Epidemiology and reporting of osteoporotic vertebral fractures in patients with long-term hospital records based on routine clinical CT imaging.

Osteoporotic vertebral fractures signify an increased risk of future fractures and mortality and can manifest the diagnosis of osteoporosis. We investigated the prevalence of vertebral fractures in routine CT of patients with long-term hospital records. Three out of ten patients showed osteoporotic vertebral fractures (VFs) corresponding to the highest rates reported in European population-based studies. INTRODUCTION: VFs are a common manifestation of osteoporosis, which influences future fracture risk. Their epidemiology has been investigated in population-based studies. However, few studies report the prevalence of osteoporotic VF in patients seen in clinical routine and include all common fracture levels of the thoracolumbar spine. The purpose of this study was to investigate the prevalence of osteoporotic VF in patients with CT scans and long-term hospital records and identify clinical factors associated with prevalent VFs. METHODS: All patients aged 45 years and older with a CT scan and prior hospital record of at least 5 years that were seen in the study period between September 2008 and May 2017 were reviewed. Imaging requirements were a CT scan with sagittal reformations including at least T6-L4. Patients with multiple myeloma were excluded. Fracture reading was performed using the Genant semi-quantitative method. Medical notes were reviewed for established diagnoses of osteoporosis and clinical information. Clinical factors (e.g. drug intake, chemotherapy, and mobility level) associated with prevalent VF were identified in logistic regression. RESULTS: The study population consisted of 718 patients (228 women and 490 men; mean age 69.3 ± 10.1 years) with mainly cancer staging and angiography CT imaging. The overall prevalence of VFs was 30.5%, with non-significantly more men showing a fracture (32.5%) compared to women (26.3%; p > 0.05). Intake of metamizole for ≥ 3 months was significantly associated with a prevalent VF. Medical records did not include information about bone health in 90% of all patients. CT reports did mention a VF in only 24.7% of patients with a prevalent VF on CT review. CONCLUSION: Approximately 30% of elderly patients with CT imaging and long-term hospital records showed VFs. Only one-quarter of these patients had VFs mentioned in CT reports. Osteoporosis management could be improved by consequent reporting of VFs in CT, opportunistic bone density measurements, and early involvement of fracture liaison services.

[Tumor vaccines-therapeutic vaccination against cancer]. / Tumorvakzinierung ­ therapeutische Vakzinierung gegen Krebs.

Tumor cells always exhibit differences to normal cells. These differences can be recognized by the immune system, enabling the destruction of tumor cells by T cells, as was impressively demonstrated by the success of immune checkpoint inhibition, e.g., in malignant melanoma. Many cancers, however, do not respond to this kind of therapy. In these cases, vaccination against tumor antigens could be very helpful. Nevertheless, all of the efforts made in this respect during the past 30 years have been virtually futile. With current knowledge and technology there is new hope.

X-ray-based quantitative osteoporosis imaging at the spine.

Osteoporosis is a metabolic bone disease with a high prevalence that affects the population worldwide, particularly the elderly. It is often due to fractures associated with bone fragility that the diagnosis of osteoporosis becomes clinically evident. However, early diagnosis would be necessary to initiate therapy and to prevent occurrence of further fractures, thus reducing morbidity and mortality. X-ray-based imaging plays a key role for fracture risk assessment and monitoring of osteoporosis. Whereas over decades dual-energy X-ray absorptiometry (DXA) has been the main method used and still reflects the reference standard, another modality reemerges with quantitative computed tomography (QCT) because of its three-dimensional advantages and the opportunistic exploitation of routine CT scans. Against this background, this article intends to review and evaluate recent advances in the field of X-ray-based quantitative imaging of osteoporosis at the spine. First, standard DXA with the recent addition of trabecular bone score (TBS) is presented. Secondly, standard QCT, dual-energy BMD quantification, and opportunistic BMD screening in non-dedicated CT exams are discussed. Lastly, finite element analysis and microstructural parameter analysis are reviewed.

Opportunistic osteoporosis screening in multi-detector CT images via local classification of textures.

Our study proposed an automatic pipeline for opportunistic osteoporosis screening using 3D texture features and regional vBMD using multi-detector CT images. A combination of different local and global texture features outperformed the global vBMD and showed high discriminative power to identify patients with vertebral fractures. INTRODUCTION: Many patients at risk for osteoporosis undergo computed tomography (CT) scans, usable for opportunistic (non-dedicated) screening. We compared the performance of global volumetric bone mineral density (vBMD) with a random forest classifier based on regional vBMD and 3D texture features to separate patients with and without osteoporotic fractures. METHODS: In total, 154 patients (mean age 64 ± 8.5, male; n = 103) were included in this retrospective single-center analysis, who underwent contrast-enhanced CT for other reasons than osteoporosis screening. Patients were dichotomized regarding prevalent vertebral osteoporotic fractures (noFX, n = 101; FX, n = 53). Vertebral bodies were automatically segmented, and trabecular vBMD was calculated with a dedicated phantom. For 3D texture analysis, we extracted gray-level co-occurrence matrix Haralick features (HAR), histogram of gradients (HoG), local binary patterns (LBP), and wavelets (WL). Fractured vertebrae were excluded for texture-feature and vBMD data extraction. The performance to identify patients with prevalent osteoporotic vertebral fractures was evaluated in a fourfold cross-validation. RESULTS: The random forest classifier showed a high discriminatory power (AUC = 0.88). Parameters of all vertebral levels significantly contributed to this classification. Importantly, the AUC of the proposed algorithm was significantly higher than that of volumetric global BMD alone (AUC = 0.64). CONCLUSION: The presented classifier combining 3D texture features and regional vBMD including the complete thoracolumbar spine showed high discriminatory power to identify patients with vertebral fractures and had a better diagnostic performance than vBMD alone.

Clinical, pathological and genetic features of follicular lymphoma grade 3A: a joint analysis of the German low-grade and high-grade lymphoma study groups GLSG and DSHNHL.

BACKGROUND: Histologically, follicular lymphoma (FL) grades 1, 2 and 3A are composed of two distinct cell types, centroblasts and centrocytes. FL grade 3B is composed only of centroblasts and has been shown to differ in immunophenotype and genetics from FL that contain centrocytes. We aimed to understand the pathogenetic and clinical relation between FL grade 3A to FL grade 1/2 on the one hand and FL grade 3B on the other hand. PATIENTS AND METHODS: Trial patients with long-term follow-up and diagnosis of FL grade 3 were selected and samples underwent a second central pathological review using a multiple-observer approach to assess grading. RESULTS: Interobserver variability for diagnosing FL grade 3 was high. FL grade 3A frequently harbored areas of FL grade 1/2 within the same tissue specimen. FL grade 3B rarely coexisted with grade 1/2 or 3A, suggesting divergent pathogenesis. There was no statistically significant difference in outcome between 47 cases of FL grade 3A and 14 cases of grade 3B. Compared with grade 1/2 FL, both groups showed longer progression-free survival without late events, especially after immunochemotherapy; this outcome difference was retained after adjustment for clinical prognostic factors. The subgroup of FL grade 3A with an additional FL grade 1/2 component or a translocation t(14;18) showed a poorer outcome. In contrast, the FL grade 3A lacking t(14;18) and of localized stage resembled the pediatric type of FL and showed a very good outcome. FL3 with MYC breaks showed a poor outcome. CONCLUSIONS: The results suggest that first-line immunochemotherapy might allow long-lasting remissions in a subgroup of FL grade 3A similar to diffuse large B-cell lymphoma. Within FL3A, prognostic subgroups can be identified by analyzing for coexisting FL1/2 and MYC breaks.

Complementary therapy use in patients with glioma: an observational study.

OBJECTIVE: Despite novel multimodal therapeutic approaches, the vast majority of glial tumors are not curable. Patients may search for complementary therapies in order to contribute to the fight against their disease or to relieve symptoms induced by their brain tumor. The extent of the use of complementary or alternative therapies, the patients' rationale behind it, and the cost of complementary therapy for gliomas are not known. We used a questionnaire and the database of the German Glioma Network to evaluate these questions. METHODS: A total of 621 questionnaires were available for evaluation from patients with glial tumors of WHO grades II to grade IV. The patients were recruited from 6 neuro-oncologic centers in Germany. Complementary therapy was defined as methods or compounds not used in routine clinical practice and not scientifically evaluated. RESULTS: Forty percent of the responding patients reported the use of complementary therapies. Significant differences between the group of complementary therapy users and nonusers were seen with respect to age (younger > older), gender (female > male), and education (high education level > low education level). The motivation for complementary therapy use was not driven by unsatisfactory clinical care by the neuro-oncologists, but by the wish to add something beneficial to the standard of care. CONCLUSIONS: In clinical practice, patients' use of complementary therapies may be largely overseen and underestimated. The major motivation is not distrust in conventional therapies. Neuro-oncologists should be aware of this phenomenon and encourage an open but critical dialogue with their patients.

Mesenchymal stem cells in cartilage repair: state of the art and methods to monitor cell growth, differentiation and cartilage regeneration.

Degenerative joint diseases caused by rheumatism, joint dysplasia or traumata are particularly widespread in countries with high life expectation. Although there is no absolutely convincing cure available so far, hyaline cartilage and bone defects resulting from joint destruction can be treated today by appropriate transplantations. Recently, procedures were developed based on autologous chondrocytes from intact joint areas. The chondrocytes are expanded in cell culture and subsequently transplanted into the defect areas of the affected joints. However, these autologous chondrocytes are characterized by low expansion capacity and the synthesis of extracellular matrix of poor functionality and quality. An alternative approach is the use of adult mesenchymal stem cells (MSCs). These cells effectively expand in 2D culture and have the potential to differentiate into various cell types, including chondrocytes. Furthermore, they have the ability to synthesize extracellular matrix with properties mimicking closely the healthy hyaline joint cartilage. Beside a more general survey of the architecture of hyaline cartilage, its composition and the pathological processes of joint diseases, we will describe here which advances were achieved recently regarding the development of closed, aseptic bioreactors for the production of autologous grafts for cartilage regeneration based on MSCs. Additionally, a novel mathematical model will be presented that supports the understanding of the growth and differentiation of MSCs. It will be particularly emphasized that such models are helpful to explain the well-known fact that MSCs exhibit improved growth properties under reduced oxygen pressure and limited supply with nutrients. Finally, it will be comprehensively shown how different analytical methods can be used to characterize MSCs on different levels. Besides discussing methods for non-invasive monitoring and tracking of the cells and the determination of their elastic properties, mass spectrometric methods to evaluate the lipid compositions of cells will be highlighted.

Are patients with Hodgkin lymphoma and high-grade non-Hodgkin lymphoma in clinical therapy optimization protocols representative of these groups of patients in Germany?

BACKGROUND: Improvement of lymphoma therapy is largely driven by clinical therapy optimization protocols (TOPs). It is unclear, however, whether the patients treated in clinical TOP are representative for all patients. PATIENTS AND METHODS: TOP participants were compared with nonstudy patients in a population-based approach. The study included patients with Hodgkin lymphoma (HL) and high-grade non-Hodgkin lymphoma (hgNHL). Incident cases (N = 743) were ascertained in a large population-based epidemiologic survey. Each patient's status with respect to exclusion criteria of the pertinent TOP was abstracted from primary data sources. TOP participants were identified on the basis of the trial databases. Baseline characteristics and risk factor prevalence were compared between nonstudy and TOP patients. RESULTS: Eligible for the respective TOPs were 64.1% of all incident HL patients and 29.6% of all hgNHL patients in the population. Main exclusion criterion was age (HL: 15.2%; hgNHL: 27.4%). Only 71 HL patients (23.0%) and 11 hgNHL patients (3.4%) had actually been enrolled in the respective TOPs. CONCLUSIONS: TOP participants do not represent all patients with hgNHL and HL in the population. TOP inclusion criteria caused considerable selection among the participants. Further investigation is required to clarify possible limitations for the application of the outcomes observed in TOP patients for all patients with these diseases.

The interleukin-10 gene promoter polymorphism -1087AG does not correlate with clinical outcome in non-Hodgkin's lymphoma.

The Interleukin 10 (IL-10) gene is highly polymorphic, and the IL-10(-1087AG) (rs1800896) gene variation is the only so far studied intensively in association with certain diseases. Conflicting data have been published about an association of IL-10(-1087AG) gene variation with lower rates of complete remission and lower overall survival (OS) in patients with diffuse large B-cell lymphoma. To further investigate this in malignant lymphoma, we established the IL-10 genotypes in patients from the NHL-B1/ B2 studies from the German High-Grade Non-Hodgkin's Lymphoma Study Group. In our study, allele frequencies of lymphoma patients are comparable as in healthy controls. No increase of IL-10(-1087G) alleles was found. In addition we did not find any difference in OS or event-free survival between patients with IL-10(-1087AA) and the other genotypes. Comparable results were obtained for the IL-10 loci at -3538 (A/T), -1354 (A/G), -824 (C/T) and -597 (A/C) (rs1800890, rs1800893, rs1800871 and rs1800872).

High diagnostic value of 18F-FDG-PET in pediatric patients with chronic inflammatory bowel disease.

Diagnosis of chronic inflammatory bowel disease (IBD) in children requires noninvasive, atraumatic diagnostic tools that depict localization and acuity of inflammation and yield only a low radiation dose. This retrospective analysis evaluates the diagnostic potential of FDG-PET. Twenty-six consecutive FDG-PET scans of 23 patients (age: 2-16, years, 14 M, 9 F) with suspected IBD were analyzed in this retrospective study. Results were compared to endoscopic, histologic, and abdominal ultrasound (US) finding. In these examinations, presence of inflammation was evaluated in each patient in 8 bowel segments (score 1-4). Standardized uptake values (SUVs) for FDG-PET were measured for all segments. Sensitivity, specificity, and accuracy were calculated using histology as the standard of reference on a segment-based analysis (pathologic if inflammation score > or = 3 or SUV(max)/SUV(liver)>1.2). With histology as the standard of reference, FDG-PET showed a sensitivity/specificity/accuracy of 98%/68%/8%/3 as compared to endoscopy (90%/75%/82%) and US (56%/92%/75%). For the small bowel, FDG-PET was even more reliable (100%/86%/90%). Because of its high sensitivity and accuracy,FDG-PET is an excellent, noninvasive diagnostic tool for IBD. Depicting inflammation in the whole bowel, while being not traumatic, it is attractive for use especially in children. FDG-PET is especially reliable for the small bowel and can inform application of topical therapy.

A prospectively randomized trial carried out by the German Hodgkin Study Group (GHSG) for elderly patients with advanced Hodgkin's disease comparing BEACOPP baseline and COPP-ABVD (study HD9elderly).

In contrast to younger patients, the prognosis of elderly patients with advanced Hodgkin's disease (HD) has not improved substantially over the last 20 years. We thus carried out a prospectively randomized study (HD9(elderly)) to compare the BEACOPP regimen in this setting against standard COPP-ABVD. Between February 1993 and 1998, 75 patients aged 66-75 years with newly diagnosed HD in advanced stages were recruited into the HD9 trial as a separate stratum (HD9(elderly)). Patients were assigned to eight alternating cycles of COPP and ABVD or eight cycles of BEACOPP in baseline doses. Radiotherapy was given to initial bulky or residual disease. In total, 68 of 75 registered patients were assessable: 26 were treated with COPP-ABVD and 42 with BEACOPP baseline. There were no significant differences between COPP-ABVD and BEACOPP in terms of complete remission (76%), overall survival (50%) and freedom from treatment failure (FFTF) (46%) at 5 years. At a median follow-up of 80 months, a total of 37 patients died: 14/26 patients (54%) treated with COPP-ABVD and 23/42 patients (55%) with BEACOPP. Two patients (8%) treated with COPP-ABVD and nine patients (21%) treated with BEACOPP died of acute toxicity. Hodgkin-specific FFTF at 5 years was 55% after COPP-ABVD and 74% after BEACOPP (P=0.13). Thus, there are no differences in survival between these regimens in elderly patients.

[Quality standards and their assurance for study centers in the competence network malignant lymphoma]. / Qualitätsstandards und ihre Sicherung für Studienzentralen im Kompetenznetz Maligne Lymphome.

BACKGROUND: The aim of most of the trials of the Competence Network Malignant Lymphoma is to optimize the standard treatment of lymphoma using only registered drugs in the case of medicinal therapies (quality assurance protocols). In contrast to regulatory trials, special regulations for quality assurance protocols are not given by the legislature. However, there is agreement that also for this type of studies the declaration of Helsinki and the Guidelines of the International Conference on Harmonisation (ICH) are relevant. The ICH Guidelines must be formulated to take into account the specific situation of quality assurance protocols and to ensure at the same time efficiency and transparency of these studies. This is the aim of the quality management of the study groups in the Competence Network Malignant Lymphoma. METHOD: The quality assurance measures already established in the study groups are being expanded to a comprehensive quality management concept in agreement with the ICH Guidelines and allowing for the situation of quality assurance protocols. To this end, a working group for quality management (AG-QM) has been set up to define and establish general quality standards for all aspects of planning, executing and evaluating quality assurance protocols in study centers. RESULTS: The AG-QM has developed a system of Standard Operating Procedures (SOPs) reflecting all working procedures of the study centers. Furthermore, evaluation parameters for the quality of trial execution have been identified and the harmonisation of documentation parameters has been initiated. Term definitions are collected and their harmonisation coordinated. CONCLUSIONS: Development of quality standards is the first step of quality management. To ensure the realisation of these standards in practice, the AG-QM will establish quality assurance measures including continuous reevaluation of quality criteria and actualization of quality standards if necessary.

Iodine excretion during stimulation with rhTSH in differentiated thyroid carcinoma.

AIM: Elevated iodine intake is a serious problem in the diagnostic and therapeutic application of (131)iodine in patients with differentiated thyroid cancer. Therefore, iodine avoidance is necessary 3 months in advance. Additionally, endogenous stimulation requires withdrawal of thyroid hormone substitution for 4 weeks. Exogenous stimulation using recombinant human TSH (rhTSH) enables the continuous substitution of levothyroxine, which contains 65.4% of its molecular weight in iodine. Thus, a substantial source of iodine intake is maintained during exogenous stimulation. Although this amount of stable iodine is comparable to the iodine intake in regions of normal iodine supply, it may reduce the accumulation of radioiodine in thyroid carcinoma tissue. The aim of this study was to assess the iodine excretion depending on different ways of stimulation. METHODS: Iodine excretion was measured in 146 patients in the long term follow up after differentiated thyroid carcinoma. Patients were separated into 2 groups, those on hormone withdrawal (G I) and rhTSH-stimulated patients on hormone substitution (G II). RESULTS: Iodine excretion was significantly lower in hypothyroid patients (G I, median 50 micro g/l, range: 25-600 micro g/l) than in those under levothyroxine medication (G II, median 75 micro g/l, 25-600 micro g/l, p <0.027). TSH in G I (median 57.0 micro U/ml, range: 14.4-183 micro U/ml) was significantly lower (p <0.001) than in G II (117 micro U/ml, 32.2-281 micro U/ml). CONCLUSION: Iodine excretion was higher in patients under rhTSH-stimulation than after hormone withdrawal. This may indicate an increased iodine pool in rhTSH-stimulated patients (deiodination of levothyroxine), thus limiting the sensitivity of radioiodine scanning to the level of endogenous stimulation despite significantly higher TSH levels during rhTSH-stimulation.

Malignant melanoma and (18)F-FDG-PET: Should the whole body scan include the legs?

AIM: (18)F-FDG-PET (FDG-PET) is established in staging and follow-up of malignant melanoma. The legs are affected in 10-40% at time of diagnosis even if the primary is at the arms and torso. Imaging including the legs may detect distant manifestations but increases duration of the scan by approximately 30 min. We intended to disclose the diagnostic benefit of scanning the legs and to evaluate the therapeutic benefit resulting. PATIENTS, METHODS: In this retrospective analyse 213 consecutive PET studies of 153 patients with suspected or recent malignant melanoma were re-evaluated for metastatic spread by a blinded investigator. Histopathological follow-up was assessed for confirmation. RESULTS: Suspicious findings at the legs were depicted in 53 patients on 76 occasions. 38/53 showed pathologic uptake in the torso as well. In 15/53 patients it was restricted to the legs. One of them had a hitherto unknown, clinically relevant finding that was not apparent in palpation and inspection. In 6 other patients with primary location at the legs a validation of the positive PET findings was not possible up to now. CONCLUSION: Metastases and local recurrence of malignant melanoma at the legs were found in 41% of women and 27% of men. However, a long scan does not yield relevant additional data. We found isolated new manifestations at the legs in only 1/153 patients. We recommend performing a long scan only in patients with previous melanoma manifestations restricted to the legs. In all other cases a short scan of the torso and proximal thighs is sufficient. This allows a higher number of PET-scans without loss of diagnostic power and a shorter examination time.

[Medical counseling of public health-insurances in questions of occupational diseases in regard to economical benefits--project of cooperation between AOK Hesse and the Medical Advisory and Expertising Service Hesse]. / Okonomischer Nutzen der Beratung gesetzlicher Krankenkassen in Berufskrankheitenfragen.

Since September 1998 exists a project of cooperation and consultation between the AOK Hesse and the Medical Advisory and Expertising Service Hesse with the aim to identify occupational diseases and to survey decisions of the Employer's Liability Insurance Association. The procedure is based on a computer-added recognition-system, a profound preparation of the single cases by the employees of the health-insurance and a very intensively carried out deliberation by Medical Doctors of occupational medicine. In a period of four and a half year 8391 cases have been reviewed of which 4859 have already been determined. An approval as occupational disease by the Employer's Liability Insurance Association has been determined in 1954 cases, in 2905 cases the acknowledgement has not been determined. Regarding the determined cases a recourse of 10,078,922.27 EUR has been realized. In regard to the invested small resources of personnel the procedure has proved itself as highly effective to discover and to assert recourses. Beside the economical aspects for the public health-insurance, other results of the project were the assurance of the entitlement to benefits of people coming down with occupational diseases or their relatives. New insights about the actual development of occupational diseases in Germany als well as their prevention can be proceeded.

Detection and location of the enzymes of de novo pyrimidine biosynthesis in mammalian spermatozoa.

Enzymes of the pathway for de novo biosynthesis of pyrimidine nucleotides have been reported in spermatozoa from fruitfly and mammals. The aim of the present study was to test the hypothesis that the enzymes for biosynthesis of uridine monophosphate (UMP) are concentrated near the mitochondria, which are segregated in the mid-piece of spermatozoa. Baby hamster kidney fibroblasts were compared with spermatozoa from rams, boars, bulls and men. Antibodies raised against synthetic peptides from sequences of the multienzyme polypeptides containing glutamine-dependent carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase (CAD) and UMP synthase, which catalyse reactions 1-3 and 5-6, respectively, were used, together with an affinity-purified antibody raised against dihydroorotate dehydrogenase (DHODH), the mitochondrial enzyme for step 4. Western blot analysis, immunofluorescent microscopy and immunoelectron microscopy confirmed that CAD and UMP synthase are found in the cytoplasm around and outside the mitochondria; DHODH is found exclusively inside the mitochondria. CAD was also located in the nucleus, where it has been reported in the nuclear matrix, and in the cytoplasm, apparently associated with the cytoskeleton. It is possible that CAD in the cytoplasm has a role unconnected with pyrimidine biosynthesis.

[Diagnosis of vesico-intestinal fistulas by contrast medium enhanced 3-D ultrasound]. / Diagnostik von Harnblasen-Darm-Fisteln durch kontrastmittelunterstützte 3D-Sonographie.

AIM: Standard diagnostic tools for vesico-intestinal fistulas are cystoscopy, cystography, colonoscopy, and contrast enema. The aim of our study was to evaluate the efficacy of transrectal 3D-ultrasound with contrast media in these patients. METHOD: From 5/98 to 12/99 we examined 10 patients with symptoms of a vesico-intestinal fistula (pneumaturia, faecaluria). After placement of a transurethral catheter a transabdominal ultrasound examination (Kretz Combison 530) was performed with the bladder half full to evaluate the bladder wall. Then the bladder was filled with diluted ultrasound contrast media (Levovist 40 mg/ml) to visualize the flow from the bladder towards the fistula. To verify a flow through the bladder wall a colour Doppler sonography of the region of interest was added. To evaluate form and extent of the fistula a transrectal ultrasound with 3D-image assessment was performed. RESULTS: Using this technique it was possible to demonstrate a vesico-intestinal fistula in 9 of 10 patients. In all cases these findings were confirmed by the standard diagnostic procedures. The fistulas were caused by: bladder carcinoma (n = 1), carcinoma of the colon (n = 2), Crohn's disease (n = 3) and diverticulitis of the sigma (n = 3). One patient presented with a neovesico-intestinal fistula in an irradiated local recurrence of bladder carcinoma. In one patient with Crohn's disease whose only symptom was pneumaturia all diagnostic tools failed to provide the diagnosis. CONCLUSION: For the first time vesico-intestinal fistulas could be demonstrated by ultrasound with 3D-image assessment using contrast media. This technique might be an effective addition to the standard diagnostics of vesico-intestinal fistulas reducing the exposure to radiation.

Recombinant expression of N-terminal truncated mutants of the membrane bound mouse, rat and human flavoenzyme dihydroorotate dehydrogenase. A versatile tool to rate inhibitor effects?

Mammalian dihydroorotate dehydrogenase, the fourth enzyme of pyrimidine de novo synthesis is an integral protein of the inner mitochondrial membrane that faces the intermembrane space and is functionally connected to the respiratory chain via ubiquinone. Here, we describe the first cloning and analyzing of the complete cDNA of mouse dihydroorotate dehydrogenase. Based on our recent functional expression of the full-length rat and human dihydroorotate dehydrogenase, here we expressed N-terminal-truncated C-terminal-histidine-tagged constructs of the mouse, rat and human enzymes in Escherichia coli. These proteins were devoid of the N-terminal bipartite sequence consisting of the mitochondrial targeting sequence and adjacent hydrophobic domain necessary for import and proper location and fixation of the enzyme in the inner mitochondrial membrane. By employing metal-chelate affinity chromatography under native conditions, the enzymes were purified without detergents to a specific activity of more than 100 micromol x min(-1) x mg(-1) at pH optimum of 8.0--8.1. Flavin analyses by UV-visible spectrometry of the native enzymes gave fairly stoichiometric ratios of 0.6--1.2 mol flavin per mol protein. The kinetic constants of the truncated rat enzyme (K(m) = 11 microM dihydroorotate; K(m) = 7 microM ubiquinone) and human enzyme (K(m) = 10 microM dihydroorotate; K(m) = 14 microM ubiquinone) were very close to those recently reported for the full-size enzymes. The constants for the mouse enzyme, K(m) = 26 microM dihydroorotate and K(m) = 62 microM ubiquinone, were slightly elevated in comparison to those of the other species. The three truncated enzymes were tested for their efficacy with five inhibitors of topical clinical relevance against autoimmune disorders and tumors. Whereas the presence of the N-terminus of dihydroorotate dehydrogenase was essentially irrelevant for the efficacy of the malononitrilamides A77-1726, MNA715 and MNA279 with the rat and human enzyme, the N-termini were found to be important for the efficacy of the dianisidine derivative redoxal. Moreover, the complete N-terminal part of the human enzyme seemed to be of crucial importance for the 'slow-binding' features of the cinchoninic acid derivative brequinar, which was suggested to be one of the reasons for the narrow therapeutic window reported from clinical trials on its anti-proliferative and immunosuppressive action.

Immunocytochemical detection of mitochondrial dihydroorotate dehydrogenase in human spermatozoa.

In mammalian cells the requirement for pyrimidines is met by uridine phosphate (UMP) de novo synthesis and, to a greater or lesser extent, by salvage of free nucleosides. The fourth enzyme of the de novo synthesis, the mitochondrially bound dihydroorotate dehydrogenase (DHODH) was the focus of the present study. Rabbit anti-DHODH IgG, which was generated using an immunization protocol with truncated recombinant human DHODH protein and purified by an immunosorbent method, was used for immunocytochemical detection and localization of this enzyme in ejaculated human spermatozoa. The presence of DHODH protein was demonstrated by Western blotting of solubilized membrane fractions with peroxidase conjugated anti-rabbit IgG in combination with chemiluminescence detection. Indirect immunofluorescence microscopy, using Cy3-conjugated anti-rabbit IgG, revealed specific binding in the midpiece of spermatozoa. As these cells no longer have a demand for de novo biosynthesis of pyrimidines, we hypothesize that the pathway could serve a specialized function in nitrogen or zinc metabolism during the process of spermiogenesis and/or epididymal maturation.