Mass Spectrometry Imaging (MSI) Delineates Thymus-Centric Metabolism In Vivo as an Effect of Systemic Administration of Dexamethasone

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is increasingly used in a broad range of research due to its ability to visualize the spatial distribution of metabolites in vivo. Here, we have developed a method, named thoracic Mass Spectrometry Imaging (tMSI), as a standard protocol of molecular imaging of whole-animal sectioning in various settings of mice in vivo. Further application of the strategy that involved the systemic administration of dexamethasone (DEX) in mice, enabled a dynamic shift in the energy status of multiple thoracic organs to be visualized, based on tMSI data of purine and pyrimidine metabolites. Furthermore, with the introduction of uniform manifold approximation and projection (UMAP) for tMSI data, metabolic profiles normally localized in the cortex and cortico-medullary junction (CMJ) of the thymus were drastically shifted as minor profiles into the medulla of DEX-treated thymus. As a massive apoptotic cell death in the thymic cortex was noticeable, a single molecule, which was upregulated in the cortex of the thymus, enabled us to predict ongoing immunosuppression by in vivo DEX-administration.

Toxic effect of titanium dioxide nanoparticles on morphological characteristics of thymus

Aim. Study of effects of titanium dioxide nanoparticles administered orally on the rodent thymus morphological and functional state. Methods. The study used classical morphological approaches and specific markers of proliferation - Ki-67, apoptosis - p53 protein and macrophages - CD68, which allow adequately identifying not only the cells themselves, but also their functional state. The rodent thymus was analyzed after oral administration (10 mg/kg of body weight of the animal, 28 days) of the TiO2 nanoparticles (rutile form, 40-60 nm) obtained by diluting the TiO2 powder in distilled water. Aggregation of the nanoparticles was prevented by processing a suspension of nanodispersed TiO2 in an ultrasonic bath. The rats of the control group were orally injected with distilled water in the same volume. Serial paraffin sections of the thymus were stained with hematoxylin-eosin, picrofuxin according to Van Gieson, immunohistochemical staining was performed with antibodies to Ki-67, CD68, p53. Results. Significant changes of the tissue of thymus gland under the influence of TiO2 nanoparticles were revealed. In the thymus of experimental animals, a decrease in the proportion of cortex was found to be 17.6 %, a significant decrease in the density of the cell population due to decrease in the number of thymocytes was demonstrated. Immunohistochemical typing revealed that under the influence of TiO2 nanoparticles, a decrease in the number of Ki-67-positive cells in the cortex of the lobule of the thymus gland was observed, which indicates the inhibition of proliferation in these conditions. Under the influence of TiO2 nanoparticles, an increase of the amount of cells entering apoptosis in the cortex of the thymus segment of the experimental group by 5.18 times was observed, as evidenced by the results of immunohistochemical study of the expression of apoptosis marker p53 protein. Perhaps, as a compensatory mechanism there is a pronounced increase in the number of macrophages, as indicated by an increase in the average number of CD68 immunopositive cells in the cortex of the thymus of the test group by 2.61, and in the brain substance by 1.35. Conclusion. The revealed morphological and functional changes of the thymus with oral administration of TiO2 nanoparticles indicate their immunosuppressive effect.

Immunohistochemical characterization of a 183 KD myeloid-specific-DNA- binding protein in B5 fixed, paraffin-embedded tissues, and bone marrow aspirates by monoclonal antibody BM-1

A monoclonal antibody, designated BM-1, which is reactive in B5 formalin-fixed, paraffin-embedded tissues, has been generated against a cytoplasmic and nuclear antigen expressed in human myeloid precursor cells and derived leukemias. Using the avidin-biotin-complex immunoperoxidase procedure, BM-1 was found to stain selectively myeloid precursor cells in normal bone marrow and mature granulocytes in the blood. In a screen of 26 normal adult and fetal human organs fixed in B5 formalin, BM-1 was negative in all nonhematopoietic tissues with the exception of tissue granulocytes and scattered cells in the peripheral cortex of the thymus. Likewise a screen of 30 solid tumor cell lines including a spectrum of carcinomas, sarcomas, and neural-derived tumors was negative. BM-1 was also negative with 21 T and B cell lymphomas and 11 Hodgkin's disease tumors. A preliminary study of tumors of the hematopoietic system revealed that BM-1 was reactive with M2 and M3 acute myelogenous leukemias (AML), chronic myelogenous leukemias (CML) and myelomonocytic leukemias, and granulocytic sarcomas. M1, M4, M5, and M6 AML clot preparations were negative in this study, indicating that BM-1 may have a role in the histopathologic diagnosis of myelogenous leukemia. Myeloid leukemic cell lines HL-60, ML-2, KG1, and TPH-1-O showed BM-1 nuclear and/or cytoplasmic reactivity in a subpopulation of cells, but erythroid and lymphoid leukemias and all lymphoma cell lines were negative. Immunoperoxidase studies of a panel of fetal tissues showed BM-1 positive cells in the peripheral cortex of the thymus and portal myelopoietic regions of the liver at 18 weeks gestation. Finally, DNA-cellulose and solid phase radioimmunoassay (RIA) techniques developed in our laboratory demonstrate that the BM-1 antigenic domain is reactive only after binding to eukaryotic but not prokaryotic single- or double-stranded DNA. Immunoblot techniques using a DNA-cellulose purified protein sample revealed that BM-1 recognizes a 183 kD protein. These studies indicate that BM-1 is recognizing a myeloid-specific antigen that, because of its DNA binding characteristics, may have an important role in the differentiation of myeloid cells at the molecular level.

Immunohistochemical characterization of a 183 KD myeloid-specific-DNA- binding protein in B5 fixed, paraffin-embedded tissues, and bone marrow aspirates by monoclonal antibody BM-1

A monoclonal antibody, designated BM-1, which is reactive in B5 formalin-fixed, paraffin-embedded tissues, has been generated against a cytoplasmic and nuclear antigen expressed in human myeloid precursor cells and derived leukemias. Using the avidin-biotin-complex immunoperoxidase procedure, BM-1 was found to stain selectively myeloid precursor cells in normal bone marrow and mature granulocytes in the blood. In a screen of 26 normal adult and fetal human organs fixed in B5 formalin, BM-1 was negative in all nonhematopoietic tissues with the exception of tissue granulocytes and scattered cells in the peripheral cortex of the thymus. Likewise a screen of 30 solid tumor cell lines including a spectrum of carcinomas, sarcomas, and neural-derived tumors was negative. BM-1 was also negative with 21 T and B cell lymphomas and 11 Hodgkin's disease tumors. A preliminary study of tumors of the hematopoietic system revealed that BM-1 was reactive with M2 and M3 acute myelogenous leukemias (AML), chronic myelogenous leukemias (CML) and myelomonocytic leukemias, and granulocytic sarcomas. M1, M4, M5, and M6 AML clot preparations were negative in this study, indicating that BM-1 may have a role in the histopathologic diagnosis of myelogenous leukemia. Myeloid leukemic cell lines HL-60, ML-2, KG1, and TPH-1-O showed BM-1 nuclear and/or cytoplasmic reactivity in a subpopulation of cells, but erythroid and lymphoid leukemias and all lymphoma cell lines were negative. Immunoperoxidase studies of a panel of fetal tissues showed BM-1 positive cells in the peripheral cortex of the thymus and portal myelopoietic regions of the liver at 18 weeks gestation. Finally, DNA-cellulose and solid phase radioimmunoassay (RIA) techniques developed in our laboratory demonstrate that the BM-1 antigenic domain is reactive only after binding to eukaryotic but not prokaryotic single- or double-stranded DNA. Immunoblot techniques using a DNA-cellulose purified protein sample revealed that BM-1 recognizes a 183 kD protein. These studies indicate that BM-1 is recognizing a myeloid-specific antigen that, because of its DNA binding characteristics, may have an important role in the differentiation of myeloid cells at the molecular level.