Impact of Tyrosine Kinase Inhibitors on the Immune Response to SARS-CoV-2 Vaccination in Patients with Non-Small Cell Lung Cancer.

Immune dysregulation and cancer treatment may affect SARS-CoV-2 vaccination protection. Antibody production by B-cells play a vital role in the control and clearance of the SARS-CoV-2 virus. This study prospectively explores B-cell seroconversion following SARS-CoV-2 immunization in healthy individuals and non-small cell lung cancer (NSCLC) patients undergoing oncological treatment. 92 NSCLC patients and 27 healthy individuals' blood samples were collected after receiving any COVID-19 vaccine. Serum and mononuclear cells were isolated, and a serum surrogate virus neutralization test kit evaluated SARS-CoV-2 antibodies. B-cell subpopulations on mononuclear cells were characterized by flow cytometry. Patients were compared based on vaccination specifications and target mutation oncological treatment. A higher percentage of healthy individuals developed more SARS-CoV-2 neutralizing antibodies than NSCLC patients (63% vs. 54.3%; p = 0.03). NSCLC patients receiving chemotherapy (CTX) or tyrosine kinase inhibitors (TKIs) developed antibodies in 45.2% and 53.7%, of cases, respectively, showing an impaired antibody generation. CTX patients exhibited trends towards lower median antibody production than TKIs (1.0, IQR 83 vs. 38.23, IQR 89.22; p = 0.069). Patients receiving immunotherapy did not generate antibodies. A sub-analysis revealed that those with ALK mutations exhibited non-significant trends towards higher antibody titers (63.02, IQR 76.58 vs. 21.78, IQR 93.5; p = 0.1742) and B-cells quantification (10.80, IQR 7.52 vs. 7.22, IQR 3.32; p = 0.1382) against the SARS-CoV-2 spike protein than EGFR patients; nonetheless, these differences were not statistically significant. This study shows that antibodies against SARS-CoV-2 may be impaired in patients with NSCLC secondary to EGFR-targeted TKIs compared to ALK-directed treatment.

A Review Study of the Participation of Late Domains in Sorting and Transport of Viral Factors to Exosomes.

Cellular communication depends heavily on the participation of vesicular systems generated by most cells of an organism. Exosomes play central roles in this process. Today, these vesicles have been characterized, and it has been determined that the cargo they transport is not within a random system. In fact, it depends on various molecular signals and the recruitment of proteins that participate in the biogenesis of exosomes. It has also been shown that multiple viruses can recruit these vesicles to transport viral factors such as genomes or proteins. It has been shown that the late domains present in viral proteins are critical for the exosomal selection and biogenesis systems to recognize these viral proteins and introduce them into the exosomes. In this review, the researchers discuss the evidence related to the characterization of these late domains and their role in exosome recruitment during viral infection.

Mycobacterium tuberculosis Cell Wall Antigens Induce the Formation of Immune Complexes and the Development of Vasculitis in an Experimental Murine Model.

Tuberculosis (TB) of the central nervous system (CNS) presents high mortality due to brain damage and inflammation events. The formation and deposition of immune complexes (ICs) in the brain microvasculature during Mycobacterium tuberculosis (Mtb) infection are crucial for its pathobiology. The relevance of ICs to Mtb antigens in the pathogenesis of CNS-TB has been poorly explored. Here, we aimed to establish a murine experimental model of ICs-mediated brain vasculitis induced by cell wall antigens of Mtb. We administered a cell wall extract of the prototype pathogenic Mtb strain H37Rv to male BALB/c mice by subcutaneous and intravenous routes. Serum concentration and deposition of ICs onto blood vessels were determined by polyethylene glycol precipitation, ELISA, and immunofluorescence. Histopathological changes in the brain, lung, spleen, liver, and kidney were evaluated by hematoxylin and eosin staining. Our results evidenced that vasculitis developed in the studied tissues. High serum levels of ICs and vascular deposition were evident in the brain, lung, and kidneys early after the last cell wall antigen administration. Cell wall Mtb antigens induce strong type III hypersensitivity reactions and the development of systemic vasculitis with brain vascular changes and meningitis, supporting a role for ICs in the pathogenesis of TB.

Novel clinical and immunological features associated with persistent post-acute sequelae of COVID-19 after six months of follow-up: a pilot study.

BACKGROUND: Currently, there is scant information regarding the features associated to the persistence of post-COVID-19 syndrome, which is the main aim of the present study. METHODS: A cohort study of 102 COVID-19 patients was conducted. The post-COVID-19 symptoms were assessed by a standardised questionnaire. Lymphocyte immunophenotyping was performed by flow cytometry and chemokines/cytokines, neutrophil extracellular traps, the tripartite motif 63, anti-cellular, and anti-SARS-CoV-2 IgG antibodies were addressed in serum. The primary outcome was the persistence of post-COVID-19 syndrome after six months follow-up. RESULTS: Thirteen patients (12.7%) developed the primary outcome and had a more frequent history of post-COVID-19 syndrome 3 months after infection onset (p = .044), increased levels of IL-1α (p = .011) and IP-10 (p = .037) and increased CD57 expression in CD8+ T cells (p = .003). There was a trend towards higher levels of IFN-γ (p = .051), IL-1ß (p = .062) and IL-6 (p = .087). The history of post COVID-19 in the previous 3 months, obesity, baseline serum MIP-1α and IP-10, and CD57 expression in CD8+ T cells were independently associated with the persistence of post-COVID-19 syndrome. CONCLUSION: Our data suggest an important relationship between a pro-inflammatory state mediated through metabolic pathways related to obesity and increased cellular senescence as a key element in the persistence of post-COVID-19 syndrome at six months of follow-up.

Peripheral expansion of myeloid-derived suppressor cells is related to disease activity and damage accrual in inflammatory myopathies.

OBJECTIVE: To assess the proportion of myeloid-derived suppressor cells (MDSCs), their expression of arginase-1 and programmed cell death ligand 1 (PD-L1) and their relationship with the clinical phenotype of patients with idiopathic inflammatory myopathies (IIMs). METHODS: We recruited 37 IIM adult patients and 10 healthy donors in Mexico City. We evaluated their clinical features, the proportion of MDSCs and their expression of PD-L1 and arginase-1 by flow cytometry. Polymorphonuclear (PMN)-MDSCs were defined as CD33dim, CD11b+ and CD66b+ while monocytic (M)-MDSCs were CD33+, CD11b+, HLA-DR- and CD14+. Serum cytokines were analysed with a multiplex assay. We compared the quantitative variables with the Kruskal-Wallis and Mann-Whitney U tests and assessed correlations with Spearman's ρ. RESULTS: Most patients had dermatomyositis [n = 30 (81.0%)]. IIM patients had a peripheral expansion of PMN-MDSCs and M-MDSCs with an enhanced expression of arginase-1 and PD-L1. Patients with active disease had a decreased percentage {median 1.75% [interquartile range (IQR) 0.31-5.50 vs 10.71 [3.16-15.58], P = 0.011} of M-MDSCs and a higher absolute number of PD-L1+ M-MDSCs [median 23.21 cells/mm3 (IQR 11.16-148.9) vs 5.95 (4.66-102.7), P = 0.046] with increased expression of PD-L1 [median 3136 arbitrary units (IQR 2258-4992) vs 1961 (1885-2335), P = 0.038]. PD-L1 expression in PMN-MDSCs correlated with the visual analogue scale of pulmonary disease activity (r = 0.34, P = 0.040) and damage (r = 0.36, P = 0.031), serum IL-5 (r = 0.55, P = 0.003), IL-6 (r = 0.46, P = 0.003), IL-8 (r = 0.53, P = 0.018), IL-10 (r = 0.48, P = 0.005) and GM-CSF (r = 0.48, P = 0.012). M-MDSCs negatively correlated with the skeletal Myositis Intention to Treat Index (r = -0.34, P = 0.038) and positively with IL-6 (r = 0.40, P = 0.045). CONCLUSION: MDSCs expressing arginase-1 and PD-L1 are expanded in IIM and correlate with disease activity, damage accrual and serum cytokines.

Clinical and immunological features associated to the development of a sustained immune humoral response in COVID-19 patients: Results from a cohort study.

Background: Until now, most of the research addressing long-term humoral responses in coronavirus disease 2019 (COVID-19) had only evaluated the serum titers of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgGs, without the assessment of the baseline antiviral clinical and immune profile, which is the aim of this study and may be the key factor leading to a broad and sustained antibody response. Methods: We included 103 patients with COVID-19. When the patients sought medical attention (baseline), a blood sample was drawn to perform immunophenotype of lymphocytes by flow cytometry. The patients were assessed 15 days after baseline and then every month until the third month, followed by a last visit 6 months after recruitment. We evaluated the anti-SARS-COV-2 IgG at all time points, and the serum levels of cytokines, chemokines, anti-cellular (AC) antibodies and neutrophil extracellular traps were also assessed during the follow-up. The primary outcome of the study was the presence of a sustained immune humoral response, defined as an anti-SARS-CoV-2 IgG titer >4.99 arbitrary units/mL in at least two consecutive measures. We used generalized lineal models to assess the features associated with this outcome and to assess the effect of the changes in the cytokines and chemokines throughout time on the development of a sustained humoral immune response. Results: At baseline the features associated to a sustained immune humoral response were the diagnosis of critical disease, absolute number of lymphocytes, serum IP-10, IL-4, IL-2, regulatory T cells, CD8+ T cells, and positive AC antibodies. Critical illness and the positivity of AC antibodies were associated with a sustained humoral immune response after 3 months, whilst critical illness and serum IL-13 were the explanatory variables after 6 months. Conclusion: A sustained immune humoral response is strongly related to critical COVID-19, which is characterized by the presence of AC antibodies, quantitative abnormalities in the T cell compartment, and the serum cytokines and chemokines during acute infection and throughout time.

Isthmin 1 is Expressed by Progenitor-Like Cells in the Lung: Phenotypical Analysis of Isthmin 1+ Hematopoietic Stem-Like Cells in Homeostasis and during Infection.

The process by which blood cells are generated has been widely studied in homeostasis and during pathogen-triggered inflammatory response. Recently, murine lungs have been shown to be a significant source of hematopoietic progenitors in a process known as extramedullary hematopoiesis. Using multiparametric flow cytometry, we have identified mesenchymal, endothelial, and hematopoietic progenitor cells that express the secreted small protein Isthmin 1 (ISM1). Further characterization of hematopoietic progenitor cells indicated that ISM1+ Lineage- Sca-1+ c-kit+ (ISM1+ LSK) cells are enriched in short-term hematopoietic stem cells (ST-HSCs). Moreover, most Sca-1+ ISM1+ cells express the residence marker CD49a, and this correlated with their localization in the extravascular region of the lung, indicating that ISM1+ cells are lung-resident cells. We also observed that ISM1+ cells express TLR4, TLR5, and TLR9, and, in a mouse model of sepsis induced by P. aeruginosa, we observed that all the LSK and ISM1+LSK cells were affected. We conclude that ISM1 is a novel biomarker associated with progenitor-like cells. ISM1+ cells are involved in the response to a bacterial challenge, suggesting an association between ISM1-producing cells and dangerous inflammatory responses like sepsis.

Low-Density Granulocytes and Neutrophil Extracellular Traps as Biomarkers of Disease Activity in Adult Inflammatory Myopathies.

BACKGROUND/OBJECTIVE: Biomarkers for disease activity and damage accrual in idiopathic inflammatory myopathies (IIMs) are currently lacking. The purpose of this cross-sectional study is to analyze the relationship among low-density granulocytes (LDGs), neutrophil extracellular traps (NETs), and clinical and immunological features of patients with IIM. METHODS: We assessed disease activity, damage accrual, amount of LDGs, NETs, expression of LL-37, and serum cytokines in 65 adult patients with IIM. Differences between groups and correlations were assessed by Kruskal-Wallis, Mann-Whitney U, and Spearman ρ tests. The association between LDGs, NETs, disease activity, calcinosis, and cutaneous ulcers was assessed by logistic regression. To address the capacity of LDGs and NETs to diagnose disease activity, we used receiving operating characteristic curves. RESULTS: Low-density granulocytes were higher in patients with active disease, ulcers, calcinosis, and anti-MDA5 antibodies, which correlated with serum levels of IL-17A and IL-18. Neutrophil extracellular traps were higher in patients with calcinosis, elevated titers of antinuclear antibodies, and positive anti-PM/Scl75 tests. The combination of a high proportion of both total LDGs and NETs was associated with the presence of calcinosis and cutaneous ulcers. LL-37 was higher in NETs originating from LDGs. Normal-density neutrophils were elevated in patients with active dermatomyositis. CONCLUSIONS: Low-density granulocytes and NETs containing LL-37 are increased in patients with IIM and active disease, and correlate with proinflammatory cytokines. Both total and CD10+ LDGs are potential biomarkers for disease activity and, in combination with NETs, have the potential to detect patients who are at risk for cutaneous ulcers and calcinosis.

Neutrophil Extracellular Traps Contribute to COVID-19 Hyperinflammation and Humoral Autoimmunity.

The coronavirus disease 2019 (COVID-19) is related to enhanced production of NETs, and autoimmune/autoinflammatory phenomena. We evaluated the proportion of low-density granulocytes (LDG) by flow cytometry, and their capacity to produce NETs was compared with that of conventional neutrophils. NETs and their protein cargo were quantified by confocal microscopy and ELISA. Antinuclear antibodies (ANA), anti-neutrophil cytoplasmic antibodies (ANCA) and the degradation capacity of NETs were addressed in serum. MILLIPLEX assay was used to assess the cytokine levels in macrophages' supernatant and serum. We found a higher proportion of LDG in severe and critical COVID-19 which correlated with severity and inflammatory markers. Severe/critical COVID-19 patients had higher plasmatic NE, LL-37 and HMGB1-DNA complexes, whilst ISG-15-DNA complexes were lower in severe patients. Sera from severe/critical COVID-19 patients had lower degradation capacity of NETs, which was reverted after adding hrDNase. Anti-NET antibodies were found in COVID-19, which correlated with ANA and ANCA positivity. NET stimuli enhanced the secretion of cytokines in macrophages. This study unveils the role of COVID-19 NETs as inducers of pro-inflammatory and autoimmune responses. The deficient degradation capacity of NETs may contribute to the accumulation of these structures and anti-NET antibodies are related to the presence of autoantibodies.

Redefining COVID-19 Severity and Prognosis: The Role of Clinical and Immunobiotypes.

Background: Most of the explanatory and prognostic models of COVID-19 lack of a comprehensive assessment of the wide COVID-19 spectrum of abnormalities. The aim of this study was to unveil novel biological features to explain COVID-19 severity and prognosis (death and disease progression). Methods: A predictive model for COVID-19 severity in 121 patients was constructed by ordinal logistic regression calculating odds ratio (OR) with 95% confidence intervals (95% CI) for a set of clinical, immunological, metabolomic, and other biological traits. The accuracy and calibration of the model was tested with the area under the curve (AUC), Somer's D, and calibration plot. Hazard ratios with 95% CI for adverse outcomes were calculated with a Cox proportional-hazards model. Results: The explanatory variables for COVID-19 severity were the body mass index (BMI), hemoglobin, albumin, 3-Hydroxyisovaleric acid, CD8+ effector memory T cells, Th1 cells, low-density granulocytes, monocyte chemoattractant protein-1, plasma TRIM63, and circulating neutrophil extracellular traps. The model showed an outstanding performance with an optimism-adjusted AUC of 0.999, and Somer's D of 0.999. The predictive variables for adverse outcomes in COVID-19 were severe and critical disease diagnosis, BMI, lactate dehydrogenase, Troponin I, neutrophil/lymphocyte ratio, serum levels of IP-10, malic acid, 3, 4 di-hydroxybutanoic acid, citric acid, myoinositol, and cystine. Conclusions: Herein, we unveil novel immunological and metabolomic features associated with COVID-19 severity and prognosis. Our models encompass the interplay among innate and adaptive immunity, inflammation-induced muscle atrophy and hypoxia as the main drivers of COVID-19 severity.

Size Determination and Phenotypic Analysis of Urinary Extracellular Vesicles using Flow Cytometry.

Extracellular vesicles, EVs, are a heterogeneous complex of lipidic membranes, secreted by any cell type, in any fluid such as urine. EVs can be of different sizes ranging from 40-100 nm in diameter such as in exosomes to 100-1000 nm in microvesicles. They can also contain different molecules that can be used as biomarkers for the prognosis and diagnosis of many diseases. Many techniques have been developed to characterize these vesicles. One of these is flow cytometry. However, there are no existing reports to show how to quantify the concentration of EVs and differentiate them by size, along with biomarker detection. This work aims to describe a procedure for the isolation, quantification, and phenotypification of urinary extracellular vesicles, uEVs, using a conventional cytometer for the analysis without any modification to its configuration. The method's limitations include staining a maximum of four different biomarkers per sample. The method is also limited by the amount of EVs available in the sample. Despite these limitations, with this protocol and its subsequent analysis, we can obtain more information on the enrichment of EVs markers and the abundance of these vesicles present in urine samples, in diseases involving kidney and brain damage.

A Novel Mutation in the FSH Receptor (I423T) Affecting Receptor Activation and Leading to Primary Ovarian Failure.

CONTEXT: Follicle-stimulating hormone (FSH) plays an essential role in gonadal function. Loss-of-function mutations in the follicle-stimulating hormone receptor (FSHR) are an infrequent cause of primary ovarian failure. OBJECTIVE: To analyze the molecular physiopathogenesis of a novel mutation in the FSHR identified in a woman with primary ovarian failure, employing in vitro and in silico approaches, and to compare the features of this dysfunctional receptor with those shown by the trafficking-defective D408Y FSHR mutant. METHODS: Sanger sequencing of the FSHR cDNA was applied to identify the novel mutation. FSH-stimulated cyclic adenosine monophosphate (cAMP) production, ERK1/2 phosphorylation, and desensitization were tested in HEK293 cells. Receptor expression was analyzed by immunoblotting, receptor-binding assays, and flow cytometry. Molecular dynamics simulations were performed to determine the in silico behavior of the mutant FSHRs. RESULTS: A novel missense mutation (I423T) in the second transmembrane domain of the FSHR was identified in a woman with normal pubertal development but primary amenorrhea. The I423T mutation slightly impaired plasma membrane expression of the mature form of the receptor and severely impacted on cAMP/protein kinase A signaling but much less on ß-arrestin-dependent ERK1/2 phosphorylation. Meanwhile, the D408Y mutation severely affected membrane expression, with most of the FSH receptor located intracellularly, and both signal readouts tested. Molecular dynamics simulations revealed important functional disruptions in both mutant FSHRs, mainly the loss of interhelical connectivity in the D408Y FSHR. CONCLUSIONS: Concurrently, these data indicate that conformational differences during the inactive and active states account for the distinct expression levels, differential signaling, and phenotypic expression of the I423T and D408Y mutant FSHRs.

Conformational changes in myeloperoxidase induced by ubiquitin and NETs containing free ISG15 from systemic lupus erythematosus patients promote a pro-inflammatory cytokine response in CD4+ T cells.

BACKGROUND: Neutrophil extracellular traps (NETs) from patients with systemic lupus erythematosus (SLE) are characterized by lower ubiquitylation and myeloperoxidase (MPO) as a substrate. The structural and functional effect of such modification and if there are additional post-translational modifications (PTMs) are unknown. METHODS: To assess the expression and functional role of PTMs in NETs of patients with SLE; reactivation, proliferation and cytokine production was evaluated by flow cytometry using co-cultures with dendritic cells (DC) and CD4+ from SLE patients and healthy controls. The impact of ubiquitylation on MPO was assessed by molecular dynamics. The expression of ISG15 in NETs was evaluated by immunofluorescence and Western Blot. RESULTS: Fifteen patients with SLE and ten healthy controls were included. In the co-cultures of CD4+ lymphocytes with DC stimulated with ubiquitylated MPO or recombinant MPO, a higher expression of IFNγ and IL-17A was found in CD4+ from SLE patients (p < 0.05). Furthermore, with DC stimulated with ubiquitylated MPO a trend towards increased expression of CD25 and Ki67 was found in lupus CD4+ lymphocytes, while the opposite was documented in controls (p < 0.05). Through molecular dynamics we found the K129-K488-K505 residues of MPO as susceptible to ubiquitylation. Ubiquitylation affects the hydration status of the HEME group depending on the residue to which it is conjugated. R239 was found near by the HEME group when the ubiquitin was in K488-K505. In addition, we found greater expression of ISG15 in the SLE NETs vs controls (p < 0.05), colocalization with H2B (r = 0.81) only in SLE samples and increased production of IFNγ in PBMCs stimulated with lupus NETs compared to healthy controls NETs. CONCLUSION: The ubiquitylated MPO has a differential effect on the induction of reactivation of CD4+ lymphocytes in patients with SLE, which may be related to structural changes by ubiquitylation at the catalytic site of MPO. Besides a lower ubiquitylation pattern, NETs of patients with SLE are characterized by the expression of ISG15, and the induction of IFNγ by Th1 cells.

Effect of Interleukin-17 in the Activation of Monocyte Subsets in Patients with ST-Segment Elevation Myocardial Infarction.

Interleukin- (IL-) 17 is increased in acute myocardial infarction (AMI) and plays a key role in inflammatory diseases through its involvement in the activation of leukocytes. Here, we describe for the first time the effect of IL-17 in the migration and activation of monocyte subsets in patients during ST-segment elevation myocardial infarction (STEMI) and post-STEMI. We analyzed the circulating levels of IL-17 in patient plasma. A gradual increase in IL-17 was found in STEMI and post-STEMI patients. Additionally, IL-17 had a powerful effect on the recruitment of CD14++CD16+/CD14+CD16++ monocytes derived from patients post-STEMI compared with the monocytes from patients with STEMI, suggesting that IL-17 recruits monocytes with inflammatory activity post-STEMI. Furthermore, IL-17 increased the expression of TLR4 on CD14 + CD16 - and CD14++CD16+/CD14+CD16++ monocytes post-STEMI and might enhance the response to danger-associated molecular patterns post-STEMI. Moreover, IL-17 induced secretion of IL-6 from CD14++CD16- and CD14++CD16+/CD14+CD16++ monocytes both in STEMI and in post-STEMI, which indicates that IL-17 has an effect on the secretion of proinflammatory cytokines from monocytes during STEMI and post-STEMI. Overall, we demonstrate that in STEMI and post-STEMI, IL-17 is increased and induces the migration and activation of monocyte subsets, possibly contributing to the inflammatory response through TLR4 and IL-6 secretion.

The role of B cells in heart failure and implications for future immunomodulatory treatment strategies.

Despite numerous demonstrations that the immune system is activated in heart failure, negatively affecting patients' outcomes, no definitive treatment strategy exists directed to modulate the immune system. In this review, we present the evidence that B cells contribute to the development of hypertrophy, inflammation, and maladaptive tissue remodelling. B cells produce antibodies that interfere with cardiomyocyte function, which culminates as the result of recruitment and activation of a variety of innate and structural cell populations, including neutrophils, macrophages, fibroblasts, and T cells. As B cells appear as active players in heart failure, we propose here novel immunomodulatory therapeutic strategies that target B cells and their products.

Prokaryotic Expression of the Immunoglobulin's Domains of CRTAM to Characterize a Monoclonal Antibody.

Class-I restricted T cell-associated molecule (CRTAM) is a member of the immunoglobulin superfamily, and it is closely related to nectin-like protein. CRTAM is expressed in activated CD8 T cells, NKT cells, NK cells and in a subpopulation CD4 T cells. In this study, we produce as recombinant proteins, the Ig-domains of CRTAM (IgV-IgC), the IgV, and the IgC. These proteins were successfully purified in the soluble fraction only if the stalk region was included. The recombinant CRTAM recognizes its ligand nectin-like 2 in a cell-free system. We also demonstrate that the IgC domain of CRTAM is recognized by the anti-hCRTAM monoclonal antibody C8 with a 0.62 nM affinity. In conclusion, the stalk region of CRTAM provides solubility for the expression of its Ig-domains as recombinant proteins.

In Vitro Impact of FSH Glycosylation Variants on FSH Receptor-stimulated Signal Transduction and Functional Selectivity.

FSH exists as different glycoforms that differ in glycosylation of the hormone-specific ß-subunit. Tetra-glycosylated FSH (FSH24) and hypo-glycosylated FSH (FSH18/21) are the most abundant glycoforms found in humans. Employing distinct readouts in HEK293 cells expressing the FSH receptor, we compared signaling triggered by human pituitary FSH preparations (FSH18/21 and FSH24) as well as by equine FSH (eFSH), and human recombinant FSH (recFSH), each exhibiting distinct glycosylation patterns. The potency in eliciting cAMP production was greater for eFSH than for FSH18/21, FSH24, and recFSH, whereas in the ERK1/2 activation readout, potency was highest for FSH18/21 followed by eFSH, recFSH, and FSH24. In ß-arrestin1/2 CRISPR/Cas9 HEK293-KO cells, FSH18/21 exhibited a preference toward ß-arrestin-mediated ERK1/2 activation as revealed by a drastic decrease in pERK during the first 15-minute exposure to this glycoform. Exposure of ß-arrestin1/2 KO cells to H89 additionally decreased pERK1/2, albeit to a significantly lower extent in response to FSH18/21. Concurrent silencing of ß-arrestin and PKA signaling, incompletely suppressed pERK response to FSH glycoforms, suggesting that pathways other than those dependent on Gs-protein and ß-arrestins also contribute to FSH-stimulated pERK1/2. All FSH glycoforms stimulated intracellular Ca2+ (iCa2+) accumulation through both influx from Ca2+ channels and release from intracellular stores; however, iCa2+ in response to FSH18/21 depended more on the latter, suggesting differences in mechanisms through which glycoforms promote iCa2+ accumulation. These data indicate that FSH glycosylation plays an important role in defining not only the intensity but also the functional selectivity for the mechanisms leading to activation of distinct signaling cascades.

DNA damage induced during mitosis undergoes DNA repair synthesis.

Understanding the mitotic DNA damage response (DDR) is critical to our comprehension of cancer, premature aging and developmental disorders which are marked by DNA repair deficiencies. In this study we use a micro-focused laser to induce DNA damage in selected mitotic chromosomes to study the subsequent repair response. Our findings demonstrate that (1) mitotic cells are capable of DNA repair as evidenced by DNA synthesis at damage sites, (2) Repair is attenuated when DNA-PKcs and ATM are simultaneously compromised, (3) Laser damage may permit the observation of previously undetected DDR proteins when damage is elicited by other methods in mitosis, and (4) Twenty five percent of mitotic DNA-damaged cells undergo a subsequent mitosis. Together these findings suggest that mitotic DDR is more complex than previously thought and may involve factors from multiple repair pathways that are better understood in interphase.

[Regulation of immune responses by exosomes derived from antigen presenting cells]. / Los exosomas de las células presentadoras de antígeno y su papel en la regulación de las respuestas inmunológicas.

Cells release several biomolecules to the extracellular environment using them as a communication alternative with neighbor cells. Besides these molecules, cells also release more complex elements, like vesicles; structures composed of a lipidic bilayer with transmembrane proteins that protect a hydrophilic content. Exosomes are a small subtype of vesicles (30-150 nm), produced by many cell types, such as tumor cells, neurons, epithelial cells and immune cells. Included in this last group, antigen presenting cells produce exosomes that contain different types of molecules depending on their activation and/or maturation state. In recent years there has been an exponential interest in exosomes due to the recent evidences that show the immunomodulatory properties of these vesicles and therefore, their great potential in diagnostic approaches and development of therapies for different inflammation-associated pathologies.

Las células liberan biomoléculas de diversa naturaleza a su entorno para comunicarse con las células vecinas. Además de dichas moléculas, secretan también elementos más complejos como las vesículas; estructuras compuestas por bicapas lipídicas con proteínas transmembranales que encierran un contenido hidrofílico. Los exosomas son un subtipo pequeño de estas vesículas (de 30 a 150 nm), producidos por una amplia variedad de tipos celulares incluyendo las neuronas, células tumorales, células epiteliales y células del sistema inmunológico. De entre estas últimas, las células presentadoras de antígeno se han caracterizado como productoras de exosomas con contenido variable, tanto en condiciones de reposo como en aquellas que derivan de su estimulación o maduración. En los últimos años, el estudio de los exosomas ha aumentado debido a que se ha demostrado que dichas vesículas poseen propiedades inmunomoduladoras, razón por la que ostentan un gran potencial en aplicaciones de diagnóstico y desarrollo de terapias en diferentes patologías con componentes inflamatorios.