Microglia and macrophages appear to be the most common cells in the GBM microenvironment. In the present study we investigated the status of macrophages/microglia activation in surgical specimens from 41 patients diagnosed with grade IV GBM. For each patient we analyzed both the center of tumor and the parenchyma surrounding the tumor. The specimens were stained for: i) IBA1, a 17-kDa EF hand protein specifically expressed in microglia/macrophages ii) CD163, a cell surface antigen associated with M2 phenotype; iii) iNOS, taken as a functional marker of M1 phenotype, and iv) ARG-I, taken as a functional marker of M2 phenotype. Staining was scored in a double-blinded score on a scale from 0 to 5. Our results suggest that CD163 expression is higher within the tumor than in surrounding periphery in both male and female patients; while iNOS is higher within the tumor in males, no significant difference was found for ARG-1. In addition, analyzing the data in TGCA database, we found that CD163 expression was significantly and inversely correlated with mean survival times, with average survival times ranging from 448days in patients having low expression, to 319 in mid, and 353 in patients with high CD163 expressing tumors. In contrast, no significant association was found between survival time and ARG-1 or iNOS expression.

The glioblastoma (GB) microenvironment includes cells of the innate immune system identified as glioma-associated microglia/macrophages (GAMs) that are still poorly characterized. A potential role on the mechanisms regulating GAM activity might be played by the endoplasmic reticulum protein ERp57/PDIA3 (protein disulfide-isomerase A3), the modulation of which has been reported in a variety of cancers. Moreover, by using The Cancer Genome Atlas database, we found that overexpression of PDIA3 correlated with about 55% reduction of overall survival of glioma patients. Therefore, we analyzed the expression of ERp57/PDIA3 using specimens obtained after surgery from 18 GB patients. Immunohistochemical analysis of tumor samples revealed ERp57/PDIA3 expression in GB cells as well as in GAMs. The ERp57/PDIA3 levels were higher in GAMs than in the microglia present in the surrounding parenchyma. Therefore, we studied the role of PDIA3 modulation in microglia–glioma interaction, based on the ability of conditioned media collected from human GB cells to induce the activation of microglial cells. The results indicated that reduced PDIA3 expression/activity in GB cells significantly limited the microglia pro-tumor polarization towards the M2 phenotype and the production of pro-inflammatory factors. Our data support a role of PDIA3 expression in GB-mediated protumor activation of microglia.

The characterization of human microglia has been hampered by poor availability of human cell sources. However, microglia is involved in the physiopathology of multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, HIV dementia, retinal degenerative diseases, cancer, and many other conditions. Therefore, there is an important need to have experimental paradigms of human microglia characterized and usable to study the role of microglia in the different pathologies in which it is involved. In the present work, we carried out an extensive characterization of Immortalized Human Microglia—SV40 cell line (IMhu), marketed by Applied Biological Material. The functional response of IMhu to a large variety of stimuli was studied. In particular, we investigated morphology, mortality, and changes in the production of different cytokines and chemokines, both under basal conditions and after stimulation. Moreover, western blotting analysis was conducted on phospho-mTOR (Ser 2448) and downstream parameters, p-P70S6K and 4EBP1, in order to understand if IMhu can be used for evaluations of mTOR pathway. In conclusion, IMhu cells proved to be a useful experimental model to investigate the physiopathology of inflammatory disease that involved microglia cells, including pathological conditions that involved the mTOR pathway.

An ultra-performance liquid chromatography-tandem mass spectrometry method was developed for the quantification of clotrimazole (CTZ) plasma levels after intravaginal administration of the drug given at approved dosages. Plasma samples were extracted by liquid–liquid extraction and a single chromatographic run could be completed within about 2 min. The method was linear over the investigated range (0.488–250 ng/mL) with all the correlation coefficients, R², greater than 0.9903. All data were in the range of ±15.0% with respect to the nominal concentration for high QC and medium QC, and in the range ±20% with respect to the nominal concentration for low QC. This rapid and sensitive method was validated and could be applied to human plasma samples from a healthy volunteer, showing that the assay is able to detect plasma concentrations of CTZ in the range of those found after the administration of the drug at approved dosages in the clinical setting.