The RSD code: proposal for a nomenclature of allelic configurations in STR-PCR-based chimerism testing after allogeneic stem cell transplantation.
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Alleles↗
Biomedical subjects
F Watzinger
Publications and source records attributed to F Watzinger.
Detection and monitoring of virus infections by real-time PCR.
The employment of polymerase chain reaction (PCR) techniques for virus detection and quantification offers the advantages of high sensitivity and reproducibility, combined with an extremely broad dynamic range. A number of qualitative and quantitative PCR virus assays have been described, but commercial PCR kits are available for quantitative analysis of a limited number of clinically important viruses only. In addition to permitting the assessment of viral load at a given time point, quantitative PCR tests offer the possibility of determining the dynamics of virus proliferation, monitoring of the response to treatment and, in viruses displaying persistence in defined cell types, distinction between latent and active infection. Moreover, from a technical point of view, the employment of sequential quantitative PCR assays in virus monitoring helps identifying false positive results caused by inadvertent contamination of samples with traces of viral nucleic acids or PCR products. In this review, we provide a survey of the current state-of-the-art in the application of the real-time PCR technology to virus analysis. Advantages and limitations of the RQ-PCR methodology, and quality control issues related to standardization and validation of diagnostic assays are discussed.
Computer Systems↗
Dysgnathia, orthognathic surgery and spinal posture.
The aim of this study was to evaluate the spine by video rasterstereography before and after orthognathic surgery. Twenty-nine patients (17 patients with a skeletal class III, 7 patients with a skeletal class II, and 5 patients with mandibular asymmetry) were evaluated preoperatively and 1 year postoperatively. Video rasterstereography is a method of back surface measurement and shape analysis using the moire topography. Orthognathic surgery in cases of class III and asymmetry did not lead to significant changes in body posture. In class II patients it led to some changes in body posture, but without orthopaedic consequences. It is concluded that orthognathic surgery causes minimal or no change in body posture.
Adolescent↗
Aggressive fibromatosis of the mandible: a case report.
An extensive tumour in a 7-year-old girl, leading to severe disfigurement, proved to be an aggressive fibromatosis on histological examination. Eighteen months after surgery there was no evidence of recurrent disease. This suggests that tumour resection and reconstruction of the mandible had been successful. Contrary to some reports, tumour resection led to curative therapy whereas radiotherapy failed.
Bone Transplantation↗
Molecular detection and quantitative analysis of the entire spectrum of human adenoviruses by a two-reaction real-time PCR assay.
Adenoviruses (AdV) can cause life-threatening infections in immunosuppressed patients. Reliable diagnostic tests are therefore of paramount importance. Apparently, any of the six AdV species (A to F), currently comprising 51 different serotypes, can play a clinically important role in patients with impaired immune response. It is imperative therefore that diagnostic assays cover the entire spectrum of these viruses. We have sequenced presumably conserved regions of the adenoviral genome in all AdV serotypes. Based on the complete sequence information of the hexon gene, we were able to develop a two-reaction real-time PCR assay covering all human adenoviruses with equally high specificity and sensitivity. The detection systems were tested using reference strains for all 51 serotypes and >1,000 clinical samples derived from peripheral blood and stool specimens from pediatric patients after allogeneic stem cell transplantation. The two-reaction assay presented permits highly specific detection and quantification of adenoviral DNA of any serotype. From the perspective of routine clinical diagnosis, the assay represents an important improvement over existing approaches by providing a sensitive and economic technique for early detection and monitoring of adenoviral infections.
Adenovirus Infections, Human↗
Real-time quantitative PCR assays for detection and monitoring of pathogenic human viruses in immunosuppressed pediatric patients.
A panel of 23 real-time PCR assays based on TaqMan technology has been developed for the detection and monitoring of 16 different viruses and virus families including human polyomaviruses BK virus and JC virus, human herpesviruses 6, 7, and 8, human adenoviruses, herpes simplex viruses 1 and 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, parvovirus B19, influenza A and B viruses, parainfluenza viruses 1 to 3, enteroviruses, and respiratory syncytial virus. The test systems presented have a broad dynamic range and display high sensitivity, reproducibility, and specificity. Moreover, the assays allow precise quantification of viral load in a variety of clinical specimens. The ability to use uniform PCR conditions for all assays permits simultaneous processing and detection of many different viruses, thus economizing the diagnostic work. Our observations based on more than 50,000 assays reveal the potential of the real-time PCR tests to facilitate early diagnosis of infection and to monitor the kinetics of viral proliferation and the response to treatment. We demonstrate that, in immunosuppressed patients with invasive virus infections, surveillance by the assays described may permit detection of increasing viral load several days to weeks prior to the onset of clinical symptoms. In virus infections for which specific treatment is available, the quantitative PCR assays presented provide reliable diagnostic tools for timely initiation of appropriate therapy and for rapid assessment of the efficacy of antiviral treatment strategies.
Adolescent↗
Gene symbol: IRF6. Disease: Van der Woude syndrome.
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Cleft Lip↗
[Evidence of osteocalcin expression in osteoblast cells of mandibular origin growing on biomaterials with RT-PCR and SDS-PAGE/Western blotting].
A new approach to addressing difficult tissue reconstructive or replacement problems in the oral cavity is to engineer new tissue by using selective cell transplantation on polymer scaffolds. The current study characterized the osteoblastic nature of adherent mandibular cells on biomaterials, which could have a potential use as scaffolds for tissue engineering strategies. Cells of mandibular origin from one patient were cultivated on three different biomaterials (PepGen P-15 trade mark, Frios Algipore, and OsteoGraf/LD-700) for 7 and 14 days and osteocalcin expression was demonstrated by RT-PCR and SDS-PAGE/Western blotting. In order to explicitly characterize only the adherent cells on the biomaterials, we first separated the biomaterials with adherent cells from the culture plate before trypsinization. We could demonstrate that cell growth of adherent mandibular osteoblast-like cells was significantly higher on biomaterials with an organic component (PepGen P-15 trade mark ) in comparison to Frios Algipore and OsteoGraf/LD-700, respectively. In conclusion, only the explicit study of adherent cells at the gene and protein levels gives information about the osteoconductivity of biomaterials.
Alginates↗
Quantitative analysis of chimerism after allogeneic stem cell transplantation by PCR amplification of microsatellite markers and capillary electrophoresis with fluorescence detection: the Vienna experience.
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Alleles↗
Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using 'real-time' quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR) - a Europe against cancer program.
Real-time quantitative RT-PCR (RQ-PCR) is a sensitive tool to monitor minimal residual disease (MRD) in leukemic patients through the amplification of a fusion gene (FG) transcript. In order to correct variations in RNA quality and quantity and to calculate the sensitivity of each measurement, a control gene (CG) transcript should be amplified in parallel to the FG transcript. To identify suitable CGs, a study group within the Europe Against Cancer (EAC) program initially focused on 14 potential CGs using a standardized RQ-PCR protocol. Based on the absence of pseudogenes and the level and stability of the CG expression, three genes were finally selected: Abelson (ABL), beta-2-microglobulin (B2M), and beta-glucuronidase (GUS). A multicenter prospective study on normal (n=126) and diagnostic leukemic (n=184) samples processed the same day has established reference values for the CG expression. A multicenter retrospective study on over 250 acute and chronic leukemia samples obtained at diagnosis and with an identified FG transcript confirmed that the three CGs had a stable expression in the different types of samples. However, only ABL gene transcript expression did not differ significantly between normal and leukemic samples at diagnosis. We therefore propose to use the ABL gene as CG for RQ-PCR-based diagnosis and MRD detection in leukemic patients. Overall, these data are not only eligible for quantification of fusion gene transcripts, but also for the quantification of aberrantly expressed genes.
Archives↗
Impact of hemoglobin level and use of recombinant erythropoietin on efficacy of preoperative chemoradiation therapy for squamous cell carcinoma of the oral cavity and oropharynx.
PURPOSE: We assessed the influence of hemoglobin level and r-HuEPO administration on response to chemoradiotherapy, locoregional tumor control, and overall survival in patients treated with neoadjuvant chemoradiotherapy and surgery for a squamous cell carcinoma of the oral cavity or oropharynx. METHODS AND MATERIALS: The 191 study patients were treated with mitomycin C (15 mg/m(2) day 1), 5-fluorouracil (750 mg/m(2)/day, days 1-5), and radiotherapy (50 Gy in 25 fractions weeks 1-5), followed by resection of the primary tumor bed and neck dissection at the General Hospital Vienna, Austria, between November 1989 and October 1998 for a T2-4, N0-3, M0 SCC of the oral cavity or oropharynx. Starting in May 1996, patients with a low hemoglobin (Hgb) before or during chemoradiotherapy received r-HuEPO 10,000 IU/kg s.c. 3-6 times/week until the week of surgery. RESULTS: On multivariate analysis, Hgb level and use of r-HuEPO were independent prognostic factors for response to chemoradiotherapy and locoregional tumor control (p < 0.01). Pathologic response to neoadjuvant therapy was also predictive of locoregional control (p < 0.001). Patients with a pretreatment Hgb > or = 14.5 g/dL had significantly higher complete response, locoregional control, and survival rates than the patients with a pretreatment Hgb < 14.5 g/dL who did not receive r-HuEPO (p < 0.05). The response, control, and survival rates in patients with a pretreatment Hgb < 14.5 g/dL given r-HuEPO were significantly higher than in low Hgb patients not given r-HuEPO (p < or = 0.001) and equivalent to patients with a pretreatment Hgb > 14.5 g/dL (p > or = 0.3). CONCLUSION: Low pretreatment Hgb is a negative prognostic factor for oral cavity and oropharyngeal SCCA patients, but was completely abrogated by r-HuEpo administration during neoadjuvant chemoradiotherapy. Randomized trials of radiation and/or chemotherapy with or without r-HuEPO for patients whose Hgb level is either low at the start of therapy or is anticipated to become low during therapy are indicated.
Antineoplastic Combined Chemotherapy Protocols↗
Quantification of mRNA expression by competitive PCR using non-homologous competitors containing a shifted restriction site.
Despite the recent introduction of real-time PCR methods, competitive PCR techniques continue to play an important role in nucleic acid quantification because of the significantly lower cost of equipment and consumables. Here we describe a shifted restriction-site competitive PCR (SRS-cPCR) assay based on a modified type of competitor. The competitor fragments are designed to contain a recognition site for a restriction endonuclease that is also present in the target sequence to be quantified, but in a different position. Upon completion of the PCR, the amplicons are digested in the same tube with a single restriction enzyme, without the need to purify PCR products. The generated competitor- and target-specific restriction fragments display different sizes, and can be readily separated by electrophoresis and quantified by image analysis. Suboptimal digestion affects competitor- and target-derived amplicons to the same extent, thus eliminating the problem of incorrect quantification as a result of incomplete digestion of PCR products. We have established optimized conditions for a panel of 20 common restriction endonucleases permitting efficient digestion in PCR buffer. It is possible, therefore, to find a suitable restriction site for competitive PCR in virtually any sequence of interest. The assay presented is inexpensive, widely applicable, and permits reliable and accurate quantification of nucleic acid targets.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Comparative analysis of ras proto-oncogene mutations in selected mammalian tumors.
Point mutations within ras proto-oncogenes are frequently detected in human malignancies and in different types of experimentally induced tumors in animals. In contrast to findings in experimental animal models of carcinogenesis, little is known about the incidence of ras mutations in naturally occurring animal tumors. In the present study, we investigated whether point mutations, particularly within the mutational hot-spot codons 12, 13, and 61, occur at comparable frequencies in human malignancies and spontaneously occurring tumors in other mammalian species. Two hundred seventy-nine of the most frequent canine and feline neoplasms were analyzed for changes in mutational hot-spot regions of the N-, Ki-, and Ha-ras genes. DNA fragments from exons 1 and 2 of all three ras genes were amplified by polymerase chain reaction, and the presence of point mutations was assessed by single-strand conformation polymorphism analysis and direct sequencing of amplified products. Only one sample, a case of canine melanoma, exhibited an Ha-ras mutation. Thus, our data strongly suggested that ras mutations at the hot-spot loci are apparently very rare and do not play a major role in the pathogenesis of the spontaneously occurring canine and feline tumors investigated. These observations were in marked contrast to those in experimental rodent models of carcinogen-induced mammary and skin tumors that described a consistent association with Ha- or Ki-ras activation. The role of ras oncogene activation in related human malignancies therefore cannot be readily inferred from studies of experimental carcinogenesis in animal models.
Animals↗
Preprosthetic vertical distraction osteogenesis of the mandible using an L-shaped osteotomy and titanium membranes for guided bone regeneration.
PURPOSE: Vertical osteodistraction is a new alternative method for alveolar ridge augmentation of the mandible. The purpose of this article is describe a technique using an L-shaped osteotomy and titanium membranes for guided bone regeneration (GBR) in the distraction gap. PATIENTS AND METHODS: Ten patients with severe atrophy of the edentulous molar region of the mandible underwent vertical callus distraction in 13 sites using intraoral microplate distractors. An L-shaped osteotomy with a short vertical part mesially and a longer horizontal part ending in the retromolar region was made, and the osteotomized segment was fixed to the mandibular ramus at its distal edge by a microplate, which became the center of rotation when distraction began. In this way, more callus generation could be achieved mesially than in the distal molar region. Follow-up computed tomography (CT) scans reconstructed axially to the axis of the mandible revealed semilunar excavations of the generated bone buccally in the distraction gap in the first cases. Clinical inspection on removal of the distractors showed fibrous connective tissue in the gap. Therefore, to prevent this from happening, titanium membranes covering the distraction gap were applied in subsequent cases. RESULTS: Ten patients (13 sites) were treated by vertical callus distraction. In 4 cases, GBR was achieved using titanium membranes. In all cases, the increase in alveolar height was sufficient to make dental implantation possible. In 1 patient, a fracture of the distractor occurred, and dehiscence was observed in 2 cases. These complications did not change the plan of therapy nor did they influence the results. The CT scans showed a homogenous surface on the regenerated mandible in the cases of GBR application. CONCLUSION: Both an L-shaped osteotomy and the application of titanium membranes for GBR in the distraction gap are of great value for mandibular augmentation, producing a physiologically shaped alveolar ridge.
Adult↗
Principles of computer-assisted arthroscopy of the temporomandibular joint with optoelectronic tracking technology.
PURPOSE: This preliminary clinical study evaluated the applicability, accuracy, and benefits of computer-assisted arthroscopy of the temporomandibular joint (TMJ) with optoelectronic tracking technology. MATERIALS AND METHODS: A hybrid of reality and virtual reality is built as a composite-reality environment by extracting 3-dimensional anatomical structures through use of computed tomography, magnetic resonance imaging, radiography, and other types of imaging procedures commonly used in clinical praxis. These various independent sources of imaging data of a particular patient can be combined with and complemented by complex graphic simulations. Intraoperatively they are merged with online position data of surgical instruments inside the patient's TMJ. This hybrid model of detailed anatomical structures, guidelines, and real-time instrument positions allows the surgeon to accurately plan the arthroscopic intervention as well as to navigate effectively intraoperatively. RESULTS: In the first 10 cases of computer-assisted TMJ arthroscopy, composite reality environment technology permitted the online visualization of TMJ structures, puncture sites, instrument positions, and virtual pathways in relation to anatomical landmarks with high spatial accuracy (minimum, 0.0 mm; maximum, 2.5 mm; mean, 1.4 mm; SD, 0.6 mm) and high temporal resolution (100 ms). Past, present, and possible future instrument positions can be displayed. The application of computer-assisted arthroscopy caused little immobility for either surgeon or patient. CONCLUSION: Even experienced surgeons profit from improved precision in the handling of the arthroscope; thus this technology was found to be particularly useful in degenerative temporomandibular disorders and for triangulation procedures.
Arthroscopes↗
Placement of endosteal implants in the zygoma after maxillectomy: a Cadaver study using surgical navigation.
Endosteal implants facilitate obturator prosthesis fixation in tumor patients after maxillectomy. Previous clinical studies have shown, however, that the survival of implants placed into available bone after maxillectomy is generally poor. Nevertheless, implants positioned optimally in residual zygomatic bone provide superior stability from a biomechanical point of view. In a pilot study, the authors assessed the precision of VISIT, a computer-aided surgical navigation system dedicated to the placement of endosteal implants in the maxillofacial area. Five cadaver specimens underwent hemimaxillectomy. The cadaver head was matched to a preoperative high-resolution computed tomograph by using implanted surgical microscrews as fiducial markers. The position of a surgical drill relative to the cadaver head was determined with an optical tracking system. Implants were placed into the zygomatic arch, where maximum bone volume was available. The results were assessed using tests for localization accuracy and postoperative computed tomographic scans of the cadaver specimens. The localization accuracy of landmarks on the bony skull was 0.6 +/- 0.3 mm (average +/- SD), as determined with a 5-df pointer probe; the localization accuracy of the tip of the implant burr was 1.7 +/- 0.4 mm. The accuracy of the implant position compared with the planned position was 1.3 +/- 0.8 mm for the external perforation of the zygoma and 1.7 +/-1.3 mm for the internal perforation. Eight of 10 implants were inserted with maximal contact to surrounding bone, and two implants were located unfavorably. Reliable placement of implants in this region is difficult to achieve. The technique described in this article may be very helpful in the management of patients after maxillary resection with poor support for obturator prostheses.
Cadaver↗
Cryptic splice site activation during RNA processing of MLL/AF4 chimeric transcripts in infants with t(4;11) positive ALL.
Co-expression of multiple variants of the MLL/AF4 fusion transcript is a common phenomenon in patients with acute lymphoblastic leukemia (ALL) with t(4;11)(q21;q23). Different transcriptional and post-transcriptional mechanisms were found to contribute to the heterogeneity of the chimeric transcripts. Multiple splice variants are generated by utilizing alternative splice sites that result in the joining of different MLL-exons within the breakpoint cluster region to one of three exons in the AF4 fusion partner. To address the question of how splice site selection occurs during RNA processing, we investigated der(11) transcripts in 10 infants with t(4;11) positive ALL. Specific RT-PCR products were analyzed by Southern blot hybridization, SSCP, endonuclease digestion, cloning and sequencing. In patients co-expressing as many as six different chimeric mRNA species, activation of cryptic splice sites has been detected in MLL-exons 8 and 10. This led to the formation of four novel transcript variants, three of which maintained open reading frames (ORFs). Patients with cryptic donor site activation in MLL-exon 8 did not have any MLL-exon 8/AF4 transcripts using the authentic 5' splice site, although this site is 100% homologous to the consensus sequence. However, since MLL-exon 8 does not end in-phase, the use of the authentic splice site would result in loss of the ORF of the fusion message. The activated cryptic splicing sites are located in the vicinity of the polypurine stretches present in MLL-exons 8 and 10, which are known to function as splicing enhancers recognized by SR proteins. We postulate that both the nonsense-mediated decay eliminating correctly spliced MLL-exon 8/AF4 mRNAs and activation of suboptimal splicing sites contribute to the diversity of MLL/AF4 RNA species.
Alternative Splicing↗
[Telenavigation and expert consultation using a stereotaxic surgical videoserver].
The exponential increase of medical information creates a need for new methods in the visualization of medical imaging modalities for diagnosis and therapy. In this sense, visualization includes the display of medical image data and image-guided stereotaxic navigation as well as the advice of an expert. The Artma Virtual Patient System enables a remote expert to observe the surgical procedure via the Internet and interactively modify the interoperative visualization from the remote location. The expert in the remote location receives the planning data almost in real time over TCP/IP from a stereotaxic videoserver. In addition to live video streaming, stereotaxic navigation data are sent over the network as rigid body coordinates. The expert modifies the surgical simulation on the remote computer and the modified operating plan is sent back to the operating site. By teleconsulting, the composite images and overlapping graphics--instruments, target structures, landmarks, contour--can be seen in affiliated clinics with the possibility of interactive graphical assistance. With this image fusion technology the knowledge of a remote expert is included in virtual data structures and visualized by the overlay with live video data (augmented reality) in real time during surgery.
Humans↗