VH gene usage and CDR3 analysis of B cell receptor in the peripheral blood of patients with PBC

We have analyzed the IgM, IgG and IgA BCR repertoire in PBC patients by means of quantitative RT-PCR and CDR3-spectratyping with immunoscope technology. PBMC from 35 PBC patients and 18 normal controls were analyzed. Quantitative B cell repertoire analysis of IgM from healthy donors showed the preferential usage of VH3a, VH3b and VH4 families. Very similar VH family usage was observed in IgM B cells from PBC patients. CDR3 spectratyping of IgM BCR rearrangements showed a Gaussian distribution for dominant VH families in control donors, and similar diversity was found for the VH3b family in PBC patients. In contrast, VH3a and VH4 families showed oligoclonal expansions in some patients. Quantitative B cell repertoire analysis of IgG and IgA did not reveal any difference in VH chain distribution in PBC patients as compared to the control donors. Immunoscope profiles of CDR3 length distribution showed several peak expansions in B cells from control donors, particularly for the VH3a and VH4 families. CDR3 length distribution profiles of IgG and IgA from PBC patients were oligoclonal too, with expansions throughout the various VH chains. However, no common expansions within the CDR3 region were found intraindividually between IgG, IgA and IgM, and between patients. In conclusion, immunoscope technology does provide, for the first time, a sensitive and rapid method for detailed immunoglobulin gene usage analysis in peripheral B cells from PBC patients. This study failed to demonstrate preferential B cell rearrangements in the blood of patients with PBC, but this technology may be more successful if applied to the analysis of compartmental B cells (i.e. liver infiltrating B cells).     

Prophylactic Insulin Treatment of Diabetes-prone BB/OK Rats by Application of a Sustained Release Insulin Implant

Normoglycemic diabetes-prone BB/OK rats aged 33, 45 or 75 days were subjected to prophylactic insulin treatment by means of a single subcutaneous application of a sustained release insulin implant. The single application of a sustained release insulin implant decreased the incidence of diabetes or delayed the onset of the disease in BB/OK rats of all treatment groups. Prophylactic insulin administration caused a transient hypoglycemic period accompanied by an inhibition of glucose stimulated insulin secretion and a decrease of the insulin content of Langerhans' islets as detectable in vitro . Compared to islets of normoglycemic controls pancreatic islets isolated from hypoglycemic BB/OK rats within 7-21 days after the insulin application at 45 days of age displayed a decreased susceptibility of the cells to complement-dependent cytotoxicity of the monoclonal islet cell surface antibody (ICSA) K14D10 but not to the cytotoxic effect of the ICSA M3aG8. The appearance of complement-dependent antibody-mediated cytotoxicity to islet cells and pancreatic exocrine cells in serum regarded as a sign of immune dysregulation in BB/OK rats seems not to be affected by insulin prophylaxis and was detectable during hypoglycemia as well as in the subsequent normoglycemic state. In conclusion, BB/OK rats of different age can be protected from diabetes by a single application of a sustained release insulin implant. Insulin and/or hypoglycemia seem to influence the expression of cell surface antigens, thus render the islets of Langerhans less vulnerable to immune cytolysis, whereas the appearance of humoral immunological abnormalites is not affected.     

High frequency of disease progression in pediatric spinal cord low-grade glioma (LGG): management strategies and results from the German LGG study group

BackgroundKnowledge on management of pediatric spinal cord low-grade glioma (LGG) is scarce.MethodsWe analyzed clinical datasets of 128 pediatric patients with spinal LGG followed within the prospective multicenter trials HIT-LGG 1996 (n = 36), SIOP-LGG 2004 (n = 56), and the subsequent LGG-Interim registry (n = 36).ResultsSpinal LGG, predominantly pilocytic astrocytomas (76%), harbored KIAA1549-BRAF fusion in 14/35 patients (40%) and FGFR1-TACC1 fusion in 3/26 patients (12%), as well as BRAFV600E mutation in 2/66 patients (3%). 10-year overall survival (OS) and event-free survival (EFS) was 93% ± 2% and 38% ± 5%, respectively. Disseminated disease (n = 16) was associated with inferior OS and EFS, while age ≥11 years and total resection were favorable factors for EFS. We observed 117 patients following total (n = 24) or subtotal/partial resection (n = 74), biopsy (n = 16), or radiologic diagnosis only (n = 3). Eleven patients were treated first with chemotherapy (n = 9) or irradiation (n = 2). Up to 20.8 years after diagnosis/initial intervention, 73/128 patients experienced one (n = 43) or up to six (n = 30) radiological/clinical disease progressions. Tumor resections were repeated in 36 patients (range, 2-6) and 47 patients required nonsurgical treatment (chemotherapy, n = 20; radiotherapy, n = 10; multiple treatment lines, n = 17). Long-term disease control for a median of 6.5 (range, 0.02-20) years was achieved in 73/77 patients following one (n = 57) or repeated (n = 16) resections, and in 35/47 patients after nonsurgical treatment.ConclusionsThe majority of patients experienced disease progression, even after years. Multiple interventions were required for more than a third, yet multimodal treatment enabled long-term disease control. Molecular testing may reveal therapeutic targets.     

Tumor–microenvironment interactions studied by zonal transcriptional profiling of squamous cell lung carcinoma

Invasion is a critical step in lung tumor progression. The interaction between tumor cells and their surroundings may play an important role in tumor invasion and metastasis. To better understand the mechanisms of tumor invasion and tumor–microenvironment interactions in lung tumors, total RNA was isolated from the inner tumor, tumor invasion front, adjacent lung, and distant normal lung tissue from 17 patients with primary squamous cell lung carcinoma using punch‐aided laser capture microdissection. Messenger RNA expression profiles were obtained by microarray analysis, and microRNA profiles were generated from eight of these samples using TaqMan Low Density Arrays. Statistical analysis of the expression data showed extensive changes in gene expression in the inner tumor and tumor front compared with the normal lung and adjacent lung tissue. Only a few genes were differentially expressed between tumor front and the inner tumor. Several genes were validated by immunohistochemistry. Evaluation of the microRNA data revealed zonal expression differences in nearly a fourth of the microRNAs analyzed. Validation of selected microRNAs by in situ hybridization demonstrated strong expression of hsa‐miR‐196a in the inner tumor; moderate expression of hsa‐miR‐224 in the inner tumor and tumor front, and strong expression of hsa‐miR‐650 in the adjacent lung tissue. Pathway analysis placed the majority of genes differentially expressed between tumor and nontumor cells in intrinsic processes associated with inflammation and extrinsic processes related to lymphocyte physiology. Genes differentially expressed between the inner tumor and the adjacent lung/normal lung tissue affected pathways of arachidonic acid metabolism and eicosanoid signaling. © 2012 Wiley Periodicals, Inc.     

Histone methyltransferase Suv39h1 deficiency prevents Myc‐induced chromosomal instability in murine myeloid leukemias

Suv39h1 mediates heterochromatin formation in pericentric and telomeric regions by trimethylation of lysine 9 of histone 3 (H3K9me3). Yet, its role in the induction of chromosomal instability is poorly understood. We established a leukemia model by retrovirally expressing Myc in wild‐type and histone methyltransferase Suv39h1‐deficient hematopoietic cells and characterized the resulting leukemias for chromosomal instability. All mice that received cells overexpressing Myc developed myeloid leukemia with a median survival of 44 days posttransplantation. Myc‐overexpressing wild‐type leukemias demonstrated clones with numerical chromosomal aberrations (5/16). In secondary transplantations of these leukemic cells, structural changes, mostly end‐to‐end fusions of chromosomes, appeared (10/12). In contrast, leukemic cells overexpressing Myc with reduced or no Suv39h1 expression had a normal karyotype in primary, secondary, and tertiary transplantations (16/16). Myc‐transduced Suv39h1‐deficient cells showed less critically short telomeres (P < 0.05) compared with Myc‐transduced wild‐type bone marrow cells. Gene expression analysis showed upregulation of genes involved in the alternative lengthening of telomeres (ALT) mechanism. Thus, we hypothesize that loss of Suv39h1 implies activation of the ALT mechanism, in turn ensuring telomere length and stability. Our data show for the first time that Suv39h1 deficiency may prevent chromosomal instability by more efficient telomere stabilization in hematopoietic bone marrow cells overexpressing Myc. © 2013 Wiley Periodicals, Inc.     

Inhibition of apoptosis by the intrinsic but not the extrinsic apoptotic pathway in myocardial ischemia-reperfusion

SummaryThe detailed molecular mechanisms following activation of apoptosis in ischemia-reperfusion injury are unknown. This study using different transgenic mouse models provided first evidence that apoptosis in myocardial ischemia-reperfusion injury is rather linked to the mitochondrial pathway than to death receptor pathway.IntroductionThere is a wealth of evidence for activation of apoptosis in ischemia-reperfusion injury. However, the understanding of detailed molecular mechanism is lacking.MethodsThe extent of myocardial infarction after ligation of the left anterior descending artery in mice carrying different transgenes for inhibition of either the intrinsic or the extrinsic or a combination of both apoptotic cascades was evaluated. The extent of myocardial damage was assessed by echocardiographic determination of left ventricular (LV) ejection fraction, LV hemodynamics, troponin T, and histology. The rate of apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and caspase-3 staining.ResultsHighest perioperative rate of death was observed in the dominant-negative form of a truncated Fas-associated death domain (FADD-DN) group. Infarction size by 2,3,5-triphenyltetrazolium chloride (TTC) staining was smaller in the Bcl-2, but not in the other groups as compared to wild-type mice. This was accompanied by lower troponin T values in Bcl-2 transgenic mice as compared to the all other groups. Troponin T correlated well with macroscopic extent of myocardial infarction by TTC staining. A lower decline of LV ejection fraction was seen in the Bcl-2 as compared to wild-type or FADD-DN mice. A smaller number of TUNEL- and caspase-3-positive myocyte nuclei were observed in the Bcl-2 and FADD-DN group as compared to wild-type mice.ConclusionsWe provide first evidence for protective effects on the myocardium in a transgenic mouse model of myocardial ischemia-reperfusion due to inhibition of the Bcl-2, but not the FADD pathway despite that reduced apoptotic cells were observed in both groups as compared to wild-type mice.     

Dissecting the Structure and Mechanism of a Complex Duplication–Triplication Rearrangement in the DMD Gene

Pathogenic complex genomic rearrangements are being increasingly characterized at the nucleotide level, providing unprecedented opportunities to evaluate the complexities of mutational mechanisms. Here, we report the molecular characterization of a complex duplication–triplication rearrangement involving exons 45–60 of the DMD gene. Inverted repeats facilitated this complex rearrangement, which shares common genomic organization with the recently described duplication‐inverted triplication–duplication (DUP–TRP/INV‐DUP) events; specifically, a 690‐kb region comprising DMD exons from 45 to 60 was duplicated in tandem, and another 46‐kb segment containing exon 51 was inserted inversely in between them. Taking into consideration (1) the presence of a predicted PRDM9 binding site in the near vicinity of the junction involving two inverted L1 elements and (2) the inherent properties of X–Y chromosome recombination during male meiosis, we proposed an alternative two‐step model for the generation of this X‐linked DMD DUP–TRP/INV‐DUP event.