Diagnostic significance of blood lymphocyte activation markers in pre-eclampsia.

The adaptive and innate immune system is important in both initiating and preventing functional disorders during pregnancy, one of which is pre-eclampsia. The research aims to conduct the comparative quantification of selected subpopulations of peripheral blood immunoregulatory cells in pregnant women with pre-eclampsia in the third trimester. The marker receptors CD4, CD8, CD95, CD25, and CD27 and the marker antigen HLA-DR were considered. The screening was performed by flow cytometry with dual phenotyping using phycoerythrin- and fluorescein-isothiocyanate-labeled monoclonal antibodies. Data processing consisted in calculating a likelihood value to assess the statistical significance of the difference between the samples. A statistically significant decrease in the subpopulation titer of T and B lymphocytes with marker receptors CD4, CD8, and CD19 was found in pre-eclampsia patients. In the CD4 carrier T-lymphocyte population, there was an increased expression of the CD25/CD95 activation and apoptosis markers. In the CD8 T-killer population, a decreased representation of the CD27/CD25/CD95 markers of differentiation, activation, and apoptosis was deterministic. The expression pattern of the major histocompatibility complex antigen HLA-DR did not change significantly in normality and pathology. The titer of peripheral natural killer cells carrying the CD56 marker increased in patients with various degrees of disease severity, while the number of CD16 natural killer remained at the level of the control group. The research results suggest that a change in the ratio of the above receptors is a diagnostic indicator for pre-eclampsia.

Assessment of inter-operator variability in peripheral monocyte subset gating strategy using flow cytometry in patients with suspected acute stroke.

BACKGROUND: Innovative tools to reliably identify patients with acute stroke are needed. Peripheral monocyte subsets, that is, classical-Mon1, intermediate-Mon2, and non-classical-Mon3, with their activation marker expression analyzed using flow-cytometry (FCM) could be interesting cell biomarker candidates. AIM: To assess the inter-operator variability in a new peripheral monocyte subset gating strategy using FCM in patients with suspected acute stroke. METHODS: In BOOST-study ("Biomarkers-algOrithm-for-strOke-diagnoSis-and Treatment-resistance-prediction," NCT04726839), patients ≥18 years with symptoms suggesting acute stroke within the last 24 h were included. Blood was collected upon admission to emergency unit. FCM analysis was performed using the FACS-CANTO-II® flow-cytometer and Flow-Jo™-software. Analyzed markers were CD45/CD91/CD14/CD16 (monocyte backbone) and CD62L/CD11b/HLA-DR/CD86/CCR2/ICAM-1/CX3CR1/TF (activation markers). Inter-operator agreement (starting from raw-data files) was quantified by the measure distribution and, for each patient, the coefficient of variation (CV). RESULTS: Three operators analyzed 20 patient blood samples. Median inter-operator CVs were below the pre-specified tolerance limits (10% [for Mon1 counts], 20% [Mon2, Mon3 counts], 15% [activation marker median-fluorescence-intensities]). We observed a slight, but systematic, inter-operator effect. Overall, absolute inter-operator differences in fractions of monocyte subsets were <0.03. CONCLUSION: Our gating strategy allowed monocyte subset gating with an acceptable inter-operator variability. Although low, the inter-operator effect should be considered in monocyte data analysis of BOOST-patients.

Circulating T cells in sarcoidosis have an aberrantly activated phenotype that correlates with disease outcome.

RATIONALE: Disease course in sarcoidosis is highly variable. Bronchoalveolar lavage fluid and mediastinal lymph nodes show accumulation of activated T cells with a T-helper (Th)17.1 signature, which correlates with non-resolving sarcoidosis. We hypothesize that the peripheral blood (PB) T cell phenotype may correlate with outcome. OBJECTIVES: To compare frequencies, phenotypes and function of circulating T cell populations in sarcoidosis patients with healthy controls (HCs) and correlate these parameters with outcome. METHODS: We used multi-color flow cytometry to quantify activation marker expression on PB T cell subsets in treatment-naïve patients and HCs. The disease course was determined after 2-year follow-up. Cytokine production was measured after T cell stimulation in vitro. MEASUREMENTS AND MAIN RESULTS: We observed significant differences between patients and HCs in several T cell populations, including CD8+ and CD4+ T cells, Th1/Th17 subsets, CD4+ T memory stem cells, regulatory T cells (Tregs) and γδ T cells. Decreased frequencies of CD4+ T cells and increased frequencies of Tregs and CD8+ γδ T cells correlated with worse outcome. Naïve CD4+ T cells displayed an activated phenotype with increased CD25 expression in patients with active chronic disease at 2-year follow-up. A distinctive Treg phenotype with increased expression of CD25, CTLA4, CD69, PD-1 and CD95 correlated with chronic sarcoidosis. Upon stimulation, both naïve and memory T cells displayed a different cytokine profile in sarcoidosis compared to HCs. CONCLUSIONS: Circulating T cell subpopulations of sarcoidosis patients display phenotypic abnormalities that correlate with disease outcome, supporting a critical role of aberrant T cell activation in sarcoidosis pathogenesis.

Immune regulation by activation markers at feto-maternal interface in infection-associated spontaneous preterm birth.

BACKGROUND: Spontaneous preterm birth (sPTB) is a global health concern. Studies reveal infections are majorly responsible for sPTB and immune activation markers play a role in regulation of maternal immune responses against pathogens during sPTB. AIM: To study the mRNA expression and correlation of activation markers (CD66a, ICAM1, ITGB1, TIM3, CD25, CD95) and associated cytokines (IL-1ß and IL-17)/prostaglandin receptors (EP2 and IP) in the placenta of Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum-infected sPTB women. METHODS: Placental samples were collected from 160 sPTB and 160 term birth women. PCR was used for the detection of C. trachomatis, M. hominis, U. urealyticum. The mRNA expression of activation markers, cytokines and prostaglandin receptors was evaluated by real-time qPCR. RESULTS: The fold-change expression of CD66a, ICAM1, TIM3, CD25 and CD95 was 2.89, 5.5, 4.95, 6.44 and 6.95-fold (p < 0.001), respectively; while for cytokines- IL-1ß and IL-17 was 5.41 and 4.71-fold (p < 0.001), respectively and for prostaglandin receptors- EP2 and IP was 5.5 and 5-fold (p < 0.001) upregulated, respectively in infected sPTB women. Significant positive correlation was obtained among ICAM-1 and IL-1ß/EP2/IL-17, TIM3 and IP/IL-17. Significant negative correlation was obtained between CD66a and EP2/IL-17, CD25 and IL-1ß/EP2, CD95 and IL-1ß/EP2 in infected sPTB women. CONCLUSIONS: CD66a, ICAM1 and TIM3 may play role in inflammation and have potential for the clinical beginning of preterm labour during infection while CD25 and CD95 are possibly involved in immunotolerance at feto-maternal interface during C. trachomatis, M. hominis and U. urealyticum infection.

Factors Associated With Systemic Immune Activation Indices in a Global Primary Cardiovascular Disease Prevention Cohort of People With Human Immunodeficiency Virus on Antiretroviral Therapy.

BACKGROUND: Among antiretroviral therapy (ART)-treated people with human immunodeficiency virus (PWH), persistent systemic immune activation contributes to atherogenesis atherosclerotic, cardiovascular disease (CVD) events, and mortality. Factors associated with key immune activation indices have not previously been characterized among a global primary CVD prevention cohort of PWH. METHODS: Leveraging baseline Randomized Trial to Prevent Vascular Events in HIV (REPRIEVE) data, we evaluated factors associated with soluble CD14 (sCD14) and oxidized low-density lipoprotein (oxLDL). RESULTS: The primary analysis cohort included 4907 participants from 5 global-burden-of-disease regions (38% female, 48% Black, median age 50 years). In fully adjusted models for sCD14, female sex and White race (among those in high-income regions) were associated with higher sCD14 levels, while higher body mass index (BMI) and current use of nucleoside reverse transcriptase inhibitor + integrase strand transfer inhibitor ART were associated with lower sCD14 levels. In fully adjusted models for oxLDL, male sex, residence in high-income regions, White race (among those in high-income regions), and higher BMI were associated with higher oxLDL levels. In a subanalysis cohort of 1396 women with HIV, increased reproductive age was associated with higher sCD14 levels but not with higher oxLDL levels. CONCLUSIONS: Factors associated with sCD14 and oxLDL, 2 key indices of immune-mediated CVD risk, differ. Future studies will elucidate ways in which medications (eg, statins) and behavioral modifications influence sCD14 and oxLDL and the extent to which dampening of these markers mediates CVD-protective effects. CLINICAL TRIALS REGISTRATION: NCT0234429.

Helminth antigens differentially modulate the activation of CD4+ and CD8+ T lymphocytes of convalescent COVID-19 patients in vitro.

BACKGROUND: The coronavirus disease 2019 (COVID-19) is a respiratory disease caused by SARS-CoV-2, a recently discovered strain of coronavirus. The virus has spread rapidly, causing millions of death worldwide. Contrary to the predictions, prevalence and mortality due to COVID-19 have remained moderate on the African continent. Several factors, including age, genetics, vaccines, and co-infections, might impact the course of the pandemic in Africa. Helminths are highly endemic in Sub-Saharan Africa and are renowned for their ability to evade, skew, and suppress human immune responses through various immune-modulatory mechanisms. Such effects will likely impact SARS-CoV-2 transmission and disease progression. METHODS: Here, we analyzed in vitro the impact of antigen extracts from three major helminth parasites, including Onchocerca volvulus, Brugia malayi, and Ascaris lumbricoides, on the immune reactivity to SARS-CoV-2 peptides in COVID-19 patients. Activation of CD4+ and CD8+ T cells was investigated using flow cytometry to monitor the expression of CD137 (4-1BB) and CD69. Cytokine expression, including IL-6, IL-10, IFN-γ, and TNFα, was measured by Luminex in cell culture supernatants. RESULTS: We observed that helminth antigens significantly reduced the frequency of SARS-CoV-2-reactive CD4+ T helper cells. In contrast, the expression of SARS-CoV-2-reactive CD8+ T cells was not affected and even significantly increased when PBMCs from COVID-19 patients living in Benin, an endemic helminth country, were used. In addition, stimulation with helminth antigens was associated with increased IL-10 and a reduction of IFNγ and TNFα. CONCLUSIONS: Our data offer a plausible explanation for the moderate incidence of COVID-19 in Africa and support the hypothesis that helper T cell-mediated immune responses to SARS-CoV-2 are mitigated in the presence of helminth antigens, while virus-specific cytotoxic T cell responses are maintained.

Refining the risk of HTLV-1-associated myelopathy in people living with HTLV-1: identification of a HAM-like phenotype in a proportion of asymptomatic carriers.

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), ß2-microglobulin (ß2M) 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), ß2M 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 ß2M. 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.

Monocyte count and soluble markers of monocyte activation in people living with HIV and uninfected controls.

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] × 109/L) lower monocyte count than uninfected controls, and in adjusted analyses HIV status was independently associated with - 0.035 [- 0.045, - 0.025] × 109/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.

The impact of anti-endothelial cell antibodies (AECAs) on the development of blood vessel damage in patients with systemic lupus erythematosus: the preliminary study.

Vascular injury represents one of the most frequent lesions in systemic lupus erythematosus (SLE). The aim of the study was to assess the influence of anti-endothelial cell antibodies (AECAs) on the development of endothelial cell (EC) activation, dysfunction and subsequent vasculitis in women with SLE. Fifty six women with SLE were divided into 2 subgroups, i.e. subjects with positive AECAs (+) and those with negative AECAs (-). The control group consisted of 25 healthy women. Clinical characteristics, routine laboratory tests and circulating markers of EC activation/dysfunction, i.e. monocyte-chemotactic protein-1 (MCP-1), soluble E- and P-selectin, vascular and intercellular adhesion molecule-1 (sVCAM-1, sICAM-1), von Willebrand factor (vWF), pentraxin 3 (the marker of vasculitis) the indicator of procoagulant activity i.e. prothrombin fragment 1 + 2 (F1 + 2) were detected using ELISA and compared between patients with AECA (+), AECA (-) and control subgroups. Serum concentrations of AECAs in AECA(+), AECA(-) and control groups were 4.58 ± 2.97, 0.92 ± 0.50 and 0.72 ± 0.28 AU/ml, respectively (p < 0.001). The study showed significant increases in EC activation markers, i.e. MCP-1, sE-selectin, sVCAM-1 and F1 + 2 in SLE AECA(+) compared to SLE AECA(-) and control groups. However, the indicator of vasculitis (PTX3) was significantly lower in SLE AECA(+). Moreover, multivariate analysis of variance showed a positive correlation between AECAs and sE-selectin and sVCAM-1 levels, but not with PTX3. AECAs were involved in the initial stages of vascular damage in SLE, i.e. in EC activation and dysfunction. However, they did not play a role in the development of vasculitis.

Platelet Activation Markers in Children with Pulmonary Arterial Hypertension Associated with Congenital Heart Disease.

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.

CD62L on blood basophils: a first pre-treatment predictor of remission in severe lupus nephritis.

BACKGROUND: Basophils were recently shown to contribute to lupus nephritis (LN). This study assessed blood basophil activation markers (BAMs) for the diagnosis of LN severity and as pre-treatment prognostic markers of the response to treatment in patients with severe LN. METHOD: The diagnostic study included all the patients of a monocentric prospective observational cohort built with consecutive patients diagnosed with LN on the basis of a renal biopsy. The prognostic study selected patients of this cohort according to the following inclusion criteria: ≥18 years old, Class III or IV A ± C ± Class V or pure Class V LN at the time of inclusion and treated with an induction treatment for LN. Clinical data and BAMs were obtained at the time of the kidney biopsy. LN remission status was recorded 12 months after induction therapy initiation. Associations between baseline data and histological severity of LN or LN remission were assessed using logistic regression. RESULTS: No significant association was found between BAMs and the histological severity of LN in 101 patients. Among the 83 patients included in the prognostic study, 64 reached renal remission. CD62L expression on blood basophils at baseline was independently negatively associated with remission at 12 months [odds ratio = 0.26, 95% confidence interval 0.08-0.82, P = 0.02 for quantitative CD62L expression >105 (geometric fluorescent intensity) gMFI]. CD62L <105 gMFI was associated with a probability of 0.87 of LN remission in the next 12 months after the start of induction therapy. CONCLUSION: Pre-treatment CD62L expression on blood basophils could be a first predictive biomarker of renal response to induction therapy at 12 months in patients with severe LN.

Thrombopoietin Contributes to Enhanced Platelet Activation in Patients with Type 1 Diabetes Mellitus.

Atherosclerotic cardiovascular disease is the major cause of morbidity and mortality in patients with type 1 diabetes mellitus (T1DM). Enhanced platelet reactivity is considered a main determinant of the increased atherothrombotic risk of diabetic patients. Thrombopoietin (THPO), a humoral growth factor able to stimulate megakaryocyte proliferation and differentiation, also modulates the response of mature platelets by enhancing both activation and binding to leukocytes in response to different agonists. Increased THPO levels have been reported in different clinical conditions characterized by a generalized pro-thrombotic state, from acute coronary syndromes to sepsis/septic shock, and associated with elevated indices of platelet activation. To investigate the potential contribution of elevated THPO levels in platelet activation in T1DM patients, we studied 28 T1DM patients and 28 healthy subjects. We measured plasma levels of THPO, as well as platelet-leukocyte binding, P-selectin, and THPO receptor (THPOR) platelet expression. The priming activity of plasma from diabetic patients or healthy subjects on platelet-leukocyte binding and the role of THPO on this effect was also studied in vitro. T1DM patients had higher circulating THPO levels and increased platelet-monocyte and platelet-granulocyte binding, as well as platelet P-selectin expression, compared to healthy subjects, whereas platelet expression of THPOR did not differ between the two groups. THPO concentrations correlated with platelet-leukocyte binding, as well as with fasting glucose and Hb1Ac. In vitro, plasma from diabetic patients, but not from healthy subjects, primed platelet-leukocyte binding and platelet P-selectin expression. Blocking THPO biological activity using a specific inhibitor prevented the priming effect induced by plasma from diabetic patients. In conclusion, augmented THPO may enhance platelet activation in patients with T1DM, potentially participating in increasing atherosclerotic risk.

Soluble CD163 and mannose receptor as markers of liver disease severity and prognosis in patients with primary biliary cholangitis.

INTRODUCTION: In primary biliary cholangitis (PBC), macrophages are involved in liver inflammation and fibrosis. The macrophage activation markers, soluble (s)CD163 and mannose receptor (sMR) are associated with liver disease severity and prognosis in other chronic liver diseases. We aimed to investigate sCD163 and sMR in patients with PBC. METHODS: We investigated PBC patients from the Italian PBC Study Group cohort and measured macrophage activation markers in serum at study enrolment. Patients were followed from enrolment until they experienced an event or were censored at their last visit. Events were defined as follows: (a) death from a liver-related cause; or (b) liver transplantation (LT) for PBC. We used Cox regression to investigate the association between sCD163 and sMR and long-term prognosis. RESULTS: In total, 202 PBC patients were included. Median age was 62 years (interquartile range (IQR), 53-71) at enrolment and 93% were women. Median sCD163 was 3.43 mg/L (IQR 2.48-5.35) and median sMR was 0.35 mg/L (IQR 0.28-0.45). There was an increase in sCD163 and sMR with increasing alkaline phosphatase. Two hundred and one patients were followed for a median of 8.6 years, and sCD163 and sMR predicted long-term risk of liver-related death or LT in univariate analyses, while sCD163 was also associated with outcome after confounder adjusting (adjusted HR = 1.14, 95% CI 1.00-1.30). Finally, we showed an increase in the prediction accuracy of poor outcome by adding sCD163 to the UK-PBC risk score. CONCLUSION: The macrophage activation markers sCD163 and sMR represent a non-invasive measure of PBC disease severity that provides useful long-term prognostic information.

Possible role of plasma Galectin-9 levels as a surrogate marker of viremia in HIV infected patients on antiretroviral therapy in resource-limited settings.

BACKGROUND: Early detection of viremia in HIV infected patients on anti-retroviral therapy (ART) is important to prevent disease progression as well as accumulation of drug resistance mutations. This makes HIV viral load (VL) monitoring indispensable in HIV infected patients on ART. However VL, being an expensive test, results in heavy financial burden on health services. Hence, cheaper surrogate markers of viremia are desired to reduce overall cost of management of HIV infected patients. METHODS: We enrolled aviremic (n = 63, M:F = 31:32) and viremic (n = 43, M:F = 21:22) HIV infected patients at 1 year after ART initiation. Viremic individuals were identified as those having a plasma VL of more than 1000 copies/µl and aviremic individuals as less than 40 copies/µl. The study participants also included immuno-virologically discordant patients as they demonstrate differential degrees of immune-reconstitution and are likely to harbour concomitant infections influencing levels of immune-activation markers screened as the surrogate markers. Immune activation markers viz. plasma hs-CRP, soluble-CD14 and Galectin-9 levels were estimated by ELISA, IL-6 by luminex assay and percentages of CD38+ CD8+ cells were determined by flow cytometry. The levels were compared between viremic and aviremic patients and correlated with plasma viral load. Receiver operated curve (ROC) analysis was done for plasma Galectin-9 levels. RESULTS: Viremic patients had significantly higher levels of Galectin-9 and %CD38+ CD8+ cells (p values < 0.0001) than aviremic patients. Levels of the other activation markers did not differ between viremic and aviremic individuals. Galectin-9 levels (r = 0.76) and %CD38+ CD8+ cells (r = 0.39) correlated positively with VL. Area under curve for Galectin-9 levels for distinguishing between viremic and aviremic individuals was 0.98. Youden index, sensitivity, specificity, positive predictive value and negative predictive value for Galectin-9 levels were 0.87, 0.97, 0.90, 0.87 and 0.98, respectively, at the cut-off value of 5.79 ng/ml. CONCLUSIONS: Plasma Galectin-9 levels could identify viremic individuals with sensitivity and specificity of more than 90%. Thus, they showed a potential to serve as a surrogate marker of viremia in HIV infected patients on ART and would have cost implications on HIV management especially in resource-limited settings. However, the findings need to be confirmed in the patients on ART for different durations of time.

The Effect of Helminths on Granulocyte Activation: A Cluster-Randomized Placebo-Controlled Trial in Indonesia.

BACKGROUND: Eosinophils are a prominent cell type in the host response to helminths, and some evidence suggests that neutrophils might also play a role. However, little is known about the activation status of these granulocytes during helminth infection. METHODS: We analyzed the expression of eosinophil and neutrophil activation markers in peripheral blood by flow cytometry and measured serum levels of eosinophil granule proteins in 300 subjects residing in an area endemic for soil-transmitted helminths (STH). The data generated are on samples before and after 1 year of 3-monthly albendazole treatment. RESULTS: Anthelmintic treatment significantly reduced the prevalence of STH. While eosinophil numbers were significantly higher in STH-infected compared to uninfected subjects and significantly decreased following albendazole treatment, there was no effect exerted by the helminths on either eosinophil nor neutrophil activation. Although at baseline eosinophil granule protein levels were not different between STH-infected and uninfected subjects, treatment significantly reduced the levels of eosinophil-derived neurotoxin (EDN) in those infected at baseline. CONCLUSIONS: These results show that besides decreasing eosinophil numbers, anthelmintic treatment does not significantly change the activation status of eosinophils, nor of neutrophils, and the only effect seen was a reduction in circulating levels of EDN. CLINICAL TRIALS REGISTRATION: http://www.isrctn.com/ISRCTN75636394.

OMIP-060: 30-Parameter Flow Cytometry Panel to Assess T Cell Effector Functions and Regulatory T Cells.

We developed this comprehensive 28-color flow cytometry panel with the aim to measure a variety of T cell effector functions in combination with T cell differentiation markers (CCR7, CD27, CD28, CD45RO, CD95) in γδ T cells and CD4+ and CD8+ αß T cells (Table 1). The effector functions measured in this panel include activation and co-stimulatory molecules (CD69, CD137, and CD154), cytokines (IL-2, IL-13, IL-17A, IL-21, IL-22, TNF, and IFNγ), the chemokine IL-8, cytotoxic molecules (perforin and granzyme B), and the degranulation marker CD107a. In addition, Ki67 enables the identification and analysis of recently activated T cells. To characterize regulatory T cells (Tregs ), we included CD25, CD39, and the canonical Tregs transcription factor FoxP3. We developed and optimized this panel for cryopreserved human peripheral blood mononuclear cells (PBMC) and stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. However, we successfully tested other types of stimulation such as staphylococcus enterotoxin B (SEB) or a mix of immunodominant peptides (CEF peptide pool) from cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.

Changes in FGF-23, Neutrophil/Platelet Activation Markers, and Angiogenin in Advanced Chronic Kidney Disease and Their Effect on Arterial Stiffness.

AIMS: The aims of this study were to measure changes in fibroblast growth factor 23 (FGF-23), neutrophil (elastase, lactoferrin)/platelet activation marker (mean platelet volume-to-platelet count ratio [MPR]), and angiogenin according to the stage of chronic kidney disease (CKD), and to evaluate the association of FGF-23, elastase, lactoferrin, MPR, and angiogenin with arterial stiffness using brachial-ankle pulse wave velocity (ba-PWV) in CKD patients. METHODS: According to the estimated glomerular filtration rate (eGFR) calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation, the patients were allocated to five groups: (1) normal controls (eGFR ≥90 mL/min/1.73 m2 without pathologic, urine [proteinuria], blood [electrolyte], and imaging abnormalities; n = 22); (2) CKD stage 2 (eGFR 60-89 mL/min/1.73 m2; n = 17); (3) CKD stage 3 (eGFR 30-59 mL/min/1.73 m2; n = 22); (4) CKD stage 4 (eGFR 15-30 mL/min/1.73 m2; n = 17); and (5) CKD stage 5-hemodialysis (HD) (n = 30). All the patients were free of clinically apparent cardiovascular disease. Serum FGF-23, elastase, lactoferrin, and angiogenin concentrations and the MPR were measured to study the association of the above parameters with the clinical (age, sex, presence of diabetes mellitus, and blood pressure), biochemical (calcium, phosphorus, uric acid, intact parathyroid hormone [PTH], low-density lipoprotein cholesterol, and high-sensitivity C-reactive protein), and ba-PWV values of the CKD patients. RESULTS: (1) The mean ba-PWV values were 1,497.2 ± 206.4 cm/s in the controls, 1,649.0 ± 247.9 cm/s in the CKD stage 2 group (p < 0.05 vs. controls), 1,655.8 ± 260.3 cm/s in the CKD stage 3 group (p < 0.05 vs. controls), 1,823.0 ± 402.4 cm/s in the CKD stage 4 group (p < 0.05 vs. controls and CKD stages 2 and 3), and 1,905.2 ± 374.1 cm/s in the CKD stage 5-HD group (p < 0.05 vs. controls and CKD stage 2). (2) The mean log10(FGF-23) concentration values were 0.77 ± 0.27, 0.97 ± 0.48, 1.10 ± 0.35 (p < 0.05 vs. controls and CKD stage 2), 1.35 ± 0.48 (p < 0.05 vs. controls and CKD stages 2 and 3), and 2.12 ± 0.82 (p < 0.05 vs. controls and CKD stages 2-4); the mean angiogenin levels were 230.6 ± 70.5 pg/mL, 283.0 ± 53.5 pg/mL (p < 0.05 vs. controls), 347.3 ± 76.9 pg/mL (p < 0.05 vs. controls and CKD stage 2), 445.9 ± 90.6 pg/mL (p < 0.05 vs. controls and CKD stages 2 and 3), and 370.9 ± 142.4 pg/mL (p < 0.05 vs. controls and CKD stages 2 and 3). (3) In the stage 3-4 CKD/HD patients, the mean elastase-to-neutrophil and lactoferrin-to-neutrophil ratios were significantly lower than in the controls and the stage 2 CKD patients. (4) Our multivariate linear regression analyses showed that age, pulse pressure, mean arterial pressure, PTH, and FGF-23 were independently associated with ba-PWV values. CONCLUSIONS: Circulating FGF-23 and angiogenin concentrations gradually increased as CKD advanced, whereas neutrophil activation markers were significantly lower in the stage 3-4 CKD/HD patients than in the controls and stage 2 CKD patients. FGF-23 was weakly associated with ba-PWV values in patients with CKD/HD and no previous cardiovascular disease.

In vitro evaluation of pathogen inactivated platelet quality: An 8 year experience of routine use in Galicia, Spain.

BACKGROUND: Platelet concentrates (PCs) treated by the pathogen inactivation technology (PI) using amotosalen and UVA illumination (PI-PCs) can be manufactured in additive solutions (PAS-III and PAS-IIIM) or in 100% Plasma. Quality control (QC) is an integral part of the production. We capitalized on our ongoing QC program to capture 8 years-worth of data on parameters related to the quality of 116,214 PI-PCs produced under different manufacturing methods. MATERIALS AND METHODS: Selected in vitro parameters of metabolism, activation, and storage were analyzed for the different manufacturing periods to compare PI-PCs versus conventional PCs (C-PCs) resuspended in different PAS. RESULTS AND DISCUSSION: All BC-PCs met quality standards for pH and dose and residual leucocytes. As expected, storage time correlated with increased lactate, LDH, Annexin V, CD62, sCD40 L levels and decreased glucose and pH. With PAS-IIIM, higher levels of glucose were observed toward the end of shelf life (p < 0.0001) with lower platelet activation markers Annexin V (p = 0.038) and CD62 (p = 0.0006). Following PI implementation, a low expire rate of <0.5% was observed. While a 2.3% mean increase in the production of PCs occurred from 2011 to 2015, the distribution of red blood cell concentrates dropped by 4.4%. A mean incidence of 0.14% for transfusion-related adverse reaction was observed while PI-PCs were distributed, similar to the one observed with C-PCs. Overall, PI-PCs prepared in additive solutions consistently met quality standards. Those prepared in PAS-IIIM appeared to have better retention of in vitro characteristics compared to PAS-III though all demonstrated functionality and clinical effectiveness.

Blood derived products in pediatrics: New laboratory tools for optimizing potency assignment and reducing side effects.

Neonates and children can develop rare bleeding disorders due to congenital/acquired coagulation Factor deficiencies, or allo-immune/autoimmune complications, or can undergo surgeries at high haemorrhagic risk. They then need specialized transfusion of blood components/products, or purified blood extracted products or recombinant proteins. Blood-derived therapies conventionally used for management of affected infants with genetic/acquired deficiencies, bleeding problems (coagulation Factor reduced or missing) or thrombotic disorders (reduced or missing anticoagulant proteins) pose some additional risks. These remedial therapies can cause tolerance when used very early in life and, sometimes needed, repeatedly. The introduction of recombinant proteins has allowed manufacturers to produce large amounts of the proteins usually present at very low concentration in blood. This has also changed the risk pattern of plasma-extracted products, especially in terms of continual reduction of viral transmission. Many efforts have been made over these past decades to reduce the risks associated with the use of all these products in terms of viral and bacterial safety, as well as immune disorders but they are not the objective of this article. Other associated side effects are the presence of undesired activities in blood products, which can produce thrombotic events or adverse reactions. The progressive introduction of blood derived products has greatly improved the prognosis and quality of life of affected patients. This concerns whole blood, but also blood cell concentrates, mainly platelets and red blood cells, plasma, while the blood extracted products are increasingly replaced by recombinant proteins. All these therapeutic products, i.e. blood extracted drugs, improve health and quality of life for hemophiliac's A or B, or patients with auto/allo-immune thrombocytopenias or with rare bleeding disorders, and those with thrombotic events occurring in childhood, which are mainly due to Protein C or Protein S deficiencies (congenital or acquired). Progress in analytical methods and biotechnology allow better control of the manufacturing processes for all blood derived or plasma extracted products and recombinant proteins, and contribute to improved manufacturing processes to minimize the occurrence of side effects. These adverse events can be due to the aging of the blood cell concentrate with release of their granule content, and generation of EVs, which can produce anaphylactic reactions and risk of thrombosis, but also to the presence of activated coagulation Factors in purified products, such as Factor Xia as recently identified in immunoglobulin concentrates. Characterization and measurement of contaminant products is of special usefulness during product preparation and for optimization of manufacturing processes for purified extracted products, but also for recombinant proteins. The pharmaceutical industry introduces these new methods for validating manufacturing processes, or for quality control assessments. The objective is first to warrant the full quality and safety of the lots produced, and assure the highest efficacy with the lowest risks when used in patients. For cell concentrates and fresh blood, storage conditions are critical and measurement of analytes such as EVs or Annexin V allows evaluation of quality of each individual transfused pouch. In addition to all the rules around viral and bacterial transmission risk, and immune tolerance, our available laboratory methods contribute to reducing the side effects of blood cell concentrates and derived plasma products, as well as those of the therapeutic recombinant proteins.