Metrological traceability in flow cytometry? Evaluation of a new volumetric method for lymphocyte subsets.

INTRODUCTION: Lymphocyte subset enumeration by flow cytometry is important for the therapeutic monitoring of a range of conditions. However, current bead-based methodologies do not produce metrologically traceable results. Here we compare an established bead-based methodology with a volumetric-based system traceable to an internationally recognised reference method. METHOD: A total of 118 samples received for lymphocyte subset analysis were tested using an established bead-based technique (BD Multitest™ 6-colour TBNK assay using Trucount™ tubes on a BD FACSLyric flow cytometer), followed by a volumetric method on the Sysmex XF-1600 flow cytometer using Exbio Kombitest 6-colour TBNK reagent. All samples were tested in accordance with the manufacturer's instructions. RESULTS: Absolute count values from both methodologies for CD3+, CD3 + CD4+, CD3 + CD8+, CD19+ and CD3-CD16+/CD56+ lymphocyte populations were compared using linear regression (R2 for all parameters >0.95) and Bland-Altman analysis. There was no significant bias (where p < 0.05) for absolute CD3 + CD4+ lymphocytes in the defined therapeutic range of 0-250 cells/µL (mean bias: 0.27 cells/µL). Although positive biases were seen for CD3 + CD4+ lymphocytes (over the entire range tested: 14-1798 cells/µL) and CD3-CD16+/CD56+ lymphocytes (mean bias: 10.83 cells/µL and 6.79 cells/µL, respectively). Negative biases were seen for CD3 + CD8+ and CD19+ lymphocytes (mean bias: -29.17 cells/µL and - 18.76 cells/µL, respectively). CONCLUSION: A high degree of correlation was found for results from both methodologies and observed bias was within the limits of clinical acceptability for all populations. This shows that the metrologically traceable lymphocyte subset absolute counts produced by the Sysmex XF-1600 are robust within clinically required limits.

Utility of Pretreatment Blood Platelet-To-Lymphocyte Ratio in Prediction of Clinical Outcomes and Chemosensitivity in Patients with Advanced Gastric Cancer: A Meta-Analysis.

BACKGROUND The results of previous studies that evaluated the association between pretreatment blood platelet-to-lymphocyte ratio (PLR) and clinical outcomes and chemosensitivity in patients with advanced gastric cancer are inconsistent. Therefore, this study was designed to investigate the association between pretreatment blood PLR and clinical outcomes and chemosensitivity in advanced gastric cancer patients. MATERIAL AND METHODS We performed a systematic literature search in PubMed, Web of Science, EMBASE, and the Cochrane Library up to Mar 9, 2021. Hazard ratios (HRs) for overall survival (OS) and disease-free survival (DFS) were pooled for meta-analysis. The quality of the included studies was measured by the Newcastle-Ottawa Quality Assessment Scale. RESULTS We included 17 studies comprising 3499 patients with advanced GC in this meta-analysis. Pooled results demonstrated that high PLR was correlated with poor OS (HR=1.429, 95% CI=1.246-1.639, P<0.001) and DFS (HR=1.47, 95% CI=1.14-1.88, P=0.003) compared with low PLR in patients with advanced GC. Moreover, high PLR was associated with a lower response to chemotherapy in patients with advanced GC (OR=1.395, 95% CI=1.056-1.841, P=0.019). However, there was no significant correlation between PLR and clinicopathological features. CONCLUSIONS This meta-analysis suggests that high PLR is a risk factor for unfavorable OS, DFS, and chemosensitivity in patients with advanced GC.

Cytokine profiles in the detection of severe lung involvement in hospitalized patients with COVID-19: The IL-8/IL-32 axis.

Coronavirus disease 2019 (COVID-19) is an infectious respiratory disorder caused by a new coronavirus called SARS-CoV-2. The pathophysiology of severe COVID-19 is associated with a "cytokine storm". IL-32 is a key modulator in the pathogenesis of various clinical conditions and is mostly induced by IL-8. IL-32 modulates important inflammatory pathways (including TNF-α, IL-6 and IL-1b), contributing to the pathogenesis of inflammatory diseases. Il-32 was never evaluated before in COVID-19 patients stratifying as mild-moderate and severe patients. A total of 64 COVID-19 patients, 27 healthy controls were consecutively enrolled in the study. Serum concentrations of biomarkers including IL-1ß, IL-10, IFN-γ, TNF-α and IL-6 were quantified by bead-based multiplex analysis and Serum concentration of IL-8 and IL-32 were determined by enzyme-linked immunosorbent assay (ELISA) kits. Interestingly, among the blood parameters, neutrophil and lymphocyte counts were significantly lower in severe COVID-19 patients than in the other, on the contrary, CRP was significantly higher in severe patients than in other groups. The cytokines that best distinguished controls from COVID-19 patients were IL-8 and IL-32, while IL-6 resulted the better variables for discriminate severe group. The best model performance for severe group was obtained by the combination of IL-32, IL-6, IFN-γ, and CRP serum concentration showing an AUC = 0.83. A cut off of 15 pg/ml of IL-6 greatly discriminate survivor from death patients. New insights related to the cytokine storm in COVID-19 patients, highlighting different severity of disease infection.

The kinase complex mTORC2 promotes the longevity of virus-specific memory CD4+ T cells by preventing ferroptosis.

Antigen-specific memory CD4+ T cells can persist and confer rapid and efficient protection from microbial reinfection. However, the mechanisms underlying the long-term maintenance of the memory CD4+ T cell pool remain largely unknown. Here, using a mouse model of acute infection with lymphocytic choriomeningitis virus (LCMV), we found that the serine/threonine kinase complex mammalian target of rapamycin complex 2 (mTORC2) is critical for the long-term persistence of virus-specific memory CD4+ T cells. The perturbation of mTORC2 signaling at memory phase led to an enormous loss of virus-specific memory CD4+ T cells by a unique form of regulated cell death (RCD), ferroptosis. Mechanistically, mTORC2 inactivation resulted in the impaired phosphorylation of downstream AKT and GSK3ß kinases, which induced aberrant mitochondrial reactive oxygen species (ROS) accumulation and ensuing ferroptosis-causative lipid peroxidation in virus-specific memory CD4+ T cells; furthermore, the disruption of this signaling cascade also inhibited glutathione peroxidase 4 (GPX4), a major scavenger of lipid peroxidation. Thus, the mTORC2-AKT-GSK3ß axis functions as a key signaling hub to promote the longevity of virus-specific memory CD4+ T cells by preventing ferroptosis.

Chronic obstructive pulmonary disease is characterized by reduced levels and defective suppressive function of regulatory B cells in peripheral blood.

Chronic obstructive pulmonary disease (COPD) is characterized by a progressive, persistent immune response to cigarette smoke, and it has been suggested that immune dysregulation is involved in its pathogenesis. A subset of regulatory B cells (Bregs) with high levels of the surface markers CD24 and CD38 (CD24hiCD38hi) has previously been shown to exert an immunosuppressive function. This study investigated the levels and activity of CD24hiCD38hi Bregs in stable COPD (sCOPD). Testing the peripheral blood from 65 patients with sCOPD and 39 control subjects for CD24hiCD38hi Breg subsets by flow cytometry showed that the patients with sCOPD had significantly lower levels of CD24hiCD38hi Bregs and IL-10+ B cells. The patients with sCOPD had lower serum interleukin-10 levels than the controls. The patients with most severe sCOPD had the lowest levels of CD24hiCD38hi Bregs. Spearman correlation analysis showed that the levels of CD24hiCD38hi Bregs in the patients with sCOPD positively correlated with serum interleukin-10 concentrations but not with levels of C-reactive protein. Compared to healthy controls, functional studies showed that Breg cells from patients with sCOPD exhibit a decreased suppressive function. We conclude that sCOPD is characterized by the exhaustion of CD24hiCD38hi regulatory B cells compartment. Therefore, CD24hiCD38hi Bregs may contribute to the pathogenesis of sCOPD.

High CD4-to-CD8 ratio identifies an at-risk population susceptible to lethal COVID-19.

Around half of people with severe COVID-19 requiring intensive care unit (ICU) treatment will survive, but it is unclear how the immune response to SARS-CoV-2 differs between ICU patients that recover and those that do not. We conducted whole-blood immunophenotyping of COVID-19 patients upon admission to ICU and during their treatment and uncovered marked differences in their circulating immune cell subsets. At admission, patients who later succumbed to COVID-19 had significantly lower frequencies of all memory CD8+ T cell subsets, resulting in increased CD4-to-CD8 T cell and neutrophil-to-CD8 T cell ratios. ROC and Kaplan-Meier analyses demonstrated that both CD4-to-CD8 and neutrophil-to-CD8 ratios at admission were strong predictors of in-ICU mortality. Therefore, we propose the use of the CD4-to-CD8 T cell ratio as a marker for the early identification of those individuals likely to require enhanced monitoring and/or pro-active intervention in ICU.

Platelet-lymphocyte ratio as a new predictor of in-hospital mortality in cardiac intensive care unit patients.

It has been discovered that both inflammation and platelet aggregation could cause crucial effect on the occurrence and development of cardiovascular diseases. As a combination of platelet and lymphocyte, platelet-lymphocyte ratio (PLR) was proved to be correlated with the severity as well as prognosis of cardiovascular diseases. Exploring the relationship between PLR and in-hospital mortality in cardiac intensive care unit (CICU) patients was the purpose of this study. PLR was calculated by dividing platelet count by lymphocyte count. All patients were grouped by PLR quartiles and the primary outcome was in-hospital mortality. The independent effect of PLR was determined by binary logistic regression analysis. The curve in line with overall trend was drawn by local weighted regression (Lowess). Subgroup analysis was used to determine the relationship between PLR and in-hospital mortality in different subgroups. We included 5577 CICU patients. As PLR quartiles increased, in-hospital mortality increased significantly (Quartile 4 vs. Quartile 1: 13.9 vs. 8.3, P < 0.001). After adjusting for confounding variables, PLR was proved to be independently associated with increased risk of in-hospital mortality (Quartile 4 vs. Quartile 1: OR 95% CI 1.55, 1.08-2.21, P = 0.016, P for trend < 0.001). The Lowess curves showed a positive relationship between PLR and in-hospital mortality. The subgroup analysis revealed that patients with low Acute Physiology and Chronic Health Evaluation IV (APACHE IV) or with less comorbidities had higher risk of mortality for PLR. Further, PLR quartiles had positive relation with length of CICU stay (Quartile 4 vs. Quartile 1: 2.7, 1.6-5.2 vs. 2.1, 1.3-3.9, P < 0.001), and the length of hospital stay (Quartile 4 vs. Quartile 1: 7.9, 4.6-13.1 vs. 5.8, 3.3-9.8, P < 0.001). PLR was independently associated with in-hospital mortality in CICU patients.

Prognostic value of preoperative neutrophil-to-lymphocyte ratio in histological variants of non-muscle-invasive bladder cancer.

PURPOSE: Many studies identified that the preoperative neutrophil-to-lymphocyte ratio (PNLR) was associated with patient prognosis in non-muscle-invasive bladder cancer (NMIBC). We hypothesized that PNLR could be prognostic in patients with histological variants of NMIBC (VH-NMIBC). MATERIALS AND METHODS: This retrospective study included patients with VH-NMIBC admitted at our center between January 2009 and May 2019. The best cut-off value of NLR was measured by the receiver operating characteristic curve and Youden index. The Kaplan-Meier method and Cox proportional hazard regression models were employed to evaluate the association between PNLR and disease prognosis, including recurrence-free survival (RFS), progression-free survival (PFS), cancer-specific survival (CSS), and overall survival (OS). RESULTS: A total of 243 patients with VH-NMIBC were enrolled in our study. According to the Kaplan-Meier method results, patients with PNLR ≥2.2 were associated with poor RFS (p<0.001), PFS (p<0.001), CSS (p<0.001), and OS (p<0.001). Multivariable analyses indicated that PNLR ≥ 2.2 was an independent prognostic factor of RFS (hazard ratio [HR], 2.11; 95% confidence interval [CI, 1.57-1.83; p<0.001), PFS (HR, 2.34; 95% CI, 1.70-3.21; p<0.001), CCS (HR, 2.87; 95% CI, 1.96-4.18; p< 0.001), and OS (HR, 2.83; 95% CI, 1.96-4.07; p<0.001). CONCLUSIONS: This study identified that PNLR ≥2.2 was usually associated with a poor prognosis for patients with VH-NMIBC.

Neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios as predictors of outcomes in inflammatory breast cancer.

Background: Inflammatory breast cancer (IBC) is uncommon, aggressive and associated with poor survival outcomes. The lack of prognostic biomarkers and therapeutic targets specific to IBC is an added challenge for clinical practice and research. Inflammatory biomarkers such as neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios (NLR and PLR) demonstrated independent prognostic impact for survival in breast cancer. In our study, these biomarkers were investigated in a cohort of patients with nonmetastatic IBC. Methods: A retrospective cohort of 102 IBC patients with nonmetastatic disease was conducted at the Mohammed VI University Hospital (Oujda, Morocco) between January 2010 and December 2014. NLR and PLR were obtained from blood cell count at baseline before neoadjuvant chemotherapy (NACT) from patients' medical records. The receiver operating characteristic was used to find the optimal cut-off. Correlation between these blood-based biomarkers and response to NACT was analyzed by Chi-squared and Fisher's exact test. Their prognostic value for predicting disease-free survival (DFS) and overall survival (OS) was performed based on Cox regression models. Results: Totally, 102 patients with IBC were included in the analysis. Pathologic complete response (pCR) after NACT, defined by the absence of an invasive tumor in the breast tissues and nodes after surgery (ypT0 ypN0), was observed in eight patients (7.8%). NACT response was found to be associated with menopausal status (p = 0.039) and nodal status (p < 0.001). Patients with a low NLR had a higher pCR rate as compared with the high-NLR group (p = 0.043). However, the pCR rate was not significantly associated with age (p = 0.122), tumor side (p = 0.403), BMI (p = 0.615), histological grade (p = 0.059), hormone receptors status (p = 0.206), HER2 (p = 0.491) and PLR (p = 0.096). Pre-treatment blood-based NLR of 2.28 was used as the cut-off value to discriminate between high and low NLR according to the receiver operating characteristic curves. Similarly, a value of 178 was used as the cut off for PLR. Patients with low-NLR had a significantly better 5-year DFS (p < 0.001) and OS (p < 0.001) than the high-NLR group. Moreover, low-PLR was significantly associated with higher DFS (p = 0.001) and OS (p = 0.003). The NLR showed a significant prognostic impact for DFS (HR: 2.57; 95% CI: 1.43-4.61; p = 0.01) and for OS (HR: 2.92; 95% CI: 1.70-5.02; p < 0.001). Similarly, a meaningful association between PLR and 5-year DFS (HR: 1.95; 95% CI: 1.10-3.46; p = 0.021) and OS (HR: 1.82; 95% CI: 1.06-3.14; p = 0.03) was noticed. Conclusions: High NLR and PLR were found associated with reduced DFS and OS in nonmetastatic IBC. Further studies are awaited to confirm these findings.

Complete blood cell count-derived ratios can be useful biomarkers for neurological diseases.

Complete blood cell count-derived parameters such as neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and lymphocyte-to-monocyte ratio (LMR) have recently shown to be highly sensitive biomarkers. Their usefulness has been proven as prognostic factors in several cancers, in the stratification of mortality in major cardiac events, as predictors and markers of infectious or inflammatory pathologies, and in many other conditions. Surprisingly, the study of these biomarkers in neurological diseases is somewhat limited. This paper aims to take stock of the data present in the literature regarding the complete blood cell count-derived ratios in this group of pathologies and to formulate a hypothesis, based on the most recent data concerning innate and acquired immunity, on which diseases of the nervous system could benefit in diagnostic and prognostic terms from the in-depth study of these new biomarkers.

A novel prognostic biomarker for cutaneous malignant melanoma: red cell distribution width (RDW) to lymphocyte ratio.

It is well-known that inflammation plays a significant role in cancer formation and prognosis. Both lymphocyte count and red cell distribution width (RDW) has been used to predict prognosis in various cancers as an indicator of inflammation. Yet, the role of RDW-lymphocyte ratio (RLR) in determining prognosis is still unknown. We aimed to determine the prognostic role of RLR in cutaneous malignant melanoma (MM). One hundred fifteen patients with MM were included in the study retrospectively. The relationship of the clinical-pathological data with overall survival (OS) and progression-free survival (PFS) was analyzed using Kaplan-Meier curves. The cut-off values of neutrophil to lymphocyte ratio, systemic immune-inflammation index (SII), prognostic nutritional index (PNI) and RLR were determined as 2, 487, 51.5 and 6.52, respectively. OS was significantly longer in the low SII, high PNI, low RLR group, while PFS was longer in groups with high PNI and low RLR. In univariate analysis, it was determined that PFS was significantly correlated with Eastern Cooperative Oncology Group (ECOG) performance, TNM stage, PNI and RLR. Moreover, in univariate analysis, a significant correlation was determined between OS and age, ECOG performance, TNM stage, adjuvant interferon, SII, PNI and RLR. In multivariate analysis, ECOG performance, TNM stage and RLR were determined as independent prognostic factors for PFS, while TNM stage and RLR were found to be independent prognostic factors for OS. RLR could be a novel prognostic marker for both PFS and OS in patients with cutaneous MM.

Coeliac Disease in Elderly Patients: Value of Coeliac Lymphogram for Diagnosis.

(1) Background: Although a meta-analysis reported that the sensitivity of CD3+ TCRγδ+ cells for coeliac disease diagnosis was >93%, a recent study has suggested that sensitivity decreased to 65% in elderly patients. (2) Aim: To evaluate whether the sensitivity of intraepithelial lymphocyte cytometric patterns for coeliac disease diagnosis changes with advanced age. (3) Methods: We performed a multicentre study including 127 coeliac disease patients ≥ 50 years: 87 with baseline cytometry (45 aged 50-59 years; 23 aged 60-69 years; 19 aged ≥ 70 years), 16 also with a follow-up cytometry (on a gluten-free diet); and 40 with only follow-up cytometry. (4) Results: In Marsh 3 patients, a sensitivity of 94.7%, 88.9% and 86.7% was observed for each age group using a cut-off value of TCRγδ+ >10% (p = 0.27); and a sensitivity of 84.2%, 83.4% and 53.3% for a cut-off value >14% (p = 0.02; 50-69 vs. ≥70 years), with difference between applying a cut-off of 10% or 14% (p = 0.008). The TCRγδ+ count in the ≥70 years group was lower than in the other groups (p = 0.014). (5) Conclusion: In coeliac patients ≥ 70 years, the TCRγδ+ count decreases and the cut-off point of >10% is more accurate than >14%.

ST-2191, an Anellated Bismorpholino Derivative of Oxy-Fingolimod, Shows Selective S1P1 Agonist and Functional Antagonist Potency In Vitro and In Vivo.

Sphingosine 1-phosphate (S1P) is an extensively studied signaling molecule that contributes to cell proliferation, survival, migration and other functions through binding to specific S1P receptors. The cycle of S1P1 internalization upon S1P binding and recycling to the cell surface when local S1P concentrations are low drives T cell trafficking. S1P1 modulators, such as fingolimod, disrupt this recycling by inducing persistent S1P1 internalization and receptor degradation, which results in blocked egress of T cells from the secondary lymphoid tissues. The approval of these compounds for the treatment of multiple sclerosis has placed the development of S1PR modulators in the focus of pharmacological research, mostly for autoimmune indications. Here, we report on a novel anellated bismorpholino derivative of oxy-fingolimod, named ST-2191, which exerts selective S1P1 agonist and functional antagonist potency. ST-2191 is also effective in reducing the lymphocyte number in mice, and this effect is not dependent on phosphorylation by sphingosine kinase 2 for activity. These data show that ST-2191 is a novel S1P1 modulator, but further experiments are needed to analyze the therapeutic impact of ST-2191 in animal models of autoimmune diseases.

Increased Blood Monocytic Myeloid Derived Suppressor Cells but Low Regulatory T Lymphocytes in Patients with Mild COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may produce a systemic disease, the coronavirus disease-19 (COVID-19), with high morbidity and mortality. Even though we do not fully understand the interaction of innate and adaptive immunity in the control and complications of the viral infection, it is well recognized that SARS-CoV-2 induces an immunodepression that impairs the elimination of the virus and favors its rapid dissemination in the organism. Even less is known about the possible participation of inhibitory cells of the innate immune system, such as the myeloid-derived suppressor cells (MDSCs), or the adaptive immune system, such as the T regulatory cells (Tregs). That is why we aimed to study blood levels of MDSCs, as well as lymphocyte subpopulations, including Tregs, and activated (OX-40+) and inhibited (PD-1) T lymphocytes in patients with mild COVID-19 in comparison with data obtained from control donors. We have found that 20 hospitalized patients with COVID-19 and no health history of immunosuppression had a significant increase in the number of peripheral monocytic MDSCs (M-MDSC), but a decrease in Tregs, as well as an increase in the number of inhibited or exhausted T cells, whereas the number of activated T cells was significantly decreased compared with that from 20 healthy controls. Moreover, there was a significant negative correlation (r = 0.496) between the number of M-MDSC and the number of activated T cells. Therefore, M-MDSC rather than Tregs may contribute to the immunosuppression observed in patients with COVID-19.

Changes in peripheral lymphocyte populations in patients with advanced/recurrent ovarian cancer undergoing splenectomy during cytoreductive surgery.

BACKGROUND: To investigate changes in peripheral lymphocyte subsets after splenectomy during cytoreductive surgery for advanced or recurrent ovarian cancers. METHODS: We enrolled 83 patients with advanced or recurrent ovarian cancer who underwent cytoreductive surgery. Twenty patients who also underwent splenectomy were assigned to the splenectomy cohort and the rest were assigned to the non-splenectomy cohort. Flow cytometry was used to measure peripheral lymphocyte subsets consisting of T cells, regulatory T cells, natural killer cells, B cells, and activation antigens before and after surgery. RESULTS: There was no difference in the number and distribution of peripheral lymphocyte subsets between the two cohorts before surgery. After surgery, we observed elevated levels of T cells (CD3+, CD3+CD8+) in the splenectomy cohort compared to those in the non-splenectomy cohort, and the difference was statistically significant. CD8+CD28+ T cells had a significant decreasing tendency (P = 0.011) while CD3+/HLA-DR+ T cells showed the opposite trend (P = 0.001) in the splenectomy cohort. The proportion of Tregs (P = 0.005) and B cells (P < 0.001) including CD3-/HLA-DR+ B cells (P = 0.007) increased after surgery, and the absolute number of T cells and NK cells decreased to different extents (P < 0.001) in the non-splenectomy cohort. The post-operative percentage of CD8+CD28+ T cells was less than the pre-operative percentage (P = 0.022), which was similar to the splenectomy cohort. There was no significant difference in progression-free survival or overall survival between the groups after a median follow-up time of 41 months. CONCLUSIONS: The changes in peripheral lymphocyte populations were different between patients with and those without splenectomy during cytoreductive surgery for ovarian cancers. T cells were increased and activated in the splenectomy cohort, whereas, B cells were increased and activated in the non-splenectomy cohort.

Lead exposure induced inflammation in bursa of Fabricius of Japanese quail (C. japonica) via NF-κB pathway activation and Wnt/ß-catenin signaling inhibition.

Bursa of Fabricius (BF), one of primary lymphoid organ, is unique to birds. Meanwhile, lead (Pb) is well known for its high toxicology to birds. Therefore, this study aimed to examine the chronic toxic effects of lead exposure on BF in Japanese quails (C. japonica) and the underlying mechanism of lead immunotoxicity. One-week old male quails were exposed to 0 ppm, 50 ppm, 500 ppm and 1000 ppm Pb concentrations by drinking water for four weeks. The results showed that Pb accumulation in BF increased in a dose dependent way. The growth and development of BF was retarded in 500 ppm and 1000 ppm Pb groups. The number of lymphocytes was decreased and the release of immunoglobulin G and M (IgG, IgM), complement 3 and 4 (C3, C4) was inhibited by Pb exposure. Lead exposure also caused oxidative stress and increasing apoptosis in BF. Moreover, histopathological damages characterized by inflammatory hyperemia and inflammatory cell infiltration and ultrastructural injury featured by mitochondrial vacuole, cristae fracture and chromatin concentration were found in BF of 500 ppm and 1000 ppm Pb groups. Furthermore, RNA sequencing based transcriptomic analysis revealed that molecular signaling and functional pathways in BF were disrupted by lead exposure. In addition, the activation of Nuclear Factor kappa B (NF-κB) pathway while the inhibition of wingless integrated/catenin beta 1 (Wnt/ß-catenin) signaling by Pb exposure were confirmed by quantitative real-time PCR (qPCR). Our study may benefit to understand potential mechanistic pathways of developmental immunotoxicology under Pb stress.

Single-Cell RNA-Seq of T Cells in B-ALL Patients Reveals an Exhausted Subset with Remarkable Heterogeneity.

Characterization of functional T cell clusters is key to developing strategies for immunotherapy and predicting clinical responses in leukemia. Here, single-cell RNA sequencing is performed with T cells sorted from the peripheral blood of healthy individuals and patients with B cell-acute lymphoblastic leukemia (B-ALL). Unbiased bioinformatics analysis enabled the authors to identify 13 T cell clusters in the patients based on their molecular properties. All 11 major T cell subsets in healthy individuals are found in the patients with B-ALL, with the counterparts in the patients universally showing more activated characteristics. Two exhausted T cell populations, characterized by up-regulation of TIGIT, PDCD1, HLADRA, LAG3, and CTLA4 are specifically discovered in B-ALL patients. Of note, these exhausted T cells possess remarkable heterogeneity, and ten sub-clusters are further identified, which are characterized by different cell cycle phases, naïve states, and GNLY (coding granulysin) expression. Coupled with single-cell T cell receptor repertoire profiling, diverse originations of the exhausted T cells in B-ALL are suggested, and clonally expanded exhausted T cells are likely to originate from CD8+ effector memory/terminal effector cells. Together, these data provide for the first-time valuable insights for understanding exhausted T cell populations in leukemia.

An optimized intracerebroventricular injection of CD4+ T cells into mice.

The blood-brain barrier acts as a major barrier for the entrance of most therapeutics into the brain, impeding treatment for neurological disorders. Intracerebroventricular (ICV) injection of T cells is a useful tool for cell therapy of neurological disorders including neurodegenerative and neuropsychiatric diseases and brain tumors. Here, we present an optimized ICV injection of T cells with improved injection efficiency at pathological sites within the brain parenchyma. We describe details of the surgical procedure and verification of injection via immunohistochemistry. For complete details on the use and execution of this protocol, please refer to Fisher et al. (2014); Strominger et al., (2018); Mittal et al. (2019); Eremenko et al. (2019).

Prognostic value of platelet-to-lymphocyte ratio in neoadjuvant chemotherapy for solid tumors: A PRISMA-compliant meta-analysis.

INTRODUCTION: Previous research indicates that the platelet-to-lymphocyte ratio (PLR) may be an indicator of poor prognosis in many tumor types. However, the PLR is rarely described in patients undergoing neoadjuvant chemotherapy (NAC) for solid tumors. Thus, we performed a meta-analysis to investigate the prognostic value of this ratio for patients with solid tumors treated by NAC. METHODS: A comprehensive search of the literature was conducted using the PubMed, EMBASE, Cochrane Library, and Web of Science databases, followed by a manual search of references from the retrieved articles. Pooled hazard ratios (HRs) with 95% confidence interval (CIs) were used to evaluate the association between PLR and 3 outcomes, namely, overall survival, disease-free survival, and pathological complete response rate after NAC. RESULTS: Eighteen studies published no earlier than 2014 were included in our study. A lower PLR was associated with better overall survival (HR = 1.46, 95% CI, 1.11-1.92) and favorable disease-free survival (HR = 1.81, 95% CI, 1.27-2.59). A PLR that was higher than a certain cutoff was associated with a lower pathological complete response rate in patients with cancer who received NAC (Odds ratio = 1.93, 95% CI, 1.40-2.87). CONCLUSION: Elevated PLR is associated with poor prognosis in various solid tumors. PLR may be a useful biomarker in delineating those patients with poorer prognoses who may benefit from neoadjuvant therapies.

White blood cell subsets in HER2-positive breast cancer patients treated with trastuzumab in relation to clinical outcome.

To examine whether HER2+ breast cancer patients who have decreased immune effector cells could respond well to trastuzumab, we evaluated the alterations in circulating immune system cell subsets: CD16+ and/or CD56+ lymphocytes, lymphocytes and granulocytes in these patients before and after treatment with trastuzumab-based regimens in relation to clinical response to therapy. The study involved 55 patients with HER2+ breast cancer before and 2 months after the initiation of the therapy. Progressive disease was confirmed in nine out of 55 patients (non-responders), while other patients achieved complete or partial response, or stable disease (responders). Control group consisted of up to 52 healthy individuals. Significantly lower percentages of total lymphocytes, CD16+, CD56+, and CD16+CD56+ lymphocytes as well as higher percentage of granulocytes and a higher ratio of granulocyte to lymphocyte percentages were found in patients before therapy and 2 months after the initiation of the therapy, compared with those in healthy individuals. Responder subgroup showed significantly lower percentages of CD16+, CD56+, and CD16+CD56+ lymphocytes before therapy, compared with those in healthy controls. Two months after the initiation of the therapy, the percentages of immune cell subsets remained significantly lower in responders in comparison with those in the healthy donors, while a significantly decreased percentages of CD56+ and CD16+CD56+ lymphocytes were observed in non-responders, in comparison with those in healthy controls. Our study demonstrated that HER2+ breast cancer patients who have decreased percentages of CD16+, CD56+, and CD16+CD56+ lymphocytes may achieve response to trastuzumab-containing treatment.