Pregnancy and Neuromyelitis Optica Spectrum Disorder - Reciprocal Effects and Practical Recommendations: A Systematic Review.

Introduction: Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory disorder of the central nervous system characterized by severe, antibody-mediated astrocyte loss with secondary demyelination and axonal damage, predominantly targeting optic nerves and the spinal cord. Recent publications have alluded to increased disease activity during pregnancy, and adverse maternal and fetal outcomes in patients with NMOSD. Our objective was to systematically review published literature to help counsel and manage women with NMOSD contemplating pregnancy. Methods: We searched five databases including MEDLINE and EMBASE, for English-language publications describing pregnancies in women with NMOSD. Article selection, data extraction, and risk-of-bias assessment using Joanna Briggs' critical appraisal tool for case reports and case series, were performed in duplicate. Pooled incidences were calculated where possible, and a narrative summary was provided. Results: Of 2,118 identified titles, 22 case reports and seven case series, representing 595 pregnancies in 389 women, were included. The mean maternal age was 28.12 ± 5.19 years. At least 20% of cases were first diagnosed during pregnancy. There were no maternal deaths. Pooled estimates for clinical outcomes could not be obtained due to inadequate reporting. NMOSD-related disability and relapses increased considerably during pregnancy and especially in the immediate postpartum period. Although a high proportion of early pregnancy losses were reported, an association with disease activity or therapeutic interventions could not be established. Apart from one publication which reported an increased risk of preeclampsia, there was no increase in adverse obstetric outcomes including preterm birth, fetal growth restriction or congenital malformations. Initial attacks and relapses were successfully managed with oral or intravenous corticosteroids and immunosuppressants, and refractory cases with immunoglobulin, plasma exchange and immunoadsorption. Conclusion: Increased NMOSD-related disability and relapses during pregnancy the postpartum period may respond to aggressive management with corticosteroids and immunosuppressants such as azathioprine, which are safely administered during pregnancy and lactation. Emerging safety data on monoclonal antibodies during pregnancy, make these attractive options, while intravenous immunoglobulin, plasma exchange and immunoadsorption can be safely used to treat severe relapses. The complex interplay between NMOSD and pregnancy outcomes would be best understood through prospective analysis of data collected through an international registry. Disclosure: Dalia Rotstein has served as a consultant or speaker for Alexion and Roche. She has received research support from Roche Canada. Rohan D'Souza has served as a consultant and speaker for Ferring Canada Inc and Ferring Global Inc, on topics unrelated to this manuscript. The other authors have no relevant relationships to disclose.

Interplay between cytoskeletal polymerization and the chondrogenic phenotype in chondrocytes passaged in monolayer culture.

Tubulin and actin exist as monomeric units that polymerize to form either microtubules or filamentous actin. As the polymerization status (monomeric/polymeric ratio) of tubulin and/or actin have been shown to be important in regulating gene expression and phenotype in non-chondrocyte cells, the objective of this study was to examine the role of cytoskeletal polymerization on the chondrocyte phenotype. We hypothesized that actin and/or tubulin polymerization status modulates the chondrocyte phenotype during monolayer culture as well as in 3D culture during redifferentiation. To test this hypothesis, articular chondrocytes were grown and passaged in 2D monolayer culture. Cell phenotype was investigated by assessing cell morphology (area and circularity), actin/tubulin content, organization and polymerization status, as well as by determination of proliferation, fibroblast and cartilage matrix gene expression with passage number. Bovine chondrocytes became larger, more elongated, and had significantly (P < 0.05) increased gene expression of proliferation-associated molecules (cyclin D1 and ki67), as well as significantly (P < 0.05) decreased cartilage matrix (type II collagen and aggrecan) and increased fibroblast-like matrix, type I collagen (COL1), gene expression by passage 2 (P2). Although tubulin polymerization status was not significantly (P > 0.05) modulated, actin polymerization was increased in bovine P2 cells. Actin depolymerization, but not tubulin depolymerization, promoted the chondrocyte phenotype by inducing cell rounding, increasing aggrecan and reducing COL1 expression. Knockdown of actin depolymerization factor, cofilin, in these cells induced further P2 cell actin polymerization and increased COL1 gene expression. To confirm that actin status regulated COL1 gene expression in human P2 chondrocytes, human P2 chondrocytes were exposed to cytochalasin D. Cytochalasin D decreased COL1 gene expression in human passaged chondrocytes. Furthermore, culture of bovine P2 chondrocytes in 3D culture on porous bone substitute resulted in actin depolymerization, which correlated with decreased expression of COL1 and proliferation molecules. In 3D cultures, aggrecan gene expression was increased by cytochalasin D treatment and COL1 was further decreased. These results reveal that actin polymerization status regulates chondrocyte dedifferentiation. Reorganization of the cytoskeleton by actin depolymerization appears to be an active regulatory mechanism for redifferentiation of passaged chondrocytes.

Efficient, Low-Cost Nucleofection of Passaged Chondrocytes.

Nucleofection of chondrocytes has been shown to be an adequate method of transfection. Using Amaxa's nucleofection system, transfection efficiencies up to 89% were achievable for vector (pmaxGFP) and 98% for siRNA (siGLO) into passaged chondrocytes. However, such methods rely on costly commercial kits with proprietary reagents limiting its use in basic science labs and in clinical translation. Bovine-passaged chondrocytes were plated in serum reduced media conditionsand then nucleofected using various in laboratory-produced buffers. Cell attachment, confluency, viability, and transfection efficiency was assessed following nucleofection. For each parameter the buffers were scored and a final rank for each buffer was determined. Buffer denoted as 1M resulted in no significant difference for cell attachment, confluency, and viability as compared to non-nucleofected controls. Nucleofection in 1M buffer, in the absence of DNA vectors, resulted in increased col2, ki67, ccnd1 mRNA levels, and decreased col1 mRNA levels at 4 days of culture. Flow cytometry revealed that the transfection efficiency of 1M buffer was comparable to that obtained using the Amaxa commercial kit. siRNA designed against lamin A/C resulted in an average reduction of lamin A and C proteins to 19% and 8% of control levels, respectively. This study identifies a cost-effective, efficient method of nonviral nucleofection of bovine-passaged chondrocytes using known buffer formulations. Human-passaged chondrocytes could also be successfully nucleofected in 1M buffer. Thus this method should facilitate cost-efficient gene targeting of cells used for articular cartilage repair in a research setting.

Expression of type I collagen and tenascin C is regulated by actin polymerization through MRTF in dedifferentiated chondrocytes.

This study examined actin regulation of fibroblast matrix genes in dedifferentiated chondrocytes. We demonstrated that dedifferentiated chondrocytes exhibit increased actin polymerization, nuclear localization of myocardin related transcription factor (MRTF), increased type I collagen (col1) and tenascin C (Tnc) gene expression, and decreased Sox9 gene expression. Induction of actin depolymerization by latrunculin treatment or cell rounding, reduced MRTF nuclear localization, repressed col1 and Tnc expression, and increased Sox9 gene expression in dedifferentiated chondrocytes. Treatment of passaged chondrocytes with MRTF inhibitor repressed col1 and Tnc expression, but did not affect Sox9 expression. Our results show that actin polymerization regulates fibroblast matrix gene expression through MRTF in passaged chondrocytes.