In vivo intervertebral disc mechanical deformation following a treadmill walking "stress test" is inversely related to T1rho relaxation time.

OBJECTIVE: To assess the in vivo relationship between the mechanical response of intervertebral discs (IVDs) to dynamic activity and IVD biochemical composition assessed via T1rho relaxation imaging. DESIGN: Eighteen asymptomatic participants with no history of low back pain (LBP), injury, or surgery underwent magnetic resonance (MR) imaging of their lumbar spine prior to and immediately following a treadmill walking "stress test." Anatomic (SPACE, FLASH) MR images were obtained pre- and post-exercise and utilized to measure IVD mechanical deformation. Quantitative (T1rho) imaging was performed pre-exercise to reflect IVD composition. Pre-exercise anatomic images were also utilized to assess IVD degenerative status based on the modified Pfirrmann scale. To quantify mechanical response, 3D surface models of the L1-L2-L5-S1 IVDs were created from manual segmentations of pre- and post-exercise anatomic images and utilized to assess changes in IVD height. IVD strain (%) was defined as change in IVD height normalized to pre-activity height. Linear mixed models were used to assess the relationships between IVD mechanical deformation (strain), composition (T1rho relaxation time), and degenerative status (Pfirrmann grade). RESULTS: Increased compressive IVD strain was associated with lower T1rho relaxation times in the nucleus pulposus (NP) of the disc (ßT1rho=5.07,CI:[1.52,7.77],Rmarg2=0.52,p=0.005). Thus, an inverse relationship between IVD strain and NP T1rho relaxation time was observed. CONCLUSION: The in vivo mechanical response of the IVD to the "stress test" was sensitive to differences in NP composition. The results of this study suggest that quantification of in vivo IVD mechanical function and composition may provide insight into IVD health.

Deep intronic 'mutations' cause hemophilia A: application of next generation sequencing in patients without detectable mutation in F8 cDNA.

BACKGROUND: In a small group of typical hemophilia A (HA) patients no mutations in the F8 coding sequence (cDNA) could be found. In the current study, we performed a systematic screening of genetic and non-genetic parameters associated with reduced FVIII:C levels in a group of mostly mild HA (only one moderate) patients with no detectable mutations in F8 cDNA. METHODS: We determined FVIII and VWF activity and antigen levels and performed VWF-FVIII binding (VWF:FVIIIB) and VWF-collagen binding assays (VWF:CB) as well as VWF multimer analysis. VWF was completely sequenced to exclude mutations. The F8 locus, including the introns, was sequenced using overlapping long-range PCRs (LR-PCRs) combined with a next generation sequencing (NGS) approach. Moreover, the F8 mRNA was analyzed quantitatively and qualitatively by real-time PCR (qRT) and overlapping reverse transcription (RT) PCRs, respectively. RESULTS: All VWF tests were normal. The LR-PCRs demonstrated the integrity of the F8 locus. Eight unique polymorphisms were found in the patients, with two being recurrent. Furthermore, RT-PCRs analysis confirmed that two of the unique variants create detectable new cryptic splice sites in the patients that result in the introduction of intronic DNA sequences into the mRNA and create premature stop codons. CONCLUSION: By systematically excluding all possible causes of HA, we could with great certainty conclude that deep intronic mutations in F8, although rare, cause abnormal mRNA splicing, leading to mild HA.

Comparative performance of mildly impaired patients with Alzheimer's disease and multiple cerebral infarctions on tests of memory and functional capacity.

There is increasing evidence that mildly impaired patients with Alzheimer's disease (AD) may be distinguished from mildly impaired patients with multi-infarct cognitive disorder (MICD) by their degree of semantic memory impairment. However, despite these observed group differences, it is unknown whether AD and MICD patients differ in their ability to perform a broad array of functional activities required for daily living and the degree to which severity of cognitive impairment is associated with functional deficits. Using a measure assessing numerous functional domains within the clinical setting, we were able to directly compare the functional capacity of mildly impaired AD and MICD patients, as well as a more cognitively impaired AD group. Although mildly impaired AD patients scored significantly lower on tests of semantic memory relative to their mildly impaired MICD counterparts, deficits in functional capacity were relatively equivalent. The AD group with more severe cognitive impairment scored lower on both memory and functional measures. A relatively high proportion of mildly impaired AD and MICD subjects evidenced impairment across a number of functional domains, suggesting that functional impairment may occur with relatively high frequency in these patient groups.

Immunophenotyping of mitotic cells from long-term cultures of chorionic villi.

Chromosomal mosaicism in chorionic villus samples (CVS) may arise from different sources, such as clonal diversity within the chorionic tissue or contamination with maternal cells. To determine the origin of karyotyped cells, we compared the immunocytochemical features of mitotic cells in CVS long-term cultures with histological sections of their tissue of origin, i.e. chorionic villi. Immunolabelling of intermediate filaments specific for epithelial cells (cytokeratin) and mesenchymal cells (vimentin) established that mitoses yielded from CVS long-term cultures indeed stem from independently growing clones derived from both the epithelial and mesenchymal parts of the chorionic villi. Thus, mosaicism in CVS cultures may reflect true genetic heterogeneity within the biopsy. However, epithelial chorionic cells undergo in vitro metaplasia leading to co-expression of cytokeratins and vimentin. Fetal-specific immune markers (beta-HCG and SP1-glycoprotein) are not reliably expressed in CVS cell culture.