groups in other V(III), V(IV), and V(V) compounds.

Journal Article↗

PubMed2002

Field trial of the brucellosis fluorescence polarization assay.

Fluorescence polarization assay (FPA) is a homogeneous technique which was applied to the serological diagnosis of bovine brucellosis. Because of its simplicity and because it may be performed very rapidly, it was an ideal test to adapt to field use. The FPA was used to test cattle on six dairy farms in Baja California, Mexico. Anticoagulated blood, serum, and milk were collected from each animal. The anticoagulated blood was tested immediately on the farm while serum and milk were tested subsequently in the laboratory. Cattle on one farm (n = 140) were thought not to be infected with Brucella abortus and the other farms were thought to have high prevalence of the infection. The whole blood FPA (FPA(bld)) did not detect antibody in any of the cattle on the first premise. This finding was confirmed using a number of other serological tests, including the buffered antigen plate agglutination test, the complement fixation test, the indirect and competitive enzyme immunoassays, and the FPA using serum and milk. Cattle on the other premises (n = 1122) were tested in a similar fashion. The sensitivity of the FPA(bld), relative to the serum FPA (considered the definitive test), was 99.1% and the relative specificity of the FPA(bld) was 99.6%. These results compared favourably with those obtained using the other serological tests.

Animals↗

PubMed2002

Antibody dynamics in Holstein Friesian heifers vaccinated with Brucella abortus strain 19, using seven serological tests.

The serological response induced by Brucella abortus strain 19 was evaluated in 52 Holstein females from a brucellosis-free herd using seven serological tests. Each calf was vaccinated at an age of 4 and 8 months old with 3 x 10(10) CFU B. abortus S19 and the antibody response was determined as the proportion of positive results to each test. The antibody dynamics, measured with the buffered plate antigen (BPA) test and the rapid automated presumptive (RAP) test, were similar. The proportion of positive reactions in these tests reached 100% one week after vaccination and remained at this level for seven weeks, after which the proportion of positive samples slowly declined to 8% (BPA) and 2% (RAP) at week 50. The response in the indirect enzyme immunoassay (i-ELISA) was similar, but shorter than that observed with the BPA/RAP. The antibody dynamic, measured using the seroagglutination test (SAT) in parallel with the 2-mercaptoethanol (2-Me) test and the complement fixation test (CFT) were similar to the RAP/BPA, but of slightly shorter duration. The competitive ELISA (c-ELISA) was positive in all animals for 3 weeks, followed by a rapid decline. The fluorescence polarization assay (FPA) reached a maximum of 68.5% positive animals at week 4 and then declined. Based on these data, the c-ELISA and FPA discriminated residual antibody activity due to vaccination more efficiently than the other tests.

Animals↗

PubMed2002

Fluorescence polarization assay for detection of Brucella abortus antibodies in bulk tank bovine milk samples.

A simple, rapid, inexpensive fluorescence polarization assay for the detection of antibodies to Brucella abortus in bulk tank milk samples at the farm level or at dairies with a sensitivity and specificity of 100 and 95.9%, respectively, is described. The assay detects antibodies to B. abortus in 15 min by testing undiluted whey produced by chemical and physical manipulation of milk from bulk tanks. This sampling is noninvasive and therefore costs less and is less stressful than blood-based tests. The assay is specific and can detect antibodies at levels below that of the indirect enzyme immunoassay for milk and the fluorescence polarization assay for individual milk samples. Use of this test would make programs for surveillance of dairy animals and eradication of B. abortus more cost-effective.

Animals↗

PubMed2001

Comparison of an indirect ELISA with the Brucella milk ring test for detection of antibodies to Brucella abortus in bulk milk samples.

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of Brucella abortus antibodies in bovine bulk milk samples was evaluated. About 31 individual milk samples from B. abortus infected cows were diluted into bulk milk from a brucellosis free herd. Individual milk samples obtained from 96 negative or positive herds to ELISA or Brucella ring test (BRT), were tested by ELISA. All positive cows were bled and serum samples were tested by the complement-fixation test (CFT) which was considered the definitive test. A herd was considered infected if at least, one cow was positive in the CFT. Four samples were negative in the BRT at the dilution 1:10 but positive in the ELISA. For samples positive in both tests, BRT titers ranged from 1:10 to 1:480 while ELISA titers ranged from 1:10 to 1:3200. Using bulk milk samples, the sensitivity of the ELISA (98.1%) was higher than the BRT (72.2%) but the specificity of BRT (90.5%) was not statistically different (P=1.0) from the ELISA (88.1%). The implications of the results for brucellosis control are discussed.

Animals↗

PubMed2001

Fluorescence polarization assay for the diagnosis of bovine brucellosis: adaptation to field use.

A fluorescence polarization assay (FPA) was used to test whole blood samples prepared by mixing blood cells from cattle without exposure to Brucella abortus (B. abortus) with sera from animals with confirmed (bacteriologically) infection. A cut-off value between negative and positive values was initially established to be 87.2mP. This value was changed to 95mP to increase assay specificity without loss of sensitivity when testing blood samples from negative animals. The FPA technology was applied to whole blood samples in the field and to stored whole blood samples using two diluent buffers. Relative sensitivity and specificity values for the FPA performed in the field, based on buffered antigen plate agglutination test and competitive enzyme immunoassay results were 95.3 and 97.3%, respectively. However, to obtain maximum sensitivity and specificity, a cut-off value of 105mP was determined for fresh whole blood samples. The relative sensitivity and specificity values of the FPA when testing stored whole blood samples were 100% each using a 95mP cut-off.The usefulness of the FPA for testing whole blood samples in the field was demonstrated.

Agglutination Tests↗

PubMed2001

Multiple roles for the C-terminal domain of eIF5 in translation initiation complex assembly and GTPase activation.

eIF5 stimulates the GTPase activity of eIF2 bound to Met-tRNA(i)(Met), and its C-terminal domain (eIF5-CTD) bridges interaction between eIF2 and eIF3/eIF1 in a multifactor complex containing Met-tRNA(i)(Met). The tif5-7A mutation in eIF5-CTD, which destabilizes the multifactor complex in vivo, reduced the binding of Met-tRNA(i)(Met) and mRNA to 40S subunits in vitro. Interestingly, eIF5-CTD bound simultaneously to the eIF4G subunit of the cap-binding complex and the NIP1 subunit of eIF3. These interactions may enhance association of eIF4G with eIF3 to promote mRNA binding to the ribosome. In vivo, tif5-7A eliminated eIF5 as a stable component of the pre-initiation complex and led to accumulation of 48S complexes containing eIF2; thus, conversion of 48S to 80S complexes is the rate-limiting defect in this mutant. We propose that eIF5-CTD stimulates binding of Met-tRNA(i)(Met) and mRNA to 40S subunits through interactions with eIF2, eIF3 and eIF4G; however, its most important function is to anchor eIF5 to other components of the 48S complex in a manner required to couple GTP hydrolysis to AUG recognition during the scanning phase of initiation.

Codon, Initiator↗

PubMed2001

Management of anaphylactic shock evaluated using a full-scale anaesthesia simulator.

BACKGROUND: The diagnosis of an anaphylactic reaction during anaesthesia is not the first consideration for the anaesthetist and might be missed. The aim of this study was to describe anaesthetists' management of an anaphylactic reaction concerning diagnosing, treatment and application of anaesthesia crisis resource management (ACRM) in a full-scale anaesthesia simulator. METHODS: Forty-two anaesthetists in teams of two attended training sessions with a critical incident of anaphylactic shock in a full-scale simulator. Trained observers from the study group evaluated the medical treatment according to a treatment sequence developed from the literature and graded the ACRM performance on a five-point scale where 1 is bad and 5 is best. RESULTS: None of the teams made the correct diagnosis within 10 min and treatment according to the treatment sequence was not initiated. Only 6/21 teams considered the right diagnosis but first after hints from the instructor 15 min after the start of the incident. Evaluation of the use of the total ACRM concept (that is the use of all of the ACRM expressions seen in a total connection: called general impression) gave a median value of 2.0 with a range of (1-3). CONCLUSION: Anaphylactic shock was difficult to diagnose and no structured plans were used for the treatment in the simulated incident in this study.

Anaphylaxis↗

PubMed2001

The spectrum of pathogenic mutations in SPINK5 in 19 families with Netherton syndrome: implications for mutation detection and first case of prenatal diagnosis.

The Comèl-Netherton syndrome is an autosomal recessive multisystemic disorder characterized by localized or generalized congenital ichthyosis, hair shaft abnormalities, immune deficiency, and markedly elevated IgE levels. Life-threatening complications during infancy include temperature and electrolyte imbalance, recurrent infections, and failure to thrive. To study the clinical presentations of the Comèl-Netherton syndrome and its molecular cause, we ascertained 19 unrelated families of various ethnic backgrounds. Results of initial linkage studies mapped the Comèl-Netherton syndrome in 12 multiplex families to a 12 cM interval on 5q32, thus confirming genetic homogeneity of Comèl-Netherton syndrome across families of different origins. The Comèl-Netherton syndrome region harbors the SPINK5 gene, which encodes a multidomain serine protease inhibitor (LEKTI) predominantly expressed in epithelial and lymphoid tissues. Recently, recessive mutations in SPINK5 were identified in several Comèl-Netherton syndrome patients from consanguineous families. We used heteroduplex analysis followed by direct DNA sequencing to screen all 33 exons and flanking intronic sequences of SPINK5 in the affected individuals of our cohort. Mutation analysis revealed 17 distinct mutations, 15 of which were novel, segregating in 14 Comèl-Netherton syndrome families. The nucleotide changes included four non-sense mutations, eight small deletions or insertions leading to frameshift, and five splice site defects, all of which are expected to result in premature terminated or altered translation of SPINK5. Almost half of the mutations clustered between exons 2 and 8, including two recurrent mutations. Genotype-phenotype correlations suggested that homozygous nucleotide changes resulting in early truncation of LEKT1 are associated with a severe phenotype. For the first time, we used molecular data to perform prenatal testing, thus demonstrating the feasibility of molecular diagnosis in the Comèl-Netherton syndrome.

Adolescent↗

PubMed2001

Comparison of some methods for determing cutoff values for serological assays: a retrospective study using the fluorescence polarization assay.

Different methods for determining cutoff values between positive and negative results for serological assays have been developed over time. Comparisons of some these methods show that five (Receiver Operating Characteristics, Frequency Distribution, and the mean, median and mode of the 100th percentile of a disease-free group of data) resulted in similar sensitivity (99.11%) and specificity values (99.21% to 99.58%). However, the Receiver Operating Characteristic analysis was considered more suitable, due to the ease of data manipulation and computer analysis, providing flexibility for different field applications.

Fluorescence Polarization Immunoassay↗

PubMed2001

Fluorescence polarization assay for the diagnosis of brucellosis: a review.

Fluorescence polarization assay (FPA) is based on the rotational differences between a small soluble antigen molecule in solution (labelled with a fluorochrome) and the antigen molecule complexed with its antibody. A small molecule will rotate randomly at a rapid rate, resulting in rapid depolarization of light, while a larger complex molecule will rotate slower and depolarize light at a reduced rate. The rate change in depolarization can be measured. The FPA is a homogeneous assay which does not require removal of unreacted reagents and can, therefore, be performed very quickly and, given portable equipment, in the laboratory and in the field. The latter obviates the need for shipping samples and eliminates waiting for results, as well as reducing test costs. The FPA technology has been developed and validated for the serological diagnosis of brucellosis in cattle, swine, sheep, goats, bison, and cervids. Sufficient cross reactivity of the common epitopes of Brucella abortus, B. melitensis and B. suis O-polysaccharide (OPS) allowed for the use of a single antigen for all species of smooth Brucella and animals. The OPS prepared from B. abortus S1119.3 was conjugated with fluorscein isothiocyanate (FITC). The FPA was initially developed for testing serum; however, the technology has been extended to testing whole blood and milk from individual animals or bulk tank samples pooled from 2000 or fewer animals. The accuracy of the FPA equalled or exceeded those obtained using other serological tests such as the buffered antigen plate agglutination test (BPAT), the milk ring test (MRT), the complement fixation test (CFT), the indirect enzyme immunoassay (IELISA), and the competitive enzyme immunoassay (CELISA).

Animals↗

PubMed2001

Validation of the fluorescence polarization assay for detection of milk antibody to Brucella abortus.

A fluorescence polarization assay (FPA) for detection of antibody to Brucella abortus in individual milk samples was developed and validated. Samples from 190 cattle from which B. abortus was isolated; milk samples from cattle in herds infected with B. abortus (n = 1,086) and positive in the milk ring test (MRT), as well as milk samples from Canadian cattle (with no evidence of brucellosis, n = 2,974) were tested by the indirect enzyme immunoassay (IELISA) and the FPA. The sensitivity (based on samples from culture positive cattle) and specificity (based on Canadian milk samples) of the IELISA and the FPA were 100%. The relative sensitivity value obtained with milk from cattle of infected herds and the specificity values of the IELISA were 98.5 and 99.9%, respectively. The relative sensitivity and specificity of the FPA with the same samples were 82.2 and 99.4% using a cutoff value of 90 millipolarization units (mP). The low relative sensitivity value of the FPA was shown, by competitive enzyme immunoassay (CELISA), to be due to vaccinal antibody (assumed as vaccinal antibody against B. abortus Sl19 is excluded by the FPA and CELISA but not by the MRT and the IELISA), present in some of the milk samples. The FPA is a homogeneous assay which, unlike the MRT and the IELISA, may be used for testing in the field.

Animals↗

PubMed2001

Maize ribosome-inactivating protein inhibits normal development of Aspergillus nidulans and Aspergillus flavus.

The abundant maize kernel ribosome-inactivating protein 1 (RIP1) was tested for antifungal activity against Aspergillus nidulans and Aspergillus flavus. A microculture assay was developed to monitor fungal growth and development after treatment of conidia with RIP1 or control proteins. A striking decrease in hyphal proliferation was observed when conidia of A. nidulans, a genetically well-characterized nonpathogenic species, were treated with RIP1 protein. Treatment with a RIP1 mutant protein that lacked enzymatic ribosome-inactivating activity caused no observable effects. RIP1 treatment of conidia from the maize pathogen A. flavus resulted in increased hyphal branching. Examination of the branched hyphae after Congo red staining revealed only one growing hyphal tip per conidium. These results indicate that both fungi were affected by RIP1 treatment, but the lysis seen with treatment of A. nidulans was apparently avoided by A. flavus. A developmental time course revealed that both fungal species were affected by RIP1 at the postdivisional growth stage. The inhibitory activity of RIP1 against normal fungal growth is consistent with a biological function to protect the seed from fungal invasion.

Aspergillus flavus↗