Reflection of neuroblastoma intratumor heterogeneity in the new OHC-NB1 disease model

Accurate modeling of intratumor heterogeneity presents a bottleneck against drug testing. Flexibility in a preclinical platform is also desirable to support assessment of different endpoints. We established the model system, OHC-NB1, from a bone marrow metastasis from a patient diagnosed with MYCN-amplified neuroblastoma and performed whole-exome sequencing on the source metastasis and the different models and passages during model development (monolayer cell line, 3D spheroid culture and subcutaneous xenograft tumors propagated in mice). OHC-NB1 harbors a MYCN amplification in double minutes, 1p deletion, 17q gain and diploid karyotype, which persisted in all models. A total of 80–540 single-nucleotide variants (SNVs) was detected in each sample, and comparisons between the source metastasis and models identified 34 of 80 somatic SNVs to be propagated in the models. Clonal reconstruction using the combined copy number and SNV data revealed marked clonal heterogeneity in the originating metastasis, with four clones being reflected in the model systems. The set of OHC-NB1 models represents 43% of somatic SNVs and 23% of the cellularity in the originating metastasis with varying clonal compositions, indicating that heterogeneity is partially preserved in our model system.     

Comparison of three different purification methods for the routine preparation of [11C] Metomidate.

PET with (R)-[O-methyl-11C] metomidate ([11C] MTO) is an attractive method for the characterisation of adrenal masses discriminating lesions of adrenal cortical origin from noncortical lesions. [11C] MTO was prepared by the reaction of [11C] methyliodide with the corresponding free acid. Three purification methods have been compared. The method of choice uses preparative HPLC with a ready-to-use weak acidic solvent.     

Reconstruction of the anterior cruciate ligament with a patella-tendon-bone graft may lead to a permanent loss of bone mineral content due to decreased patellar tendon stiffness

Immobilisation induces bone loss. Evidence from studies in animals and healthy humans that were immobilised for a limited time indicates that, in general, bone mass may be restored even in adults. Following conservative management of partial tears of the anterior cruciate ligament (ACL), bone loss is often negligible (2-3%). After surgical reconstruction, however, there is greater bone loss (15-20%), with little or no recovery. Bones adapt to the stresses they experience. Also, the largest forces in the musculoskeletal system arise from muscle pull. Tendons transmit these forces. Many surgical techniques for ACL reconstruction use autologous tendon grafts. We hypothesise that tissue harvesting causes weakening of the formerly intact tendon, which, in turn, leads to reduced muscle pull and subsequent bone loss in those parts of the bone that are loaded by the tendon. If our hypothesis holds true, it may change patients' and surgeons' choice of management. Clinical follow-up should assess the functional result with greater scrutiny, possibly including the assessment of bone mineral content. This may be particularly important since there is accumulating evidence that a decrease in bone mineral density (BMD) preceedes, and hence may be a cause of, osteoarthritis.     

Emergence of translocation t(9;11)-positive leukemia during treatment of childhood acute lymphoblastic leukemia

Therapy-related acute myeloid leukemia (t-AML) characterized by the t(9;11)(p22;q23) translocation is one of the most frequent secondary malignancies. The timing of the initiation of translocation and of development of the malignant t(9;11) clone during chemotherapy is presently unknown. In the present study, we backtracked bone marrow samples from three children during treatment for acute lymphoblastic leukemia (ALL). Two patients developed a t(9;11)-positive t-AML 19 and 30 months after therapy start, whereas the third patient, diagnosed with a rare t(9;11)-positive ALL, suffered from an ALL relapse 23 months after initial diagnosis. The genomic MLL-MLLT3 (MLL-AF9) fusion site was amplified by a multiplex, nested long-range PCR and used as a clonal marker for quantification of the MLL-MLLT3-positive cells during chemotherapy. The t(9;11)-positive clone was detectable 13 and 18 months after therapy start in both t-AML cases, which was 6-12 months before clinical diagnosis of the secondary malignancy. In the t(9;11)-positive ALL patient, the identical leukemic clone reoccurred during maintenance therapy after a short molecular remission, 8 months before clinically overt ALL relapse. The time course and characteristics of the genomic breakpoints in the present t-AML cases support the hypothesis of translocation formation as a result of defective breakage repair after topoisomerase II cleavage.     

Gene Deletions in Mycobacterium bovis BCG Stimulate Increased CD8+ T Cell Responses

Mycobacteria, the etiological agents of tuberculosis and leprosy, have coevolved with mammals for millions of years and have numerous ways of suppressing their host''s immune response. It has been suggested that mycobacteria may contain genes that reduce the host''s ability to elicit CD8⁺ T cell responses. We screened 3,290 mutant Mycobacterium bovis bacillus Calmette Guerin (BCG) strains to identify genes that decrease major histocompatibility complex (MHC) class I presentation of mycobacterium-encoded epitope peptides. Through our analysis, we identified 16 mutant BCG strains that generated increased transgene product-specific CD8⁺ T cell responses. The genes disrupted in these mutant strains had disparate predicted functions. Reconstruction of strains via targeted deletion of genes identified in the screen recapitulated the enhanced immunogenicity phenotype of the original mutant strains. When we introduced the simian immunodeficiency virus (SIV) gag gene into several of these novel BCG strains, we observed enhanced SIV Gag-specific CD8⁺ T cell responses in vivo. This study demonstrates that mycobacteria carry numerous genes that act to dampen CD8⁺ T cell responses and suggests that genetic modification of these genes may generate a novel group of recombinant BCG strains capable of serving as more effective and immunogenic vaccine vectors.     

The variation of morphological and functional cardiac manifestation in Fabry disease: potential implications for the time course of the disease.

AIMS: The aim of this clinical cross-sectional study was to investigate the cardiac interrelation of morphological and functional abnormalities in patients with Fabry disease. METHODS AND RESULTS: Fifty-one patients (5-78 years) were compared with 25 controls (8-77 years). In all subjects, end-diastolic thickness of the left ventricle was measured by echocardiography and ultrasonic peak systolic strain rate (SR) was extracted to assess regional myocardial function. Magnetic resonance imaging was performed to assess late-enhancement for the detection of myocardial fibrosis in Fabry patients (n=39). In patients, women <20 years of age had no hypertrophy, no late-enhancement, and normal radial and longitudinal function (SR longitudinal=-1.7+/-0.5 s(-1); P=n.s. compared with controls). Ten women, >20 years of age, had no hypertrophy, no late-enhancement, normal radial and longitudinal function in the septal wall, but reduced longitudinal function in the lateral wall (SR=-1.4+/-0.5 s(-1)). All male patients without hypertrophy and no late-enhancement had normal radial function but reduced longitudinal function in both the septal and lateral walls (SR=-1.3+/-0.3 s(-1)). Patients with hypertrophy but without late-enhancement (n=13) had reduced radial and longitudinal function. Twelve patients displaying hypertrophy and late-enhancement had severely reduced radial and longitudinal function (SR=-1.1+/-0.5 s(-1)). Two of them with the worst impairment of regional function (SR=-0.8+/-0.6 s(-1)) died in the follow-up period. CONCLUSION: These results illustrate the variation of morphological changes and its functional consequences in Fabry cardiomyopathy.     

Simplifying the digital workflow of facial prostheses manufacturing using a three-dimensional (3D) database: setup,development, and aspects of virtual data validation for reproduction

PurposeTo set up the digital database (DDB) of various anatomical parts, skin details and retention elements in order to simplify the digital workflow of facial prostheses manufacturing; and to quantify the reproduction of skin wrinkles on the prostheses prototypes with stereolithography (SLA) and direct light processing (DLP) methods.MethodsTwo structured light scanners were used to obtain the nasal and auricle forms of 50 probands. Furthermore, the ala nasi and scapha areas were captured with the digital single lens reflex camera and saved in jpeg format. The four magnetic retention elements were remodeled in computer aided design (CAD) software. The 14 test blocks with embossed wrinkles of 0.05–0.8 mm were printed with SLA and DLP methods and afterwards analyzed by means of profilometry and confocal microscopy.ResultsThe introduced DDB allows for production of customized facial prosthesis and makes it possible to consider the integration of concrete retention elements on the CAD stage, which makes the prosthesis modelling more predictable and efficient. The obtained skin structures can be applied onto the prosthesis surface for customization. The reproduction of wrinkles from 0.1 to 0.8 mm in depth may be associated with the loss of 4.5%–11% of its profile with SLA or DLP respectively. Besides, the reproduction of 0.05 mm wrinkles may be met with up to 40% profile increasement.ConclusionsThe utilization of DDB may simplify the digital workflow of facial prostheses manufacturing. The transfer of digitally applied skin wrinkles till the prostheses’ prototypes may be associated with deviations from 11 to 40%.