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Objective: Biomarkers with high accuracy for identification of infection in decompensated chronic liver disease (DCLD) are urgently needed. We compared the accuracy of neutrophilic cluster of differentiation 64 (nCD64) with procalcitonin for diagnosis of bacterial infection in children with DCLD., Methods: Consecutive children admitted with DCLD were enrolled prospectively. nCD64 was assessed by flow cytometry and expressed in percentage. nCD64, procalcitonin and hemogram were measured at admission and 7-14 days after treatment in those with infection. Complete work-up for infection was done. Presence, site and severity of infection was classified as per guidelines., Results: 107 children [64 boys, age 97(18-168) months] were enrolled. 78(72.9%) had infection, 26(24%) had severe sepsis and 60(56%) had systemic inflammatory response syndrome. The commonest site of infection was ascitic fluid (n=37), followed by pneumonia (n=24), urinary tract (n=15), bacteraemia (n=10), cholangitis (n=8) and cellulitis (n=3). nCD64 (cut-off-51%, AUC-0.82) had a higher sensitivity (79.5%) and specificity (82.8%) than procalcitonin (cut-off ≥0.58ng/mL, AUC-0.74, sensitivity-76.9% and specificity-62.1%) for diagnosis of infection. nCD64 and procalcitonin correlated with infection severity, being highest in children with severe sepsis [88(71-97) %and 1.98(0.83-10.36) ng/mL], than in infection alone [72(45-84) % and 1.09(0.45-2.07) ng/mL], and no-infection [36(20.2-48) % and 0.42(0.19-1.08) ng/mL]. There was no difference in diagnostic utility of procalcitonin or nCD64 with different sites of infection. Elevation of all 3 parameters (nCD64, PCT and total leukocyte count) was uncommon but highly specific for presence of infection., Conclusion: nCD64 identifies infection better than procalcitonin and correlates well with infection severity in children with DCLD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Masson SAS.)

Visceral leishmaniasis (VL) is a potentially fatal parasitic infection caused by Leishmania donovani in India. L. donovani is an obligate intracellular protozoan residing mostly in macrophages of the reticuloendothelial system throughout chronic infection. Monocytic phagocytes are critical in the pathogenesis of different forms of leishmaniasis. Subsets of monocytes are distinguished by their surface markers into CD14+CD16- classical monocytes, CD14+CD16+ intermediate monocytes, and CD16++CD14low non-classical monocyte subsets. During cutaneous leishmaniasis (CL), intermediate monocyte are reported to be a source of inflammatory cytokines IL-1β and TNF, and they express CCR2 attracting them to sites of inflammatory pathology. We examined monocyte subsets in the blood and bone marrow of patients with VL from an endemic site in Bihar, India, and found these contrasted with the roles of monocytes in CL. During VL, intermediate and non-classical CD16+ monocyte subsets expressed instead a non-inflammatory phenotype with low CCR2, high CX3CR1 and low microbicidal oxidant generation, making them more similar to patrolling monocytes than inflammatory cells. Bone marrow CD16+ monocyte subsets expressed a phenotype that might be more similar to the inflammatory subsets of CL, although our inability to obtain bone marrow from healthy donors in the endemic region hampered this interpretation Overall the data suggest that CD16+ intermediate monocyte subsets in VL patients express a phenotypes that contributes to an immunosuppressed pathologic immune state, but in contrast to CL, these do not mediate localized inflammatory responses., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Background: Childhood immune thrombocytopenia (ITP) is an immune-mediated disease characterized by an isolated low platelet count. Pathogenesis of ITP is complex but many patients have platelet specific autoantibodies leading to accelerated clearance of opsonized platelets by Fc-gamma receptor (FcγR) bearing phagocytes, particularly in the spleen. In humans, there are three main types of Fcγ receptors: high-affinity FcγRI and low-affinity FcγRII and FcγRIII. About FcγRII, genetic variation of FCGR2B is associated with response to IVIg treatment in patients with Kawasaki disease and ITP., Methods: We used a TaqMAMA genotyping assay for detection of rs1050501 FCGR2B polymorphism in children with chronic ITP. A SNP rs1050501 (GenBank access number NG_023318.1, Homo sapiens Fc fragment of IgG receptor IIb [FCGR2B]) on chromosome 1 showing a T/C transition in position 15894 on FCGRB2 gene was chosen in this study. A series of experiments was conducted to evaluate the performance of the FCGR2B-MAMAPCR real time on a QuantStudio™ 5 Real-Time PCR System as compared to the 7500 Real-Time PCR System., Results: Background noise, genotypes discrimination, variability, allele and genotype frequencies and concordance were obtained. About clinical validation, all 60 samples collected from chronic ITP patients were analyzed. We found 53 on the 60 patients (88.4%) were homozygotes (52 TT and 1 CC) and 7/60 (11.6%) heterozygotes (TC)., Conclusions: The ability of the FCGR2B-MAMAPCR real time to detect rs1050501 FCGR2B polymorphism in children with chronic ITP on the QuantStudio™ 5 System is comparable to that on the 7500 System.

Objective: Childhood bronchial asthma (BA) is a globally significant chronic disease with major health consequences. Recently, focus on the role of the innate immune system has been highlighted. Therefore, this study explores the role of circulating monocytes and natural killer (NK) clusters in childhood asthma. Methods: This case-control study enrolled 50 children with asthma divided equally into severe and mild groups and 26 healthy children. Flow-cytometry analysis was used to identify circulating blood monocytes and natural killers' subsets. In addition, pulmonary function test (spirometry) for children with asthma was performed. Results: This study showed significant negative correlations between frequency of total circulating, classical, intermediate, and nonclassical monocytes with ratio of forced expiratory volume/forced vital capacity (FEV1/FVC) (r = -0.637, P  < 0.001; r = -0.575 , P <  0.001; r = -0.657, P  < 0.001; r = -0.329, P  = 0.004, respectively). Also, there was significant negative correlations between frequency of total NKs and CD56 dim CD16+ NK with FEV1/FVC (r = -0.584, P  < 0.001) and (r = -0.579 , P <  0.001). Significant predictors of childhood asthma severity were frequencies of total monocytes, total NKs, intermediate monocytes, and CD56 dim CD16+ NK. Conclusion: Finally, we concluded that the FEV1/FVC is linked to aberrations of monocytes' and natural killers' immunophenotypic subsets in children with asthma. The frequencies of total monocytes and NK are significant predictors of severity of childhood asthma. The frequencies of CD14 high CD16+ intermediate monocytes and CD56 dim CD16+ NK cells are the best independent predictors of severity in children with asthma.

Background: Toll-like receptors (TLRs) are pattern-recognition sensors mainly expressed in innate immune cells that directly recognize conserved pathogen structures (pathogen-associated molecular patterns-PAMPs). Natural killer (NK) cells have been described to express different endosomal TLRs triggered by RNA and DNA sequences derived from both viruses and bacteria. This study was addressed to establish which endosomal TLR could directly mediate NK activation and function after proper stimuli. It was also important to establish the most suitable TLR agonist to be used as adjuvant in tumor vaccines or in combined cancer immunotherapies., Methods: We assessed endosomal TLR expression in total NK cells by using RT-qPCR and western blotting technique. In some experiments, we purified CD56 bright CD16 - and CD56 dim CD16 + cells subsets by using NK Cell Isolation Kit Activation marker, cytokine production, CD107a expression and cytotoxicity assay were evaluated by flow cytometry. Cytokine release was quantified by ELISA. NK cells obtained from ovarian ascites underwent the same analyses., Results: Although the four endosomal TLRs (TLR3, TLR7/8, and TLR9) were uniformly expressed on CD56 bright CD16 - and CD56 dim CD16 + cell subsets, the TLR7/8 (R848), TLR3 (polyinosinic-polycytidylic acid, Poly I:C) and TLR9 (ODN2395) ligands promoted NK-cell function only in the presence of suboptimal doses of cytokines, including interleukin (IL)-2, IL-12, IL-15, and IL-18, produced in vivo by other environmental cells. We showed that R848 rather than TLR3 and TLR9 agonists primarily activated CD56 bright CD16 - NK cells by increasing their proliferation, cytokine production and cytotoxic activity. Moreover, we demonstrated that R848, which usually triggers TLR7 and TLR8 on dendritic cells, macrophages and neutrophils cells, activated CD56 bright CD16 - NK-cell subset only via TLR8. Indeed, specific TLR8 but not TLR7 agonists increased cytokine production and cytotoxic activity of CD56 bright CD16 - NK cells. Importantly, these activities were also observed in peritoneal NK cells from patients with metastatic ovarian carcinoma, prevalently belonging to the CD56 bright CD16 - subset., Conclusion: These data highlight the potential value of TLR8 in NK cells as a new target for immunotherapy in patients with cancer., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: C-reactive protein (CRP) is an independent biomarker of systemic inflammation and a predictor of future cardiovascular disease (CVD). More than just a pure bystander, CRP directly interacts with endothelial cells to decrease endothelial nitric oxide synthase (eNOS) expression and bioactivity, decrease nitric oxide (NO) production, and increase the release of vasoconstrictors and adhesion molecules. Race is significantly associated with CRP levels and CVD risks. With aerobic exercise, the vessel wall is exposed to chronic high laminar shear stress (HiLSS) that shifts the endothelium phenotype towards an anti-inflammatory, antioxidant, antiapoptotic, and antiproliferative environment. Thus, the purpose of this study was to assess the racial differences concerning the CRP-induced effects in endothelial cells and the potential role of HiLSS in mitigating these differences., Methods: Human umbilical vein endothelial cells (HUVECs) from four African American (AA) and four Caucasian (CA) donors were cultured and incubated under the following conditions: (1) static control, (2) CRP (10  μ g/mL, 24 hours), (3) CRP receptor (Fc γ RIIB) inhibitor followed by CRP stimulation, (4) HiLSS (20 dyne/cm 2 , 24 hours), and (5) HiLSS followed by CRP stimulation., Results: AA HUVECs had significantly higher Fc γ RIIB receptor expression under both basal and CRP incubation conditions. Blocking Fc γ RIIB receptor significantly attenuated the CRP-induced decrements in eNOS expression only in AA HUVECs. Finally, HiLSS significantly counteracted CRP-induced effects., Conclusion: Understanding potential racial differences in endothelial function is important to improve CVD prevention. Our results shed light on Fc γ RIIB receptor as a potential contributor to racial differences in endothelial function in AA., Competing Interests: The authors declare that there is no conflict of interest., (Copyright © 2021 Chenyi Ling et al.)

Low-affinity immunoglobulin gamma Fc region receptor III-B (FcγRIIIB) deficiency is present in ˜0·05% of the general population. Among our patients, FcγRIIIB deficiency was less frequent in those with immune-system disorders (one of 1815 patients, 0·05%) than in those with blood disorders (nine of 2147 patients, 0·42%, P = 0·023): mainly primary immune thrombocytopenia (4·34%), therapy related myeloid neoplasms (1·16%) and myelodysplastic syndrome with excess blasts (1·28%). Four of the nine (44·4%) patients with blood disorders were diagnosed with or quickly evolved to acute myeloid leukaemia (AML), suggesting that FcγRIIIB deficiency could be an adverse prognostic factor for progression to AML that should be confirmed in large multicentre studies., (© 2021 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Methods: The clinical data of 76 patients with haematological malignancies and infection who were treated in our department between January 2014 and October 2019 were retrospectively analysed. To evaluate the diagnostic value of some biomarkers, infection indexes such as white blood cell count (WBC), neutrophil count (NEUT), neutrophil CD64 and procalcitonin (PCT) were compared across the patients with confirmed infection status and infection-control status. Sensitivity, specificity and area under the receiver operating characteristic curve (AUC) were also determined., Results: The WBC and NEUT did not differ significantly, whereas the neutrophil CD64 and PCT levels were significantly elevated in patients with a confirmed infection status ( p  < 0.05), with sensitivity of 31.0%, 45.2%, 76.2% and 50%, respectively, and specificity of 90.5%, 69%, 71.4% and 64.3%, respectively. The AUC of WBC, NEUT, neutrophil CD64 and PCT was 0.528, 0.517, 0.844 and 0.599, respectively. Further highlighting their diagnostic value, the neutrophil CD64 and PCT levels in neutropenia patients were significantly upregulated in patients with infection status ( p  < 0.05) but the WBC and NEUT were unchanged, with sensitivity of 73.7%, 63.2%, 68% and 68.4%, respectively, and specificity of 68.4%, 52.6%, 57.9% and 63.2%, respectively. The AUC of neutrophil CD64, PCT, WBC and NEUT was 0.864, 0.593, 0.419 and 0.403, respectively., Conclusion: These results indicate that neutrophil CD64 is a promising biomarker with superior sensitivity and specificity for diagnosing infection in patients with haematological malignancies, especially neutropenia patients.

Patients with acute myeloid leukaemia (AML) have a five-year survival rate of 28·7%. Natural killer (NK)-cell have anti-leukaemic activity. Here, we report on a series of 13 patients with high-risk R/R AML, treated with repeated infusions of double-bright (CD56 bright /CD16 bright ) expanded NK cells at an academic centre in Brazil. NK cells from HLA-haploidentical donors were expanded using K562 feeder cells, modified to express membrane-bound interleukin-21. Patients received FLAG, after which cryopreserved NK cells were thawed and infused thrice weekly for six infusions in three dose cohorts (10 6 -10 7 cells/kg/infusion). Primary objectives were safety and feasibility. Secondary endpoints included overall response (OR) and complete response (CR) rates at 28-30 days after the first infusion. Patients received a median of five prior lines of therapy, seven with intermediate or adverse cytogenetics, three with concurrent central nervous system (CNS) leukaemia, and one with concurrent CNS mycetoma. No dose-limiting toxicities, infusion-related fever, or cytokine release syndrome were observed. An OR of 78·6% and CR of 50·0% were observed, including responses in three patients with CNS disease and clearance of a CNS mycetoma. Multiple infusions of expanded, cryopreserved NK cells were safely administered after intensive chemotherapy in high-risk patients with R/R AML and demonstrated encouraging outcomes., (© 2021 British Society for Haematology and John Wiley & Sons Ltd.)

There is considerable inter-individual and inter-population variability in response to viruses. The potential of monocytes to elicit type-I interferon responses has attracted attention to their role in viral infections. Here, we use single-cell RNA-sequencing to characterize the role of cellular heterogeneity in human variation of monocyte responses to influenza A virus (IAV) exposure. We show widespread inter-individual variability in the percentage of IAV-infected monocytes. Notably, individuals with high cellular susceptibility to IAV are characterized by a lower activation at basal state of an IRF/STAT-induced transcriptional network, which includes antiviral genes such as IFITM3 , MX1 and OAS3 . Upon IAV challenge, we find that cells escaping viral infection display increased mRNA expression of type-I interferon stimulated genes and decreased expression of ribosomal genes, relative to both infected cells and those never exposed to IAV. We also uncover a stronger resistance of CD16 + monocytes to IAV infection, together with CD16 + -specific mRNA expression of IL6 and TNF in response to IAV. Finally, using flow cytometry and bulk RNA-sequencing across 200 individuals of African and European ancestry, we observe a higher number of CD16 + monocytes and lower susceptibility to IAV infection among monocytes from individuals of African-descent. Based on these data, we hypothesize that higher basal monocyte activation, driven by environmental factors and/or weak-effect genetic variants, underlies the lower cellular susceptibility to IAV infection of individuals of African ancestry relative to those of European ancestry. Further studies are now required to investigate how such cellular differences in IAV susceptibility translate into population differences in clinical outcomes and susceptibility to severe influenza., Competing Interests: JP was employed by DIACCURATE. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 O’Neill, Quach, Pothlichet, Aquino, Bisiaux, Zidane, Deschamps, Libri, Hasan, Zhang, Zhang, Matuozzo, Cobat, Abel, Casanova, Naffakh, Rotival and Quintana-Murci.)

Many of the effector functions of antibodies rely on the binding of antibodies/immune complexes to cellular Fcγ receptors (FcγRs). Since the majority of innate immune effector cells express both activating and inhibitory Fc receptors, the outcome of the binding of immune complexes to cells of a given population is influenced by the relative affinities of the respective IgG subclasses to these receptors, as well as by the numbers of activating and inhibitory FcγRs on the cell surface. A group of immune cells that has come into focus more recently is the various subsets of tissue-resident macrophages. The central functions of FcγRs on tissue macrophages include the clearance of opsonized pathogens, the removal of small immune complexes from the circulation and the depletion of antibody-opsonized cells in the therapy of autoimmunity and cancer. Despite these essential functions of FcγRs on tissue-resident macrophages, an in-depth quantification of FcγRs is lacking. Thus, the aim of our current study was to quantify the various Fcγ receptors on macrophages in murine liver, lung, kidney, brain, skin and spleen. Our study identified a pronounced heterogeneity between FcγR expression patterns of the different tissue macrophages, which may reflect their specialized functions within their unique niches in different organ environments.

Commercially approved conventional antibody-drug conjugates (ADCs) are produced as heterogeneous mixtures containing a stochastic distribution of payloads decorating the antibody molecules resulting in decreased efficacy and thus lowering their therapeutic index. Control of the DAR and conjugation site in the development of next-generation ADCs is believed to assist in increasing the therapeutic index of these targeted biologics leading to overall enhanced clinical efficacy and reduced toxicity. A chemical site-specific conjugation technology termed AJICAP® allows ADC developers to control both the location and quantity of the payload conjugation to an antibody. Furthermore, this simplified ADC composition enables a streamlined chemical analysis. Here we report the chromatographic separation of site-specific ADCs produced by AJICAP® technology using an analytical affinity chromatography HPLC column containing a recombinant FcγIIIa receptor-ligand immobilized on a non-porous polymer resin (NPR). These HPLC analyses provided visually clear chromatogram results reflecting the heterogeneity of each ADC. The affinity strength was also measured by biolayer interferometry (BLI) and predicted by molecular structure analysis. The results indicate that AJICAP® technology is a promising solution to link hydrophobic payloads to antibodies without compromising antibody receptor function. This study also shows that FcγIIIa-NPR column can be used to characterize site-specific conjugated ADCs compared to ADCs synthesized using conventional methods., (Copyright © 2021 Elsevier B.V. All rights reserved.)

[Figure: see text].

Immune deregulation has a critical role in the pathogenesis of lower risk myelodysplastic syndromes (MDS). The cells of the macrophage/monocyte lineage have been reported to contribute to the inflammatory process in MDS through impaired phagocytosis of the apoptotic hemopoietic cells and abnormal production of cytokines. In the present study we assessed the number of peripheral blood (PB) monocyte subsets, namely the classical CD14 bright /CD16 - , intermediate CD14 bright /CD16 + and non-classical CD14 dim /CD16 + cells, in patients with lower risk (low/intermediate-I) MDS (n = 32). We also assessed the production of tumor necrosis factor (TNF)α by patient PB monocytes in response to immune stimulus as well as their transcriptome profile. Compared to age- and sex-matched healthy individuals (n = 19), MDS patients had significantly lower number of classical and increased number of intermediate monocytes. Patient intermediate monocytes displayed increased production of TNFα following stimulation with lipopolysaccharide, compared to healthy individuals. Transcriptional profiling comparison of CD16 + monocytes from patients and controls revealed 43 differentially expressed genes mostly associated with biological pathways/processes relevant to hemopoiesis, immune signaling and cell adhesion. These data provide evidence for the first-time that distinct monocyte subsets display abnormal quantitative and functional characteristics in lower risk MDS substantiating their role in the immune deregulation associated with the disease., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Immune thrombocytopenia (ITP) is thought to result from an aberrant adaptive autoimmune response, involving autoantibodies, B and T lymphocytes, directed at platelets and megakaryocytes. Previous reports have demonstrated skewed CD4 + T-helper subset distribution and enhanced production of pro-inflammatory cytokines such as interleukin 17A and interferon gamma. The role of monocytes (MCs) in ITP is less widely described, but innate immune cells have a role in shaping CD4 + T-cell phenotypes. Glucocorticoids (GCs) are commonly used for first-line ITP treatment and modulate a broad range of immune cells including T cells and MCs. Using multiparameter flow cytometry analysis, we demonstrate the expansion of intermediate MCs (CD14 ++ CD16 + ) in untreated patients with newly diagnosed ITP, with these cells displaying a pro-inflammatory phenotype, characterised by enhanced expression of CD64 and CD80. After 2 weeks of prednisolone treatment (1 mg/kg daily), the proportion of intermediate MCs reduced, with enhanced expression of the anti-inflammatory markers CD206 and CD163. Healthy control MCs were distinctly different than MCs from patients with ITP before and after GC treatment. Furthermore, the GC-induced phenotype was not observed in patients with chronic ITP receiving thrombopoietin receptor agonists. These data suggest a role of MCs in ITP pathogenesis and clinical response to GC therapy., (© 2020 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons.)

Aims: To elucidate the prognostic role of monocytes in the immune response of patients with coronary artery disease (CAD) at risk for life-threatening heart and lung injury as major complications of SARS-CoV-2 infection., Methods and Results: From February to April 2020, we prospectively studied a cohort of 96 participants comprising 47 consecutive patients with CAD and acute SARS-CoV-2 infection (CAD + SARS-CoV-2), 19 CAD patients without infections, and 30 healthy controls. Clinical assessment included blood sampling, echocardiography, and electrocardiography within 12 h of admission. Respiratory failure was stratified by the Horovitz Index (HI) as moderately/severely impaired when HI ≤200 mmHg. The clinical endpoint (EP) was defined as HI ≤200 mmHg with subsequent mechanical ventilation within a follow-up of 30 days. The numbers of CD14dimCD16+ non-classical monocytes in peripheral blood were remarkably low in CAD + SARS-CoV-2 compared with CAD patients without infection and healthy controls (P < 0.0001). Moreover, these CD14dimCD16 monocytes showed decreased expression of established markers of adhesion, migration, and T-cell activation (CD54, CD62L, CX3CR1, CD80, and HLA-DR). Decreased numbers of CD14dimCD16+ monocytes were associated with the occurrence of EP. Kaplan-Meier curves illustrate that CAD + SARS-CoV-2 patients with numbers below the median of CD14dimCD16+ monocytes (median 1443 cells/mL) reached EP significantly more often compared to patients with numbers above the median (log-rank 5.03, P = 0.025)., Conclusion: Decreased numbers of CD14dimCD16+ monocytes are associated with rapidly progressive respiratory failure in CAD + SARS-CoV-2 patients. Intensified risk assessments comprising monocyte sub- and phenotypes may help to identify patients at risk for respiratory failure., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

Fc gamma receptors (FcγRs) translate antigen recognition by immunoglobulin G (IgG) into various immune responses. A better understanding of this key element of immunity promises novel insights into mechanisms of (auto-/allo-)immune diseases and more rationally designed antibody-based drugs. Glycosylation on both IgG and FcγR impacts their interaction dramatically. Regarding FcγR glycosylation profiling, major analytical challenges are associated with the presence of multiple glycosylation sites in close proximity and large structural heterogeneity. To address these challenges, we developed a straightforward and comprehensive analytical methodology to map FcγRIIIb glycosylation in primary human cells. After neutrophil isolation and immunoprecipitation, glycopeptides containing a single site each were generated by a dual-protease in-gel digestion. The complex mixture was resolved by liquid chromatography-tandem mass spectrometry (LC-MS/MS) providing information on the level of individual donors. In contrast to recently published alternatives for FcγRIIIb, we assessed its site-specific glycosylation in a single LC-MS/MS run and simultaneously determined the donor allotype. Studying FcγRIIIb derived from healthy donor neutrophils, we observed profound differences as compared to the soluble variant and the homologous FcγRIIIa on natural killer cells. This method will allow assessment of differences in FcγRIII glycosylation between individuals, cell types, subcellular locations, and pathophysiological conditions.

A deep elucidation of the mechanisms of action of anti-CD38 monoclonal antibodies (mAbs), such as daratumumab (DARA), is required to identify patients with multiple myeloma (MM) who are more responsive to this treatment. In the present study, an autologous ex vivo approach was established, focussing on the role of the monocytes in the anti CD38-mediated killing of MM cells. In bone marrow (BM) samples from 29 patients with MM, we found that the ratio between monocytes (CD14 + ) and MM cells (CD138 + ) influences the response to DARA. Further, the exposure of the BM samples to DARA is followed by the formation of a CD138 + CD14 + double-positive (DP) population, that quantitatively correlates with the anti-MM cells killing. These effects were dependent on the presence of a CD14 + CD16 + monocyte subset and on high CD16 expression levels. Lastly, the addition of a mAb neutralising the CD47/signal-regulatory protein α (SIRPα) axis was able to increase the killing mediated by DARA. The effects were observed only in coincidence with high CD14 + :CD138 + ratio, with a significant presence of the DP population and were correlated with CD16 expression. In conclusion, the present study underlines the critical role of the CD16 + monocytes in DARA anti-MM killing effects and gives a rationale to test the combination of an anti-CD47 mAb with anti-CD38 mAbs., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

Background: Increased expressions of CD16 on classical monocytes precede their transition to intermediate monocytes. Thus far, the influence of lipids on the expression of CD14 and CD16 on monocyte subsets in coronary atherosclerosis (CA) remains unclear. The aim of this study was to investigate the underlying association between blood lipids and the expression of CD14 and CD16 on monocyte subsets. Methods: This study enrolled 112 healthy controls and 110 CA patients. Monocyte subsets [CD14 ++ CD16 - (classical), CD14 ++ CD16 + (intermediate) and CD14 + CD16 ++ (non-classical)] were analyzed by flow cytometry. Median fluorescent intensity (MFI) was used to evaluate the expression levels of CD14 and CD16 on monocyte subsets. Results: Compared with the control group, the expression of CD16 was significantly increased on all three monocyte subsets in the patient group. Correlation analysis revealed that serum HDL-C was inversely associated with the expression of CD16 on intermediate monocytes after Bonferroni correction in the control group. In addition, a significant decrease in classical monocytes and an increase in intermediate monocytes were detected in patients. In linear regression analysis, intermediate monocytes showed an inverse association with serum HDL-C in the control group. Although CD14 was correlated with serum TC and HDL-C, there was no statistical difference in CD14 expression between the two groups. Conclusion: Low serum HDL-C may induce upregulation of CD16 on classical monocytes, which may in turn lead to the increase of intermediate monocytes in coronary atherosclerosis patients., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Fc γ receptor IIIa/CD16a is an activating cell surface receptor with a well-defined role in natural killer (NK) cell and monocyte effector function. The extracellular domain is decorated with five asparagine (N)-linked glycans; N-glycans at N162 and N45 directly contribute to high-affinity antibody binding and protein stability. N-glycan structures at N162 showed significant donor-dependent variation in a recent study of CD16a isolated from primary human NK cells, but structures at N45 were relatively homogeneous. In this study, we identified variations in N45 glycan structures associated with a polymorphism coding for histidine instead of leucine at position 48 of CD16a from two heterozygous donors. It is known that H48 homozygous individuals suffer from immunodeficiency and recurrent viral infections. A mass spectrometry analysis of protein isolated from the primary natural killer cells of individuals expressing both CD16a L48 and H48 variants demonstrated clear processing differences at N45. CD16a H48 displayed a greater proportion of complex-type N45 glycans compared to the more common L48 allotype with predominantly hybrid N45-glycoforms. Structures at the four other N-glycosylation sites showed minimal differences from data collected on donors expressing only the predominant L48 variant. CD16a H48 purified from a pool of monocytes similarly displayed increased processing at N45. Here, we provide evidence that CD16a processing is affected by the H48 residue in primary NK cells and monocytes from healthy human donors., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aims: Prosthetic joint infection (PJI) remains a major clinical challenge. Neutrophil CD64 index, Fc-gamma receptor 1 (FcγR1), plays an important role in mediating inflammation of bacterial infections and therefore could be a valuable biomarker for PJI. The aim of this study is to compare the neutrophil CD64 index in synovial and blood diagnostic ability with the standard clinical tests for discrimination PJI and aseptic implant failure., Methods: A total of 50 patients undergoing revision hip and knee arthroplasty were enrolled into a prospective study. According to Musculoskeletal Infection Society (MSIS) criteria, 25 patients were classified as infected and 25 as not infected. In all patients, neutrophil CD64 index and percentage of polymorphonuclear neutrophils (PMN%) in synovial fluid, serum CRP, ESR, and serum CD64 index levels were measured preoperatively. Receiver operating characteristic (ROC) curves and the area under the curve (AUC) were analyzed for each biomarker., Results: Serum CD64 index showed no significant difference between the two groups (p = 0.091). Synovial fluid CD64 index and PMN% discriminated good differentiation between groups of PJI and aseptic failure with AUC of 0.946 (95% confidence interval (CI) 0.842 to 0.990) and 0.938 (95% CI 0.832 to 0.987) separately. The optimal threshold value of synovial CD64 index for the diagnosis of PJI was 0.85, with a sensitivity of 92.00%, a specificity of 96.00%, and diagnostic odds ratio (DOR) of 227.11., Conclusion: The present study demonstrates that CD64 index in synovial fluid could be a promising laboratory marker for screening PJI. The cut-off values of 0.85 for synovial CD64 index has the potential to distinguish aseptic failure from PJI. Cite this article: Bone Joint J 2020;102-B(4):463-469.

The present study was designed to investigate the expression of tumor-associated macrophages (TAMs) in gastric cancer and its clinicopathological relationship. In addition, we also aimed to analyze the relationship between helicobacter pylori (HP) infection and TAMs in gastric cancer.The protein expression of CD16 and CD163 in 90 gastric cancer tissues and 30 margin tissues was detected by immunohistochemistry. HP infection was detected in 90 gastric cancer tissues and 30 margin tissues by gram staining and immunohistochemistry.There was no clear correlation between CD16 macrophages and gastric cancer. The density of CD163 macrophages was not correlated with the general condition of tumor patients, but with tumor size, tumor differentiation, lymphatic metastasis, depth of invasion and TNM stage. Additionally, the infection rate of HP in gastric cancer tissues was significantly higher.In summary, TAMs are associated with tumor size, degree of differentiation, depth of invasion, lymph node metastasis and TNM stage, suggesting their critical role in the invasion and metastasis of gastric cancer.

Monocytes and macrophages are major cellular components of the innate immunity that play essential roles in tissue homeostasis. The contribution of different subsets of monocytes/macrophages to periodontal health and disease has not been fully elucidated. Type 2 diabetes mellitus (T2DM) is a risk factor for periodontitis. We hypothesized that the monocyte/macrophage signaling is perturbed in periodontitis-affected sites versus periodontally healthy sites and that this perturbation plays a critical role in the pathogenesis of periodontitis. Pairs of gingival tissue samples (each from a periodontally healthy and a periodontitis-affected site of the same patient) were harvested from 27 periodontitis patients, with and without T2DM. Each sample was processed to form a single-cell suspension, and a flow-cytometry panel was designed and validated to study monocyte and macrophage phenotypes. In separate experiments, the transcriptional changes associated with a pro-inflammatory phenotype were also examined in monocyte/macrophage subsets obtained from peripheral blood of patients with T2DM versus diabetes-free controls. A significantly higher proportion of intermediate (CD14 + CD16 + ) monocytes was observed in periodontitis-affected tissues compared to healthy tissues. These monocytes overexpressed HLA-DR and PDL1 molecules, suggesting their activated inflammatory status. PDL1 increase was specific to intermediate monocytes. The ratio of M1/M2 macrophages was also significantly higher in periodontally affected sites, signifying an imbalance between inflammatory and repair mechanisms. We found a significantly higher expression of PDL1 in overall monocytes and M1 macrophages in periodontitis-affected sites compared to controls. Importantly, we identified a subpopulation of M1 macrophages present in periodontally affected tissues which expressed high levels of CD47, a glycoprotein of the immunoglobulin family that plays a critical role in self-recognition and impairment of phagocytosis. Analysis of the transcriptional landscape of monocytes/macrophages in gingival tissue of T2DM patients with periodontitis revealed a significant disruption in homeostasis toward a proinflammatory phenotype, elevation of pro-inflammatory transcription factors STAT1 and IRF1, and repression of anti-inflammatory JMJD3 in circulating monocytes. Taken together, our results demonstrate disruption of myeloid-derived cell homeostasis in periodontitis, with or without T2DM, and highlight a potentially significant role of these cell types in its pathogenesis. The impact of macrophage and monocyte signaling pathways on the pathobiology of periodontitis should be further evaluated., (Copyright © 2020 Almubarak, Tanagala, Papapanou, Lalla and Momen-Heravi.)

Sepsis is associated with bacterial invasion and inflammation and has a high mortality rate. Previous studies have demonstrated that tripartite motif 59 (TRIM59) was involved in NF-κB signaling and could promote phagocytosis of macrophages, but the role of TRIM59 in sepsis is still unknown. In our study, we found that TRIM59 was downregulated in lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages (BMDMs). In the cecal ligation and puncture (CLP) sepsis mice model, the mortality of Trim59 flox / flox Lyz-Cre ( Trim59 -cKO) mice was higher, the immune cell infiltration and damage of liver and lung were more severe, and bacteria burden was increased. We also found that TRIM59 altered the production of pro-inflammation cytokines, as well as macrophage phagocytosis ability. Further analysis indicated that NF-κB signal pathway and Fcγ receptors might be involved in these regulations. Our study demonstrated for the first time that TRIM59 protects mice from sepsis by regulating inflammation and phagocytosis in macrophages., (Copyright © 2020 Jin, Zhu, Liu, Hou, Tang, Zhu, Tian, Li, Yan and Zhu.)

Animal models suggest postoperative cognitive dysfunction may be caused by brain monocyte influx. To study this in humans, we developed a flow cytometry panel to profile cerebrospinal fluid (CSF) samples collected before and after major noncardiac surgery in 5 patients ≥60 years of age who developed postoperative cognitive dysfunction and 5 matched controls who did not. We detected 12,654 ± 4895 cells/10 mL of CSF sample (mean ± SD). Patients who developed postoperative cognitive dysfunction showed an increased CSF monocyte/lymphocyte ratio and monocyte chemoattractant protein 1 receptor downregulation on CSF monocytes 24 hours after surgery. These pilot data demonstrate that CSF flow cytometry can be used to study mechanisms of postoperative neurocognitive dysfunction.

Background: Despite the clinical significance of red blood cell (RBC) alloantibodies, there are currently no laboratory tests available to predict which patients may be at risk of antibody formation after transfusion exposure. Given their phagocytic and inflammatory functions, we hypothesized that differences in circulating monocytes may play a role in alloimmunization., Study Design and Methods: Forty-two adults with sickle cell disease (SCD) were recruited, with data extracted from the electronic medical record and peripheral blood analyzed by flow cytometry for total monocytes, monocyte subsets (CD14 high/CD16 low+ classical monocytes, CD14 high/CD16 high+ intermediate monocytes, and CD14 intermediate/CD16 high+ non-classical/inflammatory monocytes), and FcγR1 (CD64) expression. Thirteen "non-responder" patients (non-alloimmunized patients with documented RBC transfusion at the study institution) were compared to 20 alloimmunized "responder" patients, who had a total of 44 RBC alloantibodies identified., Results: There were no significant differences in the percentages of total monocytes, monocyte subsets, or measured cytokines between non-responders and responders. However, non-responders had higher CD64 expression on classical monocytes (MFI mean 3424 ± standard deviation 1141) compared to responders (MFI mean 2285 ± 1501), p = 0.029, and on intermediate monocytes (MFI mean 3720 ± 1191) compared to responders (MFI mean 2497 ± 1640), p = 0.033., Conclusions: Monocytes and the inflammatory milieu increasingly are being appreciated to play a role in some complications of SCD. The differences in FcγR1 expression on monocyte subsets noted between responders and non-responders, which cannot be directly explained by the serum cytokines evaluated, warrant further investigation., (© 2019 AABB.)

Monocytes are a heterogeneous cell population distinguished into three subsets with distinctive phenotypic and functional properties: "classical" (CD14++CD16-), "intermediate" (CD14++CD16+), and "nonclassical" (CD14+CD16++). Monocyte subsets play a pivotal role in many inflammatory systemic diseases including atherosclerosis (ATS). Only a low number of studies evaluated monocyte behavior in patients affected by cardiovascular diseases, and data about their role in acute aortic dissection (AAD) are lacking. Thus, the aim of this study was to investigate CD14++CD16-, CD14++CD16+, and CD14+CD16++ cells in patients with Stanford-A AAD and in patients with carotid artery stenosis (CAS). Methods . 20 patients with carotid artery stenosis (CAS group), 17 patients with Stanford-A AAD (AAD group), and 17 subjects with traditional cardiovascular risk factors (RF group) were enrolled. Monocyte subset frequency was determined by flow cytometry. Results . Classical monocytes were significantly increased in the AAD group versus CAS and RF groups, whereas intermediate monocytes were significantly decreased in the AAD group versus CAS and RF groups. Conclusions . Results of this study identify in AAD patients a peculiar monocyte array that can partly explain depletion of T CD4+ lymphocyte subpopulations observed in patients affected by AAD., Competing Interests: The authors have no conflict of interest including specific financial interest and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript.

Understanding the complex immune responses in sepsis is crucial to provide insight into the clinical syndrome. We evaluated the changes in the surface receptors of the cells of innate immunity, neutrophils and monocytes, in patients with sepsis. Since sepsis remains a clinical challenge, we aimed to assess the significance of altered receptor expression in diagnosis and prognosis. Critically ill patients with sepsis (n=31) were investigated for the expression of receptors for IgG heavy chain CD64 and CD16 on neutrophils and CD64 and the lipopolysaccharide receptor CD14 on monocytes by flow cytometry and compared to 23 patients with no sepsis. Patients with sepsis had increased expression of neutrophil CD64. Neutrophil CD64 was specific for discriminating patients with sepsis but showed weak sensitivity. When integrated in a scoring system, neutrophil CD64 in combination with C-reactive protein (CRP) and SOFA score showed a diagnostic accuracy of 0.93 for sepsis and significantly predicted increased mortality risk. While neutrophil CD16 did not discriminate for sepsis, decreased expression was associated with increased mortality risk. In contrast, monocyte CD64 and CD14 expression was unaltered in sepsis and was not associated with mortality risk. Our study demonstrates that unlike monocytes, neutrophil receptor expression is altered in patients with sepsis receiving intensive care. It is promising to apply a combination approach to diagnose sepsis especially in time-limited conditions., (© FEMS 2019.)

Ionizing radiation exposure to the brain is common for patients with a variety of CNS related malignancies. This exposure is known to induce structural and functional alterations to the brain, impacting dendritic complexity, spine density and inflammation. Over time, these changes are associated with cognitive decline. However, many of these impacts are only observable long after irradiation. Extracellular vesicles (EVs) are shed from cells in nearly all known tissues, with roles in many disease pathologies. EVs are becoming an important target for identifying circulating biomarkers. The aim of this study is to identify minimally invasive biomarkers of ionizing radiation damage to the CNS that are predictors of late responses that manifest as persistent cognitive impairments. Using a clinically relevant 9 Gy irradiation paradigm, we exposed mice to cranial (head only) irradiation. Using metabolomic and lipidomic profiling, we analyzed their plasma and plasma-derived EVs two days and two weeks post-exposure to detect systemic signs of damage. We identified significant changes associated with inflammation in EVs. Whole-plasma profiling provided further evidence of systemic injury. These studies are the first to demonstrate that profiling of plasma-derived EVs may be used to study clinically relevant markers of ionizing radiation toxicities to the brain.

We report the production and application of a recombinant IgCw molecule, which is composed of only the constant domains of the heavy (C H ) and light (C L ) chains, lacking a variable (V) domain. We produced IgCw, especially human IgCw-γκ (98 kDa), composed of two human Cγ chains (37 kDa each) and two Cκ chains (12 kDa each), using HEK293F cell culture. We found that the yield of IgCw-γκ protein was ∼20 mg/L, which was comparable to that of full-size IgG; it bound to Fcγ receptor-positive cells with a low background noise on Fcγ receptor-negative cells; and IgCw-γκ can be used as a reference for measurement of Ig concentration. Moreover, Cγ and Cκ chains were easily isolated from IgCw-γκ by a single step of affinity chromatography in the presence of a reducing agent. These results demonstrate that the IgCw molecule has the potential to be used for certain in vitro and in vivo applications as an alternative to an irrelevant isotype control IgG, and to be used a favorable antigen for acquiring isotype-specific antibodies by immunizing animals., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Pneumonia is the leading infectious cause of mortality worldwide and one of the most common lower respiratory tract infections that is contributing significantly to the burden of antibiotic consumption. Due to the complexity of its pathophysiology, it is widely accepted that clinical diagnosis and prognosis are inadequate for the accurate assessment of the severity of the disease. The most challenging task for a physician is the risk stratification of patients with community-acquired pneumonia. Herein, early diagnosis is essential in order to reduce hospitalization and mortality. Procalcitonin and C-reactive protein remain the most widely used biomarkers, while interleukin 6 has been of particular interest in the literature. However, none of them appear to be ideal, and the search for novel biomarkers that will most sufficiently predict the severity and treatment response in pneumonia has lately intensified. Although our insight has significantly increased over the last years, a translational approach with the application of genomics, metabolomics, microbiomics, and proteomics is required to better understand the disease. In this review, we discuss this rapidly evolving area and summarize the application of novel biomarkers that appear to be promising for the accurate diagnosis and risk stratification of pneumonia.

The process of agglutination causes firm cream layers in bovine milk, and a functioning agglutination mechanism is paramount to the quality of non-homogenized milks. The phenomenon is not well-described, but it is believed to occur due to interactions between immunoglobulins (Ig) and milk fat globules. For the first time, this paper demonstrates how the process of agglutination can be visualized using confocal laser scanning microscopy, rhodamine red and a fluoresceinisothiocynat-conjugated immunoglobulin M antibody. The method was used to illustrate the effect on agglutination of storage temperature and pasteurization temperature. Storage at 5 °C resulted in clearly visible agglutination which, however, was markedly reduced at 15 °C. Increasing storage temperature to 20 or 37 °C cancelled any detectable interaction between IgM and milk fat globules, whereby the occurrence of cold agglutination was documented. Increasing 20 s pasteurization temperatures from 69 °C to 71 °C and further to 73 °C lead to progressively higher inactivation of IgM and, hence, reduction of agglutination. Furthermore, 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that changes in storage temperature caused a redistribution of Ig-related proteins in milk fat globule membrane isolates. Poly-immunoglobulin G receptor was present in milk fat globule preparations stored at cold (4 °C) conditions, but absent at storage at higher temperature (25 °C). The findings provide valuable knowledge to dairy producers of non-homogenized milk in deciding the right pasteurization temperature to retain the crucial agglutination mechanism.

Neutrophils are detected in inflamed colon in Crohn's disease (CD). However, whether the frequency and/or activation of circulating or gut tissue neutrophils correlate with endoscopic severity remains to be investigated. A cohort of 73 CD patients was prospectively enrolled according to endoscopic severity and treatment history. Individuals with active disease were stratified using the Montreal classification. Harvey-Bradshaw Index (HBI) and Simple Endoscopic Score for Crohn's Disease (SES-CD) were performed at the time of ileocolonoscopy. Frequency of neutrophils and their expression of CD66b and CD64 were assessed in paired blood and colonic biopsies using flow cytometry. The percentage of neutrophils increased in inflamed colon and correlated with SES-CD in the entire cohort of patients examined, as well as in the subgroup with inflammatory (B1) active disease. SES-CD further correlated with neutrophil CD66b expression in mucosa but not blood and, conversely, with neutrophil CD64 expression in blood but not mucosa. However, the evaluation of neutrophil activation in mucosa when compared to blood reflected disease activity more clearly. Finally, a neutrophil activation power index (CD66b in mucosa X CD64 in blood) that correlated with SES-CD discriminated between patients with mild and severe disease. In conclusion, the frequency and activation of colonic neutrophils correlated with SES-CD, highlighting that mucosal neutrophils are associated with disease severity in CD., (© 2018 British Society for Immunology.)

Safety and efficacy of therapeutic antibodies are often dependent on their interaction with Fc receptors for IgG (FcγRs). The Göttingen minipig represents a valuable species for biomedical research but its use in preclinical studies with therapeutic antibodies is hampered by the lack of knowledge about the porcine FcγRs. Genome analysis and sequencing now enabled the localization of the previously described FcγRIIIa in the orthologous location to human FCGR3A. In addition, we identified nearby the gene coding for the hitherto undescribed putative porcine FcγRIIa. The 1'241 bp long FCGR2A cDNA translates to a 274aa transmembrane protein containing an extracellular region with high similarity to human and cattle FcγRIIa. Like in cattle, the intracellular part does not contain an immunoreceptor tyrosine-based activation motif (ITAM) as in human FcγRIIa. Flow cytometry of the whole blood and single-cell RNA sequencing of peripheral blood mononuclear cells (PBMCs) of Göttingen minipigs revealed the expression profile of all porcine FcγRs which is compared to human and mouse. The new FcγRIIa is mainly expressed on platelets making the minipig a good model to study IgG-mediated platelet activation and aggregation. In contrast to humans, minipig blood monocytes were found to express inhibitory FcγRIIb that could lead to the underestimation of FcγR-mediated effects of monocytes observed in minipig studies with therapeutic antibodies.

Background: The malaria causing parasite Plasmodium subverts host immune responses by several strategies including the modulation of dendritic cells (DCs)., Methods: In this study, we show that Plasmodium falciparum skewed CD16+ DC cytokine responses towards interleukin (IL)-10 production in vitro, distinct to the cytokine profile induced by Toll-like receptor ligation. To determine CD16+ DC responsiveness in vivo, we assessed their function after induced P falciparum infection in malaria-naive volunteers., Results: CD16+ DCs underwent distinctive activation, with increased expression of maturation markers human leukocyte antigen (HLA)-DR and CD86, enhanced tumor necrosis factor (TNF) production, and coproduction of TNF/IL-10. In vitro restimulation with P falciparum further increased IL-10 production. In contrast, during naturally acquired malaria episode, CD16+ DCs showed diminished maturation, suggesting increased parasite burden and previous exposure influence DC subset function., Conclusions: These findings identify CD16+ DCs as the only DC subset activated during primary blood-stage human Plasmodium infection. As dual cytokine producers, CD16+ DCs contribute to inflammatory as well as regulatory innate immune processes.

The mechanism of the efficacy of Intravenous immunoglobulins (IVIG) in autoimmune and inflammatory diseases is not well understood. This study aimed at understanding mechanisms of IVIG-mediated suppression of effector cell activities of peripheral blood mononuclear cells (PBMC) in antibody-dependent cellular cytotoxicity (ADCC). We were particularly interested in CD56 dim NK cells, the main ADCC effector cells in PBMC. Exposure of PBMC to IVIG for at least 48 h induced a caspase-3-dependent apoptotic cell death of CD56 dim NK cells without affecting CD56 bright NK cells. Induction of apoptosis in CD56 dim NK cells and concomitant suppression of ADCC effector activities of PBMC was associated with the monomer fraction of IVIG. Moreover, it was independent of IgG sialyation, did not depend on engagement of FcγRIII and could not be mimicked by IVIG (Fab') 2 or IVIG Fc preparations. The described effect could contribute to the reduction of peripheral NK cells observed during IVIG therapy in patients., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective- Three distinct human monocyte subsets have been identified based on the surface marker expression of CD14 and CD16. We hypothesized that monocytes were likely more heterogeneous in composition. Approach and Results- We used the high dimensionality of mass cytometry together with the FlowSOM clustering algorithm to accurately identify and define monocyte subsets in blood of healthy human subjects and those with coronary artery disease (CAD). To study the behavior and functionality of the newly defined monocyte subsets, we performed RNA sequencing, transwell migration, and efferocytosis assays. Here, we identify 8 human monocyte subsets based on their surface marker phenotype. We found that 3 of these subsets fall within the CD16 + nonclassical monocyte population and 4 subsets belong to the CD14 + classical monocytes, illustrating significant monocyte heterogeneity in humans. As nonclassical monocytes are important in modulating atherosclerosis in mice, we studied the functions of our 3 newly identified nonclassical monocytes in subjects with CAD. We found a marked expansion of a Slan + CXCR6 + nonclassical monocyte subset in CAD subjects, which was positively correlated with CAD severity. This nonclassical subset can migrate towards CXCL16 and shows an increased efferocytosis capacity, indicating it may play an atheroprotective role. Conclusions- Our data demonstrate that human nonclassical monocytes are a heterogeneous population, existing of several subsets with functional differences. These subsets have changed frequencies in the setting of severe CAD. Understanding how these newly identified subsets modulate CAD will be important for CAD-based therapies that target myeloid cells.

Objective: To evaluate the utility of the granulocyte CD64 index as a marker of infection in patients with postoperative fever., Methods: Prospective observational study of a cohort of patients with postoperative fever (2nd-21st day after the intervention) collected during 14 months. Obtaining blood samples during the first 24 hours after the febrile peak to determine the CD64 index (ratio of fluorescence intensity, measured, in the granulocytes of the patient with respect to healthy controls), procalcitonin and C-reactive protein (CRP)., Results: During the study period, 50 patients were included, 28 patients (56%) with infection and 22 patients (44%) without evidence of infection. The PCR, procalcitonin and the CD64 index showed significantly higher values in the group of patients who suffered infection. The CD64 index showed a sensitivity of 88.9%, with a specificity of 65.2%. The positive predictive value (PPV) was 75% and the negative predictive value (NPV) was 83.3%, with an area under the curve (AUC) of 0.805 (95% CI 0.68-0.93). Procalcitonin presented a sensitivity of 53.9% and specificity of 86.4%, with NPV and PPV of 82.4% and 61.3% respectively, with AUC of 0.752 (95% CI 0.61-0.89). Regarding the PCR, it showed a sensitivity of 100%, with specificity of 4.4% with an area under the curve of 0.676 (95% CI 0.52-0.83)., Conclusions: The quantification of the CD64 index in patients who develop fever in the early postoperative period is useful to distinguish post-surgical inflammatory phenomena from episodes of established infection., (©The Author 2018. Published by Sociedad Española de Quimioterapia. This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)(https://creativecommons.org/licenses/by-nc/4.0/).)

Background: Recurrent miscarriage is considered as one of the main problems in women's reproductive health. The aim of the present study was to evaluate the natural killer cells (NK cells) and cytokines in unexplained recurrent miscarriage and fertile women., Methods: In this case-control study, 40 women with unexplained recurrent miscarriage were assigned to the case group and 40 fertile women were assigned to the control group. NK cell subsets (CD56 + CD16 + /CD56 + CD16 - ) and cytokines (IL-2/IL-12) levels in the peripheral blood (PB) were used for assessing immunologic problems. The percentage of peripheral blood NK cells (CD56 dim/bright ) was identified by flow cytometry., Results: The obtained results showed a significant difference in CD56 + CD16 + and CD56 + CD16 - between the two groups. Also, there was no significant difference in the IL-2 and IL-12 between the two groups. A cut-off value of ≥5.25% (p < 0.001) and ≥3.4% (p < 0.015) for the increased percentage of CD56 + CD16 + and CD56 + CD16 - cells in the PB become predictive of recurrent miscarriage., Conclusion: Increased NK cells in the PB of women with recurrent miscarriage strongly establish prospective researches to recognize the predictive value of these parameters in the evaluation of patients with recurrent miscarriage., (Copyright © 2018. Published by Elsevier Taiwan LLC.)

HIV infection of the CNS causes neuroinflammation and damage that contributes to the development of HIV-associated neurocognitive disorders (HAND) in greater than 50% of HIV-infected individuals, despite antiretroviral therapy (ART). Opioid abuse is a major risk factor for HIV infection. It has been shown that opioids can contribute to increased HIV CNS pathogenesis, in part, by modulating the function of immune cells. HIV enters the CNS within two weeks after peripheral infection by transmigration of infected monocytes across the blood brain barrier (BBB). CD14 + CD16 + monocytes are a mature subpopulation that is increased in number in the peripheral blood of HIV-infected people. Mature monocytes can be productively infected with HIV, and they transmigrate preferentially across the BBB in response to CCL2, a chemokine elevated in the CNS and CSF of HIV-infected people even with ART. Buprenorphine, an opioid derivate, is an opioid replacement therapy for heroin addiction. It is a partial agonist of μ-opioid receptor and full antagonist of κ-opioid receptor. The effects of buprenorphine on CCL2-mediated CD14 + CD16 + monocytes transmigration across the BBB, a critical mechanism that promotes neuroinflammation and HAND, have not been characterized. We showed for the first time that buprenorphine decreases several steps of CCL2-mediated human mature monocyte transmigration. We propose that buprenorphine treatment in the context of HIV infection could serve a dual purpose, to treat opioid addiction and also to reduce neuroinflammation. Additionally, buprenorphine may be used as a treatment for HAND not only in the context of opioid abuse., (©2018 Society for Leukocyte Biology.)

Infection with Mycobacterium tuberculosis (Mtb) is characterized by an inflammatory response resulting in the formation of granulomas. These tight aggregates of immune cells play an important role in bacterial containment and in the eventual outcome of infection. Monocytes are a major component of the early immune response to Mtb and contribute to the cellular matrix of the newly forming granuloma. Therefore, defining which monocyte subset is the target of mycobacterial infection is critical. Here, we describe a flow-cytometry-based assay to analyze infectivity in vitro of monocyte subsets by Mycobacterium bovis BCG before granuloma formation. We identified CD14+CD16- monocytes as the main target of infection in peripheral blood mononuclear cells from six healthy donors. CD14+CD16+ monocytes displayed the lowest infection rates and remained uninfected in some donors. We found that a longer infection time resulted in an increase of the percentage of monocytes infected and of the number of granulomas produced. We did not observe changes in monocyte cell death or subset expansion upon infection. Future experiments with our in vitro method could help define Mtb infectivity of monocyte subsets. Our study provides a platform to investigate how early infection of different monocyte subsets may alter granuloma formation and outcomes of Mtb infection.

The survival rate of anaplastic thyroid cancer (ATC) remains about 7% to 14%. The natural killer (NK) cells are a critical component of antitumor immunity, and their composition and function in thyroid cancer patients are investigated in this study. In healthy controls and early stage thyroid cancer patients, >90% of circulating NK cells were CD56 lo CD16 hi and fewer than 10% were CD56 hi CD16 hi/lo . However, the frequency of the CD56 hi CD16 hi/lo NK subset was significantly higher in more advanced thyroid cancer patients and further increased in ATC patients. Two members of the inhibitory KIR family, CD158a and CD158b, was significantly higher in CD56 hi CD16 hi/lo NK cells than in CD56 lo CD16 hi NK cells, while NKG2D, an activator of NK cells, was significantly lower in CD56 hi CD16 hi/lo NK cells than in CD56 lo CD16 hi NK cells. We also found that the CD56 hi CD16 hi/lo NK cells presented higher PD-1, higher Tim-3, and lower cytotoxicity against the human ATC cell line CAL-62, than the CD56 lo CD16 hi NK cells. The expression of exhaustion markers and reduction in cytotoxicity was further exacerbated in more advanced thyroid cancer patients and in ATC patients. Interestingly, PD-1 and Tim-3 blockade was effective at reinvigorating both the more impaired CD56 hi CD16 hi/lo NK cells and the less impaired CD56 lo CD16 hi NK cells from ATC patients. Together, our study identified a dysfunction of NK cells in more advanced thyroid cancer patients and ATC patients, and presented actionable targets for future development of immunotherapies in thyroid cancers., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Chronic infection with Trypanosoma cruzi leads to a constant stimulation of the host immune system. Monocytes, which are recruited in response to inflammatory signals, are divided into classical CD14hiCD16-, non-classical CD14loCD16+ and intermediate CD14hiCD16+ subsets. In this study, we evaluated the frequencies of monocyte subsets in the different clinical stages of chronic Chagas disease in comparison with the monocyte profile of seronegative heart failure subjects and seronegative healthy controls. The effect of the anti-parasite drug therapy benznidazole on monocyte subsets was also explored., Methodology/principal Findings: The frequencies of the different monocyte subsets and their phenotypes were measured by flow cytometry. Trypanosoma cruzi-specific antibodies were quantified by conventional serological tests. T. cruzi-infected subjects with mild or no signs of cardiac disease and patients suffering from dilated cardiomyopathy unrelated to T. cruzi infection showed increased levels of non-classical CD14loCD16+ monocytes compared with healthy controls. In contrast, the monocyte profile in T. cruzi-infected subjects with severe cardiomyopathy was skewed towards the classical and intermediate subsets. After benznidazole treatment, non-classical monocytes CD14loCD16+ decreased while classical monocytes CD14hiCD16-increased., Conclusions/significance: The different clinical stages of chronic Chagas disease display distinct monocyte profiles that are restored after anti-parasite drug therapy. T. cruzi-infected subjects with severe cardiac disease displayed a profile of monocytes subsets suggestive of a more pronounced inflammatory environment compared with subjects suffering from heart failure not related to T. cruzi infection, supporting that parasite persistence might also alter cell components of the innate immune system., Competing Interests: SAL and MCA are members of The National Scientific and Technical Research Council, Argentina. The authors have declared that no competing interests exist.

A recent study has pointed out to CD32a as a potential biomarker of HIV-persistent CD4 cells. We have characterized the level and phenotype of CD32+ cells contained in different subsets of CD4 T-cells and its potential correlation with level of total HIV-DNA in thirty HIV patients (10 typical progressors naïve for cART, 10 cART-suppressed patients, and 10 elite controllers). Total HIV-DNA was quantified in different subsets of CD4 T-cells: Trm and pTfh cells. Level and immunephenotype of CD32+ cells were analyzed in these same subsets by flow cytometry. CD32 expression in Trm and pTfh subsets was similar in the different groups, and there was no significant correlation between the level of total HIV-DNA and the level of CD32 expression in these subsets. However, total HIV-DNA level was correlated with expression of CD127 (rho = -0.46, p = 0.043) and of CCR6 (rho = -0.418, p = 0.027) on CD32+ cells. Our results do not support CD32 as a biomarker of total HIV-DNA content. However, analyzing the expression of certain markers by CD32+ cells could improve the utility of this marker in the clinical setting, prompting the necessity of further studies to both validate our results and to explore the potential utility of certain markers expressed by CD32+ cells.

A recent study conducted in blood has proposed CD32 as the marker identifying the "elusive" HIV reservoir. We have investigated the distribution of CD32 + CD4 T cells in blood and lymph nodes (LNs) of HIV-1-uninfected subjects and viremic untreated and long-term-treated HIV-1-infected individuals and their relationship with PD-1 + CD4 T cells. The frequency of CD32 + CD4 T cells was increased in viremic compared to treated individuals in LNs, and a large proportion (up to 50%) of CD32 + cells coexpressed PD-1 and were enriched within T follicular helper (Tfh) cells. We next investigated the role of LN CD32 + CD4 T cells in the HIV reservoir. Total HIV DNA was enriched in CD32 + and PD-1 + CD4 T cells compared to CD32 - and PD-1 - cells in both viremic and treated individuals, but there was no difference between CD32 + and PD-1 + cells. There was no enrichment of latently infected cells with inducible HIV-1 in CD32 + versus PD-1 + cells in antiretroviral therapy (ART)-treated individuals. HIV-1 transcription was then analyzed in LN memory CD4 T cell populations sorted on the basis of CD32 and PD-1 expression. CD32 + PD-1 + CD4 T cells were significantly enriched in cell-associated HIV RNA compared to CD32 - PD-1 - (averages of 5.2-fold in treated individuals and 86.6-fold in viremics), CD32 + PD-1 - (2.2-fold in treated individuals and 4.3-fold in viremics), and CD32 - PD-1 + (2.2-fold in ART-treated individuals and 4.6-fold in viremics) cell populations. Similar levels of HIV-1 transcription were found in CD32 + PD-1 - and CD32 - PD-1 + CD4 T cells. Interestingly, the proportion of CD32 + and PD-1 + CD4 T cells negatively correlated with CD4 T cell counts and length of therapy. Therefore, the expression of CD32 identifies, independently of PD-1, a CD4 T cell population with persistent HIV-1 transcription and coexpression of CD32 and PD-1, the CD4 T cell population with the highest levels of HIV-1 transcription in both viremic and treated individuals. IMPORTANCE The existence of long-lived latently infected resting memory CD4 T cells represents a major obstacle to the eradication of HIV infection. Identifying cell markers defining latently infected cells containing replication-competent virus is important in order to determine the mechanisms of HIV persistence and to develop novel therapeutic strategies to cure HIV infection. We provide evidence that PD-1 and CD32 may have a complementary role in better defining CD4 T cell populations infected with HIV-1. Furthermore, CD4 T cells coexpressing CD32 and PD-1 identify a CD4 T cell population with high levels of persistent HIV-1 transcription., (Copyright © 2018 Noto et al.)

Objective: To explore the trend of CD64 + neutrophils being present in the peripheral blood of patients with burns and verification in vitro., Method: We isolated and purified CD64 + neutrophils from healthy people, stimulated cells with inactivated Staphylococcus aureus in vitro to make them proliferate and analyzed them with Modfit 2 analysis software. Patients with burns were divided into two groups, depending on whether there was a complicated infection or not, and 35 healthy participants were enrolled as blank group. WBC and CD64 + neutrophil counts were detected and then analyzed the specificity and sensitivity according to the ROC curve., Results: The proliferation index of the CD64 + cells in the experimental wells was significantly higher than that in the control wells (P < .05). The CD64 + cell counts in the peripheral blood of patients in the infection group were significantly higher than patients without infection, but also higher than the healthy people (P < .05). The sensitivity and specificity of CD64 + cellular diagnosis for burn infection were 94.2% and 76.8%, respectively, according to the ROC curve, which were significantly better than those of the WBCs., Conclusion: CD64 + cells in the peripheral blood of patients with burns complicated by infection increased significantly, and thus, it can be regarded as an early diagnostic indicator of burns with a concurrent infection., (© 2018 John Wiley & Sons Ltd.)

Cutaneous leishmaniasis (CL) treatment is based on therapy with Glucantime ® , yet, there are few laboratory methods to monitor its success. In this study, ex vivo and in vitro evaluations of peripheral blood monocytes were performed in a longitudinal study to characterize the impact of Glucantime ® on overall phenotypic/functional features of these cells from CL patients to identify predictive biomarkers for post-therapeutic monitoring by flow cytometry. The ex vivo evaluation from CL patients demonstrated a modulatory profile before treatment, with a decrease in TLR-2, FcγRII, HLA-DR, CD86, IFN-γR, TNF, IL-12, NO, and an increase in FcγRIII and IL-10R. Conversely, treatment changes some of these biomarker expressions by decreasing FcγRIII and IL-10R and increasing IFN-γR, IL-12 and NO. Moreover, an in vitro analysis of these patients showed a reduced phagocytic capacity of Leishmania braziliensis and higher levels of IL-10 and TGF-β modulating functional profile. Regardless of the compromised L. braziliensis phagocytic capacity, treatment re-established the production of IL-12, IL-10, TGF-β and NO at the basal level. Notably, monocytes from patients with early cicatrization showed enhanced FcγRI and FcγRII expressions and reduced IL-10, which was further corroborated by a baseline fold change analysis. Finally, the logistic regression model emphasized the performance of FcγRI, FcγRII and IL-10 as robust predictive biomarkers for post-therapeutic cicatrization during cutaneous leishmaniasis., (© 2018 John Wiley & Sons Ltd.)