Regulatory T cells as metabolic sensors.

Compelling experimental evidence links immunity and metabolism. In this perspective, we propose forkhead-box-P3 (FoxP3)+CD4+CD25+ regulatory T (Treg) cells as key metabolic sensors controlling the immunological state in response to their intrinsic capacity to perceive nutritional changes. Treg cell high anabolic state in vivo, residency in metabolically crucial districts, and recirculation between lymphoid and non-lymphoid sites enable them to recognize the metabolic cues and adapt their intracellular metabolism and anti-inflammatory function at the paracrine and systemic levels. As privileged regulators at the interface between neuroendocrine and immune systems, the role of Treg cells in maintaining metabolic homeostasis makes these cells promising targets of therapeutic strategies aimed at restoring organismal homeostasis not only in autoimmune but also metabolic disorders.

Signals of pseudo-starvation unveil the amino acid transporter SLC7A11 as key determinant in the control of Treg cell proliferative potential.

Human CD4+CD25hiFOXP3+ regulatory T (Treg) cells are key players in the control of immunological self-tolerance and homeostasis. Here, we report that signals of pseudo-starvation reversed human Treg cell in vitro anergy through an integrated transcriptional response, pertaining to proliferation, metabolism, and transmembrane solute carrier transport. At the molecular level, the Treg cell proliferative response was dependent on the induction of the cystine/glutamate antiporter solute carrier (SLC)7A11, whose expression was controlled by the nuclear factor erythroid 2-related factor 2 (NRF2). SLC7A11 induction in Treg cells was impaired in subjects with relapsing-remitting multiple sclerosis (RRMS), an autoimmune disorder associated with reduced Treg cell proliferative capacity. Treatment of RRMS subjects with dimethyl fumarate (DMF) rescued SLC7A11 induction and fully recovered Treg cell expansion. These results suggest a previously unrecognized mechanism that may account for the progressive loss of Treg cells in autoimmunity and unveil SLC7A11 as major target for the rescue of Treg cell proliferation.

Metabolic Plasticity of Regulatory T Cells in Health and Autoimmunity.

Immunometabolism has been demonstrated to control immune tolerance and the pathogenic events leading to autoimmunity. Compelling experimental evidence also suggests that intracellular metabolic programs influence differentiation, phenotype, proliferation, and effector functions of anti-inflammatory CD4+CD25+Foxp3+ regulatory T (Treg) cells. Indeed, alterations in intracellular metabolism associate with quantitative and qualitative impairments of Treg cells in several pathological conditions. In this review, we summarize the most recent advances linking how metabolic pathways control Treg cell homeostasis and their alterations occurring in autoimmunity. Also, we analyze how metabolic manipulations could be employed to restore Treg cell frequency and function with the aim to create novel therapeutic opportunities to halt immune-mediated disorders.

Progression of type 1 diabetes is associated with high levels of soluble PD-1 in islet autoantibody-positive children.

AIMS/HYPOTHESIS: Type 1 diabetes is an autoimmune disorder that is characterised by destruction of pancreatic beta cells by autoreactive T lymphocytes. Although islet autoantibodies (AAb) are an indicator of disease progression, specific immune biomarkers that can be used as target molecules to halt development of type 1 diabetes have not been discovered. Soluble immune checkpoint molecules (sICM) play a pivotal role in counteracting excessive lymphocyte responses, but their role in type 1 diabetes is unexplored. In this longitudinal study, we measured sICM levels in AAb-positive (AAb+) children to identify molecules related to type 1 diabetes progression. METHODS: We measured the levels of 14 sICM in the sera of AAb+ children (n=57) compared to those with recent-onset type 1 diabetes (n=79) and healthy children (n=44), obtained from two cohorts. AAb+ children were followed up and divided based on their progression to type 1 diabetes (AAbP) or not (AAbNP) (if they lost islet autoimmunity and did not develop disease in subsequent years). sICM were also measured in the sample taken at the visit closest to disease onset in AAbP children. RESULTS: We found that AAb+ children had a distinct sICM profile compared with healthy children and those with recent-onset type 1 diabetes. In addition, AAb+ children who progressed to type 1 diabetes (AAbP) had higher sICM concentrations than non-progressors (AAbNP). Further, sICM levels decreased in AAbP children close to disease onset. Application of Cox regression models highlighted that high concentrations of soluble programmed cell death protein 1 (sPD-1) are associated with type 1 diabetes progression (HR 1.71; 95% CI 1.16, 2.51; p=0.007). CONCLUSIONS/INTERPRETATION: This study reveals an sICM profile that is dysregulated during the preclinical stage of type 1 diabetes, and identifies sPD-1 as a pathophysiologically-relevant molecule that is associated with disease progression, offering a potential target for early interventions in autoimmune diabetes.

Different experimental approaches for Fourier-transform infrared spectroscopy applications in biology and biotechnology: A selected choice of representative results.

Fourier transform infrared (FTIR) spectroscopy is a powerful tool for analyzing the biochemical properties of biological samples such as proteins, cellular materials, and tissues. It provides objective information on samples and has been adopted in many research areas of biomedical and biotechnological interest. FTIR spectroscopy can be performed using different approaches at the macro and micro levels allowing the examination of an incredibly broad class of materials. However, it has become evident that the choice of proper spectra acquisition geometries and the modalities of sample preparation in FTIR spectroscopy analysis require special consideration, especially for certain classes of materials such as cells and tissues. In the present paper, we described the different procedures used for preparing and analyzing different types of biological and biotechnological samples when the more largely available approaches are employed using a commercial FTIR spectrometer. Some basic aspects of data analysis procedures are presented in an Appendix. A certain number of our previous experimental results are reported for demonstrating once more the versatility and the potentiality of FTIR spectroscopy.

Vibrational spectroscopies for biochemical investigation of X-ray exposure effects on SH-SY5Y human neuroblastoma cells.

Neuroblastoma is the most recurring cancer in childhood and adolescence. The SH-SY5Y neuroblastoma cell line is generally adopted for elaborating new therapeutical approaches and/or elaborating strategies for the prevention of central nervous system disturbances. In fact, it represents a valid model system for investigating in vitro the effects on the brain of X-ray exposure using vibrational spectroscopies that can detect early radiation-induced molecular alterations of potential clinical usefulness. In recent years, we dedicated significant efforts in the use of Fourier-transform and Raman microspectroscopy techniques for characterizing such radiation-induced effects on SH-SY5Y cells by examining the contributions from different cell components (DNA, proteins, lipids, and carbohydrates) to the vibrational spectra. In this review, we aim at revising and comparing the main results of our studies to provide a wide outlook of the latest outcomes and a framework for future radiobiology research using vibrational spectroscopies. A short description of our experimental approaches and data analysis procedures is also reported.

A Sphingolipidomic Profiling Approach for Comparing X-ray-Exposed and Unexposed HepG2 Cells.

An analytical method based on tandem mass spectrometry-shotgun is presently proposed to obtain sphingolipidomic profiles useful for the characterization of lipid extract from X-ray-exposed and unexposed hepatocellular carcinoma cells (HepG2). To obtain a targeted lipidic profile from a specific biological system, the best extraction method must be identified before instrumental analysis. Accordingly, four different classic lipid extraction protocols were compared in terms of efficiency, specificity, and reproducibility. The performance of each procedure was evaluated using the Fourier-transform infrared spectroscopic technique; subsequently, the quality of extracts was estimated using electrospray ionization tandem mass spectrometry. The selected procedure based on chloroform/methanol/water was successfully used in mass spectrometry-based shotgun sphingolipidomics, allowing for evaluation of the response of cells to X-ray irradiation, the most common anticancer therapy. Using a relative quantitative approach, the changes in the sphingolipid profiles of irradiated cell extracts were demonstrated, confirming that lipidomic technologies are also useful tools for studying the key sphingolipid role in regulating cancer growth during radiotherapy.

High levels of blood circulating immune checkpoint molecules in children with new-onset type 1 diabetes are associated with the risk of developing an additional autoimmune disease.

AIMS/HYPOTHESIS: We assessed the levels of blood circulating immune checkpoint molecules (ICMs) at diagnosis of type 1 diabetes, and determined their association with the risk of developing an additional autoimmune disorder over time. METHODS: Children with new-onset type 1 diabetes (n = 143), without biological and/or clinical signs of additional autoimmune disorders, and healthy children (n = 75) were enrolled, and blood circulating levels of 14 ICMs were measured. The children with type 1 diabetes were divided into two groups on the basis of the development of an additional autoimmune disease in the 5 years after diabetes onset. Differences in soluble ICM levels between the groups were assessed, and a Cox regression analysis was used to evaluate their association with the risk of development of an additional autoimmune disease over time. To validate the data, circulating ICMs were measured in an independent cohort of 60 children with new-onset type 1 diabetes stratified into two groups. RESULTS: We found that the levels of circulating ICMs were significantly higher in children with new-onset diabetes compared with healthy children. Further, we observed that children with type 1 diabetes who developed a second autoimmune disease over time (T1D-AAD+ children) had higher levels of soluble ICMs than children with type 1 diabetes who did not (T1D-AAD- children). Cox regression models revealed that high circulating levels of CD137/4-1BB and PD-1 molecules at diabetes diagnosis were associated with the risk of developing an additional autoimmune disease in both type 1 diabetes cohorts. CONCLUSIONS/INTERPRETATION: Our findings suggest that soluble CD137/4-1BB and PD-1 molecules may be used as prognostic biomarkers in children with type 1 diabetes, and may pave the way for novel immunological screening at diabetes onset, allowing early identification of children at higher risk of developing other autoimmune conditions over time.

Optical Sensors Technology and Applications.

Optical methods are non-invasive tools, and their use in various fields, including sensing applications, is continuously increasing, which is thanks to the continuous development of innovative low-cost sources and detectors [...].

µ-FTIR, µ-Raman, and SERS Analysis of Amide I Spectral Region in Oral Biofluid Samples during Orthodontic Treatment.

Gingival crevicular fluid (GCF) is a site-specific exudate deriving from the epithelium lining of the gingival sulcus. GCF analysis provides a simple and noninvasive diagnostic procedure to follow-up periodontal and bone remodeling in response to diseases or mechanical stimuli such as orthodontic tooth movement (OTM). In recent years, the use of vibrational spectroscopies such as Fourier Transform Infrared and Raman microspectroscopy and Surface-Enhanced Raman spectroscopy contributed to characterizing changes in GCF during fixed orthodontic treatment. Amide I band plays a relevant role in the analysis of these changes. The aim of this study was to investigate the spectroscopy response of Amide I depending on the OTM process duration. A model based on Gaussian-Lorentzian curves was used to analyze the infrared spectra, while only Lorentzian functions were used for Raman and SERS spectra. Changes induced by the OTM process in subcomponents of the Amide I band were determined and ascribed to secondary structure modification occurring in proteins. The vibrational spectroscopies allow us to efficiently monitor the effects of the orthodontic force application, thus gaining increasing attention as tools for individual patient personalization in clinical practice.

Advanced Optical Sensing of Phenolic Compounds for Environmental Applications.

Phenolic compounds are particularly dangerous due to their ability to remain in the environment for a long period of time and their toxic effects. They enter in the environment in different ways, such as waste from paper manufacturing, agriculture (pesticides, insecticides, herbicides), pharmaceuticals, the petrochemical industry, and coal processing. Conventional methods for phenolic compounds detection present some disadvantages, such as cumbersome sample preparation, complex and time-consuming procedures, and need of expensive equipment. Therefore, there is a very large interest in developing sensors and new sensing schemes for fast and easy-to-use methods for detecting and monitoring the phenolic compound concentration in the environment, with special attention to water. Good analytical properties, reliability, and adaptability are required for the developed sensors. The present paper aims at revising the most generally used optical methods for designing and fabricating biosensors and sensors for phenolic compounds. Some selected examples of the most interesting applications of these techniques are also proposed.

Monitoring Biochemical and Structural Changes in Human Periodontal Ligaments during Orthodontic Treatment by Means of Micro-Raman Spectroscopy.

The aim of the study was to examine the biochemical and structural changes occurring in the periodontal ligament (PDL) during orthodontic-force application using micro-Raman spectroscopy ( µ -RS). Adolescent and young patients who needed orthodontic treatment with first premolar extractions were recruited. Before extractions, orthodontic forces were applied using a closed-coil spring that was positioned between the molar and premolar. Patients were randomly divided into three groups, whose extractions were performed after 2, 7, and 14 days of force application. From the extracted premolars, PDL samples were obtained, and a fixation procedure with paraformaldehyde was adopted. Raman spectra were acquired for each PDL sample in the range of 1000-3200 cm - 1 and the more relevant vibrational modes of proteins (Amide I and Amide III bands) and CH 2 and CH 3 modes were shown. Analysis indicated that the protein structure in the PDL samples after different time points of orthodontic-force application was modified. In addition, changes were observed in the CH 2 and CH 3 high wavenumber region due to local hypoxia and mechanical force transduction. The reported results indicated that µ -RS provides a valuable tool for investigating molecular interchain interactions and conformational modifications in periodontal fibers after orthodontic tooth movement, providing quantitative insight of time occurring for PDL molecular readjustment.

Graphene-Based and Surface-Enhanced Raman Spectroscopy for Monitoring the Physio-Chemical Response of Thermophilic Bacterial Spores to Low Temperatures Exposure.

Monitoring the spore life cycle is one of the main issues in several fields including environmental control, sustainable ecosystems, food security, and healthcare systems. In this framework, the study of the living organism resistance to extreme conditions like those mimicking space environments is particularly interesting. The assessment of the local change of the pH level can be extremely useful for this purpose. An optical physiometer method based on the Raman response of the graphene, which is able to locally sense pH of a fluid on a micrometric scale, has been recently proposed. Due to the presence of π -bonds at the surface, the electronic doping of graphene is determined by the external conditions and can be electrochemically controlled or altered by the contact with an acid or alkaline fluid. The doping level affects the vibrational energies of the graphene that can be monitored by conventional Raman spectroscopy. In addition, Surface-Enhanced Raman Spectroscopy (SERS) can give direct information on the biochemical changes occurring in spore components. In this work, we propose the joint use of Graphene-Based Raman Spectroscopy (GbRS) and SERS for the monitoring of the response of spores to exposure to low temperatures down to 100 K. The spores of the thermophilic bacterium Parageobacillus thermantarcticus isolated from an active volcano of Antarctica (Mt. Melbourne) were investigated. These spores are particularly resistant to several stressing stimuli and able to adapt to extreme conditions like low temperatures, UV irradiation, and γ -rays exposure. The results obtained showed that the joint use of GbRS and SERS represents a valuable tool for monitoring the physio-chemical response of bacterial spores upon exposure to stressing stimuli.

An insight into pH-induced changes in FAD conformational structure by means of time-resolved fluorescence and circular dichroism.

Optical properties of flavin adenine dinucleotide (FAD) moiety are widely used nowadays for biotechnological applications. Given the fundamental role played by FAD, additional structural information about this enzymatic cofactor can be extremely useful in order to obtain a greater insight into its functional role in proteins. For this purpose, we have investigated FAD behaviour in aqueous solutions at different pH values by a novel approach based on the combined use of time-resolved fluorescence and circular dichroism spectroscopies. The results showed that pH strongly affects time-resolved fluorescence emission and the analysis allowed us to detect a three-component decay for FAD in aqueous solution with pH-depending lifetimes and relative amplitudes. Circular dichroism data were analyzed by a multi-Gaussian fitting procedure and the trends of properly chosen parameters confirmed pH-depending changes. The comparison between the results obtained by these two optical techniques allowed us to improve the significance of the outcome of circular dichroism. This combined approach may provide a useful tool for biotechnological investigation.

Multivariate Analysis of Difference Raman Spectra of the Irradiated Nucleus and Cytoplasm Region of SH-SY5Y Human Neuroblastoma Cells.

Previous works showed that spatially resolved Raman spectra of cytoplasm and nucleus region of single cells exposed to X-rays evidence different features. The present work aims to introduce a new approach to profit from these differences to deeper investigate X-ray irradiation effects on single SH-SY5Y human neuroblastoma cells. For this aim, Raman micro-spectroscopy was performed in vitro on single cells after irradiation by graded X-ray doses (2, 4, 6, 8 Gy). Spectra from nucleus and cytoplasm regions were selectively acquired. The examination by interval Principal Component Analysis (i-PCA) of the difference spectra obtained by subtracting each cytoplasm-related spectrum from the corresponding one detected at the nucleus enabled us to reveal the subtle modifications of Raman features specific of different spatial cell regions. They were discussed in terms of effects induced by X-ray irradiation on DNA/RNA, lipids, and proteins. The proposed approach enabled us to evidence some features not outlined in previous investigations.

Characterization of Human Tear Fluid by Means of Surface-Enhanced Raman Spectroscopy.

Tears are exceptionally rich sources of information on the health status of the eyes, as well as of whole body functionality, due to the presence of a large variety of salts and organic components whose concentration can be altered by pathologies, eye diseases and/or inflammatory processes. Surface enhanced Raman spectroscopy (SERS) provides a unique method for analyzing low concentrations of organic fluids such as tears. In this work, a home-made colloid of gold nanoparticles has been used for preparing glass substrates able to efficiently induce an SERS effect in fluid samples excited by a He⁻Ne laser ( λ = 633 nm). The method has been preliminary tested on Rhodamine 6G aqueous solutions at different concentrations, proving the possibility to sense substance concentrations as low as few µ M, i.e., of the order of the main tear organic components. A clear SERS response has been obtained for human tear samples, allowing an interesting insight into tear composition. In particular, aspartic acid and glutamic acid have been shown to be possible markers for two important human tear components, i.e., lactoferrin and lysozyme.

Graphene-Based Raman Spectroscopy for pH Sensing of X-rays Exposed and Unexposed Culture Media and Cells.

Graphene provides a unique way of sensing the local pH level of substances on the micrometric scale, with important implications for the monitoring of cellular metabolic activities where proton excretion could occur. Accordingly, an innovative biosensing approach for the quantification of the pH value of biological fluids, to be used also with small amounts of fluids, was realized and tested. It is based on the use of micro-Raman spectroscopy to detect the modifications of the graphene doping level induced by the contact of the graphene with the selected fluids. The approach was preliminarily tested on aqueous solutions of known pH values. It was then used to quantify the pH values of cell culture media directly exposed to different doses of X-ray radiation and to media exposed to X-ray-irradiated cells. The Raman response of cells placed on graphene layers was also examined.

PET/MR in invasive ductal breast cancer: correlation between imaging markers and histological phenotype.

BACKGROUND: Differences in genetics and receptor expression (phenotypes) of invasive ductal breast cancer (IDC) impact on prognosis and treatment response. Immunohistochemistry (IHC), the most used technique for IDC phenotyping, has some limitations including its invasiveness. We explored the possibility of contrast-enhanced positron emission tomography magnetic resonance (CE-FDG PET/MR) to discriminate IDC phenotypes. METHODS: 21 IDC patients with IHC assessment of oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor-2 (HER2), and antigen Ki-67 (Ki67) underwent CE-FDG PET/MR. Magnetic resonance-perfusion biomarkers, apparent diffusion coefficient (ADC), and standard uptake value (SUV) were compared with IHC markers and phenotypes, using a Student's t-test and one-way ANOVA. RESULTS: ER/PR- tumours demonstrated higher Kepmean and SUVmax than ER or PR+ tumours. HER2- tumours displayed higher ADCmean, Kepmean, and SUVmax than HER2+tumours. Only ADCmean discriminated Ki67⩽14% tumours (lower ADCmean) from Ki67>14% tumours. PET/MR biomarkers correlated with IHC phenotype in 13 out of 21 patients (62%; P=0.001). CONCLUSIONS: Positron emission tomography magnetic resonance might non-invasively help discriminate IDC phenotypes, helping to optimise individual therapy options.

Optical detection of different phenolic compounds by means of a novel biosensor based on sol-gel immobilized laccase.

A novel sol-gel-based biosensor exploiting the optical absorption properties of sol-gel immobilized laccase has been constructed to increase enzyme specificity toward different phenolic substrates. Laccase from Trametes versicolor has been immobilized in optically transparent sol-gel matrices. Using Fourier transform infrared spectroscopy and data analysis based on a wavelet algorithm, a successful enzyme immobilization has been determined. The changes in the optical absorption spectra of laccase reaction products at 425, 375, and 400 nm have been used to determine hydroquinone, resorcinol, and catechol concentrations, respectively. Owing to the slow response time of the hydroquinone-laccase reaction, our optical biosensor has been tested with resorcinol and catechol. Linear ranges up to 1.4 and 0.2 mM, limit-of-detection (LOD) of 4.5 and 0.6 µΜ, have been evidenced for resorcinol and catechol, respectively. Data for determining the resorcinol concentration have been particularly interesting since no other biosensor device has been reported in the literature. In comparison with other biosensors using laccase from the same native source, our biosensor has been characterized by larger linear ranges, significant sensitivities, and good LODs. To challenge our biosensor with real samples, tap water samples spiked with known amount of catechol and resorcinol have been employed.