Sequence and Copy Number Analyses of HEXB Gene in Patients Affected by Sandhoff Disease: Functional Characterization of 9 Novel Sequence Variants

Sandhoff disease (SD) is a lysosomal disorder caused by mutations in the HEXB gene. To date, 43 mutations of HEXB have been described, including 3 large deletions. Here, we have characterized 14 unrelated SD patients and developed a Multiplex Ligation-dependent Probe Amplification (MLPA) assay to investigate the presence of large HEXB deletions. Overall, we identified 16 alleles, 9 of which were novel, including 4 sequence variation leading to aminoacid changes [c.626C>T (p.T209I), c.634C>A (p.H212N), c.926G>T (p.C309F), c.1451G>A (p.G484E)] 3 intronic mutations (c.1082+5G>A, c.1242+1G>A, c.1169+5G>A), 1 nonsense mutation c.146C>A (p.S49X) and 1 small in-frame deletion c.1260_1265delAGTTGA (p.V421_E422del). Using the new MLPA assay, 2 previously described deletions were identified. In vitro expression studies showed that proteins bearing aminoacid changes p.T209I and p.G484E presented a very low or absent activity, while proteins bearing the p.H212N and p.C309F changes retained a significant residual activity. The detrimental effect of the 3 novel intronic mutations on the HEXB mRNA processing was demonstrated using a minigene assay. Unprecedentedly, minigene studies revealed the presence of a novel alternative spliced HEXB mRNA variant also present in normal cells. In conclusion, we provided new insights into the molecular basis of SD and validated an MLPA assay for detecting large HEXB deletions.     

Immortalization of antigen specific, helper T cell lines by transformation with the radiation leukemia virus (RadLV)

Antigen-specific immune T lymphocytes of male C57BL/6 mice were enriched in vitro on monolayers of antigen-pulsed syngeneic macrophages. The cells were treated in vitro with RadLV and inoculated intrathymically into irradiated female C56BL/6 animals. Thymomas arising in the inoculated recipients were characterized as donor- (male) type according to their karyotype. In vivo and in vitro cell lines were established from the primary lymphomas, two of which (designated ROT/6.1 and ROT/6.2) were capable of providing antigen- (carrier) specific help in normal or preimmunized mice. None of the lymphomas could induce antigen-specific DTH reaction. Five months after their establishment, ROT/6.2 alone retained its carrier specificity. ROT/6.2 consisted mainly of Lyt-1+ cells, whereas the ROT/6.1 population was more heterogeneous and contained Lyt-1+, Lyt-2+, and Lyt-3+ cells. The carrier specificity of the latter may have been lost due to selection against the specific helper cells during prolonged passages.     

Beta7 integrin deficiency suppresses B cell homing and attenuates chronic ileitis in SAMP1/YitFc mice

Lymphocyte recruitment to intestinal tissues depends on β(7) integrins. In this study, we studied disease severity and lymphocyte recruitment into the small intestine in SAMP1/YitFc mice, which develop chronic ileitis with similarity to human Crohn's disease. To assess the role of β(7) integrins in chronic ileitis, we generated SAMP1/YitFc lacking β(7) integrins (SAMP1/YitFc Itgb7(-/-)) using a congenic strain developed via marker-assisted selection. We analyzed ileal inflammation in SAMP1/YitFc and SAMP1/YitFc Itgb7(-/-) mice by histopathology and the distribution of T and B lymphocytes in the mesenteric lymph nodes (MLNs) by flow cytometry. Short-term (18 h) adoptive transfer experiments were used to study the in vivo homing capacity of T and B lymphocytes. In both young (<20 wk) and old (20-50 wk) SAMP1/YitFc Itgb7(-/-) mice, ileitis was reduced by 30-50% compared with SAMP1/YitFc mice. SAMP1/YitFc Itgb7(-/-) mice showed a dramatic 67% reduction in the size of their MLNs, which was caused by a 85% reduction in lymphocyte numbers and reduced short-term B cell homing. Flow cytometric analysis revealed a highly significant decrease in the percentage of B cells in MLNs of SAMP1/YitFc Itgb7(-/-) mice. Cotransfer of SAMP1/YitFc MLN B cells but not SAMP1/YitFc Itgb7(-/-) MLN B cells along with CD4(+) T cells resulted in exacerbated ileitis severity in SCID mice. Our findings suggest that β(7) integrins play an essential role in spontaneous chronic ileitis in vivo by promoting homing of disease-exacerbating B cells to MLNs and other intestinal tissues.     

A new trogiid genus from lower Eocene Fushun amber (Insecta: Psocodea: Trogiomorpha)

A new fossil brachypterous psocid tax on belonging to the family Trogiidae is characterized, illustrated and described from the early Eocene Fushun amber. Paralepinotus fushunensis nov. gen., nov. sp., shares several characters with the genus Lepinotus von Heyden, 1850, but differs from it mainly by the presence of tibial spurs other than apical ones and the absence of a forked sensillum on the last maxillary palp. This fossil taxon is also characterized by relatively developed elytriform forewing but still not reaching the tip of the abdomen. A checklist of all known Trogiidae is provided.