Additional file 2: of Gemcitabine and docetaxel combination chemotherapy for advanced bone and soft tissue sarcomas: protocol for an open-label, non-randomised, Phase 2 study

Procedure for implementation of study monitoring. (DOCX 19 kb

Additional file 1: of Gemcitabine and docetaxel combination chemotherapy for advanced bone and soft tissue sarcomas: protocol for an open-label, non-randomised, Phase 2 study

Data manegement and informed consent procedure. (DOCX 23 kb

Transcutaneous Application of Carbon Dioxide (CO₂) Induces Mitochondrial Apoptosis in Human Malignant Fibrous Histiocytoma <em>In Vivo</em>

<div><p>Mitochondria play an essential role in cellular energy metabolism and apoptosis. Previous studies have demonstrated that decreased mitochondrial biogenesis is associated with cancer progression. In mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) regulates the activities of multiple nuclear receptors and transcription factors involved in mitochondrial proliferation. Previously, we showed that overexpression of PGC-1α leads to mitochondrial proliferation and induces apoptosis in human malignant fibrous histiocytoma (MFH) cells <em>in vitro</em>. We also demonstrated that transcutaneous application of carbon dioxide (CO₂) to rat skeletal muscle induces PGC-1α expression and causes an increase in mitochondrial proliferation. In this study, we utilized a murine model of human MFH to determine the effect of transcutaneous CO₂ exposure on PGC-1α expression, mitochondrial proliferation and cellular apoptosis. PGC-1α expression was evaluated by quantitative real-time PCR, while mitochondrial proliferation was assessed by immunofluorescence staining and the relative copy number of mitochondrial DNA (mtDNA) was assessed by real-time PCR. Immunofluorescence staining and DNA fragmentation assays were used to examine mitochondrial apoptosis. We also evaluated the expression of mitochondrial apoptosis related proteins, such as caspases, cytochorome c and Bax, by immunoblot analysis. We show that transcutaneous application of CO₂ induces PGC-1α expression, and increases mitochondrial proliferation and apoptosis of tumor cells, significantly reducing tumor volume. Proteins involved in the mitochondrial apoptotic cascade, including caspase 3 and caspase 9, were elevated in CO₂ treated tumors compared to control. We also observed an enrichment of cytochrome c in the cytoplasmic fraction and Bax protein in the mitochondrial fraction of CO₂ treated tumors, highlighting the involvement of mitochondria in apoptosis. These data indicate that transcutaneous application of CO₂ may represent a novel therapeutic tool in the treatment of human MFH.</p> </div

Evaluation of mitochondrial induced apoptosis in CO₂ or control treated tumors.

<p>(A) DNA fragmentation analysis of tumor samples from CO₂ treated and control mice two weeks post-treatment by immunofluorescence. (<i>Blue</i>, nuclear; <i>Green</i>, apoptosis nuclear) (B) DNA fragmentation was assessed by flow cytometry in CO₂ treated tumors (<i>Blue dots</i>) and control tumors (<i>Red</i>) two weeks post-treatment. (C) Immunoblot analyses determined that increased expression of the cleavage products of caspase 3 and 9, and PARP occurred in the CO₂ treated tumors compared to the control tumors. Tubulin was used as an endogenous loading control. (D) Immunoblot analysis of cytochrome c and Bax in mitochondrial and cytoplasmic fractions of CO₂ treated and control tumors. Tubulin was used as an endogenous loading control. (C, D) Positive bands in immunoblot analyses were semiquantified using densitometrical analyses using the Image J program (NIH, USA, <a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a>).</p

Effect of transcutaneous application of CO₂ on MFH cell growth <i>in vivo</i>.

<p>Mice were treated with CO₂ or control air three days after MFH cell implantation. Treatment was administered twice weekly for two weeks. (A) MFH tumors in CO₂ treated and control mice, two weeks post-implantation. (B) Tumor volume (mm³) in CO₂ treated or control mice was monitored for two weeks post-implantation. (C) Body weight (g) of CO₂ treated or control mice was monitored for two weeks post-implantation. Data represent the mean ± S.E of at least three independent experiments (*<i>p</i><0.05, **<i>p</i><0.01).</p

Effect of transcutaneous application of CO₂ treatment on mitochondrial proliferation in tumors.

<p>qRT-PCR for <i>PGC-1α</i> (A) and <i>TFAM</i> (B) in CO₂ treated or control tumor specimens collected two weeks post-treatment. Expression was normalized to <i>β-actin</i> control. Data represent the mean ± S.E of at least three independent experiments (*<i>p</i><0.05). (C) mtDNA was measured in CO₂ treated or control tumor samples by PCR and the relative copy number was determined by normalizing to nDNA. Data represent the mean ± S.E. of at least three independent experiments (*<i>p</i><0.05). (D) Immunofluorescence staining of mitochondria in CO₂ treated or control tumors after two weeks (<i>Blue</i>, nuclear; <i>Red</i>, mitochondria).</p

Effect of transcutaneous CO₂ application on intracellular Ca²⁺ concentration in a mouse model of human MFH.

<p>Implanted tumors were isolated from mice at 0 (n = 12), 6 (n = 6) and 24 hours (n = 12) after transcutaneous CO₂ exposure, and the intracellular Ca²⁺ concentration was assessed using the Calcium Assay Kit. Data represent the mean ± S.E. of at least three independent experiments (*<i>p</i><0.05, **<i>p</i><0.01).</p

DataSheet_1_Effect of SARS-CoV-2 BNT162b2 mRNA vaccine on thyroid autoimmunity: A twelve-month follow-up study.pdf

ObjectivesGraves’ disease (GD) has been highlighted as a possible adverse effect of the respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccine. However, it is unknown if the SARS-CoV-2 vaccine disrupts thyroid autoimmunity. We aimed to present long-term follow-up of thyroid autoimmunity after the SARS-CoV-2 BNT162b2 mRNA vaccine.MethodsSerum samples collected from seventy Japanese healthcare workers at baseline, 32 weeks after the second dose (pre-third dose), and 4 weeks after the third dose of the vaccine were analyzed. The time courses of anti-SARS-CoV-2 spike immunoglobulin G (IgG) antibody, thyroid-stimulating hormone receptor antibody (TRAb), and thyroid function were evaluated. Anti-thyroglobulin antibodies (TgAb) and anti-thyroid peroxidase antibodies (TPOAb) were additionally evaluated in thirty-three participants.ResultsThe median age was 50 (IQR, 38-54) years and 69% were female. The median anti-spike IgG antibody titer was 17627 (IQR, 10898-24175) U/mL 4 weeks after the third dose. The mean TRAb was significantly increased from 0.81 (SD, 0.05) IU/L at baseline to 0.97 (SD, 0.30) IU/L 4 weeks after the third dose without functional changes. An increase in TRAb was positively associated with female sex (β = 0.32, P = 0.008) and low basal FT4 (β = -0.29, P = 0.02) and FT3 (β = -0.33, P = 0.004). TgAb was increased by the third dose. Increase in TgAb was associated with history of the thyroid diseases (β = 0.55, P ConclusionsSARS-CoV-2 BNT162b2 mRNA vaccine can disrupt thyroid autoimmunity. Clinicians should consider the possibility that the SARS-CoV-2 vaccine may disrupt thyroid autoimmunity.</p

DataSheet_2_Effect of SARS-CoV-2 BNT162b2 mRNA vaccine on thyroid autoimmunity: A twelve-month follow-up study.docx

ObjectivesGraves’ disease (GD) has been highlighted as a possible adverse effect of the respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccine. However, it is unknown if the SARS-CoV-2 vaccine disrupts thyroid autoimmunity. We aimed to present long-term follow-up of thyroid autoimmunity after the SARS-CoV-2 BNT162b2 mRNA vaccine.MethodsSerum samples collected from seventy Japanese healthcare workers at baseline, 32 weeks after the second dose (pre-third dose), and 4 weeks after the third dose of the vaccine were analyzed. The time courses of anti-SARS-CoV-2 spike immunoglobulin G (IgG) antibody, thyroid-stimulating hormone receptor antibody (TRAb), and thyroid function were evaluated. Anti-thyroglobulin antibodies (TgAb) and anti-thyroid peroxidase antibodies (TPOAb) were additionally evaluated in thirty-three participants.ResultsThe median age was 50 (IQR, 38-54) years and 69% were female. The median anti-spike IgG antibody titer was 17627 (IQR, 10898-24175) U/mL 4 weeks after the third dose. The mean TRAb was significantly increased from 0.81 (SD, 0.05) IU/L at baseline to 0.97 (SD, 0.30) IU/L 4 weeks after the third dose without functional changes. An increase in TRAb was positively associated with female sex (β = 0.32, P = 0.008) and low basal FT4 (β = -0.29, P = 0.02) and FT3 (β = -0.33, P = 0.004). TgAb was increased by the third dose. Increase in TgAb was associated with history of the thyroid diseases (β = 0.55, P ConclusionsSARS-CoV-2 BNT162b2 mRNA vaccine can disrupt thyroid autoimmunity. Clinicians should consider the possibility that the SARS-CoV-2 vaccine may disrupt thyroid autoimmunity.</p