Biology subjects

Bussolati, B.

Publications and source records attributed to Bussolati, B..

4 recordsLinked to original sources

bioRxiv2023

NON-TOXIC ACID-FREE GLYOXAL FIXATIVE FOR VETERINARY HISTOPATHOLOGY, IMMUNOHISTOCHEMISTRY AND MOLECULAR ANALYSIS

Formaldehyde fixation is worldwide the most used system for histopathological examination. However, its toxicity is well known, and preservation of proteins and nucleic acids is not optimal. Alternative fixatives warranting similar morphological quality of tissues and costs, but lacking toxicity and allowing better preservation of proteins and nucleic acids would therefore increase both safety of operators and quality of molecular analysis in pathology. This multi-institutional study aimed to compare the morphological, histochemical, immunohistochemical (IHC), and molecular analyses outcomes of a newly patented, non-toxic, acid-free Glyoxal (GAF) fixative with neutral buffered formaldehyde (NBF). Tissues from a total of 73 subjects were analyzed, including 13 necropsies. Gross features were preserved after GAF fixation, with no tissue hardening or discoloration. Cellular ultrastructure was also better preserved with GAF and histology and histochemistry on GAF-fixed samples showed good results when compared to NBF-fixed samples, with the exception of loss of tinctorial affinity of erythrocytes and mast cell granules. IHC analyses also showed comparable results with only slight and rare protocol adjustment. DNA and RNA yields were higher from GAF-fixed samples (P<0.05) and the tested genes (p53 and COX1) were better amplified. RNA scope showed positive results for c-KIT expression in GAF-fixed mast cell tumors. Based on these data, the non-toxic GAF fixative allows good macroscopical, histological and immunohistochemical analyses of tissue samples, including on-field application, and better molecular analyses when compared to NBF. This represents a promising possibility for teaching, diagnostic, and research in veterinary pathology.

pathology↗

bioRxiv2022

Goat PRP14 (gPRP14) has xeno-antigenic properties and works as a vaccine in preclinical models of cancer.

We studied the activity of recombinant goat PRP14 (gPRP14), a member of the RID protein family, as a xeno-antigen in preclinical models of cancer. Antisera from rabbits and mice immunized with gPRP14 showed strong reactivity against several tumor cell types, which was absent towards normal cells: the tumor selectivity was related to surface and intra-cellular expression in tumor cells, and to an exclusively intra-cellular localization in normal cells. In vitro, binding to tumor cells was followed by cytotoxicity which could be rescued by the addition of excess soluble antigen. In vivo, an anti-tumor activity of immunization with gPRP14 was observed in murine syngeneic models of breast cancer and melanoma: the anti-tumor response was present when gPRP14 was administered in a preventive setting, and persisted upon repeated challenges with tumor cells in long-term survivor mice. Finally, we showed that both the humoral and T-cell mediated responses are needed for the optimal anti-tumor effect in the murine melanoma model. Thus, we have performed an initial characterization of gPRP14 as a cancer vaccine, which -given the potential wide range of tumor cells positive for the antigen-appears as a promising, novel immunotherapy.

cancer biology↗

bioRxiv2022

Single extracellular vesicle analysis in human amniotic fluid shows evidence of phenotype alterations in preeclampsia

Amniotic fluid surrounding the developing fetus is a complex biological fluid rich in metabolically active bio-factors. The presence of extracellular vesicles (EVs) in amniotic fluid has been mainly related to fetal urine. We here characterized EVs from term amniotic fluid in terms of surface marker expression using different orthogonal techniques. EVs appeared to be a heterogeneous population expressing markers of renal, placental, epithelial and stem cells. Moreover, we compared amniotic fluid EVs from normal pregnancies with those of preeclampsia, a hypertensive disorder affecting up to 8% of pregnancies worldwide. An increase of CD105 (endoglin) expressing EVs was observed in preeclamptic amniotic fluid by bead-based cytofluorimetric analysis, and further confirmed using a chip-based analysis. HLA-G, a typical placental marker, was not co-expressed by the majority of CD105+ EVs, suggesting their origin from amniotic fluid cells. At a functional level, preeclampsia-derived EVs, but not normal pregnancy EVs, showed an antiangiogenic effect, possibly due to the decoy effect of endoglin. In addition, several miRNAs were differentially expressed in preeclampsia-derived EVs and directly related to the modulation of angiogenesis and trophoblast function. Our results provide a characterization of term amniotic fluid-EVs, supporting their origin from fetal and placental cells. In preeclampsia, the observed antiangiogenic characteristics of amniotic fluid-EVs may reflect the hypoxic and antiangiogenic microenvironment and could possibly impact on the developing fetus or on the surrounding fetal membranes.

molecular biology↗

bioRxiv2021

Surface marker expression in small and medium/large mesenchymal stromal cell-derived extracellular vesicles in naive or apoptotic condition using orthogonal techniques

Extracellular vesicles released by mesenchymal stromal cells (MSC EVs) are a promising resource for regenerative medicine. In particular, small MSC EVs represent the active EV fraction for therapeutic applications. A bulk analysis is applied to characterize MSC EVs identity and purity, coupled with the assessment of single EV morphology, size and integrity using electron microscopy. We here applied different orthogonal methods to provide a quantitative analysis of size and surface marker expression in medium/large and small fractions, namely 10k and 100k fractions, of MSC EVs obtained by sequential ultracentrifugations. Bone marrow, adipose tissue, and umbilical cord MSC EVs were compared, in naive and apoptotic conditions. The 100k EV size <100 nm, as detected by electron microscopy, was confirmed by super-resolution microscopy and ExoView. Quantitative single-vesicle imaging using super-resolution microscopy revealed heterogeneous patterns of tetraspanin expressions, being all MSC EV fractions single, double and triple positive, in variable proportions, for CD63, CD81 and CD9. Moreover, ExoView analysis allowed a comparative multiplex screening of single MSC EV tetraspanin and mesenchymal marker levels. Finally, a semiquantitative bead based cytofluorimetric analysis showed the segregation of immunological and pro-coagulative markers on the 10k MSC EV fraction. Apoptotic MSC EVs were released in higher number, without significant differences from the naive fractions in surface marker expression. These results indicate that a consistent profile of MSC EV fractions among the different MSC sources, and a safer profile of the 100k MSC EV population for clinical application. Finally, our study identified suitable applications for different EV analytical techniques.

cell biology↗