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Systemic lupus erythematosus (SLE) is an autoimmune disorder that often leads to kidney injury, known as lupus nephritis (LN). Although renal biopsy is the primary way to diagnose LN, it is invasive and not practical for regular monitoring. As an alternative, several groups have proposed urinary extracellular vesicles (uEVs) as potential biomarkers for LN, as recent studies have shown their significance in reflecting kidney-related diseases. As a result, we developed a flow cytometry approach that allowed us to determine that LN patients exhibited a significantly higher total uEV concentration compared to SLE patients without kidney involvement. Additionally, an analysis of different-sized uEV subsets revealed that microvesicles ranging from 0.3 to 0.5 μm showed the most promise for distinguishing LN. These findings indicate that evaluating uEV concentration and size distribution could be a valuable diagnostic and monitoring tool for LN, pending further validation in more comprehensive studies. [ABSTRACT FROM AUTHOR]

The coronavirus disease 2019 (COVID-19) survivors are frequently observed to present persistent symptoms constituting what has been called "post-acute COVID-19 syndrome" (PACS) or "long COVID-19". Some clinical risk factors have been identified to be associated with PACS development; however, specific mechanisms responsible for PACS pathology remain unknown. This study investigates clinical, immunological, and metabolomic risk factors associated with post-acute COVID-19 syndrome (PACS) in 51 patients, assessed 7–19 months after acute infection. Among the participants, 62.7% were male and 37.2% were female, with an average age of 47.8 years. At the follow-up, 37.2% met the criteria for PACS, revealing significant differences in immunological and metabolomic profiles at the time of acute infection. Patients with PACS were characterized by elevated levels of mature low-density granulocytes (LDGs), interleukin-8 (IL-8), pyruvate, pseudouridine, and cystine. Baseline multivariate analysis showed increased pyruvate and decreased alpha tocopherol levels. At follow-up, there was a decrease in absolute B lymphocytes and an increase in non-classical monocytes and 3-hydroxyisovaleric acid levels. These findings suggest that specific immunological and metabolomic markers during acute infection can help identify patients at higher risk of developing persistent PACS. [ABSTRACT FROM AUTHOR]

Idiopathic inflammatory myopathies are a heterogeneous group of rare autoimmune disorders characterized by progressive muscle weakness and the histopathologic findings of inflammatory infiltrates in muscle tissue. Although their pathogenesis remains indefinite, the association of autoantibodies with clinical manifestations and the evidence of high effectiveness of depleting therapies suggest that B cells could be implicated. Therefore, we explored the landscape of peripheral B cells in this disease by multiparametric flow cytometry, finding significant numerical decreases in memory and double-negative subsets, as well as an expansion of the naive compartment relative to healthy controls, that contribute to defining disease-associated B-cell subset signatures and correlating with different clinical features of patients. Additionally, we determined the potential value of these subsets as diagnostic biomarkers, thus positioning B cells as neglected key elements possibly participating in idiopathic inflammatory myopathy onset or development. [ABSTRACT FROM AUTHOR]

The contribution of B cells in COVID‐19 pathogenesis, beyond the production of specific antibodies against SARS‐CoV‐2, is still not well understood. Since one of their most relevant functional roles includes their immune‐suppressive mechanisms, we decided to evaluate one of the most recognized human B regulatory subpopulations: the IL‐10+ B10 cells, during COVID‐19 onset. After stimulation of PBMCs for IL‐10 induction, we employed multiparametric flow cytometry to determine B10 frequencies in severe and critical COVID‐19 patients and then correlated those with clinical and laboratory parameters. Compared with healthy individuals, we detected a significant reduction in the B10 subset in both patient groups, which correlates with some inflammatory parameters that define the disease severity. This evidence suggests an aberrant role of B10 cells in immune responses against SARS‐CoV‐2 that needs to be further explained. [ABSTRACT FROM AUTHOR]

Lupus nephritis (LN) is one of the most common manifestations of systemic lupus erythematosus (SLE), characterized by abnormal B cell activation and differentiation to memory or plasma effector cells. However, the role of these cells in the pathogenesis of LN is not fully understood, as well as the effect of induction therapy on B cell subsets, possibly associated with this manifestation, like aged-associated B cells (ABCs). Consequently, we analyzed the molecules defining the ABCs subpopulation (CD11c, T-bet, and CD21) through flow cytometry of blood samples from patients with lupus presenting or not LN, following up a small sub-cohort after six months of induction therapy. The frequency of ABCs resulted higher in LN patients compared to healthy subjects. Unexpectedly, we identified a robust reduction of a CD21hi subset that was almost specific to LN patients. Moreover, several clinical and laboratory lupus features showed strong and significant correlations with this undefined B cell subpopulation. Finally, it was observed that the induction therapy affected not only the frequencies of ABCs and CD21hi subsets but also the phenotype of the CD21hi subset that expressed a higher density of CXCR5. Collectively, our results suggest that ABCs, and more importantly the CD21hi subset, may work to assess therapeutic response since the reduced frequency of CD21hi cells could be associated with the onset of LN. [ABSTRACT FROM AUTHOR]

Objectives: The role of B cells in COVID‐19, beyond the production of specific antibodies against SARS-CoV-2, is still not well understood. Here, we describe the novel landscape of circulating double-negative (DN) CD27− IgD− B cells in COVID‐19 patients, representing a group of atypical and neglected subpopulations of this cell lineage. Methods: Using multiparametric flow cytometry, we determined DN B cell subset amounts from 91 COVID-19 patients, correlated those with cytokines, clinical and laboratory parameters, and segregated them by principal components analysis. Results: We detected significant increments in the DN2 and DN3 B cell subsets, while we found a relevant decrease in the DN1 B cell subpopulation, according to disease severity and patient outcomes. These DN cell numbers also appeared to correlate with pro- or anti-inflammatory signatures, respectively, and contributed to the segregation of the patients into disease severity groups. Conclusion: This study provides insights into DN B cell subsets' potential role in immune responses against SARS‐CoV‐2, particularly linked to the severity of COVID‐19. [ABSTRACT FROM AUTHOR]

The immune response plays a critical role in the pathophysiology of SARS‐CoV‐2 infection ranging from protection to tissue damage and all occur in the development of acute respiratory distress syndrome (ARDS). ARDS patients display elevated levels of inflammatory cytokines and innate immune cells, and T and B cell lymphocytes have been implicated in this dysregulated immune response. Mast cells are abundant resident cells of the respiratory tract and are able to release different inflammatory mediators rapidly following stimulation. Recently, mast cells have been associated with tissue damage during viral infections, but their role in SARS‐CoV‐2 infection remains unclear. In this study, we examined the profile of mast cell activation markers in the serum of COVID‐19 patients. We noticed that SARS‐CoV‐2‐infected patients showed increased carboxypeptidase A3 (CPA3) and decreased serotonin levels in their serum when compared with symptomatic SARS‐CoV‐2‐negative patients. CPA3 levels correlated with C‐reactive protein, the number of circulating neutrophils, and quick SOFA. CPA3 in serum was a good biomarker for identifying severe COVID‐19 patients, whereas serotonin was a good predictor of SARS‐CoV‐2 infection. In summary, our results show that serum CPA3 and serotonin levels are relevant biomarkers during SARS‐CoV‐2 infection. This suggests that mast cells and basophils are relevant players in the inflammatory response in COVID‐19 and may represent targets for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Background: SARS-CoV-2 infection represents a global health problem that has affected millions of people. The fine host immune response and its association with the disease course have not yet been fully elucidated. Consequently, we analyze circulating B cell subsets and their possible relationship with COVID-19 features and severity. Methods: Using a multiparametric flow cytometric approach, we determined B cell subsets frequencies from 52 COVID-19 patients, grouped them by hierarchical cluster analysis, and correlated their values with clinical data. Results: The frequency of CD19+ B cells is increased in severe COVID-19 compared to mild cases. Specific subset frequencies such as transitional B cell subsets increase in mild/moderate cases but decrease with the severity of the disease. Memory B compartment decreased in severe and critical cases, and antibody-secreting cells are increased according to the severity of the disease. Other non-typical subsets such as double-negative B cells also showed significant changes according to disease severity. Globally, these differences allow us to identify severity-associated patient clusters with specific altered subsets. Finally, respiratory parameters, biomarkers of inflammation, and clinical scores exhibited correlations with some of these subpopulations. Conclusions: The severity of COVID-19 is accompanied by changes in the B cell subpopulations, either immature or terminally differentiated. Furthermore, the existing relationship of B cell subset frequencies with clinical and laboratory parameters suggest that these lymphocytes could serve as potential biomarkers and even active participants in the adaptive antiviral response mounted against SARS-CoV-2. [ABSTRACT FROM AUTHOR]

B lymphocytes are a leukocyte subset capable of developing several functions apart from differentiating into antibody‐secreting cells. These processes are triggered by external activation signals that induce changes in the plasma membrane properties, regulated by the formation of different lipid‐bilayer subdomains that are associated with the underlying cytoskeleton through different linker molecules, thus allowing the functional specialization of regions within the membrane. Among these, there are tetraspanin‐enriched domains. Tetraspanins constitute a superfamily of transmembrane proteins that establish lateral associations with other molecules, determining its activity and localization. In this study, we identified TSPAN33 as an active player during B‐lymphocyte cytoskeleton and plasma membrane‐related phenomena, including protrusion formation, adhesion, phagocytosis, and cell motility. By using an overexpression model of TSPAN33 in human Raji cells, we detected a specific distribution of this protein that includes membrane microvilli, the Golgi apparatus, and extracellular vesicles. Additionally, we identified diminished phagocytic ability and altered cell adhesion properties due to the aberrant expression of integrins. Accordingly, these cells presented an enhanced migratory phenotype, as shown by its augmented chemotaxis and invasion rates. When we evaluated the mechanic response of cells during fibronectin‐induced spreading, we found that TSPAN33 expression inhibited changes in roughness and membrane tension. Contrariwise, TSPAN33 knockdown cells displayed opposite phenotypes to those observed in the overexpression model. Altogether, our data indicate that TSPAN33 represents a regulatory element of the adhesion and migration of B lymphocytes, suggesting a novel implication of this tetraspanin in the control of the mechanical properties of their plasma membrane. [ABSTRACT FROM AUTHOR]

B lymphocytes are recognized for their crucial role in the adaptive immunity since they represent the only leukocyte lineage capable of differentiating into Ab‐secreting cells. However, it has been demonstrated that these lymphocytes can exert several Ab‐independent functions, including engulfing and processing Ags for presentation to T cells, secreting soluble mediators, providing co‐stimulatory signals, and even participating in lymphoid tissues development. Beyond that, several reports claiming the existence of multiple B cell subsets contributing directly to innate immune responses have appeared. These "innate‐like" B lymphocytes, whose phenotype, development pathways, tissue distribution, and functions are in most cases notoriously different from those of conventional B cells, are crucial to early protective responses against pathogens by exerting "crossover" defensive strategies that blur the established boundaries of innate and adaptive branches of immunity. Examples of these mechanisms include the rapid secretion of the polyspecific natural Abs, increased susceptibility to innate receptors‐mediated activation, cytokine secretion, downstream priming of other innate cells, usage of specific variable immunoglobulin gene‐segments, and other features. As these new insights emerge, it is becoming preponderant to redefine the functionality of B cells beyond their classical adaptive‐immune tasks. [ABSTRACT FROM AUTHOR]

Tilmicosin is an antimicrobial agent used to treat intramammary infections against Staphylococcus aureus and has clinical anti-inflammatory effects. However, the mechanism by which it modulates the inflammatory process in the mammary gland is unknown. We evaluated the effect of tilmicosin treatment on the modulation of the mammary innate immune response after S. aureus infection and its effect on casein production in mammary epithelial cells. To achieve this goal, we used immortalized mammary epithelial cells (MAC-T), pretreated for 12 h or treated with tilmicosin after infection with S. aureus (ATCC 27543). Our data showed that tilmicosin decreases intracellular infection (P < 0.01) and had a protective effect on MAC-T reducing apoptosis after infection by 80% (P < 0.01). Furthermore, tilmicosin reduced reactive oxygen species (ROS) (P < 0.01), IL-1β (P < 0.01), IL-6 (P < 0.01), and TNF-α (P < 0.05) production. In an attempt to investigate the signaling pathways involved in the immunomodulatory effect of tilmicosin, mitogen-activated protein kinase (MAPK) phosphorylation was measured by fluorescent-activated cell sorting. Pretreatment with tilmicosin increased ERK1/2 (P < 0.05) but decreased P38 phosphorylation (P < 0.01). In addition, the anti-inflammatory effect of tilmicosin helped to preserve casein synthesis in mammary epithelial cells (P < 0.01). This result indicates that tilmicosin could be an effective modulator inflammation in the mammary gland. Through regulation of MAPK phosphorylation, ROS production and pro-inflammatory cytokine secretion tilmicosin can provide protection from cellular damage due to S. aureus infection and help to maintain normal physiological functions of the bovine mammary epithelial cell. [ABSTRACT FROM AUTHOR]

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Using a comprehensive microarray database of human gene expression, we identified that in mammals, a secreted protein known as isthmin 1 (ISM1) is expressed in skin, mucosal tissues, and selected lymphocyte populations. ISM1 was originally identified in Xenopus brain during development, and it encodes a predicted ∼50-kDa protein containing a signal peptide, a thrombospondin domain, and an adhesion-associated domain. We confirmed the pattern of expression of ISM1 in both human and mouse tissues. ISM1 is expressed by DX5+ lung lymphocytes that include NK and NKT-like cells, and is also expressed by some CD4+ T cells upon activation but its expression increases significantly when CD4+ T cells were polarized to the Th17 lineage in vitro. The presence of IFN-γ during CD4+ T cell polarization inhibits ISM1 expression. Given that ISM1 has been reported to have anti-angiogenic properties, these observations suggest that ISM1 is a mediator of lymphocyte effector functions and may participate in both innate and acquired immune responses. [ABSTRACT FROM AUTHOR]

Chemokines are a superfamily of chemotactic cytokines that direct the movement of cells throughout the body under homeostatic and inflammatory conditions. The mucosal chemokine CXCL17 was the last ligand of this superfamily to be characterized. Several recent studies have provided greater insight into the basic biology of this chemokine and have implicated CXCL17 in several human diseases. We sought to better characterize CXCL17's activity in vivo. To this end, we analyzed its chemoattractant properties in vivo and characterized a Cxcl17-/- mouse. This mouse has a significantly reduced number of macrophages in its lungs compared with wild-type mice. In addition, we observed a concurrent increase in a new population of macrophage-like cells that are F4/80+CDIIcmid. These results indicate that CXCL17 is a novel macrophage chemoattractant that operates in mucosal tissues. Given the importance of macrophages in inflammation, these observations strongly suggest that CXCL17 is a major regulator of mucosal inflammatory responses. [ABSTRACT FROM AUTHOR]

Myosin 1g (Myo1g) is a hematopoietic-specific myosin expressed mainly by lymphocytes. Here, we report the localization of Myo1g in B-cell membrane compartments such as lipid rafts, microvilli, and membrane extensions formed during spreading. By using Myo1g-deficient mouse B cells, we detected abnormalities in the adhesion ability and chemokine-induced directed migration of these lymphocytes. We also assessed a role for Myo1g in phagocytosis and exocytosis processes, as these were also irregular in Myo1g-deficient B cells. Taken together, our results show that Myo1g acts as a main regulator of different membrane/cytoskeleton-dependent processes in B lymphocytes. [ABSTRACT FROM AUTHOR]

Bruton agammaglobulinemia tyrosine kinase (BTK) is a key protein in the B-cell receptor (BCR) signaling pathway and plays an essential role in the differentiation of B lymphocytes. X-linked agammaglobulinemia (XLA) is a primary humoral immunodeficiency caused by mutations in the gene encoding BTK. Previously, we identified two novel variations, L111P and E605G, in BTK; these are localized within the pleckstrin homology and Src homology 1 domains, respectively. In the present study, we evaluated the potential effects of these variations on the structural conformation and the function of BTK. Using in silico methods, we found that the L111P and E650G variations are not located directly in protein-protein interfaces but close to them. They distorted the native structural conformation of the BTK protein, affecting not only its geometry and stability but also its ability for protein recognition and in consequence its functionality. To confirm the results of the in silico assays , WT BTK, L111P, and E650G variants were expressed in the BTK-deficient DT40 cell line. The mutant proteins exhibited an absence of catalytic activity, aberrant redistribution after BCR-crosslinking, and deficient intracellular calcium mobilization. This work demonstrates that L111 and E605 residues are fundamental for the activation and function of BTK. © 2012 IUBMB Life, 64(4): 346-353, 2012 [ABSTRACT FROM AUTHOR]

Myosin 1c (Myo1c) is a member of the unconventional class I myosins of vertebrates, which directly link the plasma membrane with the microfilament cortical web. Although this molecular motor has been implicated in cell functions such as cytoskeleton organization, cell motility, nuclear transcription, and endocytosis, its role in hematopoietic cells is largely unknown. In this study, we show that Myo1c is abundantly expressed in murine B lymphocytes and is preferentially located at the plasma membrane, especially in peripheral processes such as microvilli. We observed that this motor concentrates at the growing membrane protrusions generated during B cell spreading and that it is actively recruited to the immune synapse. Interestingly, Myo1c was detected in lipid rafts of B cells and showed strong colocalization with MHC-II, particularly after cross-linking of these molecules. By transfection of a dominant negative form of Myo1c or specific siRNA, we also detected alterations in the spreading and Ag-presenting ability of these cells. The data suggest that Myo1c is involved in the cytoskeleton dynamics and membrane protein anchoring or sorting in B lymphocytes. [ABSTRACT FROM AUTHOR]

Pancreatic cancer remains one of the most lethal diseases with dismal five-year survival rates. Although mutant KRas protein-driven activation of downstream MAPK Raf/MEK/ERK and PI3K/Akt signaling pathways represent major oncogenic alterations, signaling blockade with MEK and PI3K inhibitors has shown that intrinsic resistance may hamper the effectiveness of this targeted approach. However, there have been no mass spectrometry-based proteomic studies for in-depth comparison of protein expression differences between pancreatic cancer cells with sensitivity and resistance to MEK and PI3K kinase inhibitors. In this work, we compared PANC-1 and MIA PaCa-2 pancreatic cancer cells which are, respectively, resistant and sensitive to MEK- and PI3K-targeted therapy. We conducted a label-free data-independent acquisition mass spectrometry (SWATH-MS) study with extensive peptide fractionation to quantitate 4808 proteins and analyze differential expression of 743 proteins between resistant and sensitive cells. This allowed identification of the tumor suppressor protein phosphatase 2A (PP2A) and proteins from mitochondrial respiratory complex I implicated in oxidative phosphorylation as alternative candidate drug targets for cells resistant to MEK and PI3K inhibition. PP2A activator DT-061 decreased viability of PANC-1 cells and this was accompanied by reduced expression of c-Myc. PANC-1 cells also showed response to metformin and the novel complex I inhibitor IACS-010759. These findings provide insights into the distinct cellular proteomes and point out alternative pharmacological targets for MEK and PI3K inhibition-resistant pancreatic cancer cells. • First proteomic study of pancreatic cancer cells resistant to MEK and PI3K inhibition. • Activation of protein phosphatase PP2A overcomes resistance. • PP2A activation leads to decreased c-Myc expression. • Resistant cells have higher respiration activity, linked to oxidative phosphorylation. • Mitochondrial complex I is another drug target for resistant cells. [ABSTRACT FROM AUTHOR]