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Regulatory T cells are a class of T lymphocytes which respond to activation signals by expanding their cell numbers, and whose culturing and expansion are of significant clinical interest. Cellular activation states are used to inform process control decisions such as restimulation and can be probed with experimental measurements of cell surface markers. However, these measurements are expensive, time-consuming, and invasive, and an urgent need exists for devising a non-invasive method for activation state monitoring that could be deployed on-line. Raman spectroscopy is a label-free and information-rich optical method that, when coupled to data analytical methods, can ameliorate these experimental issues. In this work, we quantitatively estimated experimental measurements of regulatory T cell activation markers with high accuracy. We simulated a clinical manufacturing setting by building an -regularized least-squares model with spectroscopic data from six regulatory T cell donors. Then, we validated the constructed model by accurately estimating different experimental measurements of biomarker values from two external donors, unseen by the model. We have devised a robust program to effectively estimate the activation state of regulatory T cells. We anticipate our method to be used with on-line Raman probes integrated into cell manufacturing devices for label-free monitoring of these processes.

In humans, spinal cord lesions induce not only major motor and neurovegetative deficits but also severe neuropathic pain which is mostly resistant to classical analgesics. Better treatments can be expected from precise characterization of underlying physiopathological mechanisms. This led us to thoroughly investigate (i) mechanical and thermal sensory alterations, (ii) responses to acute treatments with drugs having patent or potential anti-allodynic properties and (iii) the spinal/ganglion expression of transcripts encoding markers of neuronal injury, microglia and astrocyte activation in rats that underwent complete spinal cord transection (SCT). SCT was performed at thoracic T8-T9 level under deep isoflurane anaesthesia, and SCT rats were examined for up to two months post surgery. SCT induced a marked hyper-reflexia at hindpaws and strong mechanical and cold allodynia in a limited (6 cm2) cutaneous territory just rostral to the lesion site. At this level, pressure threshold value to trigger nocifensive reactions to locally applied von Frey filaments was 100-fold lower in SCT- versus sham-operated rats. A marked up-regulation of mRNAs encoding ATF3 (neuronal injury) and glial activation markers (OX-42, GFAP, P2×4, P2×7, TLR4) was observed in spinal cord and/or dorsal root ganglia at T6-T11 levels from day 2 up to day 60 post surgery. Transcripts encoding the proinflammatory cytokines IL-1β, IL-6 and TNF-α were also markedly but differentially up-regulated at T6-T11 levels in SCT rats. Acute treatment with ketamine (50 mg/kg i.p.), morphine (3-10 mg/kg s.c.) and tapentadol (10-20 mg/kg i.p.) significantly increased pressure threshold to trigger nocifensive reaction in the von Frey filaments test, whereas amitriptyline, pregabalin, gabapentin and clonazepam were ineffective. Because all SCT rats developed long lasting, reproducible and stable allodynia, which could be alleviated by drugs effective in humans, thoracic cord transection might be a reliable model for testing innovative therapies aimed at reducing spinal cord lesion-induced central neuropathic pain.

Background Monocytes play an important role in inflammation, and monocytosis and monocyte activation are features of chronic inflammation. We aimed to investigate if HIV status was associated with monocyte count and monocyte activation and to assess the relationship between monocyte count and monocyte activation markers and HIV-related factors. Methods Persons living with HIV (PLWH) with measured monocyte count and sCD14 and sCD163 were included from the Copenhagen Comorbidity in HIV infection (COCOMO) study and matched 1:5 on sex and age with uninfected controls. In addition, 74 uninfected individuals from COCOMO with measured sCD14 and sCD163 were included. Identical protocols and equipment were used to determine monocyte counts and monocyte activation in PLWH and uninfected controls. Linear regression adjusted for age, sex, smoking and waist-to-hip-ratio was used to analyze the association between possible risk factors and monocyte outcomes. Results We included 871 PLWH and 4355 uninfected controls. PLWH had − 0.021 [− 0.031 − 0.011] × 10 9 /L) lower monocyte count than uninfected controls, and in adjusted analyses HIV status was independently associated with − 0.035 [− 0.045, − 0.025] × 10 9 /L lower monocyte count. In contrast, PLWH had higher sCD163 and sCD14 concentrations than uninfected controls. After adjustment, HIV-status was associated with higher sCD14 and sCD163 concentrations (588 [325, 851] ng/ml, and 194 [57, 330] ng/ml, respectively). Conclusion PLWH had lower monocyte counts than controls, but the absolute difference was small, and any clinical impact is likely limited. In contrast, concentrations of monocyte activation markers, previously implicated as drivers of non-AIDS comorbidity, were higher in PLWH than in controls.

Maraviroc (MVC) is an antiretroviral drug capable of binding to CCR5 receptors and block HIV entry into target cells. Moreover, MVC can activate NF-kB pathway and induce viral transcription in HIV-infected cells, being proposed as a latency reversal agent (LRA) in HIV cure strategies. However, the evaluation of immunological and metabolic parameters induced by MVC concentrations capable of inducing HIV transcription have not been explored in depth. We cultured isolated CD4 T cells in the absence or presence of MVC, and evaluated the frequency of CD4 T cell subpopulations and activation markers levels by flow cytometry, and the oxidative and glycolytic metabolic rates of CD4 T cells using a Seahorse Analyzer. Our results indicate that a high concentration of MVC did not increase the levels of activation markers, as well as glycolytic or oxidative metabolic rates in CD4 T cells. Furthermore, MVC did not induce significant changes in the frequency and activation levels of memory cell subpopulations. Our data support a safety profile of MVC as a promising LRA candidate since it does not induce alterations of the immunological and metabolic parameters that could affect the functionality of these immune cells.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections worldwide, particularly affecting infants, older adults, and immunocompromised individuals. Understanding the cellular immune response to RSV infection is essential for developing effective treatments for infection and its complications. In this study, we investigated the susceptibility of blood-derived primary monocytes and monocytic THP-1 cells to infection with a contemporary RSV A-ON1 strain and characterized the subsequent cytokine and chemokine secretion, as well as the expression of surface markers involved in antigen presentation. Our findings demonstrate that primary monocytes and related THP-1 cells are permissive to abortive infection by RSV, leading to increased expression of proinflammatory cytokines and chemokines, including IP-10, IL-6, and CCL2. Furthermore, primary monocytes expressed CD80, CD86, and HLA-DR upon direct infection or through potential paracrine stimulation. Collectively, these findings demonstrate the activation of monocytes by RSV infection, suggesting their contributory role in orchestrating early immune responses during infection.

CD (cluster of differentiation) 69 and CD25 are considered early and late markers of the activation of lymphocytes, respectively. CD25 is a part of the IL-2 receptor and is present on the surface of immune and non-immune cells, with high amounts on activated lymphocytes and regulatory T cells. CD69 is expressed on various types of white blood cells, including newly activated lymphocytes, lymphocytes infiltrating tissues isolated from subjects with chronic auto-inflammatory diseases, several subtypes of memory T cells and regulatory T cells. Primarily, CD69 was considered to be an early marker of the activation of lymphocytes, but, right now, data derived from in vitro and in vivo studies have revealed the immunomodulatory role of this surface antigen. In 84 patients with psoriasis, of whom 28 were treated with different biologic drugs, as well as in 29 healthy control subjects, the expression of CD25 and CD69 on different subtypes of peripheral blood mononuclear cells (PBMCs) was studied with the use of flow cytometry. Significantly higher levels of CD3/CD69-, CD8/CD69- and CD19/CD69-positive PBMCs as well as within CD3+ cells were present in subjects suffering from psoriasis when compared to healthy controls. In patients with psoriasis who were treated with biologic drugs, the levels of CD3/CD69-, CD4/CD69- and CD19/CD69-positive PBMCs, and CD3/CD69 within CD3+ cells, CD4/CD69 within CD4+ cells, CD4/CD25 within CD4+ cells and CD19/CD69 within CD19+ cells were significantly higher than before therapy. Our results support a role for activation markers, especially CD69, in psoriasis. Further research is warranted to fully clarify their significance in this common dermatosis, especially during biologic treatment.

Obesity is characterized by a chronic low-grade inflammation and, paradoxically, is also associated with immune cells dysfunction. In this study, we analyzed peripheral blood Invariant Natural killer T cells (iNKT) in individuals with or without obesity. These unconventional T cells recognize lipid antigens presented by the monomorphic CD1d MHC I-like protein. We demonstrated an activation of iNKT cells in individuals with obesity associated with both increased apoptosis and dysfunction as assessed by the lack of responsiveness to PMA/Ionomycin stimulation. This disruption mainly affects the CD4 − subset, more dedicated to pro-inflammatory cytokines release and cytotoxicity. Such impact could therefore be involved in the loss of immunosurveillance observed in obesity. Interestingly, CD1d is upregulated on intermediate and non-classical monocytes from individuals with obesity and its expression on both monocyte subsets is correlated with iNKT cell dysfunction. Both the activation and hypo-responsiveness of iNKT cells as well as CD1d modulation on monocytes are significantly reversed after bariatric surgery. Altogether, these data suggest that increased CD1d expression may enhance the presentation of endogenous lipid antigens, thereby contributing to iNKT cell activation in the context of obesity.

The diagnosis of celiac disease (CD) relies on the presence of serum antibodies against type 2‐tissue transglutaminase (tTG2) and endomysium, which in adult patients must be confirmed by assessing small intestinal mucosa damage through esophagogastroduodenoscopy. We aim to establish a non‐invasive method focused on detecting activated CD4+ T cells that are reactive to gliadin in the peripheral blood of patients with untreated CD. Peripheral blood mononuclear cells from 20 patients with untreated CD, 17 patients on a gluten‐free diet and 10 healthy donors were stimulated in vitro with whole gliadin or immunodominant peptides. After 48 h, cells were stained with fluorochrome‐conjugated monoclonal antibodies against OX40 and 4‐1BB surface activation markers. Gliadin‐specific, activated CD4+ T cells were detected through multiparametric flow cytometry. OX40 and 4‐1BB activation markers are upregulated on CD4+ T cells following the engagement of the HLA‐DQ2.5‐gliadin complex with the T‐cell receptor (TCR). The frequency of gliadin‐specific, CD3+/CD4+/OX40+/4‐1BB+ cells is significantly higher in untreated than in treated patients and healthy controls and shows a positive correlation with the anti‐tTG2 antibody titers. This assay, defined as the G.A.T.CD4 (Gliadin activated CD4+ T cells) method, might support CD diagnosis, particularly in doubtful cases having low autoantibody serum titers. G.A.T.CD4 can be of help in monitoring disease progression, as the pathogenic T cells expressing OX40 and 4‐1BB activation markers are undetectable in the blood of treated patients. In the future, we aim to propose the G.A.T.CD4 instead of esophagogastroduodenoscopy to perform accurate and less invasive diagnosis.

Up to 3.8% of human T-lymphotropic virus type-1 (HTLV-1)-infected asymptomatic carriers (AC) eventually develop HTLV-1-associated myelopathy (HAM). HAM occurs in patients with high (> 1%) HTLV proviral load (PVL). However, this cut-off includes more than 50% of ACs and therefore the risk needs to be refined. As HAM is additionally characterised by an inflammatory response to HTLV-1, markers of T cell activation (TCA), β 2 -microglobulin (β 2 M) and neuronal damage were accessed for the identification of ACs at high risk of HAM. Retrospective analysis of cross-sectional and longitudinal routine clinical data examining differences in TCA (CD4/CD25, CD4/HLA-DR, CD8/CD25 & CD8/HLA-DR), β 2 M and neurofilament light (NfL) in plasma in ACs with high or low PVL and patients with HAM. Comparison between 74 low PVL ACs, 84 high PVL ACs and 58 patients with HAM revealed a significant, stepwise, increase in TCA and β 2 M. Construction of receiver operating characteristic (ROC) curves for each of these blood tests generated a profile that correctly identifies 88% of patients with HAM along with 6% of ACs. The 10 ACs with this ‘HAM-like’ profile had increased levels of NfL in plasma and two developed myelopathy during follow-up, compared to none of the 148 without this viral-immune-phenotype. A viral-immuno-phenotype resembling that seen in patients with HAM identifies asymptomatic carriers who are at increased risk of developing HAM and have markers of subclinical neuronal damage.

The study aimed to evaluate mean platelet volume (MPV), platelet distribution width (PDW), and platecrit in children with pulmonary arterial hypertension associated with congenital heart disease (PAH-CHD), to assess the predictive value of these platelet activation markers for adverse outcomes, and to correlate their levels with various data in these patients. This prospective cohort study included 60 children with PAH-CHD as group I and 60 children with CHD and no PAH as group II. Another 60 healthy children of matched age and sex served as the control group. All included children were evaluated by echocardiography. MPV, PDW, and platecrit were also measured using an automated blood counter. All patients were followed up for death or readmission for 6 months. MPV, PDW, and platecrit were significantly higher in group I compared to group II and the control group and they correlated well with increasing severity of PAH. MPV, PDW, and platecrit positively correlated with right ventricular diameter and mean pulmonary artery pressure, however they correlated negatively with right ventricular systolic and diastolic function. The best cut-off of platelet activation markers levels to predict poor prognosis in group I was > 11.2 FL with 75% sensitivity and 96.6% specificity for MPV, > 12.7 FL with 75% sensitivity and 61.5% specificity for PDW, and > 0.505% with 75% sensitivity and 93.2% specificity for platecrit. MPV, PDW, and platecrit were elevated in children with PAH-CHD and found to be good predictive markers for poor prognosis in these children.