Newcastle disease virus vector-based SARS-CoV-2 vaccine candidate AVX/COVID-12 activates T cells and is recognized by antibodies from COVID-19 patients and vaccinated individuals.

Introduction: Several effective vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed and implemented in the population. However, the current production capacity falls short of meeting global demand. Therefore, it is crucial to further develop novel vaccine platforms that can bridge the distribution gap. AVX/COVID-12 is a vector-based vaccine that utilizes the Newcastle Disease virus (NDV) to present the SARS-CoV-2 spike protein to the immune system. Methods: This study aims to analyze the antigenicity of the vaccine candidate by examining antibody binding and T-cell activation in individuals infected with SARS-CoV-2 or variants of concern (VOCs), as well as in healthy volunteers who received coronavirus disease 2019 (COVID-19) vaccinations. Results: Our findings indicate that the vaccine effectively binds antibodies and activates T-cells in individuals who received 2 or 3 doses of BNT162b2 or AZ/ChAdOx-1-S vaccines. Furthermore, the stimulation of T-cells from patients and vaccine recipients with AVX/COVID-12 resulted in their proliferation and secretion of interferon-gamma (IFN-γ) in both CD4+ and CD8+ T-cells. Discussion: The AVX/COVID-12 vectored vaccine candidate demonstrates the ability to stimulate robust cellular responses and is recognized by antibodies primed by the spike protein present in SARS-CoV-2 viruses that infected patients, as well as in the mRNA BNT162b2 and AZ/ChAdOx-1-S vaccines. These results support the inclusion of the AVX/COVID-12 vaccine as a booster in vaccination programs aimed at addressing COVID-19 caused by SARS-CoV-2 and its VOCs.

Metabolic Recovery with the Persistence of Proinflammatory Leucocyte Dysfunction After Bariatric Intervention for Obesity.

PURPOSE: A suitable option for severe obesity treatment is a surgical approach. After surgery, metabolic markers and weight frequently return to adequate values; however, concerning systemic inflammatory mediators, the results are inconsistent. Furthermore, it has been suggested that leucocyte function may be affected even after weight normalization. This study aimed to determine if the surgical treatment of obesity influences the production of cytokines by LPS-stimulated as a function of leucocytes. MATERIALS AND METHODS: We performed a cross-sectional study that investigated the production of cytokines in response to lipopolysaccharide (LPS) along a kinetic of simulation by leucocytes recovered from individuals with normal weight (NW, n = 8), persons living with obesity (Ob, n = 7), persons living with obesity and diabetes mellitus (Ob-DM, n = 17), and persons that used to live with obesity who underwent bypass surgery (fOb + bypass, n = 8) and recover normal weigh. RESULTS: IL-6 levels were significantly higher in the Ob and fOb + bypass groups than in NW (p = 0.043). IL-10 secretion without LPS was significantly higher in the NW group than in the other groups explored (p < 0.05). When exposed to LPS, the IL-10 levels increased in all groups except the NW group. As also observed for IL-18 and IL-33, the secretion curve of the fOb + bypass group was more similar to the Ob group, even when they had reached normal weight, as opposed to the NW group. CONCLUSION: Our results show that in patients with fOb + bypass, inflammatory and anti-inflammatory cytokine production dynamics remain disrupted even with improved metabolic control and normal weight recovery.

Fc-gamma receptor expression and cytokine responses to intravenous human immunoglobulin in whole blood from non-pregnant and pregnant women and newborns.

BACKGROUND: Intravenous immunoglobulin (IVIG) can suppress the inflammatory response in adults, but its role in pregnant women and newborns is poorly studied. While the adult immune system is considered mature, it is immature in neonates and suppressed in pregnancy. Since the immune response differs in these 3 groups, the use of IVIG could differentially modulate the immune response. OBJECTIVES: We aimed to explore the effect of IVIG on myeloid blood cells from non-pregnant women, pregnant women and newborns. MATERIAL AND METHODS: Whole blood from healthy donors was incubated with lipopolysaccharide (LPS) and/or IVIG. After 0 h, 24 h and 48 h of culture, Fc-gamma receptor (CD16, CD32 and CD64) expression, monocyte and neutrophil bacterial phagocytosis, and cytokine and chemokine concentrations were determined in the supernatant. RESULTS: The baseline expression of monocyte CD16 was higher in newborns than in adult women, but the expression of CD32 and CD64 was similar between groups. Furthermore, LPS and IVIG stimulation, together or separately, did not change Fc-gamma receptor expression in monocytes or neutrophils and did not modify their phagocytosis capacity. On the other hand, IVIG did not downregulate the proinflammatory cytokine response induced by LPS in any group. Interestingly, IVIG induced a strong interleukin 8 (IL-8) response in neonates but not in non-pregnant or pregnant women. CONCLUSIONS: Our results show that IVIG did not induce changes in Fc-gamma receptor expression, phagocytic ability, or the cytokine response to LPS in blood cells from neonates, non-pregnant or pregnant women. However, IVIG induced a strong IL-8 response in neonates that could improve immunity.

Potential biomarkers for fatal outcome prognosis in a cohort of hospitalized COVID-19 patients with pre-existing comorbidities.

The difficulty in predicting fatal outcomes in patients with coronavirus disease 2019 (COVID-19) impacts the general morbidity and mortality due to severe acute respiratory syndrome-coronavirus 2 infection, as it wears out the hospital services that care for these patients. Unfortunately, in several of the candidates for prognostic biomarkers proposed, the predictive power is compromised when patients have pre-existing comorbidities. A cohort of 147 patients hospitalized for severe COVID-19 was included in a descriptive, observational, single-center, and prospective study. Patients were recruited during the first COVID-19 pandemic wave (April-November 2020). Data were collected from the clinical history whereas immunophenotyping by multiparameter flow cytometry analysis allowed us to assess the expression of surface markers on peripheral leucocyte. Patients were grouped according to the outcome in survivors or non-survivors. The prognostic value of leucocyte, cytokines or HLA-DR, CD39, and CD73 was calculated. Hypertension and chronic renal failure but not obesity and diabetes were conditions more frequent among the deceased patient group. Mixed hypercytokinemia, including inflammatory (IL-6) and anti-inflammatory (IL-10) cytokines, was more evident in deceased patients. In the deceased patient group, lymphopenia with a higher neutrophil-lymphocyte ratio (NLR) value was present. HLA-DR expression and the percentage of CD39+ cells were higher than non-COVID-19 patients but remained similar despite the outcome. Receiver operating characteristic analysis and cutoff value of NLR (69.6%, 9.4), percentage NLR (pNLR; 71.1%, 13.6), and IL-6 (79.7%, 135.2 pg/mL). The expression of HLA-DR, CD39, and CD73, as many serum cytokines (other than IL-6) and chemokines levels do not show prognostic potential, were compared to NLR and pNLR values.

Aspectos inmunológicos en preeclampsia / Immunologic Aspects in Preeclampsia

Resumen La preeclampsia es una patología con alta morbimortalidad a nivel mundial. En esta enfermedad la placenta es un órgano de choque donde la inflamación y la respuesta inmunológica generan el daño que se traduce en el cuadro clínico característico. La tríada clásica en preeclampsia está integrada por hipertensión, edema y proteinuria, por lo que se piensa que el endotelio debe estar afectado por la actividad inflamatoria-inmunológica. El sistema inmunológico actúa en el desarrollo del embarazo y lo hace a diferentes tiempos y regulando de manera fisiológica. Tanto componentes celulares como humorales de la respuesta innata y adquirida han sido estudiados en pacientes con preeclampsia y se ha determinado que su participación es decisiva en la fisiopatología de esta enfermedad. La participación del sistema inmunológico en la fisiopatología de la preeclampsia alcanza un alto nivel de complejidad pues interacciona con otros sistemas (coagulación, renal, cardiovascular y endocrinológico entre otros) favoreciendo así la enfermedad. Es por esto que el tratamiento debe ser integral, con una visión holística del padecimiento y que requiere de un equipo multidisciplinario, que actué armónicamente para así alcanzar el mayor éxito terapéutico con la menor frecuencia de secuelas para el binomio madre-feto o madre-recién nacido. En la gestación se desarrolla la denominada "tolerancia inmunológica del embarazo", en ese estado de tolerancia inmunológica las células B y T pueden reconocer antígenos específicos (por ejemplo, los paternos) y posteriormente activarse y generar la respuesta inmunológica, por lo que la preeclampsia podría ser considerada como una patología autoinmune, donde la perdida de la tolerancia inmunológica sería la piedra angular en la fisiopatología, conocer como limitar o regular esta activación celular anómala podría servir para proponer nuevos acercamientos terapéuticos y controlar así esta enfermedad.

Abstract Preeclampsia is a pathology with high morbidity and mortality worldwide. In this disease, the placenta is an organ of shock where inflammation and the immune response generate the damage that results in the characteristic clinical scenario. The classic triad in preeclampsia is made up of hypertension, edema, and proteinuria, so it is thought that the endothelium must be affected by inflammatory-immunological activity. The immune system acts in the development of pregnancy and does so at different times and regulating physiologically. Both, cellular and humoral components of the innate and acquired response have been studied in patients with preeclampsia and it has been determined that their participation is decisive in the pathophysiology of this disease. The involvement of the immune system in the pathophysiology of preeclampsia reaches a high level of complexity since it interacts with other systems (coagulation, renal, cardiovascular and endocrinological among others) thus favoring the disease. For this reason, treatment must be comprehensive, with a holistic vision of the condition and requires a multidisciplinary team that acts harmoniously to achieve the greatest therapeutic success with the least frequency of sequelae for the mother-fetus or mother-newborn dyads. During pregnancy, the so-called "immunological tolerance of pregnancy" develops, in this state of immunological tolerance the B and T cells can recognize specific antigens (for example, the paternal ones) and later activate and generate the immune response, which is why preeclampsia could being considered an autoimmune pathology, where the loss of immunological tolerance would be the cornerstone of pathophysiology, knowing how to limit or regulate this abnormal cell activation could help to propose new therapeutic approaches and thus control this disease.

Chronic Comorbidities in Middle Aged Patients Contribute to Ineffective Emergency Hematopoiesis in Covid-19 Fatal Outcomes.

BACKGROUND AND AIMS: Mexico is among the countries with the highest estimated excess mortality rates due to the COVID-19 pandemic, with more than half of reported deaths occurring in adults younger than 65 years old. Although this behavior is presumably influenced by the young demographics and the high prevalence of metabolic diseases, the underlying mechanisms have not been determined. METHODS: The age-stratified case fatality rate (CFR) was estimated in a prospective cohort with 245 hospitalized COVID-19 cases, followed through time, for the period October 2020-September 2021. Cellular and inflammatory parameters were exhaustively investigated in blood samples by laboratory test, multiparametric flow cytometry and multiplex immunoassays. RESULTS: The CFR was 35.51%, with 55.2% of deaths recorded in middle-aged adults. On admission, hematological cell differentiation, physiological stress and inflammation parameters, showed distinctive profiles of potential prognostic value in patients under 65 at 7 days follow-up. Pre-existing metabolic conditions were identified as risk factors of poor outcomes. Chronic kidney disease (CKD), as single comorbidity or in combination with diabetes, had the highest risk for COVID-19 fatality. Of note, fatal outcomes in middle-aged patients were marked from admission by an inflammatory landscape and emergency myeloid hematopoiesis at the expense of functional lymphoid innate cells for antiviral immunosurveillance, including NK and dendritic cell subsets. CONCLUSIONS: Comorbidities increased the development of imbalanced myeloid phenotype, rendering middle-aged individuals unable to effectively control SARS-CoV-2. A predictive signature of high-risk outcomes at day 7 of disease evolution as a tool for their early stratification in vulnerable populations is proposed.

Multiparameter flow cytometry analysis of leukocyte markers for diagnosis in preterm neonatal sepsis.

PURPOSE: Neonatal sepsis is an important public health concern worldwide due to its immediate lethality and long-term morbidity rates, Clinical evaluation and laboratory analyses are indispensable for diagnosis of neonatal sepsis. However, assessing multiple biomarkers in neonates is difficult due to limited blood availability. The aim is to investigate if the neonatal sepsis in preterm could be identified by multiparameter analysis with flow cytometry. MATERIALS AND METHODS: The expression of activation-related molecules was evaluated by flow cytometry in newborn with or without risk factors for sepsis. RESULTS: Our analysis revealed that several markers could be useful for sepsis diagnosis, such as CD45RA, CD45RO, or CD71 on T cells; HLA-DR on NKT or classic monocytes, and TREM-1 on non-classic monocytes or neutrophils. However, ROC analysis shows that the expression of CD45RO on T lymphocytes is the only useful biomarker for diagnosis of neonatal late-onset sepsis. Also, decision tree analyses showed that CD45RO plus CD27 could help differentiate the preterm septic neonates from those with risk factors. CONCLUSIONS: Our study shows a complementary and practical strategy for biomarker assessment in neonatal sepsis.

A Shift Towards an Immature Myeloid Profile in Peripheral Blood of Critically Ill COVID-19 Patients.

BACKGROUND: SARS-CoV-2, the etiological agent causing COVID-19, has infected more than 27 million people with over 894000 deaths worldwide since its emergence in December 2019. Factors for severe diseases, such as diabetes, hypertension, and obesity have been identified however, the precise pathogenesis is poorly understood. To understand its pathophysiology and to develop effective therapeutic strategies, it is essential to define the prevailing immune cellular subsets. METHODS: We performed whole circulating immune cells scRNAseq from five critically ill COVID-19 patients, trajectory and gene ontology analysis. RESULTS: Immature myeloid populations, such as promyelocytes-myelocytes, metamyelocytes, band neutrophils, monocytoid precursors, and activated monocytes predominated. The trajectory with pseudotime analysis supported the finding of immature cell states. While the gene ontology showed myeloid cell activation in immune response, DNA and RNA processing, defense response to the virus, and response to type 1 interferon. Lymphoid lineage was scarce. Expression of genes such as C/EBPß, IRF1and FOSL2 potentially suggests the induction of trained immunity. CONCLUSIONS: Our results uncover transcriptomic profiles related to immature myeloid lineages and suggest the potential induction of trained immunity.

Medical Outcomes in Women Who Became Pregnant after Vaccination with a Virus-Like Particle Experimental Vaccine against Influenza A (H1N1) 2009 Virus Tested during 2009 Pandemic Outbreak.

The clinical effects and immunological response to the influenza vaccine in women who later become pregnant remain to be thoroughly studied. Here, we report the medical outcomes of 40 women volunteers who became pregnant after vaccination with an experimental virus-like particle (VLP) vaccine against pandemic influenza A(H1N1)2009 (influenza A(H1N1)pdm09) and their infants. When included in the VLP vaccine trial, none of the women were pregnant and were randomly assigned to one of the following groups: (1) placebo, (2) 15 µg dose of VLP vaccine, or (3) 45 µg dose of VLP vaccine. These 40 women reported becoming pregnant during the follow-up phase after receiving the placebo or VLP vaccine. Women were monitored throughout pregnancy and their infants were monitored until one year after birth. Antibody titers against VLP were measured in the mothers and infants at delivery and at six months and one year after birth. The incidence of preeclampsia, fetal death, preterm delivery, and premature rupture of membranes was similar among groups. All vaccinated women and their infants elicited antibody titers (≥1:40). Women vaccinated prior to pregnancy had no adverse events that were different from the nonvaccinated population. Even though this study is limited by the sample size, the results suggest that the anti-influenza A(H1N1)pdm09 VLP experimental vaccine applied before pregnancy is safe for both mothers and their infants.

Infectious episodes during pregnancy, at particular mucosal sites, increase specific IgA1 or IgA2 subtype levels in human colostrum.

BACKGROUND: Colostrum is the primary source of maternal immunoglobulin A (IgA) for the newborn. IgA participates in protection and regulation mechanisms of the immune response at the neonate's mucosa. Several studies have evaluated infectious diseases and vaccine protocols effects during pregnancy on maternal milk IgA levels, with the aim to understand lactation protecting effect on newborn. However, most of their results demonstrated that there were no differences in the total IgA levels. In humans, IgA has two subclasses (IgA1 and IgA2), they have an anatomical distribution among mucosal compartments, their levels vary after antigen stimulation and are also seen to describe differential affinities in colostrum. Although there are differences between IgA subclasses in several compartments, these studies have excluded specific colostrum IgA1 and IgA2 determination. METHODS: We analyzed data from 900 women in Mexico City. With Pearson correlation, we compared the number of infectious episodes during their pregnancy that was associated with mucosal compartments (skin, respiratory and gastrointestinal tracts) and colostrum IgA subclasses. RESULTS: We show a correlation between increased colostrum IgA1 levels and the number of infectious episodes at respiratory tract and the skin. In contrast, infections at the gastrointestinal tract correlated with increased IgA2 amounts. CONCLUSIONS: Infections present during pregnancy at certain mucosal site increase specific IgA subclasses levels in human colostrum. These results will help in understanding infections and immunizations effects on maternal IgA at the mammary gland, and their impact on the development and protection of the newborn.

Function is Dissociated From Activation-Related Immunophenotype on Phagocytes From Patients With SIRS/Sepsis Syndrome.

Sepsis is a life-threatening condition associated with failure of at least one organ in the presence of infection. Along with SIRS, the acute systemic inflammatory syndrome without documented infection, sepsis represents a main health problem in intensive care units around the world. Hypercytokinemia and overexpression of activation-markers on leukocytes are frequently reported in SIRS/sepsis. Leukocyte functions including antibody mediated-phagocytosis, pathogen recognition, and migration appear to be disabled in SIRS/septic patients. Our aim was to evaluate the so-called activation immunophenotype and functions related to infection contention in phagocytes from patients with sepsis. We analyzed blood samples from 44 patients with SIRS/sepsis and 14 healthy volunteers. CD16, CD69, CD64, CCR7, and TREM-1 levels were determined on the surface of neutrophils and monocytes. Phagosome maturation and p38, STAT3, and STAT5 phosphorylation were evaluated in these phagocytes. As expected, sepsis and SIRS patients had increased serological levels of pro- and anti-inflammatory cytokines. E coli internalization was not increased in monocytes from patients with SIRS/sepsis, despite increased numbers of circulating neutrophils and monocytes (P < 0.05) and overexpression of CD64 and CD69 in neutrophils (P < 0.05), TREM-1 (P < 0.01), CD69 (P < 0.001), and CCR7 (P < 0.05). Moreover, phagosome maturation was decreased in phagocytes from patients with SIRS/sepsis syndrome (P < 0.00001). Furthermore, p38 and STAT-3 phosphorylation elicited by LPS or IL-10 (respectively) was diminished in neutrophils and monocytes from patients (P < 0.05). Our results indicate that "activation markers" may not reflect higher functionality, so a more profound analysis should be made before assuming that the activated immunophenotype means increased phagocyte responses.

High Serum Levels of High-Mobility Group Box 1 (HMGB1) and Low Levels of Heat Shock Protein 70 (Hsp70) are Associated with Poor Prognosis in Patients with Acute Pancreatitis.

INTRODUCTION: Cell damage in Acute Pancreatitis (AP) lead to release of cytokines and HMGB1 and Hsp70. While Hsp70 plays a role in cytoprotection, when released to extracellular milieu constitutes, as HMGB1, a danger signal and trigger pro-inflammatory responses. These molecules seem to be related to the clinical progression; but because no evidence exists about them as molecular network in AP development, we quantify HSP70, HMGB1, and cytokines in patients with AP and search for correlations with severity and prognosis. METHODS: Fifteen patients with AP were included. The average age was 52 years. Six patients had mild pancreatitis, 4 were moderately severe and 5 with a severe form. Blood samples were taken within the first 24 h, at 3d and 7d from the start. Serum HMGB1 and Hsp70 were determined using ELISA; TNF-α, IL-1ß, IL-6, IL-8, IL-10 and IL-12p70 were determined by bead based immuassay. RESULTS: Of all 15 patients recruited, 4 were women. Eight patients had APACHEII score higher than 8. Two patients died from AP related complications. Increase in serum HMGB1 and decrease of Hsp70 were associated with the severity and mortality. TNF-α, IL-6 and IL-8 were higher in patients that did not survive, in those with an APACHE II >8, and in those with severe AP. CONCLUSIONS: High HMGB1 and low Hsp70 were associated with poor prognosis. Hsp70 might play a protective role in AP. TNF-α, IL-6, IL-8, HMGB1 and Hsp70 during hospital admissions might serve to evaluate risk of death due to AP.

Characterization of CD127- CD25++ Treg from human colostrum.

PROBLEM: Breastfeeding's influence on the tolerance to environmental antigens is essential for short- and long-term homeostasis for children. Colostrum is rich in leucocytes, but it is unknown whether regulatory T cells (Treg) account for part of this cell population. METHOD OF STUDY: Frequencies of CD127-  CD25++ Treg and levels of immunoregulatory-associated cell markers were determined in colostrum and were compared with autologous blood cells. In addition, we evaluated whether the birth conditions can affect these features. RESULTS: Higher frequencies of CD127 - CD25++ Treg cells expressing Foxp3 and CD45RO were observed in the colostrum. The cells' CD25, CD152, CD279, and TGF-ß expression levels were greater than those in autologous blood cells. In addition, the CD279 and TGF-ß expressions of colostrum CD127-  CD25++ Treg cells were influenced by gestational age and delivery mode. CONCLUSION: The higher proportion of these cells with a function-associated phenotype may reflect certain tolerogenic effects of breastmilk on newborns and infants, contributing to immune system homeostasis.

Effect of Oral ω3-Polyunsaturated Fatty Acids as a Complement Management to Control Fistula Output and Inflammation in Patients With Digestive Fistula.

BACKGROUND: The presence of digestive fistula involves chronic inflammation and fibrosis. It has been reported that ω3-polyunsaturated fatty acids stimulate the resolution of inflammation. AIM: Determine if the administration of oral ω3 reduces fistula output and the time required for fistula closure. METHODS: Forty-nine patients with postoperative fistula were randomly divided in two groups: 26 received conventional treatment and 23 received the conventional treatment supplemented with ω3 (540 mg eicosapentaenoic acid and 360 mg docosahexaenoic acid) for 35 days. Patients were monitored daily for fistula output and spontaneous closure. Additionally, serum pro-inflammatory cytokines and C-reactive protein were quantified in four patients with conventional and in seven patients with ω3 treatment. RESULTS: Patients with ω3 had significantly decreased fistula output from days 2 to 27, compared to control (p < 0.05). Spontaneous fistula closure was achieved in 15 patients (65%) in the ω3 group and in 14 (54%) in the control group. ω3-polyunsaturated fatty intake also decreased the serum concentrations of interleukin-6 and C-reactive protein (p < 0.05). CONCLUSIONS: Our results suggest that ω3 supplementation to conventional medical treatment decreases fistula output and reduces inflammation (interleukin-6 and C-reactive protein), and these effects may increase the efficiency of conventional medical treatment.

Listeria monocytogenes induces mast cell extracellular traps.

Mast cells play an essential role in different immunological phenomena including allergy and infectious diseases. Several bacteria induce mast cell activation leading to degranulation and the production of several cytokines and chemokines. However, mast cells also have different microbicidal activities such as phagocytosis and the release of DNA with embedded granular proteins known as Mast Cell Extracellular Traps (MCETs). Although previous reports indicate that extracellular bacteria are able to induce MCETs little is known if intracellular bacteria can induce these structures. In this work, we evaluated MCETs induction by the intracellular bacteria Listeria monocytogenes. We found that mast cells released DNA after stimulation with L. monocytogenes, and this DNA was complexed to histone and tryptase. Before extracellular DNA release, L. monocytogenes induced modifications to the mast cell nuclear envelope and DNA was detected outside the nucleus. L. monocytogenes stimulated mast cells to produce significant amounts of reactive oxygen species (ROS) and blocking NADPH oxidase diminished DNA release by mast cells. Finally, MCETs showed antimicrobial activity against L. monocytogenes that was partially blocked when ß-hexosaminidase activity was inhibited. These results show that L. monocytogenes induces mast cells to produce microbicidal MCETs, suggesting a role for mast cells in containing infection beyond the induction of inflammation.

Endothelial colony-forming cells: Biological and functional abnormalities in patients with recurrent, unprovoked venous thromboembolic disease.

INTRODUCTION: Endothelial cells (ECs) are an important component of the blood coagulation system because it maintains blood fluid. Because in patients with venous thromboembolic disease (VTD) a thrombophilic condition is not found sometimes, we investigated if endothelial colony-forming cells (ECFCs) from these patients have biological and functional abnormalities. PATIENTS AND METHODS: Human mononuclear cells (MNCs) were obtained from peripheral blood from patients with VTD and controls to obtain ECFCs. These cells were assayed for their immunophenotype and electron microscopy characteristics and their ability to form capillary-like structures and to produce pro-inflammatory and pro-angiogenic cytokines and reactive oxygen species (ROS). RESULTS: ECFCs appeared at 7 and 21 days of culture in VTD patients and controls, respectively. ECFCs increased 8-fold in patients and emerged 1 week earlier. No differences in the size of the colonies of ECFCs were found. Numbers and time of appearance of ECFCs was different between groups. ECFC-derived ECs (ECFC-ECs) of both groups expressed CD31, CD34, CD146, and CD-309 but none expressed CD45, CD14, or CD90. Interest CD34 was highly expressed in ECFC-ECs from patients. In both groups, ECFC-ECs showed similar capacity to form capillary-like structures but ECFC-ECs from patients had significant abnormalities in the mitochondrial membrane. We found a significant increase in ROS production in ECFC-ECs from patients. There were significant differences in cytokine profiles between VTD patients and controls. CONCLUSIONS: We found a dysfunctional state in ECFC from VTD patients resembling some characteristics of dysfunctional ECs. These findings may help to understand some pathophysiological aspects of VTD.

Differential Immune Profiles in Two Pandemic Influenza A(H1N1)pdm09 Virus Waves at Pandemic Epicenter.

BACKGROUND AND AIMS: Severe influenza A(H1N1)pdm2009 virus infection cases are characterized by sustained immune activation during influenza pandemics. Seasonal flu data suggest that immune mediators could be modified by wave-related changes. Our aim was to determine the behavior of soluble and cell-related mediators in two waves at the epicenter of the 2009 influenza pandemic. METHODS: Leukocyte surface activation markers were studied in serum from peripheral blood samples, collected from the 1(st) (April-May, 2009) and 2(nd) (October 2009-February 2010) pandemic waves. Patients with confirmed influenza A(H1N1)pdm2009 virus infection (H1N1), influenza-like illness (ILI) or healthy donors (H) were analyzed. RESULTS: Serum IL-6, IL-4 and IL-10 levels were elevated in H1N1 patients from the 2(nd) pandemic wave. Additionally, the frequency of helper and cytotoxic T cells was reduced during the 1(st) wave, whereas CD69 expression in helper T cells was increased in the 2(nd) wave for both H1N1 and ILI patients. In contrast, CD62L expression in granulocytes from the ILI group was increased in both waves but in monocytes only in the 2(nd) wave. Triggering Receptor Expressed on Myeloid cells (TREM)-1 expression was elevated only in H1N1 patients at the 1(st) wave. CONCLUSIONS: Our results show that during the 2009 influenza pandemic a T cell activation phenotype is observed in a wave-dependent fashion, with an expanded activation in the 2(nd) wave, compared to the 1(st) wave. Conversely, granulocyte and monocyte activation is infection-dependent. This evidence collected at the pandemic epicenter in 2009 could help us understand the differences in the underlying cellular mechanisms that drive the wave-related immune profile behaviors that occur against influenza viruses during pandemics.

Prolonged exposure to neutrophil extracellular traps can induce mitochondrial damage in macrophages and dendritic cells.

Neutrophils are one the earliest, crucial innate defenses against innumerable pathogens. Their main microbicidal activities include phagocytosis and degranulation, with many pharmacologically active molecules contributing to inflammation. Recently, a novel antimicrobial mechanism was discovered; the Neutrophil Extracelullar Traps (NETs) formed by extrusion of DNA and associated molecules (histones, elastase, antimicrobial peptides, among others) which trap and kill microorganisms. Since NETs were recently described, research has focused on their induction and microbicidal properties, and recently on disease involvement. However, the functional consequences of NETs interacting with other immune cells, either resident or recruited during early inflammation, have not been assessed. We therefore investigated the consequences of exposing two major APCs, macrophages (Mfs) and conventional Dendritic Cells (cDCs) to NETs. Our data revealed that at early times (30 min), both Antigen Presenting Cells (APCs) showed induction of important costimulatory molecules (CD80, CD86). Unexpectedly, however, at later times (6 and 24 hours) NETs apparently triggered a cell death process in these APCs by a caspase- and Apoptosis induced factor (AIF)-dependent pathway, suggesting mitochondrial damage. By rhodamine-123 labelling we found that in both APCs, relatively prolonged exposure to NETs or their components importantly decreased the mitochondrial membrane potential. Ultrastructural analysis confirmed mitochondrial alterations in both APCs. Our results would suggest that early in inflammation, NETs can activate the two main APCs (Mfs and cDCs), but as the process continues, NETs can then initiate apoptosis of these cells through mitochondrial harm. Conceivable, this "late" induction of cell death in these two APCs might start limiting an ongoing inflammatory process to control it.

Activated endothelial cells limit inflammatory response, but increase chemoattractant potential and bacterial clearance by human monocytes.

Inflammation is the normal immune response of vascularized tissues to damage and bacterial products, for which leukocyte transendothelial migration (TEM) is critical. The effects of cell-to-cell contact seen in both leukocyte and endothelial cells include cytoskeleton rearrangement, and dynamic expression of adhesion molecules and metalloproteinases. TEM induces expression of anti-apoptotic molecules, costimulatory molecules associated with antigen presentation, and pattern recognition receptors (PRR), such as TLR-4, in monocytes. However, little is known about how TLR-4 increment operates in monocytes during an inflammatory response. To understand it better, we used an in vitro model in which monocytes crossed a layer of IL-1ß stimulated Human Umbilical Vein Endothelial Cells (HUVEC). After TEM, monocytes were tested for the secretion of inflammatory cytokines and chemokines, their phenotype (CD14, CD16, TLR-4 expression), and TLR-4 canonical [Nuclear Factor kappa B, (NF-κB) pathway] and non-canonical [p38, extracellular signal-regulated kinases (ERK) 1/2 pathway] signal transduction induced by lipopolysaccharide (LPS). Phagocytosis and bacterial clearance were also measured. There was diminished secretion of LPS-induced inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and higher secretion of chemokines (CXCL8/IL-8 and CCL2/MCP-1) in supernatant of TEM monocytes. These changes were accompanied by increases in TLR-4, CD14 (surfaces expression), p38, and ERK1/2 phosphorylated cytoplasmic forms, without affecting NF-κB activation. It also increased bacterial clearance after TEM by an O2 -independent mechanism. The data suggest that interaction between endothelial cells and monocytes fine-tunes the inflammatory response and promotes bacterial elimination.

Pregnant women infected with pandemic H1N1pdm2009 influenza virus displayed overproduction of peripheral blood CD69+ lymphocytes and increased levels of serum cytokines.

The first pandemic of the 21st century occurred in 2009 and was caused by the H1N1pdm influenza A virus. Severe cases of H1N1pdm infection in adults are characterized by sustained immune activation, whereas pregnant women are prone to more severe forms of influenza, with increased morbi-mortality. During the H1N1pdm09 pandemic, few studies assessed the immune status of infected pregnant women. The objective of this study was to evaluate the behavior of several immune markers in 13 H1N1pdm2009 virus-infected pregnant (PH1N1) women, in comparison to pregnant women with an influenza-like illness (ILI), healthy pregnant women (HP) and healthy non-pregnant women (HW). The blood leukocyte phenotypes and the serological cytokine and chemokine concentrations of the blood leukocytes, as measured by flow cytometry, showed that the CD69+ cell counts in the T and B-lymphocytes were significantly higher in the PH1N1 group. We found that pro-inflammatory (TNF-α, IL-1ß, IL-6) and anti-inflammatory (IL-10) cytokines and some chemokines (CXCL8, CXCL10), which are typically at lower levels during pregnancy, were substantially increased in the women in the ILI group. Our findings suggest that CD69 overexpression in blood lymphocytes and elevated levels of serum cytokines might be potential markers for the discrimination of H1N1 disease from other influenza-like illnesses in pregnant women.