Increased Plasma Levels of Mitochondrial DNA and Normal Inflammasome Gene Expression in Monocytes Characterize Patients With Septic Shock Due to Multidrug Resistant Bacteria

Introduction: The activity and regulation of inflammasome is receiving increasing attention in septic shock. Moreover, there is a growing body of evidence suggesting that mitochondrial DNA (mtDNA) can play a role as biomarker of disease severity and even mortality both in adults and children in critically ill setting. However, no data are available on the amount of circulating mtDNA and inflammasome gene expression in multi-drug resistant (MDR) bacteria septic shock. For this reason, the aim of this study was to determine whether plasma mtDNA levels and inflammasome gene expression in monocytes could be related to severity in patients admitted to intensive care unit (ICU) with septic shock due to MDR pathogens. Materials and Methods: Peripheral blood mononuclear cells (PBMC) and plasma were isolated from up to 20 ml of venous blood by density gradient centrifugation in patients admitted to ICU with the diagnosis of septic shock due to MDR-bacteria. Then, CD14+ monocytes were sorted, and RNA and DNA were extracted. NLRP3, PYCARD, AIM2 and NAIP expression level was analyzed by RT-PCR. Plasma circulating mtDNA levels were quantified by digital droplet PCR. Basal and outcome characteristics of the patients were collected. Age-matched healthy subjects were chosen as controls. Results: Nineteen patients with septic shock and 20 healthy subjects were enrolled in the study. A small trend toward an increased expression of inflammasome genes was observed in septic shock patients, who also displayed a marked tendency to an increased expression of IL-18 and IL-1\u3b2 genes. Circulating mtDNA levels were significantly higher in septic shock patients if compared to healthy subjects, and patients who died in ICU were characterized by higher level of mtDNA if compared to those who were dismissed after 7 days. No correlations were found between mtDNA and inflammasome level and other clinical variables. Conclusion: Despite many limitations, our data suggest that in patients with septic shock caused by MDR pathogens the expression of main inflammasome genes was comparable to that of healthy patients without infection. Furthermore, our data evidence a possible role of mtDNA as a prognostic marker of severity in septic shock from MDR

3D Microfluidic model for evaluating immunotherapy efficacy by tracking dendritic cell behaviour toward tumor cells

Immunotherapy efficacy relies on the crosstalk within the tumor microenvironment between cancer and dendritic cells (DCs) resulting in the induction of a potent and effective antitumor response. DCs have the specific role of recognizing cancer cells, taking up tumor antigens (Ags) and then migrating to lymph nodes for Ag (cross)-presentation to naïve T cells. Interferon-α-conditioned DCs (IFN-DCs) exhibit marked phagocytic activity and the special ability of inducing Ag-specific T-cell response. Here, we have developed a novel microfluidic platform recreating tightly interconnected cancer and immune systems with specific 3D environmental properties, for tracking human DC behaviour toward tumor cells. By combining our microfluidic platform with advanced microscopy and a revised cell tracking analysis algorithm, it was possible to evaluate the guided efficient motion of IFN-DCs toward drug-treated cancer cells and the succeeding phagocytosis events. Overall, this platform allowed the dissection of IFN-DC-cancer cell interactions within 3D tumor spaces, with the discovery of major underlying factors such as CXCR4 involvement and underscored its potential as an innovative tool to assess the efficacy of immunotherapeutic approaches

Prognostic immune markers identifying patients with severe COVID-19 who respond to tocilizumab

Introduction: A growing number of evidences suggest that the combination of hyperinflammation, dysregulated T and B cell response and cytokine storm play a major role in the immunopathogenesis of severe COVID-19. IL-6 is one of the main pro-inflammatory cytokines and its levels are increased during SARS-CoV-2 infection. Several observational and randomized studies demonstrated that tocilizumab, an IL-6R blocker, improves survival in critically ill patients both in infectious disease and intensive care units. However, despite transforming the treatment options for COVID-19, IL-6R inhibition is still ineffective in a fraction of patients. Methods: In the present study, we investigated the impact of two doses of tocilizumab in patients with severe COVID-19 who responded or not to the treatment by analyzing a panel of cytokines, chemokines and other soluble factors, along with the composition of peripheral immune cells, paying a particular attention to T and B lymphocytes. Results: We observed that, in comparison with non-responders, those who responded to tocilizumab had different levels of several cytokines and different T and B cells proportions before starting therapy. Moreover, in these patients, tocilizumab was further able to modify the landscape of the aforementioned soluble molecules and cellular markers. Conclusions: We found that tocilizumab has pleiotropic effects and that clinical response to this drug remain heterogenous. Our data suggest that it is possible to identify patients who will respond to treatment and that the administration of tocilizumab is able to restore the immune balance through the re-establishment of different cell populations affected by SARS-COV-2 infection, highlighting the importance of temporal examination of the pathological features from the diagnosis

Innate immunity changes in soccer players after whole-body cryotherapy

Whole-body cryotherapy (WBC) consists of short exposure (up to 2-3 min) to dry air at cryogenic temperatures (up to -190 degrees C) and has recently been applied for muscle recovery after injury to reduce the inflammation process. We aimed to determine the impact of cryotherapy on immunological, hormonal, and metabolic responses in non-professional soccer players (NPSPs). Nine male NPSPs (age: 20 +/- 2 years) who trained regularly over 5 consecutive days, immediately before and after each training session, were subjected to WBC treatment (WBC-t). Blood samples were collected for the evaluation of fifty analytes including hematologic parameters, serum chemistry, and hormone profiles. Monocytes phenotyping (Mo) was performed and plasmatic markers, usually increased during inflammation [CCL2, IL-18, free mitochondrial (mt)DNA] or with anti-inflammatory effects (IL2RA, IL1RN), were quantified. After WBC-t, we observed reduced levels of ferritin, mean corpuscular hemoglobin, mean platelet volume, testosterone, and estradiol, which however remain within the normal ranges. The percentage of the total, intermediates and non-classical Mo increased, while classical Mo decreased. CXCR4 expression decreased in each Mo subset. Plasma IL18 and IL2RA levels decreased, while IL1RN only exhibited a tendency to decrease and CCL2 showed a tendency to increase. Circulating mtDNA levels were not altered following WBC-t. The differences observed in monocyte subsets after WBC-t may be attributable to their redistribution into the surrounding tissue. Moreover, the decrease of CXCR4 in Mo subpopulations could be coherent with their differentiation process. Thus, WBC through yet unknown mechanisms could promote their differentiation having a role in tissue repair

Detailed characterization of SARS-CoV-2-specific T and B cells after infection or heterologous vaccination

: The formation of a robust long-term antigen (Ag)-specific memory, both humoral and cell-mediated, is created following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or vaccination. Here, by using polychromatic flow cytometry and complex data analyses, we deeply investigated the magnitude, phenotype, and functionality of SARS-CoV-2-specific immune memory in two groups of healthy subjects after heterologous vaccination compared to a group of subjects who recovered from SARS-CoV-2 infection. We find that coronavirus disease 2019 (COVID-19) recovered patients show different long-term immunological profiles compared to those of donors who had been vaccinated with three doses. Vaccinated individuals display a skewed T helper (Th)1 Ag-specific T cell polarization and a higher percentage of Ag-specific and activated memory B cells expressing immunoglobulin (Ig)G compared to those of patients who recovered from severe COVID-19. Different polyfunctional properties characterize the two groups: recovered individuals show higher percentages of CD4+ T cells producing one or two cytokines simultaneously, while the vaccinated are distinguished by highly polyfunctional populations able to release four molecules, namely, CD107a, interferon (IFN)-γ, tumor necrosis factor (TNF), and interleukin (IL)-2. These data suggest that functional and phenotypic properties of SARS-CoV-2 adaptive immunity differ in recovered COVID-19 individuals and vaccinated ones

Analysis of Antigen-Specific T and B Cells for Monitoring Immune Protection Against SARS-CoV-2

: Immunological memory is the basis of protection against most pathogens. Long-living memory T and B cells able to respond to specific stimuli, as well as persistent antibodies in plasma and in other body fluids, are crucial for determining the efficacy of vaccination and for protecting from a second infection by a previously encountered pathogen. Antigen-specific cells are represented at a very low frequency in the blood, and indeed, they can be considered "rare events" present in the memory T-cell pool. Therefore, such events should be analyzed with careful attention. In the last 20 years, different methods, mostly based upon flow cytometry, have been developed to identify such rare antigen-specific cells, and the COVID-19 pandemic has given a dramatic impetus to characterize the immune response against the virus. In this regard, we know that the identification, enumeration, and characterization of SARS-CoV-2-specific T and B cells following infection and/or vaccination require i) the use of specific peptides and adequate co-stimuli, ii) the use of appropriate inhibitors to avoid nonspecific activation, iii) the setting of appropriate timing for stimulation, and iv) the choice of adequate markers and reagents to identify antigen-specific cells. Optimization of these procedures allows not only determination of the magnitude of SARS-CoV-2-specific responses but also a comparison of the effects of different combinations of vaccines or determination of the response provided by so-called "hybrid immunity," resulting from a combination of natural immunity and vaccine-generated immunity. Here, we present two methods that are largely used to monitor the response magnitude and phenotype of SARS-CoV-2-specific T and B cells by polychromatic flow cytometry, along with some tips that can be useful for the quantification of these rare events. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Identification of antigen-specific T cells Basic Protocol 2: Identification of antigen-specific B cells

Study on the Public Perception of “Community-Owned Dogs” in the Abruzzo Region, Central Italy

The Abruzzo Regional Law Nr. 47/2013, following a circular from the Italian Ministry of Health and OIE recommendations, allows the local municipalities to release free-roaming dogs (FRDs) caught on the territory once the local veterinary services (LVSs) have rated the dogs as unowned and not aggressive, have neutered them, as well as identified them through a microchip and a visible collar. The responsibility of these “community-owned dogs” (CODs) falls under the mayor of the local municipality that can entrust their custody to qualified people. The present study was conducted in the Abruzzo region, located in central Italy, to investigate public perception towards CODs, and in particular whether their presence is perceived as a problem or a benefit by the local communities. The data were collected by both direct interviews and an online survey, based on a questionnaire developed by a multidisciplinary team. The questionnaire was distributed in 31 municipalities sampled on the basis of the urbanization rate, and 497 people were interviewed over a 9-month period. More than half of the respondents (54%) stated that CODs can contribute to the control of stray dogs. The majority (83%) believed that a greater commitment is needed to involve the local communities on issues regarding CODs. The findings of this study highlighted the general difficulty for people to distinguish stray dogs from CODs that are not fully known, as evidenced by the fact that 59% of the respondents did not know the aforementioned Regional Law that defines and regulates the presence of the CODs

E-cigarettes induce in vivo toxicological effects that can raise the cancer risk

Introduction: Electronic cigarettes (e-cigs) are devices thought to deliver nicotine in a vaping solution without tobacco combustion. Perceived as a safer alternative to conventional cigarettes, e-cigs are aggressively marketed as lifestyle-choice consumables, thanks to few regulatory restrictions. E-cigs have also gained popularity among never-smokers and teenagers, becoming an emergent public health issue. Despite the burgeoning worldwide consumption, their safety remains largely unproven and it is unknown whether these devices could cause in vivo toxicological effects contributing to cancer. Materials and methods: Male Sprague Dawley rats were randomly split into two groups: non-exposed and whole-body mode exposed (10 animals per group). Treated animals consumed 1 mL/day of e-liquid containing 18 mg/mL of nicotine. During the experiment the e-cigarette voltage was set at 5.5 (and the wattage was about 15). Animals were exposed for 4 hours/day for 5 consecutive days/week, and for 4 consecutive weeks. Results: Toxic aldehydes production, due to thermal decomposition of glycerol, were detected in e-cig aerosol. In exposed lung, we observed a significant increase in phase I CYP1A1/2, CYP2B1/2, CY2C11 and CYP3A enzymes. The overproduction of reactive oxygen species resulting from CYP induction, together with the free radicals present in the aerosol, was confirmed by means “Electron paramagnetic resonance-radical probe” technique. Notably, we observed that the lung antioxidant enzymes catalase, DT-diaphorase, superoxide dismutase and the conjugating phase II glutathione S-transferases were all markedly reduced in animal treated. In addition, we found a markedly reduced FRAP (ferric reducing antioxidant power) value in the lung of e-cig rodents. Moreover, plasma FRAP levels and measurement of carbonyl residues, as biomarkers of oxidative injury to proteins, were inversely correlated in e-cig vapour-exposed rats. We also found that 8-hydro-2′-deoxyguanosine markedly increased in the lungs of treated rats. Interestingly, e-cigs produce extensive DNA damage in leukocytes (Comet test). We also found that e-cig vapour determines an increase in the percentage of immature micronucleated reticulocytes over normal reticulocyte. Next, the urine of e-cig-exposed animals induced a dose-dependent increase in the number of S. typhimurium revertants in different strains. Discussion and conclusion: We found that e-cigs have a powerful booster effect on phase-I carcinogen-bioactivating enzymes, and increase oxygen free radical production and DNA oxidation to 8-hydroxy- 2′-deoxyguanosine. Furthermore, we found that e-cigs damage DNA not only at chromosomal level in peripheral blood, such as strand breaks in leucocytes and micronuclei formation in reticulocytes, but also at gene level such as point mutations in urine. As these detrimental phenomena are typically induced by conventional cigarettes, the erroneous belief that e-cigs are safe should be retracted and suitable measures implemented to protect public health. Our study should be seen as the starting point for further investigations designed to confirm the harmful health impact of e-cigs, particularly after long-term exposure and under different usage conditions

Development of Real-Time Kinematic Magnetic Resonance Imaging (kMRI) Techniques for Studying the Kinematics of the Spine and Joints in Dogs—Preliminary Study on Cadavers

Kinematic MRI (kMRI) is a novel human imaging technique that couples the excellent soft tissue contrast and multiplanar capabilities of traditional MRI with kinematic potential. The study’s goals are: (1) testing the feasibility of spinal cord and joints real-time kMRI; and (2) evaluating the quality of these kinematic studies as a new diagnostic option in veterinary medicine. Standard and real-time kinematic MRI were performed on cervical spine, elbow, and stifle joints of seven cadavers. Studies were repeated after a surgical insult aimed to create a certain degree of joint instability. A total of 56 MRI were performed—7 cervical spinal tracts, 3 elbow joints, and 4 stifle joints were examined. The technique was feasible in all the three regions examined. The images were considered of excellent quality for the stifle joint, good to fair for the cervical spine, whereas two of three elbow studies were considered to have unacceptable image quality. Additionally, real-time kMRI provided good to excellent information about stifle instability. Therefore we consider kMRI a promising technique in veterinary medicine. Further studies and an in vivo setting are needed to increase the quality of the kMRI images, and to fully evaluate clinical usefulness

Redistribution of CD8+ T cell subsets in metastatic renal cell carcinoma patients treated with anti-PD-1 therapy

Renal-cell carcinoma (RCC) is responsible for the majority of tumors arising from the kidney parenchyma. Although a progressive improvement in median overall survival has been observed after the introduction of anti-PD-1 therapy, many patients do not benefit from this treatment. Therefore, we have investigated T cell dynamics to find immune modification induced by anti-PD-1 therapy. Here, we show that, after therapy, RCC patients (5 responders and 14 nonresponders) are characterized by a redistribution of different subsets across the memory T cell compartment