The Influence of Nucleoside Reverse Transcriptase Inhibitors on Mitochondrial Activity, Lipid Content, and Fatty-Acid-Binding Protein Levels in Microglial HMC3 Cells.

Despite the availability of a wide range of preventive measures and comprehensive treatment options following infection, the development of acquired immunodeficiency syndrome (AIDS) remains a persistent challenge. Nucleoside reverse transcriptase inhibitors (NRTIs) represent the most commonly utilized therapeutic approach, despite being on the pharmaceutical market for nearly four decades. During this time, a spectrum of side effects ranging from mild discomfort and hypersensitivity reactions to the more prevalent nephrotoxicity and hepatotoxicity has been documented. In light of these considerations, our study aimed to investigate the impacts of two NRTIs, lamivudine and zidovudine, on lipid metabolism in HMC3 microglial cells. Our findings revealed statistically significant reductions in the ATP levels (nearly 8%) and increased mitochondrial superoxide levels (around 10%) after 24 h of treatment with the maximum therapeutic concentration of zidovudine compared to the untreated microglial cells. Furthermore, the concentrations of fatty-acid-binding proteins 4 and 5 were significantly lower (approximately 40%) in the microglial cells that were exposed to NRTIs than in the untreated cells. Notably, the total lipid concentration within the microglial cells markedly increased following NRTI administration with a 13% rise after treatment with 10 µM lamivudine and a remarkable 70% surge following the administration of 6 µM zidovudine. These results suggest that the prolonged administration of NRTIs may potentially lead to lipid accumulation, posing a significant risk to the delicate homeostasis of the neuronal system and potentially triggering a pro-inflammatory response in microglial cells.

Three-dimensional Collagen Scaffolds in Cultures of Olfactory Ensheathing Cells Used for Severed Spinal Cord Regeneration.

BACKGROUND/AIM: The regeneration of a completely damaged spinal cord is still a challenge in modern medicine. A promising treatment method is autologous transplantation of olfactory ensheathing cells (OECs). This study aimed primarily to test methods of culturing OECs with the use of materials and reagents that are certified for pharmaceutical use in the production of an advanced cell therapy product intended for humans. MATERIALS AND METHODS: The culture of OECs was performed using various modifications of the surface of the culture vessels (with fibronectin and poly-D-lysine). The number of cells was assessed after immunofluorescence staining using anti-fibronectin and anti-p75 NGF receptor antibodies. The study compared, in terms of surgical manipulations, scaffolds with OECs prepared based on 3 types of collagen: Acid Solubilized Telo Collagen and Pepsin Solubilized Atelocollagen, and the popular Corning collagen. RESULTS: We have shown that when suspending OECs in collagen gel, it is much better to use acid-solubilized collagen (ASC) than pepsin-solubilized collagen (PSC) because the 3D collagen scaffold from ASC provides much easier handling of the product during a surgical procedure. We also found that the OEC cultures should be grown on the surface modified with fibronectin. Furthermore, we have also shown that the optimal concentration of fetal bovine serum (FBS) for culturing these cells should be around 10%. CONCLUSION: The culture of OECs based on reagents intended for human use can be successfully carried out, obtaining sufficient OECs content in the heterogeneous cell culture to produce a functional advanced therapy medicinal product.

Effect of amyloid-ß on the redox system activity in SH-SY5Y cells preincubated with lipopolysaccharide or co-cultured with microglia cells.

Amyloid deposits and hyperphosphorylation of the tau protein are still believed to be the two main causes of Alzheimer's disease. However, newer studies show the beneficial (including antiradical and antimicrobial) effects of amyloid at physiological concentrations. Therefore, this study aimed to investigate the impact of three amyloid fragments - 25-35, 1-40, and 1-42 at concentrations close to physiological levels on the oxidative stress induced by the administration of lipopolysaccharide (LPS) or co-culturing with microglia cells. Differentiated SH-SY5Y cells were used, constituting a model of neuronal cells that were preincubated with LPS or supernatant collected from THP-1 cell culture. The cells were treated with amyloid-ß fragments at concentrations of 0.001, 0.1, and 1.0 µM, and then biological assays were carried out. The results of the study support the antioxidant properties of Aß, which may protect neurons from the damaging effects of neuroinflammation. All tested amyloid-ß fragments reduced oxidative stress and increased the levels of enzymatic stress parameters - the activity of SOD, GPx and catalase. In addition, the administration of amyloid-ß at low physiological concentrations also increased reduced glutathione (GSH) levels and the ratio between reduced and oxidized glutathione (GSH/GSSG), which is considered a good indicator of maintaining cellular redox balance. Furthermore, a stronger antioxidant effect of 1-40 fragment was observed, occurring in a wider range of concentrations, compared to the other tested fragments 25-35 and 1-42.

Molecular Mechanism of Lipotoxicity as an Interesting Aspect in the Development of Pathological States-Current View of Knowledge.

Free fatty acids (FFAs) play numerous vital roles in the organism, such as contribution to energy generation and reserve, serving as an essential component of the cell membrane, or as ligands for nuclear receptors. However, the disturbance in fatty acid homeostasis, such as inefficient metabolism or intensified release from the site of storage, may result in increased serum FFA levels and eventually result in ectopic fat deposition, which is unfavorable for the organism. The cells are adjusted for the accumulation of FFA to a limited extent and so prolonged exposure to elevated FFA levels results in deleterious effects referred to as lipotoxicity. Lipotoxicity contributes to the development of diseases such as insulin resistance, diabetes, cardiovascular diseases, metabolic syndrome, and inflammation. The nonobvious organs recognized as the main lipotoxic goal of action are the pancreas, liver, skeletal muscles, cardiac muscle, and kidneys. However, lipotoxic effects to a significant extent are not organ-specific but affect fundamental cellular processes occurring in most cells. Therefore, the wider perception of cellular lipotoxic mechanisms and their interrelation may be beneficial for a better understanding of various diseases' pathogenesis and seeking new pharmacological treatment approaches.

The Impact of High Glucose or Insulin Exposure on S100B Protein Levels, Oxidative and Nitrosative Stress and DNA Damage in Neuron-Like Cells.

Alzheimer's disease (AD) is attracting considerable interest due to its increasing number of cases as a consequence of the aging of the global population. The mainstream concept of AD neuropathology based on pathological changes of amyloid ß metabolism and the formation of neurofibrillary tangles is under criticism due to the failure of Aß-targeting drug trials. Recent findings have shown that AD is a highly complex disease involving a broad range of clinical manifestations as well as cellular and biochemical disturbances. The past decade has seen a renewed importance of metabolic disturbances in disease-relevant early pathology with challenging areas in establishing the role of local micro-fluctuations in glucose concentrations and the impact of insulin on neuronal function. The role of the S100 protein family in this interplay remains unclear and is the aim of this research. Intracellularly the S100B protein has a protective effect on neurons against the toxic effects of glutamate and stimulates neurites outgrowth and neuronal survival. At high concentrations, it can induce apoptosis. The aim of our study was to extend current knowledge of the possible impact of hyper-glycemia and -insulinemia directly on neuronal S100B secretion and comparison to oxidative stress markers such as ROS, NO and DBSs levels. In this paper, we have shown that S100B secretion decreases in neurons cultured in a high-glucose or high-insulin medium, while levels in cell lysates are increased with statistical significance. Our findings demonstrate the strong toxic impact of energetic disturbances on neuronal metabolism and the potential neuroprotective role of S100B protein.

Sirtuins as Important Factors in Pathological States and the Role of Their Molecular Activity Modulators.

Sirtuins (SIRTs), enzymes from the family of NAD+-dependent histone deacetylases, play an important role in the functioning of the body at the cellular level and participate in many biochemical processes. The multi-directionality of SIRTs encourages scientists to undertake research aimed at understanding the mechanisms of their action and the influence that SIRTs have on the organism. At the same time, new substances are constantly being sought that can modulate the action of SIRTs. Extensive research on the expression of SIRTs in various pathological conditions suggests that regulation of their activity may have positive results in supporting the treatment of certain metabolic, neurodegenerative or cancer diseases or this connected with oxidative stress. Due to such a wide spectrum of activity, SIRTs may also be a prognostic markers of selected pathological conditions and prove helpful in assessing their progression, especially by modulating their activity. The article presents and discusses the activating or inhibiting impact of individual SIRTs modulators. The review also gathered selected currently available information on the expression of SIRTs in individual disease cases as well as the biological role that SIRTs play in the human organism, also in connection with oxidative stress condition, taking into account the progress of knowledge about SIRTs over the years, with particular reference to the latest research results.

PC12 Cell Line: Cell Types, Coating of Culture Vessels, Differentiation and Other Culture Conditions.

The PC12 cell line is one of the most commonly used in neuroscience research, including studies on neurotoxicity, neuroprotection, neurosecretion, neuroinflammation, and synaptogenesis. Two types of this line are available in the ATCC collection: traditional PC12 cells grown in suspension and well-attached adherent phenotype. PC12 cells grown in suspension tend to aggregate and adhere poorly to non-coated surfaces. Therefore, it is necessary to modify the surface of culture vessels. This paper aims to characterise the use of two distinct variants of PC12 cells as well as describe their differentiation and neuronal outgrowth with diverse NGF concentrations (rat or human origin) on various surfaces. In our study, we evaluated cell morphology, neurite length, density and outgrowth (measured spectrofluorimetrically), and expression of neuronal biomarkers (doublecortin and NeuN). We found that the collagen coating was the most versatile method of surface modification for both cell lines. For adherent cells, the coating was definitely less important, and the poly-d-lysine surface was as good as collagen. We also demonstrated that the concentration of NGF is of great importance for the degree of differentiation of cells. For suspension cells, we achieved the best neuronal characteristics (length and density of neurites) after 14 days of incubation with 100 ng/mL NGF (change every 48 h), while for adherent cells after 3-5 days, after which they began to proliferate. In the PC12 cell line, doublecortin (DCX) expression in the cytoplasm and NeuN in the cell nucleus were found. In turn, in the PC12 Adh line, DCX was not expressed, and NeuN expression was located in the entire cell (both in the nucleus and cytoplasm). Only the traditional PC12 line grown in suspension after differentiation with NGF should be used for neurobiological studies, especially until the role of the NeuN protein, whose expression has also been noted in the cytoplasm of adherent cells, is well understood.

The Interplay between Diabetes and Alzheimer's Disease-In the Hunt for Biomarkers.

The brain is an organ in which energy metabolism occurs most intensively and glucose is an essential and dominant energy substrate. There have been many studies in recent years suggesting a close relationship between type 2 diabetes mellitus (T2DM) and Alzheimer's disease (AD) as they have many pathophysiological features in common. The condition of hyperglycemia exposes brain cells to the detrimental effects of glucose, increasing protein glycation and is the cause of different non-psychiatric complications. Numerous observational studies show that not only hyperglycemia but also blood glucose levels near lower fasting limits (72 to 99 mg/dL) increase the incidence of AD, regardless of whether T2DM will develop in the future. As the comorbidity of these diseases and earlier development of AD in T2DM sufferers exist, new AD biomarkers are being sought for etiopathogenetic changes associated with early neurodegenerative processes as a result of carbohydrate disorders. The S100B protein seem to be interesting in this respect as it may be a potential candidate, especially important in early diagnostics of these diseases, given that it plays a role in both carbohydrate metabolism disorders and neurodegenerative processes. It is therefore necessary to clarify the relationship between the concentration of the S100B protein and glucose and insulin levels. This paper draws attention to a valuable research objective that may in the future contribute to a better diagnosis of early neurodegenerative changes, in particular in subjects with T2DM and may be a good basis for planning experiments related to this issue as well as a more detailed explanation of the relationship between the neuropathological disturbances and changes of glucose and insulin concentrations in the brain.