Low-Input RNA-Sequencing in Patients with Cartilage Lesions, Osteoarthritis, and Healthy Cartilage.

OBJECTIVE: To analyze and compare cartilage samples from 3 groups of patients utilizing low-input RNA-sequencing. DESIGN: Cartilage biopsies were collected from patients in 3 groups (n = 48): Cartilage lesion (CL) patients had at least ICRS grade 2, osteoarthritis (OA) samples were taken from patients undergoing knee replacement, and healthy cartilage (HC) was taken from ACL-reconstruction patients without CLs. RNA was isolated using an optimized protocol. RNA samples were assessed for quality and sequenced with a low-input SmartSeq2 protocol. RESULTS: RNA isolation yielded 48 samples with sufficient quality for sequencing. After quality control, 13 samples in the OA group, 9 in the HC group, and 9 in the CL group were included in the analysis. There was a high degree of co-clustering between the HC and CL groups with only 6 genes significantly up- or downregulated. OA and the combined HC/CL group clustered significantly separate from each other, yielding 659 significantly upregulated and 1,369 downregulated genes. GO-term analysis revealed that genes matched to cartilage and connective tissue development terms. CONCLUSION: The gene expression profiles from the 3 groups suggest that there are no major differences in gene expression between cartilage from knees with a cartilage injury and knees without an apparent cartilage injury. OA cartilage, as expected, showed markedly different gene expression from the other 2 groups. The gene expression profiles resulting from this low-input RNA-sequencing study offer opportunities to discover new pathways not previously recognized that may be explored in future studies.

WT1 and interferon-ß-vitamin D association in MS: a longitudinal study.

BACKGROUND: It has been suggested that polymorphisms in the WT1 gene modulate the effect of IFN-ß treatment in multiple sclerosis (MS) through regulation of the relationship between IFN-ß and vitamin D. OBJECTIVE: To examine whether WT1 modulates the relationship between IFN-ß and vitamin D in a longitudinal study with repeated assessment of vitamin D before and after initiation of IFN-ß. METHODS: In a prospective study of 85 patients with relapsing remitting MS, 25-hydroxyvitamin D was measured at month 0, 1, 3, 6, 7, 9, 12, 18 and 24. None of the patients used any immunomodulatory treatment at inclusion, and all started IFN-ß treatment at month 6. RESULTS: The mean concentrations of seasonally adjusted 25-hydroxyvitamin increased slightly (3.1 ± 1.2 nmol/l, P = 0.008) after initiation of IFN-ß. The association between IFN-ß treatment and 25-hydroxyvitamin D was similar in patients carrying any of the two alleles in the WT1 SNPs (rs10767935 and rs5030244) recently reported to modulate this relationship. CONCLUSIONS: In this prospective study with repeated measurements of 25-hydroxyvitamin D before and during treatment with IFN-ß, we did not find that genetic variation in WT1 plays any role in regulating the relationship between IFN-ß and serum 25-hydroxyvitamin D.

Subcloning and characterization of the binding domain of fragment B of diphtheria toxin.

The binding domain (R domain) of diphtheria toxin as defined from the recently published crystal structure [Choe, Bennett, Fujii, Curmi, Kantardjieff, Collier and Eisenberg (1992) Nature (London) 357, 216-222] was subcloned. The 17 kDa peptide containing amino acids 378-535 from fragment B of diphtheria toxin preceded by the tripeptide Met-His-Gly bound specifically and with high affinity to diphtheria-toxin receptors. It efficiently inhibited the toxicity of full-length toxin. The binding domain entered the detergent phase of Triton X-114 at pH values below 6, indicating that it exposed hydrophobic regions at acidic pH.