Diversity of the 32-kilodalton protein gene may form a basis for species determination of potentially pathogenic mycobacterial species.

In this study, partial gene sequences of the mycobacterial 32-kDa protein gene were determined by PCR-based sequencing. A total of 50 strains representing 18 potentially pathogenic mycobacterial species were studied. In 10 cases, all three strains of the species studied were identical, and intraspecies variability was found in 6 cases. Thus, the 32-kDa protein gene may be a good target for identification of mycobacteria by PCR-based sequencing.     

Proteins of the retinoblastoma pathway,FEN1 and MGMT are novel potential prognostic biomarkers in pancreatic adenocarcinoma

Background

We studied the expression of some major proteins involved in cell-cycle regulation and DNA repair, the roles of which are not well known in pancreatic ductal adenocarcinoma (PDAC), but which have a significant impact on carcinogenesis of many other cancers.

Novel non‐neutral mitochondrial DNA mutations found in childhood acute lymphoblastic leukemia

Mitochondria produce adenosine triphosphate (ATP) for energy requirements via the mitochondrial oxidative phosphorylation (OXPHOS) system. One of the hallmarks of cancer is the energy shift toward glycolysis. Low OXPHOS activity and increased glycolysis are associated with aggressive types of cancer. Mitochondria have their own genome (mitochondrial DNA [mtDNA]) encoding for 13 essential subunits of the OXPHOS enzyme complexes. We studied mtDNA in childhood acute lymphoblastic leukemia (ALL) to detect potential pathogenic mutations in OXPHOS complexes. The whole mtDNA from blood and bone marrow samples at diagnosis and follow‐up from 36 ALL patients were analyzed. Novel or previously described pathogenic mtDNA mutations were identified in 8 out of 36 patients. Six out of these 8 patients had died from ALL. Five out of 36 patients had an identified poor prognosis genetic marker, and 4 of these patients had mtDNA mutations. Missense or nonsense mtDNA mutations were detected in the genes encoding subunits of OXPHOS complexes, as follows: MT‐ND1, MT‐ND2, MT‐ND4L and MT‐ND6 of complex I; MT‐CO3 of complex IV; and MT‐ATP6 and MT‐ATP8 of complex V. We discovered mtDNA mutations in childhood ALL supporting the hypothesis that non‐neutral variants in mtDNA affecting the OXPHOS function may be related to leukemic clones.     

Drug susceptibility testing of Mycobacterium tuberculosis to fluoroquinolones: first experience with a quality control panel in the Nordic-Baltic collaboration.

In the first attempt to establish a quality assurance programme for susceptibility testing of Mycobacterium tuberculosis to fluoroquinolones, 20 strains with different fluoroquinolone susceptibility patterns were distributed by the Supranational Reference Laboratory in Stockholm to the other mycobacterial reference laboratories of the Nordic and Baltic countries. Susceptibility testing to fluoroquinolones was performed according to routine procedures in each laboratory. Results were compared to sequence analysis of the gyrA gene and minimal inhibitory concentration determination. Most laboratories found identical susceptibility patterns. The two resistant strains were correctly identified by all laboratories, but three laboratories each falsely reported one susceptible strain as resistant. These results indicate that the participating laboratories yield reliable results in detection of fluoroquinolone-resistant strains, although the need for a standardised quality assurance programme for drug susceptibility testing for fluoroquinolones is stressed by the strains falsely reported as resistant.     

Immunohistochemical evaluation of the cytoarchitecture of benign and malignant breast lesions.

Fifty-three breast lesions, which had been fixed in formalin and embedded in paraffin, were immunohistochemically analyzed with monoclonal antibodies to cytokeratin subtypes 1, 5, 10, 14 (34BE12), muscle-specific actins (HHF35) and antiserum to S100 protein, all of which have been used as markers for myoepithelial cells. With these antibodies, a continuous myoepithelial cell layer could generally be seen around the benign ducts and acini. In in situ carcinomas, such a layer could still be observed, though it was usually discontinuous and sometimes absent. In infiltrating carcinomas, no myoepithelial cell layer could be observed. In intraductal hyperplasias, scattered HHF35, 34BE12 and S100-positive cells could be seen amongst the proliferating intraductal cells. In in situ and infiltrating carcinomas, however, such cells could also be observed. This was seen especially with antibodies 34BE12 and S100, and to a lesser extent also with HHF35. Morphologically these cells seemed to belong to the malignant cell population. Although myoepithelial cell preservation is an important morphological parameter in the histological evaluation of breast lesions, the results suggest that the myoepithelial cell markers 34BE12, HHF35 and S100 cannot be used in the differential diagnosis between benign and malignant breast lesions in a straightforward manner. This is because in situ carcinomas have a more or less preserved myoepithelial cell layer, and because many infiltrating and in situ carcinomas contain a subpopulation of neoplastic cells expressing these markers, possibly signifying myoepithelial cell differentiation.     

Time-resolved fluorescence in biospecific assays

Fluorescent lanthanide chelates and their detection using time-resolved fluorometry for in vitro as well as for microscopic bio-specific assays are reviewed, with reference to other fluorescent dyes and labelling materials. The minimum detectable dose of labelled components and the theoretical ultimate sensitivities of immunoassays and DNA-assays are discussed and compared with experimental results. It is concluded that the time-resolved fluorometry provides a high sensitivity labelling method in immunoassays, and a sensitivity comparable to phosphorus-32 in DNA hybridization assays. It is also concluded that lanthanide chelates are a potential alternative for organic fluorescent dyes in microscopy and that they are most useful in multiparameter assays. The combination of organic fluorescent dyes and lanthanide chelates provides the highest separation efficiency between two assay parameters.     

Feasibility of commercial interferon-γ-based methods for the diagnosis of latent Mycobacterium tuberculosis infection in Finland, a country of low incidence and high bacille Calmette–Guérin vaccination coverage

The performances of the QuantiFERON-TB Gold in Tubes (QFGT), T SPOT-TB (ELISPOT) and the Mantoux test were compared for the diagnosis of latent tuberculosis infection in Finland, a country of low tuberculosis incidence. In Cohort A (16 students), freshly isolated peripheral blood mononuclear cells (PBMCs), and in Cohort B (21 school children), cryopreserved PBMCs, were used for the ELISPOT assay. Cryopreservation of cells in fetal calf serum, but not in serum-free medium, produced false-positive results. Discrepancies between the results of the assays were observed. It was concluded that the accuracy of these ex-vivo methods needs additional evaluation.     

Time-resolved fluorometer for lanthanide chelates--a new generation of nonisotopic immunoassays

Pulsed-light time-resolved fluorometry of lanthanide chelates has proved to be very sensitive for use with nonisotopic immunoassays. We describe a manually operated fluorometer with a conventional xenon flashtube. Sensitivity for 1-s determinations is similar to that of radioisotopic methods.     

Clinical relevance of discography combined with CT scanning. A study of 100 patients

Two different classifications of discograms have been used in a prospective study of 279 injected discs in 100 patients. The five-stage classification of Adams, Dolan and Hutton (1986) showed increased degeneration in the lower lumbar discs and more degenerative changes in men than in women. Exact reproduction of the patient's pain on injection was more common in fissured or ruptured discs than in less degenerate discs, with 81% sensitivity and 64% specificity of the discogram for pain. The additional information obtained by comparing computerised tomography (CT) with discograms was minimal. Discography was found to be useful in the evaluation of chronic low back pain in patients whose ordinary CT scans, myelograms and flexion-extension radiographs were normal. In spondylolysis and spondylolisthesis, discography can disclose whether fusion needs to be extended above the lytic level, and it may show if the pain in patients who have had posterolateral fusion is discogenic. Thus, discography gives information which is useful in deciding whether to operate on patients with chronic low back pain.