The volume effect of detectors in the dosimetry of small fields used in IMRT

In this study we investigate the effect of detector size in the dosimetry of small fields and steep dose gradients with a particular emphasis on IMRT measurements. Comparisons of calculated and measured cross-profiles and absolute dose values of IMRT treatment plans are presented. As a consequence of the finite size of the detector that was used for the commissioning of the IMRT tool, local discrepancies of more than 10% are found between calculated cross-profiles of intensity modulated beams and intensity modulated profiles measured with film. Absolute dose measurements of intensity modulated fields with a 0.6 cm3 Farmer chamber show significant differences of more than 6% between calculated and measured dose values at the isocenter of an IMRT treatment plan. Differences of not more than 2% are found in the same experiment for dose values measured with a 0.015 cm3 pinpoint ion chamber. A method to correct for the spatial response of finite-sized detectors and to obtain the "real" penumbra width of cross-profiles from measurements is introduced. Output factor measurements are performed with different detectors and are presented as a function of detector size for a 1 x 1 cm2 field. Because of its high spatial resolution and water equivalence, a diamond detector is found to be suitable as an alternative to other detectors used for small field dosimetry as there are photographic and photochromic film, TLDs, or water-equivalent scintillation detectors.     

Comparative Analysis and Immunological Characterization of the Borrelia Bdr Protein Family

Multiple circular and linear plasmids of Lyme disease and relapsing fever Borrelia spirochetes carry genes for members of the Bdr (Borrelia direct repeat) protein family. To define their common and divergent attributes, we first comprehensively compared the known homologs. Bdr proteins with predicted sizes ranging from 10.7 to 30. 6 kDa formed five homology groups, based on variable numbers of short direct repeats in a central domain and diverse N- and C-terminal domains. In a further characterization, Western blots were probed with rabbit antisera raised against either of two purified recombinant Bdr proteins from Borrelia burgdorferi B31. The results showed that antibodies cross-react and several Bdr paralogs 19.5 to 30.5 kDa in size are expressed by cultured strain B31 in a temperature-independent manner. In situ proteolysis, immunofluorescence, and growth inhibition assays indicated that Bdr proteins are not surface exposed. Distinct patterns of cross-reacting proteins of 17.5 to 33 kDa were also detected in other B. burgdorferi, Borrelia garinii, and Borrelia afzelii strains as well as in relapsing fever spirochetes Borrelia hermsii and Borrelia turicatae. Last, we examined whether these proteins are antibody targets during Lyme disease. Analysis of 47 Lyme disease patient sera by immunoblotting and enzyme-linked immunosorbent assays showed that 24 (51%) and 20 (43%), respectively, had detectable antibodies to one or more of the Bdr proteins. Together, these data indicate that Bdr proteins constitute a family of cross-reactive Borrelia proteins which are expressed in the course of Lyme disease and in vitro.     

An automated GCxGC‐TOF‐MS protocol for batch‐wise extraction and alignment of mass isotopomer matrixes from differential 13C‐labelling experiments: a case study for photoautotrophic‐mixotrophic grown Chlamydomonas reinhardtii cells

Two dimensional gas chromatography coupled to time‐of‐flight mass spectrometry (GCxGC‐TOF‐MS) is a promising technique to overcome limits of complex metabolome analysis using one dimensional GC‐TOF‐MS. Especially at the stage of data export and data mining, however, convenient procedures to cope with the complexity of GCxGC‐TOF‐MS data are still in development. Here, we present a high sample throughput protocol exploiting first and second retention index for spectral library search and subsequent construction of a high dimensional data matrix useful for statistical analysis. The method was applied to the analysis of ¹³C‐labelling experiments in the unicellular green alga Chlamydomonas reinhardtii. We developed a rapid sampling and extraction procedure for Chlamydomonas reinhardtii laboratory strain (CC503), a cell wall deficient mutant. By testing all published quenching protocols we observed dramatic metabolite leakage rates for certain metabolites. To circumvent metabolite leakage, samples were directly quenched and analyzed without separation of the medium. The growth medium was adapted to this rapid sampling protocol to avoid interference with GCxGC‐TOF‐MS analysis. To analyse batches of samples a new software tool, MetMax, was implemented which extracts the isotopomer matrix from stable isotope labelling experiments together with the first and second retention index (RI1 and RI2). To exploit RI1 and RI2 for metabolite identification we used the Golm metabolome database (GMD [1] with RI1/RI2‐reference spectra and new search algorithms. Using those techniques we analysed the dynamics of ¹³CO₂ and ¹³C‐acetate uptake in Chlamydomonas reinhardtii cells in two different steady states namely photoautotroph and mixotroph growth conditions. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A randomized,double-blind,placebo-controlled trial evaluating the efficacy and safety of ABT-594 in patients with diabetic peripheral neuropathic pain

ABT-594 is a neuronal nicotinic acetylcholine receptor (NNR) agonist that exhibits potent analgesic activity in preclinical models of acute, chronic, and neuropathic pain. The purpose of this phase 2, randomized, multicenter, double-blind, placebo-controlled study was to evaluate the safety and analgesic efficacy of ABT-594 in patients with diabetic peripheral neuropathic pain (DPNP). A total of 266 DPNP patients were randomized 1:1:1:1 to receive placebo, ABT-594 150 μg BID, ABT-594 225 μg BID, or ABT-594 300 μg BID. Patients were titrated to a fixed-dose of ABT-594 over 7 days and remained at this dose for another 6 weeks. Compared to placebo, all three ABT-594 treatment groups showed significantly greater decreases on the average diary-based 0–10 Pain Rating Scale (PRS) score from baseline to final evaluation, the primary efficacy measure (placebo, −1.1; 150 μg BID, −1.9; 225 μg BID, −1.9; 300 μg BID, −2.0). The proportion of patients achieving at least a 50% improvement in the average diary-based PRS was greater in all three ABT-594 treatment groups. However, adverse event (AE) dropout rates were significantly higher in all three ABT-594 treatment groups (28% for 150 μg BID, 46% for 225 μg BID, and 66% for 300 μg BID) than for the placebo group (9%). Consistent with the expected side-effect profile of NNR agonists, the most frequently reported AEs were nausea, dizziness, vomiting, abnormal dreams, and asthenia. This study establishes proof of concept for NNR agonists as a new class of compounds for treating neuropathic pain.     

Proteome Serological Determination of Tumor-Associated Antigens in Melanoma

Proteome serology may complement expression library-based approaches as strategy utilizing the patients' immune responses for the identification pathogenesis factors and potential targets for therapy and markers for diagnosis. Melanoma is a relatively immunogenic tumor and antigens recognized by melanoma-specific T cells have been extensively studied. The specificities of antibody responses to this malignancy have been analyzed to some extent by molecular genetic but not proteomics approaches. We screened sera of 94 melanoma patients for anti-melanoma reactivity and detected seropositivity in two-thirds of the patients with 2–6 antigens per case detected by 1D and an average of 2.3 per case by 2D Western blot analysis. For identification, antigen spots in Western blots were aligned with proteins in 2-DE and analyzed by mass spectrometry. 18 antigens were identified, 17 of which for the first time for melanoma. One of these antigens, galectin-3, has been related to various oncogenic processes including metastasis formation and invasiveness. Similarly, enolase has been found deregulated in different cancers. With at least 2 of 18 identified proteins implicated in oncogenic processes, the work confirms the potential of proteome-based antigen discovery to identify pathologically relevant proteins.     

Unexpected high complication rate following internal fixation of unstable proximal humerus fractures with an angled blade plate

OBJECTIVES: The increasing number of fractures of the proximal humerus, especially in the elderly, carries with it the unsolved problem of the optimal treatment for the displaced or unstable fractures. The authors' goal was to analyze whether fixation with a bent valgus angled blade plate could improve the outcomes reported in the literature to date. DESIGN: Prospective clinical study. SETTING: Urban level 1 university trauma center. PATIENTS: Over a 27-month period, 42 consecutive patients were treated for an unstable or displaced proximal humerus fracture. INTERVENTION: Open reduction and internal fixation with a 90-degree cannulated angled blade plate prebent to 110 degrees. MAIN OUTCOME MEASUREMENTS: Active follow-up for 1 year with assessment of objective and subjective functional results (ie, motion; strength; Constant score; Disabilities of the Arm, Shoulder, and Hand (DASH) score; and visual analog scale (VAS)) and radiographic assessment (reduction, alignment, necrosis, and nonunion). RESULTS: Follow-up was completed for 86% of the patients, who achieved a mean Constant score of 66 points (82% of the contralateral side) with a mean forward flexion of 125 degrees and an average strength of 72% of the contralateral side. Low disability (mean DASH score 22) and pain values (mean VAS 2) were demonstrated after this type of stabilization. The overall complication rate was 33% (12/36), with protrusion of the blade into the glenohumeral articulation as the most frequent problem (8/36, 22%). The negative impact of an adverse event on subjective and objective outcomes was only significant for forward flexion (P = 0.02). Neither clinical outcome nor complication rate was different when compared to patients with regard to fracture type (3 versus 4 parts) or age (younger or older than 70 years). CONCLUSIONS: Fixation of displaced proximal humeral fractures with an angled blade plate provided sufficient stability. Blade perforation into the humeral joint occurred in every fourth patient and was found to be the major reason for a high complication rate. In view of this major problem, the technique described here cannot be recommended, even though the absence of nonunions in our series seems to support the low invasiveness of this surgical approach.     

Severe damage of the femoral head after dislocation and difficult reduction maneuvers after total hip arthroplasty

Introduction: Aseptic loosening associated with osteolysis and release of wear particles is the main reason for revision of total hip arthroplasty (THA). Damage of femoral heads is well known to increase the wear rate at the articulating surface. Instability and dislocation are serious complications and are the second most frequent reason for revision surgery after THA. In the present work the possible damaging of the femoral head as a result of difficult reduction maneuvers of dislocated THA should be investigated. Patients and methods: In three cases of total hip dislocation an open reduction was performed after dislocation of primary THA. Thereby one metal and two ceramic femoral heads were retrieved. Each head was analyzed macroscopically and by scanning electron microscopy (SEM) subsequently. Results: In the first case the SEM analysis revealed severe titanium deposits on the retrieved ceramic head in the case of repeated unsuccessful closed reduction maneuvers. In the second case the retrieved ceramic head showed multiple scratches and a spalling of the surface structure. In the third case the retrieved metal head presented a roughened surface and severe scratches at the articulating area. Discussion: Difficult reduction maneuvers following total hip dislocation can lead to severe damaging of the femoral head associated with an increase of the surface roughness. While repeated reduction maneuvers can lead to metallic deposits and spalling of the surface of ceramic heads, severe scratches can occur at metal heads. The damaging of the femoral head at the articulating surface can lead to increased wear and subsequent aseptic implant loosening. Therefore, open joint reduction has to be considered in cases of difficult or unsuccessful closed reduction maneuvers to prevent severe femoral head damage which may cause an increased release of wear particles. No benefits or funds were received for this work.     

Concordant loss of melanoma differentiation antigens in synchronous and asynchronous melanoma metastases: implications for immunotherapy

Because of its known heterogeneity, the analysis of antigen expression is crucial prior to the initiation of antigen-specific immunotherapy for melanoma. The melanoma differentiation antigens gp100, MART-1 and tyrosinase are involved in a common pathway of melanin synthesis. Peptides derived from these melanoma differentiation antigens are used in the immunotherapy of melanoma and antibodies recognizing these antigens are commonly applied to detect melanocytic lesions. One hundred and ninety-one paraffin-embedded melanoma metastases from 28 patients with 2-19 lesions (mean, 6.8) developing synchronously (n = 67) or asynchronously (n = 124) were analysed by immunohistochemistry for the expression of the melanoma differentiation antigens, as well as cancer/testis antigens of the melanoma antigen-A (MAGE-A) family (monoclonal antibodies 77B and 57B), anti-S100 and SM5-1. The overall reactivities were 81.6% (gp100), 79.5% (MART-1), 59.6% (tyrosinase), 59.1% (77B), 60.7% (57B), 93.2% (S100) and 91.6% (SM5-1). Twenty-seven lesions (14.1%) were positive for all tumour-associated antigens, 75 lesions (39.2%) were negative for one antigen and 87 lesions (45.5%) were negative for several tumour-associated antigens. Co-ordinated loss was found for lesions negative for gp100 and MART-1 (9.4%, P < 0.0005), gp100 and tyrosinase (11.0%, P = 0.009), MART-1 and tyrosinase (15.2%, P < 0.0005) and gp100, MART-1 and tyrosinase (8.9%, P < 0.0005), which is up to six times higher than the expected calculated loss. This co-ordinated loss of melanoma differentiation antigens in melanoma did not include cancer testis antigens and S100 or SM5-1. On average, the melanoma differentiation antigens stained 50-65% of cells within a lesion, and 10-39% of synchronous clusters were heterogeneous for melanoma differentiation antigen expression. In conclusion, broader polypeptide vaccines should be used for melanoma immunotherapy.     

Evaluation of B-cell clonality in archival skin biopsy samples of cutaneous B-cell lymphoma by immunoglobulin heavy chain gene polymerase chain reaction

Polymerase chain reaction (PCR)-based detection of clonal T- and B-cells is widely used in the diagnosis of various lymphomas, including those of the skin. A large number of corresponding methods have been published. Recently, for the first time, standardized PCR protocols were developed in common by 14 European centers of lymphoma diagnosis and research (Biomed-2 protocols). Here, we have applied Biomed-2 immunoglobulin heavy chain (IgH)-PCR for clonality detection in primary cutaneous B-cell lymphoma (CBCL) and compared it with previously established methods. The DNA of 43 paraffin-embedded lesional skin biopsies of confirmed CBCL cases [27 follicle center cell lymphoma (FCCL), 11 marginal zone B-cell lymphoma/immunocytoma (MZL/IC) and five large CBCL of the lower leg (CBCL-LL)] were amplified by the Biomed-2 IgH-PCR protocols as well as using four other assays, priming also the three IgH framework regions (FR) 1-3. All PCR products were analysed by fluorescence fragment analysis. Twenty-nine of 43 (67%) CBCL samples (5/5, 100% of CBCL-LL; six of 11, 54.5% of IC/MZL; 18 of 27, 66.7% of FCCL) showed monoclonal B-cell presence complementary in all of the IgH-PCR. The three Biomed-2 PCR indicated together clonality in 24 of 43 samples (56%). Considering each method separately, the Biomed-2 FR3-PCR showed the highest rate of clonality detection (20 of 43, 47%). In conclusion, the Biomed-2 FR3-PCR is recommended for detecting B-cell clonality in archival skin samples of CBCL but should be completed by FR1- and/or FR2-PCR.