Prdm1 (Blimp-1) and the Expression of Fast and Slow Myosin Heavy Chain Isoforms during Avian Myogenesis In Vitro

BACKGROUND. Multiple types of fast and slow skeletal muscle fibers form during early embryogenesis in vertebrates. In zebrafish, formation of the earliest slow myofibers in fin muscles requires expression of the zinc-finger transcriptional repressor Prdm1 (also known as Blimp1). To further understand how the role of Prdm1 in early myogenesis may vary through evolution and during development, we have now analyzed Prdm1 expression in the diverse types of myotubes that form in culture from somitic, embryonic, and fetal chicken myoblasts. PRINCIPAL FINDINGS. In cultures of somitic, embryonic limb, and fetal limb chicken cells, we found that Prdm1 was expressed in all of the differentiated muscle cells that formed, including those that expressed only fast myosin heavy chain isoforms, as well as those that co-expressed both fast and slow myosin heavy chain isoforms. Prdm1 was also expressed in Pax7-positive myoblasts, as well as in non-myogenic cells in the cultures. Furthermore, though all differentiated cells in control somite cultures co-expressed fast and slow myosin heavy chains, antisense knockdown of Prdm1 expression inhibited the formation of these co-expressing cells in somite cultures. CONCLUSIONS. In chicken myogenic cell cultures, Prdm1 was expressed in most Pax7-positive myoblasts and in all differentiated muscle cells, irrespective of the developmental stage of cell donor or the pattern of fast and slow myosin heavy chains expressed in the differentiated cells that were formed. Thus, Prdm1 was expressed in myogenic cells prior to terminal differentiation; and, after differentiation, Prdm1 expression was not limited to cells that expressed slow myosin heavy chain isoforms. In addition, Prdm1 appeared to be required for differentiation of the somitic myocytes, which are the earliest myocytes to form in the avian embryo.National Research Initiative of the United States Department of Agriculture Cooperative State Research, Education, and Extension Service (#2006-35206-16622); National Heart, Lung, and Blood Institute (2R01HL064641

Decreased Expression of the Protein Orhodenticle Homeobox 2 (Otx2) in Individuals With Schizophrenia: A Postmortem Investigation

The purpose of this thesis was to determine if the amount of the protein Orthodenticle homeobox 2 (Otx2) is reduced in individuals with schizophrenia. Otx2 is a transcription factor that plays an essential role in the development and maintenance of a specific class of GABAergic interneurons that express the protein parvalbumin (PV+ GABAergic interneurons). In addition, Otx2 participates in the development and maintenance of perineuronal nets. Perineuronal nets are important extracellular matrix structures that encompass PV+ GABAergic interneurons and facilitate their proper functioning (Beurdeley, 2012). It is speculated that perineuronal net structural abnormalities can lead to impaired PV+ GABAergic interneuron function and these impairments contribute to the manifestation of the condition schizophrenia (Berretta et al., 2015). Because Otx2 is needed for both healthy PV+ GABAergic interneurons and functional perineuronal nets, we postulated that insufficient levels of Otx2 in the amygdala might contribute to impaired GABAergic neurotransmission as exhibited in individuals with schizophrenia. Interestingly, PV+ GABAergic interneurons that require Otx2 do not produce Otx2. Recently, it was shown in mouse models that Otx2 is made in the choroid plexus, released into the cerebral spinal fluid and carried to cortical areas where it becomes available to be used by neurons (Spattazza et al., 2013). Similarly, in human postmortem samples, we observed the presence of Otx2 in the choroid plexus (Pantazopoulos et al., Manuscript in Preparation). Furthermore, we observed a novel phenomenon in human samples; Otx2 was found in vesicles in the cerebral spinal fluid and in the amygdala samples. We hypothesized that PV+ GABAergic interneurons located in the amygdala obtained Otx2 from the choroid plexus by delivery in vesicles via the cerebral spinal fluid. Based on this observation and building on current scientific understanding of schizophrenia, we proposed that there would be fewer vesicles in the amygdala samples donated by individuals with schizophrenia compared to samples donated by healthy controls. To test this hypothesis, the number of vesicles that contained Otx2 in amygdala samples donated by individuals diagnosed with schizophrenia (n=15) was compared to the number of vesicles that contained Otx2 in amygdala samples donated by healthy control donors (n=15). The results showed that there was not a significant decrease in the number of vesicles that contained Otx2 in amygdala samples donated by individuals with schizophrenia compared to samples donated by healthy controls (p-value > 0.05). However, an enzyme linked immunosorbent assay (ELISA) was performed to measure the amount of Otx2 inside vesicles and a significant decrease in the amount of Otx2 was observed in cerebral spinal fluid samples donated by individuals with schizophrenia (n=11) compared to healthy controls (n=15), (p-value < 0.05). In conclusion, this thesis work honed in on three major ideas. First, the method of delivery of Otx2 to neurons in the amygdala appeared to be via vesicles filled with Otx2, which originated in the choroid plexus. Second, there was not a decrease in the number of vesicles that contained Otx2 in samples from individuals with schizophrenia. Finally, preliminary experiments that measured Otx2 protein concentration inside the vesicles indicated that the amount of Otx2 contained in the vesicles was reduced in samples donated by individuals with schizophrenia compared to healthy controls. These finding are potential first steps in the development of a bioassay for the molecular characterization of the condition of schizophrenia

Effect of Neutralizing Monoclonal Antibody Treatment on Early Trajectories of Virologic and Immunologic Biomarkers in Patients Hospitalized With COVID-19

BACKGROUND: Neutralizing monoclonal antibodies (nmAbs) failed to show clear benefit for hospitalized patients with coronavirus disease 2019 (COVID-19). Dynamics of virologic and immunologic biomarkers remain poorly understood. METHODS: Participants enrolled in the Therapeutics for Inpatients with COVID-19 trials were randomized to nmAb versus placebo. Longitudinal differences between treatment and placebo groups in levels of plasma nucleocapsid antigen (N-Ag), anti-nucleocapsid antibody, C-reactive protein, interleukin-6, and D-dimer at enrollment, day 1, 3, and 5 were estimated using linear mixed models. A 7-point pulmonary ordinal scale assessed at day 5 was compared using proportional odds models. RESULTS: Analysis included 2149 participants enrolled between August 2020 and September 2021. Treatment resulted in 20% lower levels of plasma N-Ag compared with placebo (95% confidence interval, 12%-27%; P \u3c .001), and a steeper rate of decline through the first 5 days (P \u3c .001). The treatment difference did not vary between subgroups, and no difference was observed in trajectories of other biomarkers or the day 5 pulmonary ordinal scale. CONCLUSIONS: Our study suggests that nmAb has an antiviral effect assessed by plasma N-Ag among hospitalized patients with COVID-19, with no blunting of the endogenous anti-nucleocapsid antibody response. No effect on systemic inflammation or day 5 clinical status was observed. CLINICAL TRIALS REGISTRATION: NCT04501978

Expression of Prdm1 and fast and slow MyHC isoforms in E4 somite explant cultures and the somitic myotome.

<p>Double immunofluorescence analysis for fast MyHC(s) with mAb F59 (<b>A</b>, red fluorescence) and slow MyHC(s) with mAb S58 (<b>B</b>, green fluorescence) showed that fast and slow MyHCs were co-expressed by all differentiated myocytes in somite cultures. In addition, Prdm1 immunostaining (Ab from Cell Signaling Technology) was found in the nuclei of all myocytes, as well as in many MyHC-negative cells (<b>C</b>, merged image, green fluorescence  =  S58, red fluorescence  =  Prdm1, blue fluorescence  =  nuclei). <b>D</b>. Prdm1 (green fluorescence, Ab from Abcam) was expressed throughout the myotome (mt, arrows) of a mature somite at the forelimb bud level at E4. Additional Prdm1 staining was found in some cells of the sclerotome (sct) and neural tube (nt). Arrows indicate Prdm1-positive nuclei within myocytes. Bar in Panel A = 40 µm for panels A and B); 15 µm for panel C; and 50 µm for panel D.</p

Prdm1 and fast MyHC expression in cultures of E12 hindlimb cells.

<p>Prdm1 immunostaining (Ab from Cell Signaling Technology) was found in the nuclei of both mononucleate cells (<b>A, B</b>) and myotubes (<b>C, D</b>). An additional Prdm1 antibody (Abcam) produced similar nuclear staining of E12 mononucleate cells and myotubes (not shown). As found in previous studies, myotubes formed from E12 myoblasts showed immunostaining with mAb F59 (<b>E</b>), which reacts with all fast MyHC isoforms, but did not stain with mAb S58 (<b>F</b>), which reacts with the slow MyHC2 and slow MyHC3 isoforms. Bar in Panel A = 10 µm for panels A–D and 30 µm for panels E and F.</p

Expression of Prdm1 and fast and slow MyHC isoforms in cultures of E4 hindlimb cultures.

<p>As indicated, individual cultures were analyzed by triple immunostaining for fast MyHC(s) with mAb F59 (A, E, blue fluorescence), Prdm1 (B, F, green fluorescence, Ab from Cell Signaling Technology), and slow MyHC(s) with mAb S58 (C, G, red fluorescence). Merged photographs (D, H) showed that, as found previously, E4 myoblasts formed differentiated, usually mononucleate, cells of two types: those that express only fast MyHC(s) (<i>e.g.</i>, downward pointing arrows in panels E–H) and those that co-express fast and slow MyHCs (upward pointing arrows in all panels). Prdm1 immunostaining was found in the nuclei of both types of differentiated cells. An additional Prdm1 antibody (Abcam) produced similar nuclear staining of E4 cultures (not shown). Bar in Panel A = 20 µm.</p

Prdm1 mRNA and protein were expressed in chicken tissues and muscle cell cultures.

<p><b>A.</b> As indicated, RNAs or proteins were isolated from trunk tissues (Tk), hindlimbs (HL), and forelimbs (FL) of chicken embryos (E4  =  day 4 <i>in ovo</i>) and fetuses (E12), and also from differentiated cultures of mouse C₂C₁₂ myogenic cells (C₂) and primary cultures of E4 and E12 chicken hindlimb cells. RNAs were analyzed by RT-PCR with exon-spanning primers specific for chicken Prdm1 and, as a positive control, GAPDH as indicated. Proteins were analyzed by immunoblotting with antibodies specific for Prdm1 (Cell Signaling Technology) or GAPDH. Bands with the expected ∼240 bp size of the Prdm1 RT-PCR product or the expected ∼100 kDa M_r of the Prdm1 protein were obtained from all chicken embryonic and fetal tissues and from both E4 and E12 chicken myogenic cell cultures. Prdm1 mRNA and protein were not obtained from cells of the mouse C₂C₁₂ myogenic cell line, though GAPDH was found. Specificity of the amplified cDNAs for Prdm1 was confirmed by restriction enzyme analysis (not shown). Bands from some gels were re-arranged for presentation. <b>B.</b> Full-length Prdm1 immunoblot (Ab from Cell Signaling Technology) of the same tissue samples shown in panel A, demonstrating lack of non-specific bands. MW  =  molecular standards with number  =  kDa.</p

Effect of Neutralizing Monoclonal Antibody Treatment on Early Trajectories of Virologic and Immunologic Biomarkers in Patients Hospitalized With COVID-19

BACKGROUND: Neutralizing monoclonal antibodies (nmAbs) failed to show clear benefit for hospitalized patients with coronavirus disease 2019 (COVID-19). Dynamics of virologic and immunologic biomarkers remain poorly understood.METHODS: Participants enrolled in the Therapeutics for Inpatients with COVID-19 trials were randomized to nmAb versus placebo. Longitudinal differences between treatment and placebo groups in levels of plasma nucleocapsid antigen (N-Ag), anti-nucleocapsid antibody, C-reactive protein, interleukin-6, and d-dimer at enrollment, day 1, 3, and 5 were estimated using linear mixed models. A 7-point pulmonary ordinal scale assessed at day 5 was compared using proportional odds models.RESULTS: Analysis included 2149 participants enrolled between August 2020 and September 2021. Treatment resulted in 20% lower levels of plasma N-Ag compared with placebo (95% confidence interval, 12%-27%; P &lt; .001), and a steeper rate of decline through the first 5 days (P &lt; .001). The treatment difference did not vary between subgroups, and no difference was observed in trajectories of other biomarkers or the day 5 pulmonary ordinal scale.CONCLUSIONS: Our study suggests that nmAb has an antiviral effect assessed by plasma N-Ag among hospitalized patients with COVID-19, with no blunting of the endogenous anti-nucleocapsid antibody response. No effect on systemic inflammation or day 5 clinical status was observed.CLINICAL TRIALS REGISTRATION: NCT04501978.</p