The N-terminus of hTERT contains a DNA-binding domain and is required for telomerase activity and cellular immortalization

Telomerase defers the onset of telomere damage-induced signaling and cellular senescence by adding DNA onto chromosome ends. The ability of telomerase to elongate single-stranded telomeric DNA depends on the reverse transcriptase domain of TERT, and also relies on protein:DNA contacts outside the active site. We purified the N-terminus of human TERT (hTEN) from Escherichia coli, and found that it binds DNA with a preference for telomeric sequence of a certain length and register. hTEN interacted with the C-terminus of hTERT in trans to reconstitute enzymatic activity in vitro. Mutational analysis of hTEN revealed that amino acids Y18 and Q169 were required for telomerase activity in vitro, but not for the interaction with telomere DNA or the C-terminus. These mutants did not reconstitute telomerase activity in cells, maintain telomere length, or extend cellular lifespan. In addition, we found that T116/T117/S118, while dispensable in vitro, were required for cellular immortalization. Thus, the interactions of hTEN with telomere DNA and the C-terminus of hTERT are functionally separable from the role of hTEN in telomere elongation activity in vitro and in vivo, suggesting other roles for the protein and nucleic acid interactions of hTEN within, and possibly outside, the telomerase catalytic core

Distribution and efficacy of ofatumumab and ocrelizumab in humanized CD20 mice following subcutaneous or intravenous administration

Approval of B-cell-depleting therapies signifies an important advance in the treatment of multiple sclerosis (MS). However, it is unclear whether the administration route of anti-CD20 monoclonal antibodies (mAbs) alters tissue distribution patterns and subsequent downstream effects. This study aimed to investigate the distribution and efficacy of radiolabeled ofatumumab and ocrelizumab in humanized-CD20 (huCD20) transgenic mice following subcutaneous (SC) and intravenous (IV) administration. For distribution analysis, huCD20 and wildtype mice (n = 5 per group) were imaged by single-photon emission computed tomography (SPECT)/CT 72 h after SC/IV administration of ofatumumab or SC/IV administration of ocrelizumab, radiolabeled with Indium-111 (111In-ofatumumab or 111In-ocrelizumab; 5 µg, 5 MBq). For efficacy analysis, huCD20 mice with focal delayed-type hypersensitivity lesions and associated tertiary lymphoid structures (DTH-TLS) were administered SC/IV ofatumumab or SC/IV ocrelizumab (7.5 mg/kg, n = 10 per group) on Days 63, 70 and 75 post lesion induction. Treatment impact on the number of CD19+ cells in select tissues and the evolution of DTH-TLS lesions in the brain were assessed. Uptake of an 111In-labelled anti-CD19 antibody in cervical and axillary lymph nodes was also assessed before and 18 days after treatment initiation as a measure of B-cell depletion. SPECT/CT image quantification revealed similar tissue distribution, albeit with large differences in blood signal, of 111In-ofatumumab and 111In-ocrelizumab following SC and IV administration; however, an increase in both mAbs was observed in the axillary and inguinal lymph nodes following SC versus IV administration. In the DTH-TLS model of MS, both treatments significantly reduced the 111In-anti-CD19 signal and number of CD19+ cells in select tissues, where no differences between the route of administration or mAb were observed. Both treatments significantly decreased the extent of glial activation, as well as the number of B- and T-cells in the lesion following SC and IV administration, although this was mostly achieved to a greater extent with ofatumumab versus ocrelizumab. These findings suggest that there may be more direct access to the lymph nodes through the lymphatic system with SC versus IV administration. Furthermore, preliminary findings suggest that ofatumumab may be more effective than ocrelizumab at controlling MS-like pathology in the brain

The challenge of indication extrapolation for infliximab biosimilars

AbstractA biosimilar is intended to be highly similar to a reference biologic such that any differences in quality attributes (i.e., molecular characteristics) do not affect safety or efficacy. Achieving this benchmark for biologics, especially large glycoproteins such as monoclonal antibodies, is challenging given their complex structure and manufacturing. Regulatory guidance on biosimilars issued by the U.S. Food and Drug Administration, Health Canada and European Medicines Agency indicates that, in addition to a demonstration of a high degree of similarity in quality attributes, a reduced number of nonclinical and clinical comparative studies can be sufficient for approval. Following a tiered approach, clinical studies are required to address concerns about possible clinically significant differences that remain after laboratory and nonclinical evaluations. Consequently, a critical question arises: can clinical studies that satisfy concerns regarding safety and efficacy in one condition support “indication extrapolation” to other conditions? This question will be addressed by reviewing the case of a biosimilar to infliximab that was approved recently in South Korea, Europe, and Canada for multiple indications through extrapolation. The principles discussed should also apply to biosimilars of other monoclonal antibodies that are approved to treat multiple distinct conditions

Determination of CSF GFAP, CCN5, and vWF Levels Enhances the Diagnostic Accuracy of Clinically Defined MS From Non-MS Patients With CSF Oligoclonal Bands

BackgroundInclusion of cerebrospinal fluid (CSF) oligoclonal IgG bands (OCGB) in the revised McDonald criteria increases the sensitivity of diagnosis when dissemination in time (DIT) cannot be proven. While OCGB negative patients are unlikely to develop clinically definite (CD) MS, OCGB positivity may lead to an erroneous diagnosis in conditions that present similarly, such as neuromyelitis optica spectrum disorders (NMOSD) or neurosarcoidosis.ObjectiveTo identify specific, OCGB-complementary, biomarkers to improve diagnostic accuracy in OCGB positive patients.MethodsWe analysed the CSF metabolome and proteome of CDMS (n=41) and confirmed non-MS patients (n=64) comprising a range of CNS conditions routinely encountered in neurology clinics.ResultsOCGB discriminated between CDMS and non-MS with high sensitivity (85%), but low specificity (67%), as previously described. Machine learning methods revealed CCN5 levels provide greater accuracy, sensitivity, and specificity than OCGB (79%, +5%; 90%, +5%; and 72%, +5% respectively) while glial fibrillary acidic protein (GFAP) identified CDMS with 100% specificity (+33%). A multiomics approach improved accuracy further to 90% (+16%).ConclusionThe measurement of a few additional CSF biomarkers could be used to complement OCGB and improve the specificity of MS diagnosis when clinical and radiological evidence of DIT is absent

Telomere Maintenance and Survival in Saccharomyces cerevisiae in the Absence of Telomerase and RAD52

Telomeres are essential features of linear genomes that are crucial for chromosome stability. Telomeric DNA is usually replenished by telomerase. Deletion of genes encoding telomerase components leads to telomere attrition with each cycle of DNA replication, eventually causing cell senescence or death. In the Saccharomyces cerevisiae strain W303, telomerase-null populations bypass senescence and, unless EXO1 is also deleted, this survival is RAD52 dependent. Unexpectedly, we found that the S. cerevisiae strain S288C could survive the removal of RAD52 and telomerase at a low frequency without additional gene deletions. These RAD52-independent survivors were propagated stably and exhibited a telomere organization typical of recombination between telomeric DNA tracts, and in diploids behaved as a multigenic trait. The polymerase-δ subunit Pol32 was dispensable for the maintenance of RAD52-independent survivors. The incidence of this rare escape was not affected by deletion of other genes necessary for RAD52-dependent survival, but correlated with initial telomere length. If W303 strains lacking telomerase and RAD52 first underwent telomere elongation, rare colonies could then bypass senescence. We suggest that longer telomeres provide a more proficient substrate for a novel telomere maintenance mechanism that does not rely on telomerase, RAD52, or POL32

Determination of CSF GFAP, CCN5, and vWF Levels Enhances the Diagnostic Accuracy of Clinically Defined MS From Non-MS Patients With CSF Oligoclonal Bands.

Peer reviewed: TrueBACKGROUND: Inclusion of cerebrospinal fluid (CSF) oligoclonal IgG bands (OCGB) in the revised McDonald criteria increases the sensitivity of diagnosis when dissemination in time (DIT) cannot be proven. While OCGB negative patients are unlikely to develop clinically definite (CD) MS, OCGB positivity may lead to an erroneous diagnosis in conditions that present similarly, such as neuromyelitis optica spectrum disorders (NMOSD) or neurosarcoidosis. OBJECTIVE: To identify specific, OCGB-complementary, biomarkers to improve diagnostic accuracy in OCGB positive patients. METHODS: We analysed the CSF metabolome and proteome of CDMS (n=41) and confirmed non-MS patients (n=64) comprising a range of CNS conditions routinely encountered in neurology clinics. RESULTS: OCGB discriminated between CDMS and non-MS with high sensitivity (85%), but low specificity (67%), as previously described. Machine learning methods revealed CCN5 levels provide greater accuracy, sensitivity, and specificity than OCGB (79%, +5%; 90%, +5%; and 72%, +5% respectively) while glial fibrillary acidic protein (GFAP) identified CDMS with 100% specificity (+33%). A multiomics approach improved accuracy further to 90% (+16%). CONCLUSION: The measurement of a few additional CSF biomarkers could be used to complement OCGB and improve the specificity of MS diagnosis when clinical and radiological evidence of DIT is absent