Treatment-resistant CIDP in an IgG Tubulin Autoantibody Positive Patient

Objectives: To describe a case of rapidly relapsing chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) in the setting of positive serum IgG tubulin autoantibodies. Methods: We wrote a case report and performed a literature review of IgG tubulin autoantibodies and the use of rituximab in treatment resistant CIDP. Results: Our case report describes a 29-year-old woman with CIDP that was resistant to treatment with steroids, intravenous immunoglobulin, and plasma exchange. An extensive workup of her rapidly relapsing CIDP was negative, with the exception of positive serum IgG tubulin autoantibodies. She ultimately stabilized on oral steroids, plasma exchange and rituximab, with a regular recurrence of weakness occurring approximately every month that led to rehospitalization. Conclusion: Anti-tubulin antibodies could be a marker of a subtype of CIDP that is treatment resistant. We detail her clinical course to serve as an example for other cases of IgG tubulin autoantibody positive CIDP patients that could be described in the future.

New data on dinophilid neurogenesis: a variation of a common pattern

BackgroundThe structure and development of the nervous system in Lophotrochozoa species is of the most important questions for comparative neurobiology. During the last decade the number of comprehensive studies on the development of serotonergic and FMRFamidergic systems has been skyrocketing. However, the detailed research of the earliest events of Polychaeta neurogenesis is still sparce. Polychaeta is a huge taxon within Lophotrochozoa. Its representatives are widely used as model systems in developmental and physiological investigations. Dinophilidae is a unique Polychaeta group. Its representatives combine morphological traits of different lophotrochozoan taxa. Moreover, adult dinophilids demonstrate morphological similarity to a trochophore larva. This similarity may be associated with either archaic origin of this group or neoteny. The main goal of our study is to provide a detailed description of the earliest events in Dinophilus neurogenesis. These data might improve our understanding of Polychaeta development and evolution.ResultsWe have studied the earliest events in nervous system development in two relative species D. gyrociliatus and D. taeniatus using immunochemical labelling of serotonin, FMRF-amide related peptides, and acetylated tubulin. We used external ciliation as marker for staging. Both species go through the same developmental stages: prototroch, ventral ciliary field and ciliary bands. In both species the first neurons differenciate revealed by anti alpha-acetylated tubulin antibodies only and show no reaction with 5-HT or FMRFa antibodies. These neurons located at the anterior and posterior parts of the embryo in both species. In D. taeniatus embryons the anterior cell is transient and disappear just after head neuropil is constructed. On the contrary, in D. gyrociliatus embryos the anterior cell is not transient and remains at the same position during the whole life span of the specimen. Caudal cell is present during the whole embryogenesis in both species. Neurites of these early neurons surround the stomadeum and constitute anlagen of paired ventro-lateral longitudinal bundles. During the development the number of neurites increases and they form compact head neuropil, paired ventro-lateral and lateral longitudinal bundles, unpaired medial longitudinal bundle and transverse commissures in ventral hyposphere. Serotonin- and FMRFamide-immunoreactive neurons differentiate adjacent to ventro-lateral bundles and head neuropil, respectively, after the establishment of main structures of the nervous system at the ventral ciliary field and ciliary bands stages. Processes of serotonin-, FMRFamide- immunopositive neurons constitute the small portion of tubulin immunopositive neuropil at all described stages.ConclusionsWe announce a detailed data on the earliest events in D. gyrociliatus and D. taeniatus neurodevelopment based on anti-acetylated tubulin, serotonin, and FMRFamide-like immuno labeling. The first nerve elements demonstrate no 5-HT-IR and no FMRFa-IR, which differs from the most Polychaetes and even Lophotrochozoans, investigated so far. Moreover, these animals do not have a typical apical organ (or perhaps do not have it at all) and the pioneer neurons of D.gyrociliatus are also peculiar in that they join the definitive nervous system unlike other lophotrochozoans where pioneer nerons are transient. Thus, Dinophilus neurogenesis demonstrates a variation of common scheme. The reported study was funded by RFBR, project number 19-3460040.

Use of Confocal Microscopy to Evaluate Equine Zygote Development After Sperm Injection of Oocytes Matured In Vivo or In Vitro

Confocal microscopy was used to image stages of equine zygote development, at timed intervals, after intracytoplasmic sperm injection (ICSI) of oocytes that were matured in vivo or in vitro. After fixation for 4, 6, 8, 12, or 16 h after ICSI, zygotes were incubated with α/β tubulin antibodies and human anticentromere antibody (CREST/ACA), washed, incubated in secondary antibodies, conjugated to either Alexa 488 or Alexa 647, and incubated with 561-Phalloidin and Hoechst 33258. An Olympus IX81 spinning disk confocal microscope was used for imaging. Data were analyzed using χ2 and Fisher’s exact tests. Minor differences in developmental phases were observed for oocytes matured in vivo or in vitro. Oocytes formed pronuclei earlier when matured in vivo (67% at 6 h and 80% at 8 h) than in vitro (13% at 6 and 8 h); 80% of oocytes matured in vitro formed pronuclei by 12 h. More (p=0.04) zygotes had atypical phenotypes, indicative of a failure of normal zygote development, when oocyte maturation occurred in vitro versus in vivo (30 and 11%, respectively). Some potential zygotes from oocytes matured in vivo had normal phenotypes, although development appeared to be delayed or arrested. Confocal microscopy provided a feasible method to assess equine zygote development using limited samples.

MeHg Developing Exposure Causes DNA Double-Strand Breaks and Elicits Cell Cycle Arrest in Spinal Cord Cells

The neurotoxicity caused by methylmercury (MeHg) is well documented; however, the developmental neurotoxicity in spinal cord is still not fully understood. Here we investigated whether MeHg affects the spinal cord layers development. Chicken embryos at E3 were treatedin ovowith 0.1 μg MeHg/50 μL saline solution and analyzed at E10. Thus, we performed immunostaining using anti-γ-H2A.X to recognize DNA double-strand breaks and antiphosphohistone H3, anti-p21, and anti-cyclin E to identify cells in proliferation and cell cycle proteins. Also, to identify neuronal cells, we used anti-NeuN and anti-βIII-tubulin antibodies. After the MeHg treatment, we observed the increase onγ-H2A.X in response to DNA damage. MeHg caused a decrease in the proliferating cells and in the thickness of spinal cord layers. Moreover, we verified that MeHg induced an increase in the number of p21-positive cells but did not change the cyclin E-positive cells. A significantly high number of TUNEL-positive cells indicating DNA fragmentation were observed in MeHg-treated embryos. Regarding the neuronal differentiation, MeHg induced a decrease in NeuN expression and did not change the expression ofβIII-tubulin. These results showed thatin ovoMeHg exposure alters spinal cord development by disturbing the cell proliferation and death, also interfering in early neuronal differentiation.

Usefulness of Monitoring γ-H2AX and Cell Cycle Arrest in HepG2 Cells for Estimating Genotoxicity Using a High-Content Analysis System

Formation of the phosphorylated protein γ-H2AX is a well-established marker of DNA strand breakage induced by DNA-damaging compounds. Many of these genotoxic compounds also inhibit cell division, leading to arrest at specific points in the cell cycle. Detection of γ-H2AX in combination with cell cycle arrest may therefore be useful for estimating the genotoxicity of experimental compounds. In this study, we examined γ-H2AX formation and cell cycle arrest using high-content screening (HCS) as a method for determining genotoxicity. HepG2 cells were treated with a panel of compounds and then stained with Hoechst 33342 and anti-γ-H2AX, anti-phospho-histone H3, and anti-tubulin antibodies. In total, 19 genotoxic and 7 nongenotoxic compounds were tested in this study. γ-H2AX production was observed within 1 h posttreatment for the majority of Ames-positive compounds, topoisomerase inhibitors, and DNA polymerase inhibitors. Cell cycle arrest in either the S or G2 phase was detected for all DNA-damaging compounds 24 h posttreatment, whereas tubulin-targeting compounds were shown to induce cell cycle arrest in the mitotic phase. Together, these results show that HCS is a simple, rapid, and effective tool for estimating the genotoxicity of compounds through detection of γ-H2AX production and cell cycle arrest.

Changes in γ-Tubulin Protein Distribution in Zebrafish (Danio rerio) Oocytes and the Early Cleavage-Stage Embryo

We investigated the distribution of γ-tubulin in zebrafish oocytes and embryos using epifluorescent or confocal microscopy and γ-tubulin antibodies. During meiotic maturation of zebrafish oocytes, γ-tubulin begins redistribution from oocyte ooplasm and cortex to the future blastodisc region at the animal pole. In activated eggs, γ-tubulin was uniformly distributed in the enlarging blastodisc with label emanating from the yolk cell. In newly fertilized eggs, γ-tubulin was evenly distributed in blastomere cytoplasm, with the presence of pronuclei but initially lacking discernable centrosomes. During early cleavage, especially at the eight-cell stage, striking arc-shaped/rings (A/R) of putative centrosomes were detected. Decreasing γ-tubulin was seen in yolk cells while early cleavage blastomeres had strong cytoplasmic label along with obvious A/R arrays. In addition, we found the orientation of the A/R array and nuclear division alternated by about 90 degrees for each cell cycle along with alternation of punctate and A/R arrays.