Second Opinion Assessment in Diagnostic Mammography at a Breast Cancer Centre.

Purpose: The aim of this retrospective study was to evaluate the importance of second opinion assessment for diagnostic mammography and sonography in a breast cancer centre. Material and Method: We analysed a total of 374 diagnostic mammographies and sonographies. All patients had previously undergone mammography and sonography examination in different external clinics, and the findings had been classified according to the BI-RADS system. All patients underwent additional sonography investigation in the outpatient department of our university clinic with additional mammography where necessary. The final diagnosis (histological clarification in 316 cases, follow-up in 58 cases) was compared with the BI-RADS classification made by the external clinics and by the university clinic, and the correlation between their findings and the final diagnosis was analysed. Results: The final diagnosis yielded 146 benign lesions and 228 cancers. In 74 % of cases (277/374), the BI-RADS classification of the first assessment corresponded to that of the second assessment. 26/55 lesions (47 %) were upgraded at the second assessment from BI-RADS 3 to BI-RADS 4, and 71/186 findings (38 %) were downgraded at the second assessment from BI-RADS 4 to BI-RADS 3. The correlation between the initial diagnosis made in the external facilities and the final diagnosis was low (kappa: 0.263), but the correlation between the second opinion assessment and the final diagnosis was significantly (p < 0.001) higher (kappa: 0.765). The second assessment increased the sensitivity from 91 % (208/228) to 99 % (225/228) and the specificity from 32 % (46/146) to 74 % (108/146). 20 additional malignant lesions were only detected at the second assessment; however the second assessment also resulted in 3 additional false-negative findings. Surgical biopsy was prevented in 49 women after the second assessment. Conclusion: An independent second diagnostic evaluation can significantly improve the correlation between BI-RADS classification and the final diagnosis, resulting in a benefit for the patient.

Screening for atherosclerotic plaques in the abdominal aorta in high-risk patients with multicontrast-weighted MRI: a prospective study at 3.0 and 1.5 tesla.

OBJECTIVE: This prospective study compares MRI of atherosclerotic plaque in the abdominal aorta at 3 T with that at 1.5 T in patients suffering from hereditary hyperlipidaemia, a major risk factor for atherosclerosis. METHODS: MRI of the abdominal aorta at 1.5 and 3 T was performed in 21 patients (mean age 58 years). The study protocol consisted of proton density (PD), T(1), T(2) and fat-saturated T(2) weighted black blood images of the abdominal aorta in corresponding orientation. Two independent radiologists performed image rating. First, image quality was rated on a five-point scale. Second, atherosclerotic plaques were scored according to the modified American Heart Association (AHA) classification and analysed for field strength-related differences. Weighted κ statistics were calculated to assess interobserver agreement. RESULTS: Interobserver agreement was substantial for nearly all categories. MRI at 3 T offered superior image quality in all contrast weightings, most significantly in T(1) and T(2) weighted techniques. Plaque burden in the study collective was unexpectedly moderate. The majority of plaques were classified as AHA III lesions; no lesions were classified above AHA V. There was no significant influence of the field strength regarding the AHA classification. CONCLUSION: Abdominal aortal plaque screening is basically feasible at both field strengths, whereas the image quality is rated superior at 3 T. However, the role of the method in clinical practice remains uncertain, since substantial findings in the high-risk collective were scarce.

[Diagnostic mammography and sonography: concordance of the breast imaging reporting assessments and final clinical outcome]. / Diagnostische Mammographie und Sonographie: Korrelation von diagnostischer BI-RADS-Einstufung mit dem histologischen und klinischen Endbefund.

PURPOSE: The purpose of the study was to assess the final clinical outcome of BI-RADS Categories for diagnostic mammography and sonography. MATERIAL AND METHODS: We analysed 632 mammography and sonography examinations from women with diagnostic indications (age: 23 - 100, mean 58) performed during 2001 and 2003. All patients received mammography and sonography examinations at different outside facilities and all patients received an additional sonography examination at the university radiology department and if necessary supplemental mammographic views. Final clinical outcome (Histology: 554; follow-up: 78) was ascertained in each case and concordance of BI-RADS-categories for mammography and sonography and final diagnosis were analysed. RESULTS: Final diagnosis yielded 230 benign lesions (36 %) and 402 cancers (64 %). Concordance of BI-RADS Assessment and final outcome was documented in 542 cases (86 %). There were 11 correct category 1 and 2 assessments (2 %). 142 lesions were classified with BI-RADS 3 (22 %) with 5 false negative ratings. There were 264 category 4 lesions (42 %) with a PPV for a malignant lesion of 71 % (187/264) and finally 215 BI-RADS 5 lesions with a PPV of 98 % (210/215). Overall sensitivity of mammography was 92 % with specificity of 75 % and for sonography 86 % and 76 %. Mammography had a significantly higher detection rate for malignant lesions than sonography. The highest correlation between BI-RADS category and final outcome was documented for the diagnostic combination of mammography and sonography with a kappa-value of 0.817 (p < 0.001), followed by mammography (kappa: 0.684) and sonography (kappa: 0.631). The overall correlation was 0.681 (p < 0.001). CONCLUSION: BI-RADS assessments of diagnostic mammography and sonography yields in a high cancer detection rate with a justifiable part of false positive ratings.

Dentists' perception of risks for molars without antagonists. A questionnaire study of dentists in Sweden.

The aim of this questionnaire study was to investigate dentists' assessment of and the decision making process in a clinical situation with unopposed molar teeth. The questionnaire comprised, besides questions about the dentist's background, a presentation of a clinical situation with a 42-year old male patient who just had lost the left mandibular molars (teeth 36 and 37). A series of questions was provided with multiple choice answers regarding what most probably would occur with the unopposed maxillary molars within a 10-year period, what treatment to propose in such a situation, and indications for the proposed treatment. The questionnaire was sent to a randomly selected group of active members of the Göteborg Dental Society. Two hundred completed questionnaires were returned (response rate 72.5%). The great majority (85%) suggested that marked overeruption of the unopposed molars would occur, whereas 13% believed in minor changes. Almost half of the respondents (47%) proposed to wait and see before any treatment was started, whereas the remaining dentists wanted to perform some therapy as soon as possible or within a specified period of time. The most commonly suggested indications for treatment were risk for overeruption (79%), risk for impaired masticatory function (54%), and risk for development of TMD (50%). Differences in answers were found between female and male dentists, between specialists and general practitioners, and with respect to year of graduation. Most dentists believed that overeruption would occur in spite of the limited knowledge of what will happen to unopposed teeth.

Role of Stat3 in lipopolysaccharide-induced IL-10 gene expression.

IL-10 is a unique cytokine because it is anti-inflammatory and immunosuppressive. IL-10 is regulated at the level of transcription, but the critical motifs and the relevant transcription factors controlling this gene have remained elusive to date. We now report that a sequence at -120 bp in the human IL-10 promoter binds Stat3 but no other Stat proteins. Mutation of this motif abrogates LPS-induced trans-activation. Overexpression of dominant negative Stat3 suppresses promoter activity, while wild-type Stat3 leads to an enhancement of this activity. Our results show that Stat3, by binding to a single motif in the IL-10 promoter, is controlling expression of the human IL-10 gene.

Role of p52 (NF-kappaB2) in LPS tolerance in a human B cell line.

Cells of the weakly CD14 positive human B cell line RPMI 8226, clone 1, will mobilize NF-kappaB (p50/p65 and p50/p50) proteins and produce TNF mRNA when stimulated with lipopolysaccharide (LPS). When such cells are precultured with a low amount of LPS (50-250 ng/ml) for 3 - 4 days followed by a secondary stimulation with a high dose of LPS (1 microg/ml) then the cytokine expression is strongly reduced, i. e. the cells have become tolerant. Western blot analysis of proteins of the NF-kappaB/rel family demonstrates cytoplasmic p50 and p65 for naive B cells plus a low level of p52. While with tolerance induction the pattern of p50 and p65 proteins remains essentially unchanged, the LPS tolerant 8226 cells show a dramatic increase of both p52 protein and its p100 precursor in the cytosol. This p52 is found strongly upregulated in Western blots of extracts from purified nuclei of tolerant cells. Also, gelshift analysis with the -605 kappaB motif of the human TNF 5'-region shows an additional high mobility complex in LPS tolerant cells -a complex that is supershifted with an anti-p52 antibody. Functional analysis with the -1064 TNF 5'-region in front of the luciferase reporter gene demonstrates that transactivation of the TNF promoter is strongly reduced in tolerant cells. Also, overexpression of p52 will suppress activity of TNF promoter reporter gene constructs. Taken together these data show that tolerance to LPS in the human RPMI 8226 B cell line involves upregulation of the p52 (NF-kappaB2) gene, which appears to be instrumental in the blockade of TNF gene expression.

Tooth wear and temporomandibular joint morphology in a skull material from the 17th century.

Skeletal remnants from the skulls of 69 subjects from the 17th century have been studied focusing on TMJ morphology and tooth wear. Several of the skulls were damaged and altogether 68 condyles and 28 temporal components of the TMJ, and 97 dentate jaws could be examined. Tooth wear was extensive and most of the first molars in both jaws had lost most of their occlusal morphology. This is remarkable with respect to the fact that the great majority of the subjects had died before the age of 35 years, according to the age determination performed. The TMJs showed frequent remodelling but only rarely deformative changes. The frequent observation of a broken up compact bone layer on the condyle was interpreted as a post-mortem artefact. The results indicate adaptive response of the TMJs to the probably heavy masticatory function but do not support the suggested relationship between tooth wear and TMJ osteoarthrosis.

Stimulation and suppression of PCR-mediated recombination.

Recombination, or chimera formation, is known to occur between related template sequences present in a single PCR amplification. To characterize the conditions under which such recombinant amplification products form we monitored the exchange of sequence between two homologous templates carrying different restriction sites separated by 282 bp. Using a typical cycling program the rates of recombination between the two restriction sites were 1 and 7% using Taq and Vent polymerases respectively over 12 doublings. However, by using long elongation times and cycling only to the mid-point of the amplification recombination could be suppressed below visual detection with both polymerases. Conversely, cycling programs designed to promote incomplete primer elongation and subsequent template strand exchange stimulated recombination to >20%.

Fishing the best pool for novel ribozymes.

Novel RNA enzymes, or ribozymes, are sought in large pools of random RNA sequences. Because of the large number of random positions in an individual pool molecule, only a vanishingly small fraction of the possible sequences are actually present. Even so, increasing the length of the individual pool molecules significantly increases the probability of finding a particular complex ribozyme. Because ribozymes are typically composed of conserved sequences interleaved with regions that can vary in sequence and length, a longer molecule allows a greater number of possible arrangements of a given ribozyme motif, increasing the likelihood that it will be present in the pool. Once a ribozyme motif has been found, rational and irrational optimization techniques can be used to identify related ribozyme sequences with greater activity.

CCAAT/enhancer binding protein is involved in the expression of the tumour necrosis factor gene in human monocytes.

Within the human TNF promoter we have identified two sites at positions -189 and -101 that show C/EBP specific binding of nuclear proteins from cells of the human monocytic line Mono Mac 6. Supershift analysis with anti C/EBP antibodies revealed that the complexes formed consist of both C/EBP alpha and C/EBP beta. When studying reporter constructs with a 5'-deletion series of the TNF promoter in cotransfection experiments with a C/EBP beta expression plasmid, a construct with the -1064 TNF fragment gave 26-fold transactivation, the -630 fragment showed 23-fold transactivation and the -107 fragment (containing the -101 C/EBP binding motif) still gave 16-fold transactivation. Mutagenesis of the -101 site in the -630 construct resulted in a reduction of C/EBP driven transactivation from 26-fold to 7-fold. Finally, when Mono Mac 6 cells were transfected with these constructs, stimulation by LPS induced a 19-fold transactivation in the -630 wild type construct, while the -630 construct carrying the -101 mutation was transactivated only 4-fold. Hence, the data indicate that the -101 C/EBP motif is crucial for TNF gene expression in human monocytes.

The bacterial enhancer-binding protein NTRC is a molecular machine: ATP hydrolysis is coupled to transcriptional activation.

NTRC is a prokaryotic enhancer-binding protein that activates transcription by sigma 54-holoenzyme. NTRC has an ATPase activity that is required for transcriptional activation, specifically for isomerization of closed complexes between sigma 54-holoenzyme and a promoter to open complexes. In the absence of ATP hydrolysis, there is known to be a kinetic barrier to open complex formation (i.e., the reaction proceeds so slowly that the polymerase synthesizes essentially no transcripts even from a supercoiled template). We show here that open complex formation is also thermodynamically unfavorable. In the absence of ATP hydrolysis the position of equilibrium between closed and open complexes favors the closed ones. Use of linear templates with a region of heteroduplex around the transcriptional start site--"preopened" templates--does not bypass the requirement for either NTRC or ATP hydrolysis, providing evidence that the rate-limiting step in open complex formation does not lie in DNA strand denaturation per se. These results are in contrast to recent findings regarding the ATP requirement for initiation of transcription by eukaryotic RNA polymerase II; in the latter case, the ATP requirement is circumvented by use of a supercoiled plasmid template or a preopened linear template.

The C/EBP family of transcription factors.

The C/EBP proteins form a family of transcription factors with at least seven members. These proteins consist of three structural components which include a C-terminal leucine-zipper, a basic DNA-binding region and a N-terminal transactivating region. Dimerization through the leucine-zipper leads to formation of homo- and heterodimers which then bind with their two basic regions to often non-symmetric DNA-sequences in the promoter/enhancer regions of a variety of genes. Expression of C/EBP is prominent in adipocytes, hepatocytes and monocytes/macrophages, and here these proteins are involved in tissue-specific gene expression. Target genes for C/EBP include those for acute phase response genes in liver cells and for cytokine genes in monocytes/macrophages. Therefore, intervention at the level of C/EBP transcription factors may prove effective in controlling immune response.

Tolerance to lipopolysaccharide in human blood monocytes.

When monocytes are stimulated with Lipopolysaccharide (LPS), they efficiently produce cytokines like tumor necrosis factor (TNF). Upon secondary stimulation, this response is only minimal, and there is little TNF mRNA transcription, mRNA accumulation, and protein production. Studies with the monocytic cell line Mono Mac 6 have shown that in such tolerant cells the CD14 LPS receptor is still present, and the transcription factor NF-kB is still efficiently mobilized. This NF-kB complex has, however, a different composition, that does not transactivate TNF promoter reportergene constructs. We now show that similar mechanisms apply to primary blood monocytes. After primary stimulation these cells also produce high levels of TNF and then develop tolerance in that upon secondary challenge little TNF is produced. CD14 cell surface expression is unchanged or even increased in tolerant cells and NF-kB mobilization does still occur. The complex mobilized in such tolerant monocytes is, however, composed mainly of high mobility binding proteins. This indicates that p50 homodimers predominate in NF-kB complex of tolerant blood monocytes, similar to what has been reported for Mono Mac 6 cells. The data add to the notion that p50 binding to the cognate -kB DNA motif in the TNF promoter may be responsible for the unresponsiveness in LPS tolerance.

In vitro transcription close to the melting point of DNA: analysis of Thermotoga maritima RNA polymerase-promoter complexes at 75 degrees C using chemical probes.

The interaction of DNA dependent RNA polymerase of the extreme thermophile bacteria Thermotoga maritima with a promoter bearing DNA fragment was investigated in the temperature range from 20 to 85 degrees C. We show that the T. maritima RNA polymerase recognizes and utilizes the Escherichia coli T7 A1 promoter with an efficiency similar to that of the E. coli polymerase. We have investigated the interaction of both polymerases with the same promoter over a wide range of temperatures using hydroxyl radical foot-printing and osmium tetroxide probing. This study revealed that the T. maritima polymerase goes through a series of isomerisation events very similar to the E. coli polymerase, i.e. the closed, intermediate and open complexes, but the transitions themselves occur at radically different temperatures. This indicates that conformational changes in the DNA that accompany initiation of transcription such as promoter melting are determined by the polymerase rather than the DNA sequence.

Masticatory function in patients with congenital and acquired maxillofacial defects.

Ninety-eight patients who received prosthodontic treatment for maxillofacial defects were examined clinically and by means of questionnaires and registration of chewing efficiency and occlusal force. Although 30% of the patients stated that they could chew soft food, and one third could not chew the test food (almonds), only 14% said they had a poor chewing ability. The mean occlusal force was small (80 N) but the individual variation was great (median 49 N, maximum 327 N). Despite major defects and poor functional test results, most patients were remarkably well-adapted to their situation and to maxillofacial prosthodontic rehabilitation. Severe signs and symptoms of temporomandibular disorders were rare.

From initiation to elongation: comparison of transcription by prokaryotic and eukaryotic RNA polymerases.

Multisubunit RNA polymerases in prokaryotes and eukaryotes share an evolutionarily conserved core. Here, we compare the processes of promoter recognition, transcription initiation and transcript elongation by human RNA polymerase II and by the RNA polymerase of the eubacterium Escherichia coli. Although these two polymerases have diverged widely in structure, important functions have been conserved, suggesting that the basic mechanisms of RNA transcription are similar in eukaryotes and prokaryotes.

Tolerance to lipopolysaccharide involves mobilization of nuclear factor kappa B with predominance of p50 homodimers.

Stimulation of the human monocytic cell line Mono Mac 6 with lipopolysaccharide (LPS) leads to rapid and transient expression of cytokines like tumor necrosis factor (TNF). When such cells are precultured for 2 days with a low dose of LPS (20 ng/ml) followed by stimulation with a high dose of LPS (1 microgram/ml), expression of the TNF gene is minimal, i.e. the cells are tolerant. In nuclear run-on analysis, such tolerant cells show only a low degree of transcription, indicating that tolerance operates at or upstream of the transcription level. The CD14 LPS receptor is, however, up-regulated (not down-regulated) in tolerant cells, and LPS can, in fact, still lead to activation of tolerant cells as evidenced by mobilization of the transcription factor nuclear factor kappa B (NF-kappa B). Resolution of the NF-kappa B complex in gel shift analysis shows that the binding protein, mobilized in naive Mono Mac 6 cells, consists mainly of p50-p65 heterodimers, while in tolerant cells, the p50 homodimer is predominant. This increase in p50 homodimers coincides with an increase in p105 mRNA, suggestive of a transcriptional up-regulation of p50. Reporter gene analysis reveals that the NF-kappa B complex mobilized in tolerant cells is functionally inactive in that NF-kappa B-dependent luciferase constructs containing the human immunodeficiency virus long terminal repeat or the TNF 5'-region show only minimal transactivation after LPS stimulation. Similar to Mono Mac 6 cells, primary blood monocytes, when precultured with a low dose of LPS, also become tolerant and produce little TNF after LPS stimulation. The tolerant blood monocytes also up-regulate CD14, and they mobilize NF-kappa B with a predominance of p50 homodimers. Taken together, these results demonstrate that tolerance to LPS is determined by post-receptor mechanisms that involve an altered composition of the NF-kappa B complex.

Pyrrolidine dithiocarbamate inhibits NF-kappa B mobilization and TNF production in human monocytes.

The human TNF promoter contains four potential nuclear factor-kappa B (NF-kappa B)-binding sites, with the strongest binding seen for the -605 motif. Nuclear extracts from unstimulated cells of the human monocytic cell line, Mono Mac 6, contain one specific binding protein (complex II), consistent with a constitutive p50 homodimer. Stimulation of Mono Mac 6 cells with LPS will increase complex II and will strongly induce a second specific complex (complex I), which represents the p50/65 heterodimer. Treatment of Mono Mac 6 cells with pyrrolidine-dithiocarbamate (PDTC) at 300 microM will block the LPS-induced complex I almost completely and will reduce complex II to the constitutive level. Binding activity of other nuclear factors that recognize the SP-1 and c/EBP motifs of the human TNF promoter is not affected by such treatment. Northern blot analysis demonstrates that PDTC treatment will strongly reduce LPS-induced TNF transcripts. Secreted TNF protein as detected in the Wehi 164S/ActD bioassay and in a sandwich immunoassay was similarly reduced by PDTC. Kinetic analyses show that after LPS stimulation, NF-kappa B will peak at 1 h, TNF transcript prevalence at 2 h, and TNF protein at 4 h. PDTC did not shift this response to LPS to a later time, but suppressed NF-kappa B mobilization, TNF transcripts, and TNF protein over the entire 8-h observation period. Analysis of freshly isolated, LPS-stimulated blood monocytes showed a similar blockade of NF-kappa B. Furthermore, in these primary cells, induction of TNF transcripts, as determined by Northern blot analysis and by quantitative polymerase chain reaction, was prevented by PDTC as was TNF protein production. These data show that dithiocarbamates can profoundly affect cytokine expression and suggest that NF-kappa B is involved in LPS-induced TNF gene expression in human monocytes.

Oligomerization of NTRC at the glnA enhancer is required for transcriptional activation.

To activate transcription of the glnA gene, the dimeric NTRC protein (nitrogen regulatory protein C) of enteric bacteria binds to an enhancer located approximately 100 bp upstream of the promoter. The enhancer is composed of two binding sites for NTRC that are three turns of the DNA helix apart. One role of the enhancer is to tether NTRC in high local concentration near the promoter to allow for its frequent interaction with sigma 54 holoenzyme by DNA looping. We have found that a second role of the enhancer is to ensure oligomerization of NTRC into a complex of at least two dimers that is required for transcriptional activation. Formation of this complex is greatly facilitated by a protein-protein interaction between NTRC dimers that is increased when the protein is phosphorylated.