Whole Blood and Plasma-Based Lipid Profiling Reveals Distinctive Metabolic Changes in Systemic Lupus Erythematosus and Systemic Sclerosis.

Autoimmune diseases (AID), such as systemic lupus erythematosus (SLE) and systemic sclerosis (SS), are complex conditions involving immune system dysregulation. Diagnosis is challenging, requiring biomarkers for improved detection and prediction of relapses. Lipids have emerged as potential biomarkers due to their role in inflammation and immune response. This study uses an untargeted C18 RP-LC-MS lipidomics approach to comprehensively assess changes in lipid profiles in patients with SLE and SS. By analyzing whole blood and plasma, the study aims to simplify the lipidomic analysis, explore cellular-level lipids, and compare lipid signatures of SLE and SS with healthy controls. Our findings showed variations in the lipid profile of SLE and SS. Sphingomyelin and ceramide molecular species showed significant increases in plasma samples from SS patients, suggesting an atherosclerotic profile and potentially serving as lipid biomarkers. Phosphatidylserine species in whole blood from SLE patients exhibited elevated levels supporting previously reported dysregulated processes of cell death and defective clearance of dying cells in this AID. Moreover, decreased phospholipids bearing PUFA were observed, potentially attributed to the degradation of these species through lipid peroxidation processes. Further studies are needed to better understand the role of lipids in the pathological mechanisms underlying SLE and SS.

Immunoparalysis in critically ill children.

Immunoparalysis is associated with poorer outcomes in the paediatric intensive care unit (PICU) setting. We aimed to determine the group of patients with higher chances of immunoparalysis and correlate this status with increased risks of nosocomial infection and adverse clinical parameters. We conducted an exploratory study with prospective data collection in a university-affiliated tertiary medical, surgical, and cardiac PICU. Fifteen patients with multiple organ dysfunction syndrome were included over a period of 6 months. Monocyte's human leucocyte antigen (HLA)-DR expression and tumour necrosis factor (TNF)-α and interleukin (IL)-6 production were measured by flow-cytometry at three time points (T1 = 1-2 days; T2 = 3-5 days; T3 = 6-8 days). Using the paediatric logistic organ dysfunction-2 score to assess initial disease severity, we established the optimal cut-off values of the evaluated parameters to identify the subset of patients with a higher probability of immunoparalysis. A comparative analysis was performed between them. Sixty per cent were males; the median age was 4.1 years. Considering the presence of two criteria in T1 (classical monocytes mean fluorescence intensity [MFI] for HLA-DR ≤ 1758.5, area under the curve (AUC) = 0.775; and frequency of monocytes producing IL-6 ≤ 68.5%, AUC = 0.905) or in T3 (classical monocytes MFI of HLA-DR ≤ 2587.5, AUC = 0.675; and frequency of monocytes producing TNF-α ≤ 93.5%, AUC = 0.833), a variable to define immunoparalysis was obtained (100% sensitivity, 81.5% specificity). Forty per cent of patients were assigned to the immunoparalysis group. In this: a higher frequency of nosocomial infection (p = 0.011), vasoactive inotropic score (p = 0.014) and length of hospital stay (p = 0.036) was observed. In the subgroup with the diagnosis of sepsis/septic shock (n = 5), patients showed higher percentages of non-classical monocytes (p = 0.004). No mortality was recorded. A reduction in classical monocytes HLA-DR expression with lower frequencies of monocytes producing TNF-α and IL-6 during the first week of critical illness, appears to be a good marker of immunoparalysis; these findings relate to an increased risk of nosocomial infection and deleterious outcomes. The increased frequency of non-classical monocytes in patients with sepsis/septic shock is suggestive of a better prognosis.

Reduced numbers of regulatory T cells in chronic heart failure seems not to be restored by cardiac resynchronization therapy.

BACKGROUND: T cells have been implicated in the development and progression of inflammatory processes in chronic heart failure (CHF). Cardiac resynchronization therapy (CRT) has beneficial effects on symptoms and cardiac remodeling in CHF. However, its impact on the inflammatory immune response remains controversial. We aimed to study the impact of CRT on T cells in heart failure (HF) patients. METHODS: Thirty-nine HF patients were evaluated before CRT (T0) and six months later (T6). Quantification of T cells, their subsets, and their functional characterization, after in vitro stimulation, were evaluated by flow cytometry. RESULTS: T regulatory (Treg) cells were decreased in CHF patients (healthy group (HG): 1.08 ± 0.50 versus (heart failure patients (HFP)-T0: 0.69 ± 0.40, P = 0.022) and remaining diminished after CRT (HFP-T6: 0.61 ± 0.29, P = 0.003). Responders (R) to CRT presented a higher frequency of T cytotoxic (Tc) cells producing IL-2 at T0 compared with non-responders (NR) (R: 36.52 ± 12.55 versus NR: 24.71 ± 11.66, P = 0.006). After CRT, HF patients presented a higher percentage of Tc cells expressing TNF-α and IFN-γ (HG: 44.50 ± 16.62 versus R: 61.47 ± 20.54, P = 0.014; and HG: 40.62 ± 15.36 versus R: 52.39 ± 18.66, P = 0.049, respectively). CONCLUSION: The dynamic of different functional T cell subpopulations is significantly altered in CHF, which results in an exacerbated pro-inflammatory response. Even after CRT, it seems that the inflammatory condition underlying CHF continues to evolve with the progression of the disease. This could be due, at least in part, to the inability to restore Treg cells levels. TRIAL REGISTRATION: Observational and prospective study with no trial registration.

Role of monocytes and dendritic cells in cardiac reverse remodelling after cardiac resynchronization therapy.

BACKGROUND AND AIMS: Monocytes and dendritic cells (DC) are both key inflammatory cells, with recognized effects on cardiac repair. However, there are distinct subsets of monocytes with potential for beneficial or detrimental effects on heart failure (HF) pathogenesis. The connection between reverse cardiac remodelling, the potential anti-inflammatory effect of cardiac resynchronization therapy (CRT) and monocytes and DC homeostasis in HF is far from being understood. We hypothesized that monocytes and DC play an important role in cardiac reverse remodelling and CRT response. Therefore, we aimed to assess the potential role of baseline peripheral levels of blood monocytes and DC subsets and their phenotypic and functional activity for CRT response, in HF patients. As a secondary objective, we aimed to evaluate the impact of CRT on peripheral blood monocytes and DC subsets, by comparing baseline and post CRT circulating levels and phenotypic and functional activity. METHODS: Forty-one patients with advanced HF scheduled for CRT were included in this study. The quantification and phenotypic determination of classical (cMo), intermediate (iMo) and non-classical monocytes (ncMo), as well as of myeloid (mDC) and plasmacytoid DC (pDC) were performed by flow cytometry in a FACSCanto™II (BD) flow cytometer. The functional characterization of total monocytes and mDC was performed by flow cytometry in a FACSCalibur flow cytometer, after in vitro stimulation with lipopolysaccharide from Escherichia coli plus interferon (IFN)-γ, in the presence of Brefeldina A. Comparisons between the control and the patient group, and between responders and non-responders to CRT were performed. RESULTS: Compared to the control group, HF population presented a significantly lower frequency of pDC at baseline and a higher proportion of monocytes and mDC producing IL-6 and IL-1ß, both before and 6-months after CRT (T6). There was a remarkable decrease of cMo and an increase of iMo after CRT, only in responders. The responder group also presented higher ncMo values at T6 compared to the non-responder group. Both responders and non-responders presented a decrease in the expression of CD86 in all monocyte and DC populations after CRT. Moreover, in non-responders, the increased frequency of IL-6-producing DC persisted after CRT. CONCLUSION: Our study provides new knowledge about the possible contribution of pDC and monocytes subsets to cardiac reverse remodelling and response to CRT. Additionally, CRT is associated with a reduction on CD86 expression by monocytes and DC subsets and in their potential to produce pro-inflammatory cytokines, contributing, at least in part, for the well described anti-inflammatory effects of CRT in HF patients.

Dried blood spots in clinical lipidomics: optimization and recent findings.

Dried blood spots (DBS) are being considered as an alternative sampling method of blood collection that can be used in combination with lipidomic and other omic analysis. DBS are successfully used in the clinical context to collect samples for newborn screening for the measurement of specific fatty acid derivatives, such as acylcarnitines, and lipids from whole blood for diagnostic purposes. However, DBS are scarcely used for lipidomic analysis and investigations. Lipidomic studies using DBS are starting to emerge as a powerful method for sampling and storage in clinical lipidomic analysis, but the major research work is being done in the pre- and analytical steps and procedures, and few in clinical applications. This review presents a description of the impact factors and variables that can affect DBS lipidomic analysis, such as the type of DBS card, haematocrit, homogeneity of the blood drop, matrix/chromatographic effects, and the chemical and physical properties of the analyte. Additionally, a brief overview of lipidomic studies using DBS to unveil their application in clinical scenarios is also presented, considering the studies of method development and validation and, to a less extent, for clinical diagnosis using clinical lipidomics. DBS combined with lipidomic approaches proved to be as effective as whole blood samples, achieving high levels of sensitivity and specificity during MS and MS/MS analysis, which could be a useful tool for biomarker identification. Lipidomic profiling using MS/MS platforms enables significant insights into physiological changes, which could be useful in precision medicine.

Serum phospholipidomics reveals altered lipid profile and promising biomarkers in multiple sclerosis.

Multiple sclerosis is a neurodegenerative disease causing disability in young adults. Alterations in metabolism and lipid profile have been associated with this disease. Several studies have reported changes in the metabolism of arachidonic acid and the profile of fatty acids, ceramides, phospholipids and lipid peroxidation products. Nevertheless, the understanding of the modulation of circulating lipids at the molecular level in multiple sclerosis remains unclear. In the present study, we sought to assess the existence of a distinctive lipid signature of multiple sclerosis using an untargeted lipidomics approach. It also aimed to assess the differences in lipid profile between disease status (relapse and remission). For this, we used hydrophilic interaction liquid chromatography coupled with mass spectrometry for phospholipidomic profiling of serum samples from patients with multiple sclerosis. Our results demonstrated that multiple sclerosis has a phospholipidomic signature different from that of healthy controls, especially the PE, PC, LPE, ether-linked PE and ether-linked PC species. Plasmalogen PC and PE species, which are natural endogenous antioxidants, as well as PC and PE polyunsaturated fatty acid esterified species showed significantly lower levels in patients with multiple sclerosis and patients in both remission and relapse of multiple sclerosis. Our results show for the first time that the serum phospholipidome of multiple sclerosis is significantly different from that of healthy controls and that few phospholipids, with the lowest p-value, such as PC(34:3), PC(36:6), PE(40:10) and PC(38:1) may be suitable as biomarkers for clinical applications in multiple sclerosis.

Immunologic characterization of COVID-19 patients with hematological cancer.

Not available.

Immune-endocrine responses and physical performance of master athletes during the sports season.

The purpose of this study was to investigate the impact of a training season (approximately 7 months) on physiological and salivary immune-endocrine markers in master athletes. Nine male master athletes were evaluated at the beginning of the season (M1) and a week after the main official competition at the end of the sports season (M2). The controlled variables included Maximal oxygen consumption, anthropometric, physiological, and salivary immune-endocrine markers. Master athletes presented a reduced percentage of fat mass and increased lean body mass at the end of the season. VO2max values were similar at M1 and M2, while the maximal heart rate and lactate were lower at M2. No differences were observed in Immunoglobulin A and cortisol levels between moments, whereas testosterone levels and the testosterone/cortisol ratio were significantly lower at the end of the season. The results suggest that maintaining regular training throughout life has positive effects on body composition and improves physiological fitness. However, care should be taken to avoid fatigue as indicated by lower testosterone levels at the end of the season.

Amniotic membrane extract differentially regulates human peripheral blood T cell subsets, monocyte subpopulations and myeloid dendritic cells.

The discovery of the immunoregulatory potential of human amniotic membrane (hAM) propelled several studies focusing on its application for the treatment of immunological disorders. However, there is little information regarding the effects of hAM on distinct activation and differentiation stages of immune cells. Here, we aim to investigate the effect of human amniotic membrane extract (hAME) on the pattern of cytokine production by T cells, monocytes and myeloid dendritic cells (mDCs). For this purpose, peripheral blood mononuclear cells (PBMCs) from eight healthy individuals were stimulated in vitro in the presence or absence of hAME. Mitogen-induced proliferation of PBMCs and cytokine production among the distinct T cell functional compartments, monocyte subpopulations and mDCs were evaluated. hAME displayed an anti-proliferative effect and decreased the frequency of T cells producing tumor necrosis factor (TNF)α, interferon (IFN)γ and interleukin (IL)-2, for all T cell functional compartments. The frequency of IL-17 and IL-9-producing T cells was also reduced. The inhibition of mRNA expression of granzyme B, perforin and NKG2D by CD8+ T cells and γδ T cells and the augment of FoxP3 and IL-10 in CD4+ T cells and IL-10 in regulatory T cells were also observed. Furthermore, hAME inhibited IFNγ-induced protein (IP)-10 expression by classical and non-classical monocytes, without hampering the production of TNFα and IL-6 by monocytes and mDCs. These results suggest that hAME exerts an anti-inflammatory effect on T cells, still at a different extent for distinct T cell functional compartments.

Modulation of the inflammatory response of immune cells in human peripheral blood by oxidized arachidonoyl aminophospholipids.

Aminophospholipids (APL), phosphatidylethanolamine (PE) and phosphatidylserine (PS), can be oxidized upon oxidative stress. Oxidized PE and PS have been detected in clinical samples of different pathologies and may act as modulators of the inflammatory response. However, few studies have focused on the effects of oxidized APL (ox-APL) esterified with arachidonic acid, even though a considerable number of studies have assessed the modulation of the immune system by oxidized 1-palmitoyl-2-arachidonoyl-sn-3-glycerophosphocholine (OxPAPC). In the present study, we have used flow cytometry to evaluate the ability of oxidized PAPE (OxPAPE) and PAPS (OxPAPS) to promote or suppress an inflammatory phenotype on monocytes subsets and myeloid dendritic cells (mDCs). The results indicate that OxPAPE increases the frequency of all monocyte subpopulations expressing TNF-α, which promotes an inflammatory response. However, immune cell stimulation with OxPAPE in the presence of LPS results in a decrease of TNF-α expressed by classical monocytes. Incubation with OxPAPS and LPS induces a decrease in TNF-α produced by monocytes, and a significant decrease in IL-1ß expressed by monocytes and mDCs, indicating that OxPAPS reduces the LPS-induced pro-inflammatory expression in these populations. These results show the importance of OxPAPE and OxPAPS as modulators of the inflammatory response and demonstrate their possible contribution to the onset and resolution of human diseases related to oxidative stress and inflammation.

Increased frequencies of circulating CXCL10-, CXCL8- and CCL4-producing monocytes and Siglec-3-expressing myeloid dendritic cells in systemic sclerosis patients.

OBJECTIVE: To investigate the ex vivo pro-inflammatory properties of classical and non-classical monocytes as well as myeloid dendritic cells (mDCs) in systemic sclerosis (SSc) patients. METHODS: Spontaneous production of CXCL10, CCL4, CXCL8 and IL-6 was intracellularly evaluated in classical, non-classical monocytes and Siglec-3-expressing mDCs from peripheral blood of SSc patients and healthy controls (HC) through flow cytometry. In addition, production of these cytokines was determined upon toll-like receptor (TLR) 4 plus Interferon-γ (IFN-γ) stimulation. RESULTS: The frequency of non-classical monocytes spontaneously producing CXCL10 was increased in both limited (lcSSc) and diffuse cutaneous (dcSSC) subsets of SSc patients and CCL4 was augmented in dcSSc patients. The proportion of CCL4-producing mDCs was also elevated in dcSSc patients and the percentage of mDCS producing CXCL10 only in lcSSc patients. Upon stimulation, the frequency of non-classical monocytes expressing CXCL8 was increased in both patient groups and mDCs expressing CXCL8 only in lcSSc. Moreover, these parameters in unsupervised clustering analysis identify a subset of patients which are characterized by lung fibrosis and reduced pulmonary function. CONCLUSIONS: These data point towards a role of activated non-classical monocytes and mDCs producing enhanced levels of proinflammatory cytokines in SSc, potentially contributing to lung fibrosis.

Effects of lifelong training on senescence and mobilization of T lymphocytes in response to acute exercise.

BACKGROUND/PURPOSE: Ageing has profound impact on the immune system, mainly on T-cells. However, it has been suggested that chronic exercise may delay immunosenescence. Master athletes represent an interesting sub-demographic group to test this theory since they maintain a high training frequency and load throughout life. The purpose of this study was to evaluate the effects of lifelong training on the senescence and mobilization of T lymphocytes in response to acute exercise. MATERIAL AND METHODS: Nineteen athletes who regularly participated in training and competitions for more than 20 years throughout their lives and a control group of 10 healthy individuals participated in this study. All subjects performed a progressive test to exhaustion on a cycle ergometer. Blood samples were obtained before (Pre), 10 min after the test (Post) and 1 h after the test (1h). Phenotypic study of peripheral blood T-cells was performed by flow cytometry. Genes of interest expression was done on T-cells purified by cell sorting. RESULTS: Master athletes had a lower percentage of senescent naïve, central memory and effector memory CD8+ T-cells and senescent naïve and effector memory CD4+ T-cells. Age had a positive effect on SLEC CD8+ T-cells and a negative effect on naïve CD8+ T-cells. VO2max positively correlated with the proportion of naïve CD4+ T-cells and negatively correlated with the percentage of total lymphocytes. No differences were founded for CD4+ and CD8+ T-cells and their subsets between master athletes and the control group at all times of measurement. No differences were observed in the CD45RA expressing effector memory cells (EMRA) for the various study conditions. The mRNA expression of the CCR7 gene for naïve CD8+ T-cells and the Fas-L gene for effector-terminal CD8+ T-cells was not different between masters and controls and did not change in response to the maximal protocol test. CONCLUSION: In conclusion, maintaining high levels of aerobic fitness during the natural course of aging may help prevent the accumulation of senescent T-cells.

Lifelong training improves anti-inflammatory environment and maintains the number of regulatory T cells in masters athletes.

PURPOSE: The purpose of this study was to quantify and characterize peripheral blood regulatory T cells (Tregs), as well as the IL-10 plasma concentration, in Masters athletes at rest and after an acute exhaustive exercise test. METHODS: Eighteen Masters athletes (self-reported training: 24.6 ± 1.83 years; 10.27 ± 0.24 months and 5.45 ± 0.42 h/week per each month trained) and an age-matched control group of ten subjects (that never took part in regular physical training) volunteered for this study. All subjects performed an incremental test to exhaustion on a cycle ergometer. Blood samples were obtained before (Pre), 10 min into recovery (Post), and 1 h after the test (1 h). RESULTS: Absolute numbers of Tregs were similar in both groups at rest. Acute exercise induced a significant increase in absolute numbers of Tregs at Post (0.049 ± 0.021 to 0.056 ± 0.024 × 109/L, P = 0.029 for Masters; 0.048 ± 0.017 to 0.058 ± 0.020 × 109/L, P = 0.037 for control) in both groups. Treg mRNA expression for FoxP3, IL-10, and TGF-ß in sorted Tregs was similar throughout the trials in both groups. Masters athletes showed a higher percentage of subjects expressing the FoxP3 (100% for Masters vs. 78% for Controls, P = 0.038) and TGF-ß (89% for Masters vs. 56% for Controls, P = 0.002) after exercise and a higher plasma IL-10 concentration (15.390 ± 7.032 for Masters vs. 2.411 ± 1.117 for control P = 0.001, ES = 2.57) at all timepoints. KLRG1 expression in Tregs was unchanged. CONCLUSION: Our findings showed that Masters athletes have elevated anti-inflammatory markers and maintain the number of Tregs, and may be an adaptive response to lifelong training.

Subset-specific alterations in frequencies and functional signatures of γδ T cells in systemic sclerosis patients.

OBJECTIVE AND DESIGN: Here, we evaluated the distribution and functional profile of circulating CD27+ and CD27- γδ T-cell subsets in systemic sclerosis (SSc) patients to assess their potential role in this disorder. MATERIALS AND METHODS: Peripheral blood from 39 SSc patients and 20 healthy individuals was used in this study. The TCR-γδ repertoire, cytokine production and cytotoxic signatures of circulating γδ T-cell subsets were assessed by flow cytometry. Gene expression of EOMES, NKG2D and GZMA was evaluated by quantitative RT-PCR in both purified γδ T-cell subsets. RESULTS: Absolute numbers of γδ T-cell subsets were significantly decreased in SSc groups, likely reflecting their mobilization to the inflamed skin. Both γδ T-cell subsets preserved their relative proportions and Th1-type cytokine responses. However, cytotoxic properties showed significant disease-associated and subset-specific changes. SSc patients exhibited increased percentages of CD27+ γδ T cells expressing granzyme B or perforin and upregulated GZMA expression in diffuse cutaneous SSc. Conversely, EOMES and NKG2D were downregulated in both SSc γδ T-cell subsets vs. normal controls. Interestingly, patients with pulmonary fibrosis showed a biased TCR repertoire, with a selected expansion of effector Vγ9+ γδ T cells associated with increased frequency of cells expressing granzyme B, but decreased IFN-γ production. CONCLUSIONS: Significant alterations on circulating γδ T-cell subsets suggest a deregulated (increased) cytotoxic activity and thus enhanced pathogenic potential of CD27+ γδ T cells in SSc.

Subjects with chronic lymphocytic leukaemia-like B-cell clones with stereotyped B-cell receptors frequently show MDS-associated phenotypes on myeloid cells.

An increasing body of evidence suggests the potential occurrence of antigen encounter by the cell of origin in chronic lymphocytic leukaemia (CLL) and CLL-like monoclonal B-cell lymphocytosis (MBL). However, the scenario in which this event might occur remains unknown. In order to gain insight into this scenario we investigated the molecular, cytogenetic and haematological features of 223 CLL-like (n = 84) and CLL (n = 139) clones with stereotyped (n = 32) versus non-stereotyped (n = 191) immunoglobulin heavy chain variable region (IGHV) amino acid sequences. Overall, stereotyped CLL-like MBL and CLL clones showed a unique IGHV profile, associated with higher IGHV1 and lower IGHV3 gene family usage (P = 0·03), longer IGHV complementary determining region 3 (HCDR3) sequences (P = 0·007) and unmutated IGHV (P < 0·001) versus non-stereotyped clones. Whilst the overall size of the stereotyped B-cell clones in peripheral blood did not appear to be associated with the CLL-related cytogenetic profile of B-cells (P > 0·05), it did show a significant association with the presence of myelodysplastic syndrome (MDS)-associated immunophenotypes on peripheral blood neutrophils and/or monocytes (P = 0·01). Altogether our results point to the potential involvement of different selection forces in the expansion of stereotyped vs. non-stereotyped CLL and CLL-like MBL clones, the former being potentially favoured by an underlying altered haematopoiesis.

Alterations on peripheral blood B cell subsets induced by allergic rhinitis.

OBJECTIVES AND DESIGN: Here we evaluated whether allergic rhinitis to Dermatophagoides pteronyssinus induces alterations on circulating B cell subsets. MATERIALS AND METHODS: Circulating B cell subsets and isotype expression on antigen-experienced B cells from allergic patients under conventional pharmacological treatment (NO-SIT, n = 15) and under subcutaneous immunotherapy (SCIT, n = 33), and non-allergic subjects (NC, n = 25) were analyzed by flow cytometry. RESULTS: In allergic patients, we found a significant decrease in IgM(+) and IgG(+) memory B cells and an increase in IgA(+) memory B cells. Additionally, the numbers of circulating IgA(+) plasmablasts in allergic patients were also increased, while those cells expressing IgM were reduced. CONCLUSIONS: Allergic patients have a disturbed B cell subsets distribution which seems to underlie rhinitis pathogenesis and remain unchanged after SCIT.

Reduced levels of circulating endothelial progenitor cells in acute myocardial infarction patients with diabetes or pre-diabetes: accompanying the glycemic continuum.

BACKGROUND: Diabetic patients have a significantly worse prognosis after an acute myocardial infarction (AMI) than their counterparts. Previous studies have shown that the number of circulating endothelial progenitor cells (EPCs) significantly increase early after an AMI in normoglycemic patients. However, it is well known that type 2 diabetes mellitus (DM) is associated with impaired function and reduced circulating EPCs levels. Nonetheless, few studies have analyzed EPCs response of diabetics to an AMI and the EPC response of pre-diabetic patients has not been reported yet. Therefore, we hypothesized that in the acute phase of an AMI, diabetic and pre-diabetics have lower circulating EPCs levels than patients with normal glucose metabolism. We also evaluated the possible capacity of chronic antidiabetic treatment in the recovery of EPCs response to an AMI in diabetics. METHODS: One-hundred AMI patients were prospectively enrolled in the study. Using the high-performance flow cytometer FACSCanto II, circulating EPCs (CD45dimCD34+KDR+ and CD45dimCD133+KDR+ cells) were quantified, within the first 24 hours of admission. In addition, as an indirect functional parameter, we also analyzed the fraction of EPCs coexpressing the homing marker CXCR4. RESULTS: We found that in the acute phase of an AMI, diabetic patients presented significantly lower levels of circulating CD45dimCD34+KDR+ and CD45dimCD133+KDR+ EPCs by comparison with nondiabetics, with a parallel decrease in the subpopulations CXCR4+ (p < 0.001). Indeed, this study suggests that the impaired response of EPCs to an AMI is an early event in the natural history of DM, being present even in pre-diabetes. Our results, also demonstrated that numbers of all EPCs populations were inversely correlated with HbA1c (r = -0.432, p < 0.001 for CD45dimCD34+KDR+ cells). Finally, this study suggests that previous chronic insulin therapy (but not oral antidiabetic drugs) attenuate the deficient response of diabetic EPCs to an AMI. CONCLUSION: This study indicates that there is a progressive decrease in EPCs levels, from pre-diabetes to DM, in AMI patients. Moreover, glycemic control seems to be determinant for circulating EPCs levels presented in the acute phase of an AMI and chronic insulin therapy may probably attenuate the deficit in EPCs pool seen in diabetics.

Low amounts of mitochondrial reactive oxygen species define human sperm quality.

We have applied the mitochondria-specific superoxide fluorescent probe MitoSOX Red (MitoSOX) to detect mitochondria-specific reactive oxygen species (mROS) production in human sperm samples using flow cytometry. We show that human ejaculates are heterogeneous in terms of mROS production, with three subpopulations clearly detectable, comprising sperm that produce increasing amounts of mROS (MitoSOX-, MitoSOX+, and MitoSOX++). The sperm subpopulation producing the lowest amount of mROS represented the most functional subset of male gametes within the ejaculate, as it was correlated with the highest amount of live and non-apoptotic sperm and increased both in samples with better semen parameters and in samples processed by both density-gradient centrifugation and swim-up, both known to select for higher quality sperm. Importantly, the MitoSOX- subpopulation was clearly more prevalent in samples that gave rise to pregnancies following assisted reproduction. Our work, therefore, not only describe discreet human sperm heterogeneity at the mROS level but also suggests that mROS may represent a strategy to both evaluate sperm samples and isolate the most functional gametes for assisted reproduction.

Molecular and cytogenetic characterization of expanded B-cell clones from multiclonal versus monoclonal B-cell chronic lymphoproliferative disorders.

Chronic antigen-stimulation has been recurrently involved in the earlier stages of monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The expansion of two or more B-cell clones has frequently been reported in individuals with these conditions; potentially, such coexisting clones have a greater probability of interaction with common immunological determinants. Here, we analyzed the B-cell receptor repertoire and molecular profile, as well as the phenotypic, cytogenetic and hematologic features, of 228 chronic lymphocytic leukemia-like and non-chronic lymphocytic leukemia-like clones comparing multiclonal (n=85 clones from 41 cases) versus monoclonal (n=143 clones) monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The B-cell receptor of B-cell clones from multiclonal cases showed a slightly higher degree of HCDR3 homology than B-cell clones from mono clonal cases, in association with unique hematologic (e.g. lower B-lymphocyte counts) and cytogenetic (e.g. lower frequency of cytogenetically altered clones) features usually related to earlier stages of the disease. Moreover, a subgroup of coexisting B-cell clones from individual multiclonal cases which were found to be phylogenetically related showed unique molecular and cytogenetic features: they more frequently shared IGHV3 gene usage, shorter HCDR3 sequences with a greater proportion of IGHV mutations and del(13q14.3), than other unrelated B-cell clones. These results would support the antigen-driven nature of such multiclonal B-cell expansions, with potential involvement of multiple antigens/epitopes.

Epstein-Barr virus infection and clinical outcome in breast cancer patients correlate with immune cell TNF-α/IFN-γ response.

BACKGROUND: For nearly two decades now, various studies have reported detecting the Epstein-Barr virus (EBV) in breast cancer (BC) cases. Yet the results are unconvincing, and their interpretation has remained a matter of debate. We have now presented prospective data on the effect of EBV infection combined with survival in patients enrolled in a prospective study. METHODS: We assessed 85 BC patients over an 87-month follow-up period to determine whether EBV infection, evaluated by qPCR in both peripheral blood mononuclear cells (PBMCs) and tumor biopsies, interacted with host cell components that modulate the evolution parameters of BC. We also examined the EBV replicating form by the titration of serum anti-ZEBRA antibodies. Immunological studies were performed on a series of 35 patients randomly selected from the second half of the survey, involving IFN-γ and TNF-α intracellular immunostaining tests performed via flow cytometry analysis in peripheral NK and T cells, in parallel with EBV signature. The effect of the EBV load in the blood or tumor tissue on patient survival was analyzed using univariate and multivariate analyses, combined with an analysis of covariance. RESULTS: Our study represents the first ever report of the impact of EBV on the clinical outcome of BC patients, regardless of tumor histology or treatment regimen. No correlation was found between: (i) EBV detection in tumor or PBMCs and tumor characteristics; (ii) EBV and other prognostic factors. Notably, patients exhibiting anti-ZEBRA antibodies at high titers experienced poorer overall survival (p = 0.002). Those who recovered from their disease were found to have a measurable EBV DNA load, together with a high frequency of IFN-γ and TNF-α producing PBMCs (p = 0.04), which indicates the existence of a Th1-type polarized immune response in both the tumor and its surrounding tissue. CONCLUSIONS: The replicative form of EBV, as investigated using anti-ZEBRA titers, correlated with poorer outcomes, whereas the latent form of the virus that was measured and quantified using the EBV tumor DNA conferred a survival advantage to BC patients, which could occur through the activation of non-specific anti-tumoral immune responses.