Evolution of atrophied T2 lesion volume in primary-progressive multiple sclerosis: results from the phase 3 ORATORIO study.

BACKGROUND: Atrophied T2-lesion volume (aT2-LV) is an exploratory imaging marker in multiple sclerosis (MS) reflecting the volume of lesions subsumed into cerebrospinal fluid (CSF). OBJECTIVE: To investigate the effect of ocrelizumab (OCR) versus placebo (PBO) over 120 weeks on the accumulation of aT2-LV in a double-blind placebo-controlled (DBP) phase 3, primary-progressive (PP) MS study (ORATORIO; NCT01194570). METHODS: This post-hoc, MRI-blinded analysis evaluated 732 PPMS randomised to OCR (488) or PBO (244). Atrophied T2-LV was calculated by overlaying baseline T2-lesion masks on follow-up CSF maps. Clinical data from DBP and open-label extension (OLE) periods were available. Treatment effect was evaluated by a mixed-effect model with repeated measures, while logistic regression explored the association of aT2-LV at week 120 and clinical outcomes in the OLE period. RESULTS: OCR treatment significantly reduced accumulation of aT2-LV compared with PBO (319.4 mm3 vs 366.1 mm3, p=0.015) at 120 weeks. OCR showed superiority over PBO in reducing aT2-LV in patients who developed confirmed disability progression (CDP) during the DBP period at 12 (CDP12) and 24 (CDP24) weeks for the composite of Expanded Disability Status Scale (EDSS), Nine-Hole Peg Test and Timed 25-Foot Walk test. Accumulation of aT2-LV at week 120 was related to CDP12-EDSS (p=0.018) and CDP24-EDSS (p=0.022) in the OLE for the patients who were treated by PBO in the DBP only. CONCLUSIONS: OCR showed a significant effect of reducing the accumulation of aT2-LV in PPMS in the DBP period and was related to CDP-EDSS in OLE only in the PBO arm.

Site-directed mutagenesis of large DNA palindromes: construction and in vitro characterization of herpes simplex virus type 1 mutants containing point mutations that eliminate the oriL or oriS initiation function.

Technical challenges associated with mutagenesis of the large oriL palindrome have hindered comparisons of the functional roles of the herpes simplex virus type 1 (HSV-1) origins of DNA replication, oriL and oriS, in viral replication and pathogenesis. To address this problem, we have developed a novel PCR-based strategy to introduce site-specific mutations into oriL and other large palindromes. Using this strategy, we generated three plasmids containing mutant forms of oriL, i.e., pDoriL-I(L), pDoriL-I(R), and pDoriL-I(LR), containing point mutations in the left, right, and both copies, respectively, of the origin binding protein (OBP) binding site (site I) which eliminate OBP binding. In in vitro DNA replication assays, plasmids with mutations in only one arm of the palindrome supported origin-dependent DNA replication, whereas plasmids with symmetrical mutations in both arms of the palindrome were replication incompetent. An analysis of the cloned mutant plasmids used in replication assays revealed that a fraction of each plasmid mutated in only one arm of the palindrome had lost the site I mutation. In contrast, plasmids containing symmetrical mutations in both copies of site I retained both mutations. These observations demonstrate that the single site I mutations in pDoriL-I(L) and pDoriL-I(R) are unstable upon propagation in bacteria and suggest that functional forms of both the left and right copies of site I are required to initiate DNA replication at oriL. To examine the role of oriL and oriS site I in virus replication, we introduced the two site I mutations in pDoriL-I(LR) into HSV-1 DNA to yield the mutant virus DoriL-I(LR) and the same point mutations into the single site I sequence present in both copies of oriS to yield the mutant virus DoriS-I. In Vero cells and primary rat embryonic cortical neurons (PRN) infected with either mutant virus, viral DNA synthesis and viral replication were efficient, confirming that the two origins can substitute functionally for one another in vitro. Measurement of the levels of oriL and oriS flanking gene transcripts revealed a modest alteration in the kinetics of ICP8 transcript accumulation in DoriL-I(LR)-infected PRN, but not in Vero cells, implicating a cell-type-specific role for oriL in regulating ICP8 transcription.

Naive B lymphocytes undergo homeostatic proliferation in response to B cell deficit.

Naive peripheral B cells are maintained in sufficient numbers and diversity to mount effective immune responses against infectious agents. However, the size and repertoire of this B cell pool is constantly diminished by normal cell turnover and Ag activation. Homeostatic (Ag-independent) proliferation in response to B cell depletion is one mechanism to compensate for this cell loss. We have used purified CFSE-labeled B cells and an adoptive transfer model system to show that immature and mature B cells divide in a variety of B cell-deficient (scid, xid, IL-7(-/-), and sublethally irradiated) hosts. Homeostatic B cell proliferation is T cell independent, and B cells that have replicated by this mechanism retain the antigenic phenotype of naive B cells. Replication is significantly reduced in B cell-sufficient normal or B cell-reconstituted immunodeficient recipients by the action of competing mature follicular B cells. Using xid mice and transcription factor knockouts, we show that the activation signal(s) that lead to homeostatic B cell proliferation require Bruton's tyrosine kinase; however, c-Rel, a Bruton's tyrosine kinase-induced NF-kappaB/Rel transcription factor critical for Ag and mitogen stimulation, is dispensable, indicating the uniqueness of this activation pathway. Survival and replication signals can also be separated, because the transcription factor p50 (NF-kappaB1), which is required for the survival of peripheral B cells, is not necessary for homeostatic replication. Homeostatic B cell proliferation provides an Ag-independent mechanism for the maintenance and expansion of naive B cells selected into the mature B cell pool.