Maria Carla Mazzotti

HEMA but not TEGDMA induces autophagy in human gingival fibroblasts

Polymerized resin-based materials are successfully used in restorative dentistry. Despite their growing popularity, one drawback is the release of monomers from the polymerized matrix due to an incomplete polymerization or degradation processes. Released monomers are responsible for several adverse effects in the surrounding biological tissues, inducing high levels of oxidative stress. Reactive oxygen species are important signaling molecules that regulate many signal-trasduction pathways and play critical roles in cell survival, death, and immune defenses. Reactive oxygen species were recently shown to activate autophagy as a mechanism of cell survival and cell death. Although the toxicity induced by dental resin monomers is widely studied, the cellular mechanisms underlying these phenomena are still unknown. The aim of the study was to investigate the behavior of human gingival cells exposed to 2-hydroxy-ethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA) to better elucidate the mechanisms of cell survival and cell death induced by resin monomers. Primary culture of human gingival cells were exposed to 3 mmol/L of HEMA or 3 mmol/L of TEGDMA for 24, 48, and 72 h. Morphological investigations were performed by transmission electron microscopy to analyze the ultrastructure of cells exposed to the monomers. The expression of protein markers for apoptosis (caspase – 3 and PARP) and autophagy (beclin – 1 and LC3B I/II) were analyzed by western blot to investigate the influence of dental resin monomers on mechanisms underlying cell death. Results showed that HEMA treatment clearly induced autophagy followed by apoptosis while the lack of any sign of autophagy activation is observed in HGFs exposed to TEGDMA. These data indicate that cells respond to monomer-induced stress by the differential induction of adaptive mechanisms to maintain cellular homeostasis.

The tumor microenvironment promotes cancer progression and cell migration

The tumor microenvironment contributes to cancer progression, in part through interactions between tumor and normal stromal cells. This study analyzed morphological and molecular changes induced in co-cultured human fibroblasts (HFs) and the MG-63 osteosarcoma cell line. Co-cultured cell monolayers were morphologically analyzed using high resolution scanning electron microscopy (HR-SEM), and trans-well assays were performed to assess cell migration and invasion. Proteins involved in inflammatory responses, cancer cell invasion, and angiogenesis were assessed using western blotting. HR-SEM showed progressive spatial orientation loss by fibroblasts in contact with MG-63s, while MG-63s proliferated rapidly and invaded HF space. Trans-well assays showed enhanced MG-63 migration in the presence of HFs. IL-6 expression was increased in co-cultured HFs, possibly stimulated by TNF-α. HFs do not normally express YKL-40 but did so in co-culture. Band densitometric analyses showed that increasing YKL-40 expression was followed by VEGF overexpression, especially in MG-63s. Finally, our results confirmed fibroblasts as the main matrix metalloproteinase source in this tumor microenvironment. Our study sheds new light on how tumor-stroma interactions promote tumor development and progression, and may support identification of novel anti-cancer therapeutics.

A homemade furnace. Influence of occupational skills in a fire-related planned complex suicide

The term “planned complex suicide” means the combination of more than one method of suicide, planned to prevent failure of the first method to ensure a fatal outcome. Professional skills and tools are sometimes used to plan and perpetrate the suicide. A case of planned complex suicide of a mechanical engineer working with furnaces in a tube factory is herein reported. The suicide was committed in a rudimental furnace set up in the victim’s apartment using his professional skills, by assembling furniture, mattresses, books and flammable liquid present in the house. Three-dimensional models of the crime scene before and after the realization of the “homemade furnace” are proposed. The discussion will focus on the importance of a comprehensive analysis of the professional background of the victim to infer the manner of death for the identification of complex and occupation-related suicides.

HIF1α protein and mRNA expression as a new marker for post mortem interval estimation in human gingival tissue

Estimating the post mortem interval (PMI) is still a crucial step in Forensic Pathology. Although several methods are available for assessing the PMI, a precise estimation is still quite unreliable and can be inaccurate. The present study aimed to investigate the immunohistochemical distribution and mRNA expression of hypoxia inducible factor (HIF‐1α) in post mortem gingival tissues to establish a correlation between the presence of HIF‐1α and the time since death, with the final goal of achieving a more accurate PMI estimation. Samples of gingival tissues were obtained from 10 cadavers at different PMIs (1–3 days, 4–5 days and 8–9 days), and were processed for immunohistochemistry and quantitative reverse transcription‐polymerase chain reaction. The results showed a time‐dependent correlation of HIF‐1α protein and its mRNA with different times since death, which suggests that HIF‐1α is a potential marker for PMI estimation. The results showed a high HIF‐1α protein signal that was mainly localized in the stratum basale of the oral mucosa in samples collected at a short PMI (1–3 days). It gradually decreased in samples collected at a medium PMI (4–5 days), but it was not detected in samples collected at a long PMI (8–9 days). These results are in agreement with the mRNA data. These data indicate an interesting potential utility of Forensic Anatomy‐based techniques, such as immunohistochemistry, as important complementary tools to be used in forensic investigations.

Possible fatal hyperthermia involving drug abuse in a vehicle: case series

Major interplaying causes of heat stroke (HS) and fatal hyperthermia are climate, physical activity, artificial extreme ambient temperatures, confinement in a small compartment, and effects of drugs and chemicals, combined with predispositions and complications. A common and unfortunate cause of HS is vehicular hyperthermia (VH) death. Hyperthermia in a vehicle is a type of environmental thermal disorder, involving victims susceptibility, confinement with restraint, and possible influences of drugs and poisons, including exhaust gas containing complex fumes, carbon dioxide and monoxide. Whereas VH typically occurs when babies or young children are being left unattended in parked vehicles in direct sunlight, it has been reported only anecdotally for adult subjects. Three cases of adult fatal VH will be herein presented. In each presented case the corpse was found enclosed in a vehicle on spring/summer days. During crime scene investigation (CSI) psychoactive substances were found near to the corpses leading to the suspect of a suicidal fatal drug intoxication. Basing on this misleading suspect in Case 2 and 3 a forensic expert was not charged for the CSI and a fatal VH was not suspected nor properly investigated. Later, a comprehensive autopsy, including biochemical and toxicological analyses, excluded a death related to natural causes, fatal intoxications, ketoacidosis and traumas. On the other hand, the reconstruction of the temperature, the humidity, the heat index and the related risk of HS allowed the diagnosis of fatal VH in all the reported cases. In particular, death occurred because of the long-lasting stay into a hot parked vehicle which was facilitated from self-administration of psychoactive drugs with related neuro-depression. This case series confirms that a comprehensive CSI followed by an autopsy including histology, biochemical and toxicological analysis remains mandatory in cases of forensic interest, as well as when a corpse is found enclosed in a vehicle. Anyway, sometimes the diagnosis of heat-related fatalities remains a medley of investigative and medicolegal observations.

Increased MG-63s invasion potential mediated by HFs

During a malignant transformation, the crosstalk between the stroma and the cancer cells is described as a growing network of physical and paracrine signals, and it seems to have a direct influence on the phenotypic, genetic and epigenetic changes that affect the cells (1). In order to invade and metastasize to distant tissues, cancer cells transform themselves via ECM, induce tumor angiogenesis as well as undergo proliferation, detachment, migration, and invasion through secretion of various tumor derived factors (2). In this study we decided to analyze morphological and molecular aspects due to the coexistence between tumor cells MG-63s and fibroblasts HFs, verifying in particular the ability of MG-63s of invasion and microenvironment modulation. Monolayers of co-cultured cells were morphologically analyzed in time-laps by HR-SEM microscopy and a trans-well migration assay was performed over 24 h, 48 h, 72 h, and 96 h. The expression of several proteins, focusing on those involved in cancer cell invasion, inflammatory responses, and angiogenesis (TNF alpha, IL-6, YKL-40, MMP-1, MMP-9, and VEGF) was validated by Western blotting analysis. The images in time-laps for HR - SEM showed that fibroblasts in contact with MG-63 lost their spatial orientation, while the MG-63 quickly reached the confluence advancing towards HF cells, invading their space and overlying them. The increased MG-63s invasion mediated by the coexistence with HFs was confirmed by invasion assays in transwell co-culture. The protein levels of TNF-alpha, IL-6, YKL-40 and VEGF confirmed that tumor cells can regulate the development of a “tumor-stroma” via the aberrant expression of growth factors in the stromal compartment. Our results showed how tumor-stroma interactions play a significant role in tumor development and progression.

Changes in the gene expression of co-cultured human fibroblast cells and osteosarcoma cells: the role of microenvironment

The progression of malignant tumors does not depend exclusively on the autonomous properties of cancer cells; it is also influenced by tumor stroma reactivity and is under strict microenvironmental control. By themselves, stromal cells are not malignant, and they maintain normal tissue structure and function. However, through intercellular interactions or by paracrine secretions from cancer cells, normal stromal cells acquire abnormal phenotypes that sustain cancer cell growth and tumor progression. In their dysfunctional state, fibroblast and immune cells produce chemokines and growth factors that stimulate cancer cell growth and invasion. In our previous work (1), we established an in vitro model based on a monolayer co-culture system of healthy human fibroblasts (HFs) and human osteosarcoma cells (the MG-63 cell line) that simulates the microenvironment of tumor cells and healthy cells. The coexistence between MG-63 cells and HFs allowed us to identify the YKL-40 protein as the main marker for verifying the influence of tumor cells grown in contact with healthy cells. In this study, we evaluated the interactions of HFs and MG-63 cells in a transwell co-culture system over 24 h, 48 h, 72 h, and 96 h. We analyzed the contributions of these populations to the tumor microenvironment during cancer progression, as measured by multiple markers. We examined the effect of siRNA knockdown of YKL- 40 by tracking the subsequent changes in gene expression within the co-culture. We validated the expression of several genes, focusing on those involved in cancer cell invasion, inflammatory responses, and angiogenesis: TNF alpha, IL-6, MMP-1, MMP- 9, and VEGF. We compared the results to those from a transwell co-culture without the YKL-40 knockdown. In a pro-inflammatory environment promoted by TNF alpha and IL-6, siRNA knock- down of YKL-40 caused a down-regulation of VEGF and MMP-1 expression in HFs. These results suggest that the tumor microenvironment has an influence on the protein expression of healthy surrounding tissues and on the process of tumorigenicity. The mechanisms of the microenvironment are emerging as attractive targets for therapeutic strategies.

Ultrastructural changes in human gingival fibroblasts after exposure to 2-hydroxy-ethyl methacrylate.

Polymerized resin-based materials are successfully utilized in medical applications. One draw- back is the release of monomers from the matrix due to an incomplete polymerization or degradation processes. Released monomers can diffuse in the systemic circulation and induceadverse effects to biological tissues. Although there are many hypotheses about the induction of cell death by resin monomers, the underlying mechanisms are still under discussion. The aim of the study was to investigate the morphological modifications in human gingival fibroblasts exposed to 2-hydroxy-ethyl methacrylate (HEMA) to better elucidate the mechanism of cell death induced by resin monomers. Primary cultures of gingival fibroblasts were exposed to 3mM HEMA for 24 h, 72 h, 96 h. Morphological investigations were performed by scanning and transmission electron microscopy, while western blot for caspase-3 was carried out to ver- ify apoptosis. Electron microscopy images showed deep changes in the cell surface and cyto- plasm after 72 h and 96 h of HEMA treatment. Autophagic vesicles were easily observed just after 24 h. Cleaved caspase-3 was detected after 72 h of treatment. These findings suggest that resin based materials induced cell death by the cooperation of apoptosis and autophagy mecha- nisms. The understanding of these mechanisms will lead to the development of smart biomate- rials without or with low adverse effects.