Cell metabolism: Functional and phenotypic single cell approaches.

Several metabolic pathways are essential for the physiological regulation of immune cells, but their dysregulation can cause immune dysfunction. Hypermetabolic and hypometabolic states represent deviations in the magnitude and flexibility of effector cells in different contexts, for example in autoimmunity, infections or cancer. To study immunometabolism, most methods focus on bulk populations and rely on in vitro activation assays. Nowadays, thanks to the development of single-cell technologies, including multiparameter flow cytometry, mass cytometry, RNA cytometry, among others, the metabolic state of individual immune cells can be measured in a variety of samples obtained in basic, translational and clinical studies. Here, we provide an overview of different single-cell approaches that are employed to investigate both mitochondrial functions and cell dependence from mitochondria metabolism. Moreover, besides the description of the appropriate experimental settings, we discuss the strengths and weaknesses of different approaches with the aim to suggest how to study cell metabolism in the settings of interest.

Immunosenescence and vaccine efficacy revealed by immunometabolic analysis of SARS-CoV-2-specific cells in multiple sclerosis patients.

Disease-modifying therapies (DMT) administered to patients with multiple sclerosis (MS) can influence immune responses to SARS-CoV-2 and vaccine efficacy. However, data on the detailed phenotypic, functional and metabolic characteristics of antigen (Ag)-specific cells following the third dose of mRNA vaccine remain scarce. Here, using flow cytometry and 45-parameter mass cytometry, we broadly investigate the phenotype, function and the single-cell metabolic profile of SARS-CoV-2-specific T and B cells up to 8 months after the third dose of mRNA vaccine in a cohort of 94 patients with MS treated with different DMT, including cladribine, dimethyl fumarate, fingolimod, interferon, natalizumab, teriflunomide, rituximab or ocrelizumab. Almost all patients display functional immune response to SARS-CoV-2. Different metabolic profiles characterize antigen-specific-T and -B cell response in fingolimod- and natalizumab-treated patients, whose immune response differs from all the other MS treatments.

Advances and Challenges in Sepsis Management: Modern Tools and Future Directions.

Sepsis, a critical condition marked by systemic inflammation, profoundly impacts both innate and adaptive immunity, often resulting in lymphopenia. This immune alteration can spare regulatory T cells (Tregs) but significantly affects other lymphocyte subsets, leading to diminished effector functions, altered cytokine profiles, and metabolic changes. The complexity of sepsis stems not only from its pathophysiology but also from the heterogeneity of patient responses, posing significant challenges in developing universally effective therapies. This review emphasizes the importance of phenotyping in sepsis to enhance patient-specific diagnostic and therapeutic strategies. Phenotyping immune cells, which categorizes patients based on clinical and immunological characteristics, is pivotal for tailoring treatment approaches. Flow cytometry emerges as a crucial tool in this endeavor, offering rapid, low cost and detailed analysis of immune cell populations and their functional states. Indeed, this technology facilitates the understanding of immune dysfunctions in sepsis and contributes to the identification of novel biomarkers. Our review underscores the potential of integrating flow cytometry with omics data, machine learning and clinical observations to refine sepsis management, highlighting the shift towards personalized medicine in critical care. This approach could lead to more precise interventions, improving outcomes in this heterogeneously affected patient population.

Circulating and Tumor-Associated Neutrophils in the Era of Immune Checkpoint Inhibitors: Dynamics, Phenotypes, Metabolism, and Functions.

Neutrophils are the most abundant myeloid cells in the blood and are a considerable immunological component of the tumor microenvironment. However, their functional importance has often been ignored, as they have always been considered a mono-dimensional population of terminally differentiated, short-living cells. During the last decade, the use of cutting-edge, single-cell technologies has revolutionized the classical view of these cells, unmasking their phenotypic and functional heterogeneity. In this review, we summarize the emerging concepts in the field of neutrophils in cancer, by reviewing the recent literature on the heterogeneity of both circulating neutrophils and tumor-associated neutrophils, as well as their possible significance in tumor prognosis and resistance to immune checkpoint inhibitors.

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.

Patients Recovering from Severe COVID-19 Develop a Polyfunctional Antigen-Specific CD4+ T Cell Response.

Specific T cells are crucial to control SARS-CoV-2 infection, avoid reinfection and confer protection after vaccination. We have studied patients with severe or moderate COVID-19 pneumonia, compared to patients who recovered from a severe or moderate infection that had occurred about 4 months before the analyses. In all these subjects, we assessed the polyfunctionality of virus-specific CD4+ and CD8+ T cells by quantifying cytokine production after in vitro stimulation with different SARS-CoV-2 peptide pools covering different proteins (M, N and S). In particular, we quantified the percentage of CD4+ and CD8+ T cells simultaneously producing interferon-γ, tumor necrosis factor, interleukin (IL)-2, IL-17, granzyme B, and expressing CD107a. Recovered patients who experienced a severe disease display high proportions of antigen-specific CD4+ T cells producing Th1 and Th17 cytokines and are characterized by polyfunctional SARS-CoV-2-specific CD4+ T cells. A similar profile was found in patients experiencing a moderate form of COVID-19 pneumonia. No main differences in polyfunctionality were observed among the CD8+ T cell compartments, even if the proportion of responding cells was higher during the infection. The identification of those functional cell subsets that might influence protection can thus help in better understanding the complexity of immune response to SARS-CoV-2.

The multivariate approach identifies relationships between pre-slaughter factors, body lesions, ham defects and carcass traits in pigs.

Abattoir meat inspection has been proposed for the collection of welfare outcomes. The identification of suitable animal-based measures (ABM) is still a critical point that needs to be implemented to avoid collinearity among measures. The present study aims to benchmark the presence of ABM such as skin and tail lesions and ham defects in carcasses from 79 batches of Italian Heavy pigs and to identify possible relationships between the assessed ABM and pre-slaughter factors such as the season and the overnight lairage. Furthermore, the study also considers the effect of pre-slaughter conditions and ABM on carcass traits parameters (cold carcass weight and lean meat percentage). Skin and tail lesions were recorded at the slaughter line. The presence of abscesses, muscle tears and veining defects were assessed in the hams at trimming, according to the Parma Ham Consortium. A multivariate analysis was performed to identify relationships between ABM and pre-slaughter factor; therefore, a linear model was built to assess the effect on carcass weight and lean meat percentage. Main welfare issues were represented by skin and tail lesions and muscle tears (prevalence above 10%). Multivariate analysis evidenced that skin lesions and veining defects were mostly associated with the warm season. Abscesses and muscle tears in the hams were more likely related to overnight lairage, while tail lesions contributed equally to both season and lairage. Moreover, lairage related factors showed to affect lean meat percentage. The findings of the present study suggest that ham defects might be useful indicators of pre-slaughter stress. The validation of these findings with physiological parameters could be of interest for further studies.

On-Farm Welfare Assessment Protocol for Suckling Piglets: A Pilot Study.

Piglets experience welfare issues during the nursery phase. This pilot study aimed to test a protocol for identifying the main welfare issues in suckling piglets and to investigate relationships among animal-based indicators and management conditions. Litters (n = 134), composed of undocked and tail-docked piglets, were assessed at two farms. After birth, observations were made at the age of 7 days and 20 days. At each observation, housing conditions (HCs) were measured, and 13 animal-based indicators, modified from Welfare Quality, Classyfarm, Assurewel and others introduced ex novo, were recorded. A generalized linear mixed model was used, considering animal-based indicators as dependent variables and farm, piglets' age, tail docking and HCs as independent variables. The main welfare issues were lesions of the limb (32.6%) and the front area of the body (22.8%), a poor body condition score (BCS) (16.1%), ear lesions (15.5%), and tail lesions (9.7%). Negative social behaviour (e.g., fighting and biting) represented 7.0% of the active behaviour, with tail biting observed in 8.7% of the piglets. While lesions on the front areas of the body were mostly associated with the farm, tail lesions, low BCS, tear staining, and diarrhoea were associated with light and nest temperature (p < 0.05). In particular, tail biting increased with scarce light (p = 0.007). Tail docking did not influence any animal-based indicator except for tear staining which was higher in the tail-docked as compared to the undocked piglets (p = 0.05), increasing awareness on this practice as a source of negative emotion in piglets. The protocol tested may be a promising tool for assessing on-farm piglets' welfare.

Reversed arterial flow in the paramedian segments of the liver after pancreaticoduodenectomy.

Variation of splanchnic arterial anatomy is frequent and may complicate pancreatic surgery, leading to life-threatening postoperative complications. We report a case of severe hepatic dysfunction after pancreaticoduodenectomy due to accidental section of the right hepatic artery. Bedside ultrasound examination showed a reversed arterial flow in the paramedian segments of the liver. Based on intraoperative and ultrasound findings, a retrograde arterial supply of the right liver from the left hepatic artery through spontaneous anastomosis between the arteries of segment 4 and the right paramedian segments was assumed and confirmed by postoperative CT examination.

Bilateral epidermoid cysts of the testis: sonographic and MRI findings.

Epidermoid cysts of the testis are rare benign lesions, without malignant potential, that can be managed conservatively with cyst enucleation and testis-sparing surgery. Bilateral epidermoid cysts in the testes have been reported very infrequently. We report the sonographic and MRI findings in a patient who presented with a palpable nodule in only 1 testis, but in whom sonography showed bilateral lesions. On sonography, the cysts had an internal "onion-ring" structure; color Doppler signals were absent. On MRI, the cysts had a laminated appearance, with alternating low- and high-signal-intensity areas on T2-weighted images. These imaging findings and the negative results of laboratory tests for tumor markers suggested the correct diagnosis, and testis-sparing surgery could be performed.

[Focal liver lesions: characterization with quantitative analysis of T2 relaxation time in TSE sequence with double echo time].

PURPOSE: To evaluate the accuracy of calculation of T2 relaxation time with simplified algorithm in characterization of liver focal lesions with Turbo Spin Echo (TSE) T2-weighted sequences with double echo time (TE). MATERIALS AND METHODS: We carried out a retrospective analysis of 113 hyperintense hepatic focal lesions in T2-weighted sequences (52 metastases, 51 hemangiomas and 10 cysts). We have employed a single TSE T2-weighted sequence with effective TE of 83 and 165 msec and turbo factor 9. Signal intensity (IS) of each lesion was measured on different TE. Using these values, we calculated T2 relaxation time by the following simplified algorithm: T2(ms)=(TE2-TE1)/ (lnIS1-lnIS2). T2 relaxation time has been correlated with the final diagnosis, obtained by percutaneous biopsy, surgical resection or three months follow-up. RESULTS: The mean T2 relaxation time for metastases was 108,7 msec (+/-30), for hemangiomas 166,5 msec (+/- 29) and for cysts 312,4 msec (+/-19). The difference between the mean T2 relaxation time for metastases and hemangiomas was statistically significant (p<0.0001). Most metastases were best characterized between the threshold value of 130 and 140 msec. The values of specificity and sensitivity of the diagnosis of malignant hepatic lesion was respective 0,86 and 0,88. All false positive diagnoses were hemangiomas of less than one centimeter. CONCLUSIONS: Introducing double echo time TSE sequences in the protocol of liver study concurs to obtain high diagnostic accuracy in the characterization of the hepatic lesions. The quantitative analysis of T2 represents a useful additional instrument to the morphologic analysis in discrimination between hepatic lesions and can be carried out by TSE sequences with elevated reliability reducing the imaging time compared with conventional sequences. The evaluation of hepatic lesions of less than one centimeter should always include analysis of dynamic gadolinium-contrast images.

Brachial plexus sonography: a technique for assessing the root level.

OBJECTIVE: Our study was intended to establish a technique to assess the level of the roots of the brachial plexus using high-resolution sonography. MATERIALS AND METHODS: The skeleton of a cervical spine was examined in vitro to determine whether the vertebrae may be identified individually on sonography by means of the evaluation of their transverse processes. Then 20 healthy subjects and five patients who had undergone CT of the cervical spine were evaluated sonographically, and we attempted to identify the level of individual roots of the brachial plexus using the transverse processes as landmarks. To establish the reliability of this method, a blinded review of sonograms of the paravertebral area obtained at various levels was performed independently by three examiners. RESULTS: In vitro, sonography was reliable in depicting the level of the C7 vertebra because of the absence of the anterior tubercle from its transverse processes. In healthy subjects, this feature allowed us to establish the level of the roots outside the spine. In our series, the C4-C7 roots were visible sonographically in all cases, whereas the C8 and T1 levels were seen, respectively, in only 16 of 20 and eight of 20 cases. All examiners correctly identified the C7 level in the blinded review of sonograms. CONCLUSION: High-resolution sonography can reveal the level of the roots of the brachial plexus on the basis of the different morphology of the transverse processes of the vertebrae. Our study has implications for confirming the exact level of pathologic roots before surgery.