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.

Airborne pathogens diffusion: A comparison between tracer gas and pigmented aerosols for indoor environment analysis.

The evaluation of airborne pathogens diffusion is a crucial practice in preventing airborne diseases like COVID-19, especially in indoor environments. Through this transmission route, pathogens can be carried by droplets, droplet nuclei and aerosols and be conveyed over long distances. Therefore, understanding their diffusion is vital for prevention and curbing disease transmission. There are different techniques used for this purpose, and one of the most common is the utilization of tracer gas, however, it has limitations such as the difference in size between the gas molecules and the respiratory droplets, as well as its incapability to take into account evaporation. For this reason, a new method for evaluating the diffusion of respiratory droplets has been developed. This approach involves the use of an ultrasonic emitter to release and disperse pigmented aerosols, and a colorimeter for the following quantitative evaluation. A comparison with the tracer gas technique has been carried out, showing for the pigmented aerosols methodology a response that is dependent on different relative humidity conditions, while there is no clear difference in the dispersion of tracer gas at high or low humidity.

Profiling the dysregulated immune response in sepsis: overcoming challenges to achieve the goal of precision medicine.

Sepsis is characterised by a dysregulated host immune response to infection. Despite recognition of its significance, immune status monitoring is not implemented in clinical practice due in part to the current absence of direct therapeutic implications. Technological advances in immunological profiling could enhance our understanding of immune dysregulation and facilitate integration into clinical practice. In this Review, we provide an overview of the current state of immune profiling in sepsis, including its use, current challenges, and opportunities for progress. We highlight the important role of immunological biomarkers in facilitating predictive enrichment in current and future treatment scenarios. We propose that multiple immune and non-immune-related parameters, including clinical and microbiological data, be integrated into diagnostic and predictive combitypes, with the aid of machine learning and artificial intelligence techniques. These combitypes could form the basis of workable algorithms to guide clinical decisions that make precision medicine in sepsis a reality and improve patient outcomes.

Evaluating immunological and inflammatory changes of treatment-experienced people living with HIV switching from first-line triple cART regimens to DTG/3TC vs. B/F/TAF: the DEBATE trial.

Background: The aim of this randomized clinical trial (RCT) was to compare immunological changes in virally suppressed people living with HIV (PLWH) switching from a three-drug regimen (3DR) to a two-drug regimen (2DR). Methods: An open-label, prospective RCT enrolling PLWH receiving a 3DR who switched to bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF) or dolutegravir/lamivudine (DTG/3TC) was performed. Blood was taken at baseline and months 6 and 12. The primary outcome was the change in CD4+ or CD8+ T-cell counts and CD4/CD8 ratio over time points. The secondary outcomes were the changes in immunological and inflammatory parameters. Parametric mixed-linear models with random intercepts and slopes were fitted separately for each marker after controlling for potential confounders. Results: Between the two arms (33 PLWH each), there was no difference in CD4+ or CD8+ T cells, CD4/CD8 ratio, and IL-6 trajectories. PLWH switching to DTG/3TC had increased levels of both transitional memory and terminally differentiated CD4+ T cells (arm-time interaction p-value = 0.02) and to a lesser extent for the corresponding CD8+ T-cell subsets (p = 0.09). Significantly lower levels of non-classical monocytes were detected in the B/F/TAF arm at T6 (diff = -6.7 cells/mm3; 95% CI; -16, +2.6; p-value for interaction between arm and time = 0.03). All differences were attenuated at T12. Conclusion: No evidence for a difference in absolute CD4+ and CD8+ T-cell counts, CD4/CD8 ratio, and IL-6 trajectories by study arm over 12 months was found. PLWH on DTG/3TC showed higher levels of terminally differentiated and exhausted CD4+ and CD8+ T lymphocytes and non-classical monocytes at T6. Further studies are warranted to better understand the clinical impact of our results. Clinical Trial Registration: https://clinicaltrials.gov, identifier NCT04054089.

The Association of Procalcitonin and C-Reactive Protein with Bacterial Infections Acquired during Intensive Care Unit Stay in COVID-19 Critically Ill Patients.

In COVID-19 patients, procalcitonin (PCT) and C-reactive protein (CRP) performance in identifying bacterial infections remains unclear. Our study aimed to evaluate the association of PCT and CRP with secondary infections acquired during ICU stay in critically ill COVID-19 patients. This observational study included adult patients admitted to three COVID-19 intensive care units (ICUs) from February 2020 to May 2022 with respiratory failure caused by SARS-CoV-2 infection and ICU stay ≥ 11 days. The values of PCT and CRP collected on the day of infection diagnosis were compared to those collected on day 11 after ICU admission, the median time for infection occurrence, in patients without secondary infection. The receiver operating characteristic curve (ROC) and multivariate logistic model were used to assess PCT and CRP association with secondary infections. Two hundred and seventy-nine patients were included, of whom 169 (60.6%) developed secondary infection after ICU admission. The PCT and CRP values observed on the day of the infection diagnosis were larger (p < 0.001) than those observed on day 11 after ICU admission in patients without secondary infections. The ROC analysis calculated an AUC of 0.744 (95%CI 0.685-0.803) and 0.754 (95%CI 0.695-0.812) for PCT and CRP, respectively. Multivariate logistic models showed that PCT ≥ 0.16 ng/mL and CRP ≥ 1.35 mg/dL were associated (p < 0.001) with infections acquired during ICU stay. Our results indicated that in COVID-19 patients, PCT and CRP values were associated with infections acquired during the ICU stay and can be used to support, together with clinical signs, rather than predict or rule out, the diagnosis of these infections.

Africa-specific human genetic variation near CHD1L associates with HIV-1 load.

HIV-1 remains a global health crisis1, highlighting the need to identify new targets for therapies. Here, given the disproportionate HIV-1 burden and marked human genome diversity in Africa2, we assessed the genetic determinants of control of set-point viral load in 3,879 people of African ancestries living with HIV-1 participating in the international collaboration for the genomics of HIV3. We identify a previously undescribed association signal on chromosome 1 where the peak variant associates with an approximately 0.3 log10-transformed copies per ml lower set-point viral load per minor allele copy and is specific to populations of African descent. The top associated variant is intergenic and lies between a long intergenic non-coding RNA (LINC00624) and the coding gene CHD1L, which encodes a helicase that is involved in DNA repair4. Infection assays in iPS cell-derived macrophages and other immortalized cell lines showed increased HIV-1 replication in CHD1L-knockdown and CHD1L-knockout cells. We provide evidence from population genetic studies that Africa-specific genetic variation near CHD1L associates with HIV replication in vivo. Although experimental studies suggest that CHD1L is able to limit HIV infection in some cell types in vitro, further investigation is required to understand the mechanisms underlying our observations, including any potential indirect effects of CHD1L on HIV spread in vivo that our cell-based assays cannot recapitulate.

Randomized, double-blind, placebo-controlled trial of rapamycin in amyotrophic lateral sclerosis.

In preclinical studies rapamycin was found to target neuroinflammation, by expanding regulatory T cells, and affecting autophagy, two pillars of amyotrophic lateral sclerosis (ALS) pathogenesis. Herein we report a multicenter, randomized, double-blind trial, in 63 ALS patients who were randomly assigned in a 1:1:1 ratio to receive rapamycin 2 mg/m2/day,1 mg/m2/day or placebo (EUDRACT 2016-002399-28; NCT03359538). The primary outcome, the number of patients exhibiting an increase >30% in regulatory T cells from baseline to treatment end, was not attained. Secondary outcomes were changes from baseline of T, B, NK cell subpopulations, inflammasome mRNA expression and activation status, S6-ribosomal protein phosphorylation, neurofilaments; clinical outcome measures of disease progression; survival; safety and quality of life. Of the secondary outcomes, rapamycin decreased mRNA relative expression of the pro-inflammatory cytokine IL-18, reduced plasmatic IL-18 protein, and increased the percentage of classical monocytes and memory switched B cells, although no corrections were applied for multiple tests. In conclusion, we show that rapamycin treatment is well tolerated and provides reassuring safety findings in ALS patients, but further trials are necessary to understand the biological and clinical effects of this drug in ALS.

Pillars of long-term antiretroviral therapy success.

BACKGROUND: Meeting the challenge of antiretroviral therapy (ART) whose efficacy can last a lifetime requires continuous updating of the virological, pharmacological, and quality of life outcomes to be pursued and a continuous review of literature data on the efficacy and tolerability of new drugs and therapeutic strategies. METHODS: With the aim of identifying open questions and answers about the current controversies in modern ART, we adapted the Design Thinking methodology to the needs of the design phase of a scientific article, involving a team of experts in HIV care. RESULTS: Five main pillars of treatment success were discussed: sustained virologic suppression over time; immunological recovery; pharmacological attributes; long-term tolerability and safety of ART; and people's satisfaction and quality of life. The definition of the outcomes to be achieved in each thematic area and the tools to achieve them were reviewed and discussed. CONCLUSIONS: Long-term treatment success should be intended as a combination of HIV-RNA suppression, immune recovery, and high quality of life. To achieve this, the regimen should be well-tolerated, with high potency, genetic barrier, and forgiveness, and should be tailored by a person-centered perspective, based on individual needs, preferences, and therapeutic history.

Critical COVID-19 patients through first, second, and third wave: retrospective observational study comparing outcomes in intensive care unit.

Background: The time-course of the coronavirus disease 2019 (COVID-19) pandemic was characterized by subsequent waves identified by peaks of intensive care unit (ICU) admission rates. During these periods, progressive knowledge of the disease led to the development of specific therapeutic strategies. This retrospective study investigates whether this led to improvement in outcomes of COVID-19 patients admitted to ICU. Methods: Outcomes were evaluated in consecutive adult COVID-19 patients admitted to our ICU, divided into three waves based on the admission period: the first wave from February 25th, 2020, to July 6th, 2020; the second wave from September 20th, 2020, to February 13th, 2021; the third wave from February 14th, 2021 to April 30th, 2021. Differences were assessed comparing outcomes and by using different multivariable Cox models adjusted for variables related to outcome. Further sensitivity analysis was performed in patients undergoing invasive mechanical ventilation (IMV). Results: Overall, 428 patients were included in the analysis: 102, 169, and 157 patients in the first, second, and third wave. The ICU and in-hospital crude mortalities were lower by 7% and 10% in the third wave compared to the other two waves (P>0.05). A higher number of ICU- and hospital-free days at day 90 was found in the third wave when compared to the other two waves (P=0.001). Overall, 62.6% underwent invasive ventilation, with decreasing requirement during the waves (P=0.002). The adjusted Cox model showed no difference in the hazard ratio (HR) for mortality among the waves. In the propensity-matched analysis the hospital mortality rate was reduced by 11% in the third wave (P=0.044). Conclusions: With application of best practice as known by the time of the first three waves of the pandemic, our study failed to identify a significant improvement in mortality rate when comparing the different waves of the COVID-19 pandemic, notwithstanding, the sub-analyses showed a trend in mortality reduction in the third wave. Rather, our study identified a possible positive effect of dexamethasone on mortality rate reduction and the increased risk of death related to bacterial infections in the three waves.

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.

Reduced Graphene Oxide Electrolyte-Gated Transistor Immunosensor with Highly Selective Multiparametric Detection of Anti-Drug Antibodies.

The advent of immunotherapies with biological drugs has revolutionized the treatment of cancers and auto-immune diseases. However, in some patients, the production of anti-drug antibodies (ADAs) hampers the drug efficacy. The concentration of ADAs is typically in the range of 1-10 pm; hence their immunodetection is challenging. ADAs toward Infliximab (IFX), a drug used to treat rheumatoid arthritis and other auto-immune diseases, are focussed. An ambipolar electrolyte-gated transistor (EGT) immunosensor is reported based on a reduced graphene oxide (rGO) channel and IFX bound to the gate electrode as the specific probe. The rGO-EGTs are easy to fabricate and exhibit low voltage operations (≤ 0.3 V), a robust response within 15 min, and ultra-high sensitivity (10 am limit of detection). A multiparametric analysis of the whole rGO-EGT transfer curves based on the type-I generalized extreme value distribution is proposed. It is demonstrated that it allows to selectively quantify ADAs also in the co-presence of its antagonist tumor necrosis factor alpha (TNF-α), the natural circulating target of IFX.

Altered pathways of keratinization, extracellular matrix generation, angiogenesis, and stromal stem cells proliferation in patients with systemic sclerosis.

Objective: Systemic sclerosis is characterized by endothelial dysfunction, autoimmunity abnormalities, and fibrosis of the skin and internal organs. The pathogenetic mechanisms underlying systemic sclerosis vasculopathy are still not clarified. A complex cellular and extracellular network of interactions has been studied, but it is currently unclear what drives the activation of fibroblasts/myofibroblasts and the extracellular matrix deposition. Methods: Using RNA sequencing, the aim of the work was to identify potential functional pathways implied in systemic sclerosis pathogenesis and markers of endothelial dysfunction and fibrosis in systemic sclerosis patients. RNA-sequencing analysis was performed on RNA obtained from biopsies from three systemic sclerosis patients and three healthy controls enrolled in our University Hospital. RNA was used to generate sequencing libraries that were sequenced according to proper transcriptomic analyses. Subsequently, we performed gene set enrichment analysis of differentially expressed genes on the entire list of genes that compose the RNA-sequencing expression matrix. Results: Gene set enrichment analysis revealed that healthy controls were characterized by gene signatures related to stromal stem cells proliferation, cytokine-cytokine receptor interaction, macrophage-enriched metabolic network, whereas systemic sclerosis tissues were enriched in signatures associated with keratinization, cornification, retinoblastoma 1 and tumor suppressor 53 signaling. Conclusion: According to our data, RNA-sequencing and pathway analysis revealed that systemic sclerosis subjects display a discrete pattern of gene expression associated with keratinization, extracellular matrix generation, and negative regulation of angiogenesis and stromal stem cells proliferation. Further analysis on larger numbers of patients is needed; however, our findings provide an interesting framework for the development of biomarkers useful to explore potential future therapeutic approaches.

Kaspar Hauser, the Child of Europe: Are smallpox vaccination scars the clue to a 2-century-old mystery?

We have explored the 19th century mystery of the identity of Kaspar Hauser, the so-called Child of Europe, from the perspective of the smallpox vaccination. We have highlighted the improbability that he was secretly inoculated based on the vaccination policies and methodologies applied at the time. This consideration allows for a reflection on the whole case and the importance of vaccination scars in ascertaining immunization against one of humanity's deadliest killers, especially given the recent monkeypox outbreak.

Superior vena cava syndrome and gynecomastia in antiquity: Paleodermatologic considerations on aging in the past.

We explore the antiquity of two well-known conditions often associated with advancing age, namely superior vena cava syndrome and gynecomastia, through the veristic sculptural representation dating back to the classical age. The statue of the Old Fisherman from the "Paolo Orsi" Regional Archaeological Museum of Syracuse, Italy, thanks to the extremely accurate rendering of the appearance of the cutaneous tissues, makes it possible to open a window to the antiquity and morphologic presentation of pathologic phenomena that would be difficult to infer solely from the human skeleton remains. The analysis of this statue also offers an opportunity to highlight the capacity of Hellenistic art in portraying human misery and illness.

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.

Risk of SARS-CoV-2 reinfection by vaccination status, predominant variant and time from prior infection: a cohort study, Reggio Emilia province, Italy, February 2020 to February 2022.

BackgroundUnderstanding the epidemiology of reinfections is crucial for SARS-CoV-2 control over a long period.AimTo evaluate the risk of SARS-CoV-2 reinfection by vaccination status, predominant variant and time after first infection.MethodsWe conducted a cohort study including all residents in the Reggio Emilia province on 31 December 2019, followed up until 28 February 2022 for SARS-CoV-2 first infection and reinfection after 90 days. Cox models were used to compare risk of first infection vs reinfection, adjusting for age, sex, vaccine doses and comorbidities.ResultsThe cohort included 538,516 residents, 121,154 with first SARS-CoV-2 infections and 3,739 reinfections, most in the Omicron BA.1 period. In the pre-Omicron period, three doses of vaccine reduced risk of reinfection by 89% (95% CI: 87-90), prior infection reduced risk by 90% (95% CI: 88-91), while two doses and infection reduced risk by 98% (95% CI: 96-99). In the Omicron BA.1 period, protection estimates were 53% (95% CI: 52-55), 9% (95% CI: 4-14) and 76% (95% CI: 74-77). Before Omicron, protection from reinfection remained above 80% for up to 15 months; with Omicron BA.1, protection decreased from 71% (95% CI: 65-76) at 5 months to 21% (95% CI: 10-30) at 22 months from the first infection. Omicron BA.1 reinfections showed 48% (95% CI: 10-57) lower risk of severe disease than first infections.ConclusionsNatural immunity acquired with previous variants showed low protection against Omicron BA.1. Combined vaccination and natural immunity seems to be more protective against reinfection than either alone. Vaccination of people with prior infection reduced the risk of severe disease.

Healthy preterm newborns: Altered innate immunity and impaired monocyte function.

Birth prior to 37 completed weeks of gestation is referred to as preterm (PT). Premature newborns are at increased risk of developing infections as neonatal immunity is a developing structure. Monocytes, which are key players after birth, activate inflammasomes. Investigations into the identification of innate immune profiles in premature compared to full-term infants are limited. Our research includes the investigation of monocytes and NK cells, gene expression, and plasma cytokine levels to investigate any potential differences among a cohort of 68 healthy PT and full-term infants. According to high-dimensional flow cytometry, PT infants have higher proportions of CD56+/- CD16+ NK cells and immature monocytes, and lower proportions of classical monocytes. Gene expression revealed lower proportions of inflammasome activation after in vitro monocyte stimulation and the quantification of plasma cytokine levels expressed higher concentrations of alarmin S100A8. Our findings suggest that PT newborns have altered innate immunity and monocyte functional impairment, and pro-inflammatory plasmatic profile. This may explain PT infants' increased susceptibility to infectious disease and should pave the way for novel therapeutic strategies and clinical interventions.