Functional Assessment of TSC2 Variants Identified in Individuals with Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in the TSC1 or TSC2 genes. The TSC1 and TSC2 gene products, TSC1 and TSC2, form a complex that inhibits the mammalian target of rapamycin (mTOR) complex 1 (TORC1). Here, we investigate the effects of 78 TSC2 variants identified in individuals suspected of TSC, on the function of the TSC1–TSC2 complex. According to our functional assessment, 40 variants disrupted the TSC1–TSC2‐dependent inhibition of TORC1. We classified 34 of these as pathogenic, three as probably pathogenic and three as possibly pathogenic. In one case, a likely effect on splicing as well as an effect on function was noted. In 15 cases, our functional assessment did not agree with the predictions of the SIFT amino acid substitution analysis software. Our data support the notion that different, nonterminating TSC2 mutations can have distinct effects on TSC1–TSC2 function, and therefore, on TSC pathology.     

Simulated dive in rats lead to acute changes in cerebral blood flow on MRI, but no cerebral injuries to grey or white matter

In this study, the effect of a simulated dive on rat brain was investigated using several magnetic resonance imaging (MRI)-methods and immunohistochemistry. Rats were randomly assigned to a dive- or a control group. The dive group was exposed to a simulated air dive to 600 kPa for 45 min. Pulmonary artery was monitored for vascular gas bubbles by ultrasound. MRI was performed 1 h after decompression and at one and 2 weeks after the dive with a different combination of MRI sequences at each time point. Two weeks after decompression, rats were sacrificed and brains were prepared for histology. Dived rats had a different time-curve for the dynamic contrast-enhanced MRI signal than controls with higher relative signal intensity, a tendency towards longer time to peak and a larger area under the curve for the whole brain on the acute MRI scan. On MRI, 1 and 2 weeks after dive, T₂-maps showed no signal abnormalities or morphological changes. However, region of interest based measurements of T₂ showed higher T₂ in the brain stem among decompressed animals than controls after one and 2 weeks. Microscopical examination including immunohistochemistry did not reveal apparent structural or cellular injuries in any part of the rat brains. These observations indicate that severe decompression does not seem to cause any structural or cellular injury to the brain tissue of the rat, but may cause circulatory changes in the brain perfusion in the acute phase.     

Dendritic cells fused with core binding factor-beta positive acute myeloid leukaemia blast cells induce activation of cytotoxic lymphocytes

Several reports have described various strategies of dendritic cell (DC) vaccination to induce specific T-cell responses in patients with acute myeloid leukaemia (AML). About 50-60% of AML cases blasts have chromosomal abnormalities, such as inv(16) or t(8,21), which could encode for leukaemia-specific antigenic peptide sequences, possibly presented in the context of self-major histocompatibility complex (MHC) molecules. As the co-culture of AML blasts with T lymphocytes seldom resulted in T-cell stimulation, we fused AML blasts with autologous DC to enhance this effect. The fusion cells expressed MHC class I and II, CD40, B7-1, B7-2, CD209 and several adhesion molecules. In a mixed lymphocyte hybrid reaction, the fusion cells induced the proliferation of autologous T cells. Moreover, in the special case of fusion cells established from AML blasts with the chromosomal abnormality inv(16), the autologous T lymphocytes could be primed to induce cytotoxicity against up to 70% autologous AML blasts in a effector:target ratio of 20:1. Blocking assays demonstrated that the lysis was chiefly mediated by CD8(+), CCR7(-) T lymphocytes, which could be further expanded in the form of effector memory CD8(+) T cells by repeated co-cultures with the autologous fusion cells.