Microbiological characterization of biofilm from different immobilization structures used in submersed aerobic biofilters in domestic effluent treatment at the city of Joinville, Brazil.

The objective of this work was to develop a polymeric structure for a biofiltration unit of domestic effluents through microbiological immobilization, capable of promoting the efficient removal of pollutants, meeting local/national Brazilian standards and/or legislation while providing low environmental impact on their production. Four different structures were tested, namely, polypropylene casings without filling material (TF1); polypropylene casings filled with expanded polystyrene grains (TF2); polypropylene casings, filled with polyurethane foam (TF3); and polypropylene casings, filled with polyvinyl chloride pellets (TF4). A flow of 0.216 m3 d-1 was applied to the system, and the biofilters operated in sequential batches with a hydraulic retention time of 6 h. The efficiency potential of the four immobilization structures was verified regarding biochemical and chemical oxygen demand, total ammoniacal nitrogen and total phosphorus. Microbiological analysis of the formed biofilm, performed with the 16S library sequencing method, with amplification of the 16S rRNA V3 and V3-V4 genomic regions, showed a high diversity of microbiological colonization in the four immobilization structures, with better results and consequently greater community stability in TF2. It is recommended using the filter bed made up of unfilled casings, followed by the one filled with expanded polystyrene grains.

Immune cell kinetics and antibody response in COVID-19 patients with low-count monoclonal B-cell lymphocytosis.

Low-count monoclonal B-cell lymphocytosis (MBLlo ) has been associated with an underlying immunodeficiency and has recently emerged as a new risk factor for severe COVID-19. Here, we investigated the kinetics of immune cell and antibody responses in blood during COVID-19 of MBLlo versus non-MBL patients. For this study, we analyzed the kinetics of immune cells in blood of 336 COVID-19 patients (74 MBLlo and 262 non-MBL), who had not been vaccinated against SARS-CoV-2, over a period of 43 weeks since the onset of infection, using high-sensitivity flow cytometry. Plasma levels of anti-SARS-CoV-2 antibodies were measured in parallel by ELISA. Overall, early after the onset of symptoms, MBLlo COVID-19 patients showed increased neutrophil, monocyte, and particularly, plasma cell (PC) counts, whereas eosinophil, dendritic cell, basophil, and lymphocyte counts were markedly decreased in blood of a variable percentage of samples, and with a tendency toward normal levels from week +5 of infection onward. Compared with non-MBL patients, MBLlo COVID-19 patients presented higher neutrophil counts, together with decreased pre-GC B-cell, dendritic cell, and innate-like T-cell counts. Higher PC levels, together with a delayed PC peak and greater plasma levels of anti-SARS-CoV-2-specific antibodies (at week +2 to week +4) were also observed in MBLlo patients. In summary, MBLlo COVID-19 patients share immune profiles previously described for patients with severe SARS-CoV-2 infection, associated with a delayed but more pronounced PC and antibody humoral response once compared with non-MBL patients.

Development of a standardized and validated flow cytometry approach for monitoring of innate myeloid immune cells in human blood.

Innate myeloid cell (IMC) populations form an essential part of innate immunity. Flow cytometric (FCM) monitoring of IMCs in peripheral blood (PB) has great clinical potential for disease monitoring due to their role in maintenance of tissue homeostasis and ability to sense micro-environmental changes, such as inflammatory processes and tissue damage. However, the lack of standardized and validated approaches has hampered broad clinical implementation. For accurate identification and separation of IMC populations, 62 antibodies against 44 different proteins were evaluated. In multiple rounds of EuroFlow-based design-testing-evaluation-redesign, finally 16 antibodies were selected for their non-redundancy and separation power. Accordingly, two antibody combinations were designed for fast, sensitive, and reproducible FCM monitoring of IMC populations in PB in clinical settings (11-color; 13 antibodies) and translational research (14-color; 16 antibodies). Performance of pre-analytical and analytical variables among different instruments, together with optimized post-analytical data analysis and reference values were assessed. Overall, 265 blood samples were used for design and validation of the antibody combinations and in vitro functional assays, as well as for assessing the impact of sample preparation procedures and conditions. The two (11- and 14-color) antibody combinations allowed for robust and sensitive detection of 19 and 23 IMC populations, respectively. Highly reproducible identification and enumeration of IMC populations was achieved, independently of anticoagulant, type of FCM instrument and center, particularly when database/software-guided automated (vs. manual "expert-based") gating was used. Whereas no significant changes were observed in identification of IMC populations for up to 24h delayed sample processing, a significant impact was observed in their absolute counts after >12h delay. Therefore, accurate identification and quantitation of IMC populations requires sample processing on the same day. Significantly different counts were observed in PB for multiple IMC populations according to age and sex. Consequently, PB samples from 116 healthy donors (8-69 years) were used for collecting age and sex related reference values for all IMC populations. In summary, the two antibody combinations and FCM approach allow for rapid, standardized, automated and reproducible identification of 19 and 23 IMC populations in PB, suited for monitoring of innate immune responses in clinical and translational research settings.

Altered innate immune profile in blood of systemic mastocytosis patients.

Background: Mast cells (MC) from systemic mastocytosis (SM) patients release MC mediators that lead to an altered microenvironment with potential consequences on innate immune cells, such as monocytes and dendritic cells (DC). Here we investigated the distribution and functional behaviour of different populations of blood monocytes and DC among distinct diagnostic subtypes of SM. Methods: Overall, we studied 115 SM patients - 45 bone marrow mastocytosis (BMM), 61 indolent SM (ISM), 9 aggressive SM (ASM)- and 32 healthy donors (HD). Spontaneous and in vitro-stimulated cytokine production by blood monocytes, and their plasma levels, together with the distribution of different subsets of blood monocytes and DCs, were investigated. Results: SM patients showed increased plasma levels and spontaneous production by blood monocytes of IL1ß, IL6, IL8, TNFα and IL10, associated with an exhausted ability of LPS + IFNγ-stimulated blood monocytes to produce IL1ß and TGFß. SM (particularly ISM) patients also showed decreased counts of total monocytes, at the expense of intermediate monocytes and non-classical monocytes. Interestingly, while ISM and ASM patients had decreased numbers of plasmacytoid DC and myeloid DC (and their major subsets) in blood, an expansion of AXL+ DC was specifically encountered in BMM cases. Conclusion: These results demonstrate an altered distribution of blood monocytes and DC subsets in SM associated with constitutive activation of functionally impaired blood monocytes and increased plasma levels of a wide variety of inflammatory cytokines, reflecting broad activation of the innate immune response in mastocytosis.

Monocyte Subsets and Serum Inflammatory and Bone-Associated Markers in Monoclonal Gammopathy of Undetermined Significance and Multiple Myeloma.

BACKGROUND: Monocyte/macrophages have been shown to be altered in monoclonal gammopathy of undetermined significance (MGUS), smoldering (SMM) and active multiple myeloma (MM), with an impact on the disruption of the homeostasis of the normal bone marrow (BM) microenvironment. METHODS: We investigated the distribution of different subsets of monocytes (Mo) in blood and BM of newly-diagnosed untreated MGUS (n = 23), SMM (n = 14) and MM (n = 99) patients vs. healthy donors (HD; n = 107), in parallel to a large panel of cytokines and bone-associated serum biomarkers. RESULTS: Our results showed normal production of monocyte precursors and classical Mo (cMo) in MGUS, while decreased in SMM and MM (p ≤ 0.02), in association with lower blood counts of recently-produced CD62L+ cMo in SMM (p = 0.004) and of all subsets of (CD62L+, CD62L- and FcεRI+) cMo in MM (p ≤ 0.02). In contrast, intermediate and end-stage non-classical Mo were increased in BM of MGUS (p ≤ 0.03), SMM (p ≤ 0.03) and MM (p ≤ 0.002), while normal (MGUS and SMM) or decreased (MM; p = 0.01) in blood. In parallel, increased serum levels of interleukin (IL)1ß were observed in MGUS (p = 0.007) and SMM (p = 0.01), higher concentrations of serum IL8 were found in SMM (p = 0.01) and MM (p = 0.002), and higher serum IL6 (p = 0.002), RANKL (p = 0.01) and bone alkaline phosphatase (BALP) levels (p = 0.01) with decreased counts of FcεRI+ cMo, were restricted to MM presenting with osteolytic lesions. This translated into three distinct immune/bone profiles: (1) normal (typical of HD and most MGUS cases); (2) senescent-like (increased IL1ß and/or IL8, found in a minority of MGUS, most SMM and few MM cases with no bone lesions); and (3) pro-inflammatory-high serum IL6, RANKL and BALP with significantly (p = 0.01) decreased blood counts of immunomodulatory FcεRI+ cMo-, typical of MM presenting with bone lesions. CONCLUSIONS: These results provide new insight into the pathogenesis of plasma cell neoplasms and the potential role of FcεRI+ cMo in normal bone homeostasis.

Monocytes carrying GFAP detect glioma, brain metastasis and ischaemic stroke, and predict glioblastoma survival.

Diagnosis and monitoring of primary brain tumours, brain metastasis and acute ischaemic stroke all require invasive, burdensome and costly diagnostics, frequently lacking adequate sensitivity, particularly during disease monitoring. Monocytes are known to migrate to damaged tissues, where they act as tissue macrophages, continuously scavenging, phagocytizing and digesting apoptotic cells and other tissue debris. We hypothesize that upon completion of their tissue-cleaning task, these tissue macrophages might migrate via the lymph system to the bloodstream, where they can be detected and evaluated for their phagolysosomal contents. We discovered a blood monocyte subpopulation carrying the brain-specific glial fibrillary acidic protein in glioma patients and in patients with brain metastasis and evaluated the diagnostic potential of this finding. Blood samples were collected in a cross-sectional study before or during surgery from adult patients with brain lesions suspected of glioma. Together with blood samples from healthy controls, these samples were flowing cytometrically evaluated for intracellular glial fibrillary acidic protein in monocyte subsets. Acute ischaemic stroke patients were tested at multiple time points after onset to evaluate the presence of glial fibrillary acidic protein-carrying monocytes in other forms of brain tissue damage. Clinical data were collected retrospectively. High-grade gliomas (N = 145), brain metastasis (N = 21) and large stroke patients (>100 cm3) (N = 3 versus 6; multiple time points) had significantly increased frequencies of glial fibrillary acidic protein+CD16+ monocytes compared to healthy controls. Based on both a training and validation set, a cut-off value of 0.6% glial fibrillary acidic protein+CD16+ monocytes was established, with 81% sensitivity (95% CI 75-87%) and 85% specificity (95% CI 80-90%) for brain lesion detection. Acute ischaemic strokes of >100 cm3 reached >0.6% of glial fibrillary acidic protein+CD16+ monocytes within the first 2-8 h after hospitalization and subsided within 48 h. Glioblastoma patients with >20% glial fibrillary acidic protein+CD16+ non-classical monocytes had a significantly shorter median overall survival (8.1 versus 12.1 months). Our results and the available literature, support the hypothesis of a tissue-origin of these glial fibrillary acidic protein-carrying monocytes. Blood monocytes carrying glial fibrillary acidic protein have a high sensitivity and specificity for the detection of brain lesions and for glioblastoma patients with a decreased overall survival. Furthermore, their very rapid response to acute tissue damage identifies large areas of ischaemic tissue damage within 8 h after an ischaemic event. These studies are the first to report the clinical applicability for brain tissue damage detection through a minimally invasive diagnostic method, based on blood monocytes and not serum markers, with direct consequences for disease monitoring in future (therapeutic) studies and clinical decision making in glioma and acute ischaemic stroke patients.

Atomistic Modelling of Size-Dependent Mechanical Properties and Fracture of Pristine and Defective Cove-Edged Graphene Nanoribbons.

Cove-edged graphene nanoribbons (CGNR) are a class of nanoribbons with asymmetric edges composed of alternating hexagons and have remarkable electronic properties. Although CGNRs have attractive size-dependent electronic properties their mechanical properties have not been well understood. In practical applications, the mechanical properties such as tensile strength, ductility and fracture toughness play an important role, especially during device fabrication and operation. This work aims to fill a gap in the understanding of the mechanical behaviour of CGNRs by studying the edge and size effects on the mechanical response by using molecular dynamic simulations. Pristine graphene structures are rarely found in applications. Therefore, this study also examines the effects of topological defects on the mechanical behaviour of CGNR. Ductility and fracture patterns of CGNR with divacancy and topological defects are studied. The results reveal that the CGNR become stronger and slightly more ductile as the width increases in contrast to normal zigzag GNR. Furthermore, the mechanical response of defective CGNRs show complex dependency on the defect configuration and distribution, while the direction of the fracture propagation has a complex dependency on the defect configuration and position. The results also confirm the possibility of topological design of graphene to tailor properties through the manipulation of defect types, orientation, and density and defect networks.

Age Distribution of Multiple Functionally Relevant Subsets of CD4+ T Cells in Human Blood Using a Standardized and Validated 14-Color EuroFlow Immune Monitoring Tube.

CD4+ T cells comprise multiple functionally distinct cell populations that play a key role in immunity. Despite blood monitoring of CD4+ T-cell subsets is of potential clinical utility, no standardized and validated approaches have been proposed so far. The aim of this study was to design and validate a single 14-color antibody combination for sensitive and reproducible flow cytometry monitoring of CD4+ T-cell populations in human blood to establish normal age-related reference values and evaluate the presence of potentially altered profiles in three distinct disease models-monoclonal B-cell lymphocytosis (MBL), systemic mastocytosis (SM), and common variable immunodeficiency (CVID). Overall, 145 blood samples from healthy donors were used to design and validate a 14-color antibody combination based on extensive reagent testing in multiple cycles of design-testing-evaluation-redesign, combined with in vitro functional studies, gene expression profiling, and multicentric evaluation of manual vs. automated gating. Fifteen cord blood and 98 blood samples from healthy donors (aged 0-89 years) were used to establish reference values, and another 25 blood samples were evaluated for detecting potentially altered CD4 T-cell subset profiles in MBL (n = 8), SM (n = 7), and CVID (n = 10). The 14-color tube can identify ≥89 different CD4+ T-cell populations in blood, as validated with high multicenter reproducibility, particularly when software-guided automated (vs. manual expert-based) gating was used. Furthermore, age-related reference values were established, which reflect different kinetics for distinct subsets: progressive increase of naïve T cells, T-helper (Th)1, Th17, follicular helper T (TFH) cells, and regulatory T cells (Tregs) from birth until 2 years, followed by a decrease of naïve T cells, Th2, and Tregs in older children and a subsequent increase in multiple Th-cell subsets toward late adulthood. Altered and unique CD4+ T-cell subset profiles were detected in two of the three disease models evaluated (SM and CVID). In summary, the EuroFlow immune monitoring TCD4 tube allows fast, automated, and reproducible identification of ≥89 subsets of CD4+ blood T cells, with different kinetics throughout life. These results set the basis for in-depth T-cell monitoring in different disease and therapeutic conditions.

Expression profile of novel cell surface molecules on different subsets of human peripheral blood antigen-presenting cells.

Although major steps have been recently made in understanding the role of the distinct subsets of dendritic cells (DC)/antigen-presenting cells (APC), further studies are required to unravel their precise role, including in-depth immunophenotypic characterisation of these cells. Here, we used eight-colour flow cytometry to investigate the reactivity of a panel of 72 monoclonal antibodies (including those clustered in seven new Cluster of Differentiation, CD) on different subsets of APC in peripheral blood (PB) samples from five healthy adults. These experiments were performed in the context of the Tenth International Workshop on Human Leukocyte Differentiation Antigens (HLDA10). Plasmacytoid DC was the only cell population that expressed CD85g and CD195, whereas they lacked all of the other molecules investigated. In contrast, myeloid DC mostly expressed inhibitory C-type lectin receptors (CLRs) and other inhibitory-associated molecules, whereas monocytes expressed both inhibitory and activating CLRs, together with other phagocytosis-associated receptors. Within monocytes, progressively lower levels of expression were generally observed from classical monocytes (cMo) to SLAN- and SLAN+ non-classical monocytes (ncMo) for most of the molecules expressed, except for the CD368 endocytic receptor. This molecule was found to be positive only in cMo, and the CD369 and CD371 modulating/signalling receptors. In addition, the CD101 inhibitory molecule was found to be expressed at higher levels in SLAN+ vs SLAN- ncMo. In summary, the pattern of expression of the different signalling molecules and receptors analysed in this work varies among the distinct subsets of PB APCs, with similar profiles for molecules within each functional group. These findings suggest unique pattern-recognition and signalling capabilities for distinct subpopulations of APCs, and therefore, diverse functional roles.