The combined effects of binder addition and different sintering methods on the mechanical properties of bovine hydroxyapatite.

Hydroxyapatite (HA) from bovine bones has been used as a biomaterial in dentistry due to its biocompatibility and bioactivity. However, dense HA bioceramics still present inadequate properties for applications that require high mechanical performance, such as infrastructure. Microstructural reinforcements and control of ceramic processing steps are methods to improve these shortcomings. The present study assessed the effects of polyvinyl butyral (PVB) addition in combination with two sintering methodologies (2-step and conventional), on the mechanical properties of polycrystalline bovine HA bioceramics. The samples were divided into four groups (with 15 samples per group): conventional sintering with binder (HBC) and without binder (HWC) and 2-step sintering with (HB2) and without binder (HW2). HA was extracted from bovine bones, turned into nanoparticles in a ball mill, and subjected to uniaxial and isostatic pressing into discs, according to ISO 6872 standards. All groups were characterized by x-ray diffractometry (XRD), differential thermal analysis (DTA) and Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and relative density. Besides, mechanical analyses (biaxial flexural strength (BFS) and modulus of elasticity) were also performed. The characterization results demonstrated that adding agglutinants or the sintering method did not affect HA's chemical and structural characteristics. Even so, the HWC group showed the highest mechanical values for BFS and modulus of elasticity being 109.0 (98.0; 117.0) MPa and 105.17 ± 14.65 GPa, respectively. The HA ceramics submitted to conventional sintering and without the addition of binders achieved better mechanical properties than the other groups. The impacts of each variable were discussed and correlated to the final microstructures and mechanical properties.

Contrasting Relationship of True Weevils (Curculionidae) and Woody Plants Diversity in Semi-arid Landscape Units.

In general, insect herbivore diversity is positively correlated to plant diversity since plant assemblages structure different microhabitats and provide food resources for such insects. Although poorly studied, insect herbivores in tropical dry forests are positively affected by tree species richness and the structural architecture of vegetation. In this study, we analyzed whether true weevil and woody plants present similar patterns of diversity across different landscape units in a Neotropical tropical dry forests located in Brazil, and if there is a correlation among their assemblages. We used Hill numbers according to species richness and the inverse of Simpson to compare the taxonomic diversity of true weevils and plants in nine landscape units located at the São Francisco river basin in the states of Sergipe and Alagoas, north-eastern Brazil. All trees and shrubs with diameter at breast height (1.3 m) ≥ 5 cm were sampled, and true weevils were collected using a modified Malaise trap. We used co-correspondence analysis to test if plants and true weevil species tend to co-occur. A total of 538 true weevils from 60 species was collected, and a total of 1419 plants belonging to 49 species was recorded. There is no general pattern of co-occurrence and diversity among true weevils and plants, indicating that the plant community is not structuring true weevil assemblage in the Caatinga. However, there was positive relationship between Sibinia sp4 and Fabaceae-Mimosoideae. These results suggest true weevil-plant relationships in the Caatinga differ from that of more mesic ecosystems. Therefore, we observed that the mechanisms that regulate herbivore-plant diversity relationships do not always follow a positive relationship, as observed in previous studies.

Sepsis: evolving concepts and challenges.

Sepsis remains a major cause of morbidity and mortality worldwide, with increased burden in low- and middle-resource settings. The role of the inflammatory response in the pathogenesis of the syndrome has supported the modern concept of sepsis. Nevertheless, a definition of sepsis and the criteria for its recognition is a continuous process, which reflects the growing knowledge of its mechanisms and the success and failure of diagnostic and therapeutic interventions. Here we review the evolving concepts of sepsis, from the "systemic inflammatory response syndrome triggered by infection" (Sepsis-1) to "a severe, potentially fatal, organic dysfunction caused by an inadequate or dysregulated host response to infection" (Sepsis-3). We focused in the pathophysiology behind the concept and the criteria for recognition and diagnosis of sepsis. A major challenge in evaluating the host response in sepsis is to characterize what is protective and what is harmful, and we discuss that, at least in part, the apparent dysregulated host response may be an effort to adapt to a hostile environment. The new criteria for recognition and diagnosis of sepsis were derived from robust databases, restricted, however, to developed countries. Since then, the criteria have been supported in different clinical settings and in different economic and epidemiological contexts, but still raise discussion regarding their use for the identification versus the prognostication of the septic patient. Clinicians should not be restricted to definition criteria when evaluating patients with infection and should wisely use the broad array of information obtained by rigorous clinical observation.

Sepsis: evolving concepts and challenges

Sepsis remains a major cause of morbidity and mortality worldwide, with increased burden in low- and middle-resource settings. The role of the inflammatory response in the pathogenesis of the syndrome has supported the modern concept of sepsis. Nevertheless, a definition of sepsis and the criteria for its recognition is a continuous process, which reflects the growing knowledge of its mechanisms and the success and failure of diagnostic and therapeutic interventions. Here we review the evolving concepts of sepsis, from the "systemic inflammatory response syndrome triggered by infection" (Sepsis-1) to "a severe, potentially fatal, organic dysfunction caused by an inadequate or dysregulated host response to infection" (Sepsis-3). We focused in the pathophysiology behind the concept and the criteria for recognition and diagnosis of sepsis. A major challenge in evaluating the host response in sepsis is to characterize what is protective and what is harmful, and we discuss that, at least in part, the apparent dysregulated host response may be an effort to adapt to a hostile environment. The new criteria for recognition and diagnosis of sepsis were derived from robust databases, restricted, however, to developed countries. Since then, the criteria have been supported in different clinical settings and in different economic and epidemiological contexts, but still raise discussion regarding their use for the identification versus the prognostication of the septic patient. Clinicians should not be restricted to definition criteria when evaluating patients with infection and should wisely use the broad array of information obtained by rigorous clinical observation.

B-cell subsets imbalance and reduced expression of CD40 in ataxia-telangiectasia patients

BACKGROUND: Ataxia-telangiectasia (AT) is a well-known primary immunodeficiency with recurrent sinopulmonary infections and variable abnormalities in both the humoral and cellular immune system. Dysfunctions in immunoglobulin production, reduced number of B cells, and B-cell receptor excision circles copies have been reported. We aimed to understand the immunological mechanisms involving the humoral compartment in AT patients by analysing peripheral blood B cells subsets, B-T lymphocyte cooperation through the expression of CD40 and CD40 ligand (CD40L), and cytokines involved in class-switch recombination production. METHODS: We compared the proportion of B-cell subsets, the expression of CD40/CD40L, and the plasma levels of IL-6 and IFN-gamma of 18 AT patients and 15 healthy age-sex-matched controls using flow cytometry. RESULTS: We found that some steps in peripheral B cell development were altered in AT with a pronounced reduction of cell-surface CD40 expression. The proportions of transitional and naïve-mature B cells were reduced, whereas CD21-low, natural effector memory, IgM-only memory, and IgG atypical memory B cells were present in a higher proportion. CONCLUSIONS: These findings revealed a disturbed B-cell homeostasis with unconventional maturation of B lymphocyte memory cells, which can explain the consequent impairment of humoral immunity

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B-cell subsets imbalance and reduced expression of CD40 in ataxia-telangiectasia patients.

BACKGROUND: Ataxia-telangiectasia (AT) is a well-known primary immunodeficiency with recurrent sinopulmonary infections and variable abnormalities in both the humoral and cellular immune system. Dysfunctions in immunoglobulin production, reduced number of B cells, and B-cell receptor excision circles copies have been reported. We aimed to understand the immunological mechanisms involving the humoral compartment in AT patients by analysing peripheral blood B cells subsets, B-T lymphocyte cooperation through the expression of CD40 and CD40 ligand (CD40L), and cytokines involved in class-switch recombination production. METHODS: We compared the proportion of B-cell subsets, the expression of CD40/CD40L, and the plasma levels of IL-6 and IFN-γ of 18 AT patients and 15 healthy age-sex-matched controls using flow cytometry. RESULTS: We found that some steps in peripheral B cell development were altered in AT with a pronounced reduction of cell-surface CD40 expression. The proportions of transitional and naïve-mature B cells were reduced, whereas CD21-low, natural effector memory, IgM-only memory, and IgG atypical memory B cells were present in a higher proportion. CONCLUSIONS: These findings revealed a disturbed B-cell homeostasis with unconventional maturation of B lymphocyte memory cells, which can explain the consequent impairment of humoral immunity.

Attractiveness of Different Food Resources to Dung Beetles (Coleoptera: Scarabaeidae) of a Dry Tropical Area.

Excrement scarcity is one of the main historical factors leading dung beetles to adapt to other food resources. In the Caatinga, a seasonally tropical dry forest, harsh environmental conditions seem to restrict the availability of food resources. In this scenario, the aim of our study was to experimentally investigate the attractiveness of different potential food resources to these insects in the Caatinga. Field samplings were performed, and we tested five resources in pitfall baited traps: excrement, carrion, fruits of two species of columnar cacti, and seeds of one species of Euphorbiaceae (Jatropha mollissima (Pohl)). In a controlled setting, we tested dietary preferences of Deltochilum verruciferum Felsche by offering one or two resources simultaneously. In the field experiments, 297 dung beetles (9 species) were recovered from the traps, and D. verruciferum was the most abundant species. Carrion and excrement were the most attractive resources. Controlled dietary preference tests with D. verruciferum evidenced that these beetles used all tested food resources, excrement and carrion most pronouncedly. Our findings support copro-necrophagy as the main feeding habit of D. verruciferum, but also suggest that alternative resources might be utilized if preferred resources are scarce.

Inflammasome gene profile is modulated in septic patients, with a greater magnitude in non-survivors.

Inflammasome signalling induces the processing and secretion of interleukin (IL)-1ß and IL-18 which, coupled with pyroptosis, activate further the inflammatory response. In the present study we evaluated the expression of genes involved in inflammasome signalling pathways in septic patients, their interaction networks and the predicted functions modulated in survivors and non-survivors. Twenty-seven patients with sepsis secondary to community-acquired pneumonia admitted to intensive care units from three general hospitals in São Paulo were included into the study. We performed a polymerase chain reaction (PCR) array encompassing 35 genes related to the nucleotide-binding oligomerization domain and leucine-rich repeat-containing (NLR)-inflammasome in peripheral blood mononuclear cells obtained at admission and after 7 days of follow-up. Eleven healthy volunteers were used as the reference group. Increased NLRC4 and NLRP3 and decreased nucleotide-binding oligomerization domain (NOD1), and NLRP1 expression was observed in septic patients compared to healthy individuals; the IL-1ß and IL-18 expression levels were also high in the patients. The gene expression changes followed the same patterns in surviving and non-surviving patients, with higher magnitudes observed in non-survivors. Functional analyses revealed, however, that activation and inhibition intensity for representing functions were different in survivors and non-survivors, as for production of reactive oxygen species, synthesis of nitric oxide and for the control of bacterial infections. Our results showed that the genes involved in the activation of the NLR-inflammasome cascades were altered substantially in septic patients, with a higher number of altered genes and a higher intensity in the disturbance of gene expression found among patients dying of sepsis.

Persistent effects of pre-Columbian plant domestication on Amazonian forest composition.

The extent to which pre-Columbian societies altered Amazonian landscapes is hotly debated. We performed a basin-wide analysis of pre-Columbian impacts on Amazonian forests by overlaying known archaeological sites in Amazonia with the distributions and abundances of 85 woody species domesticated by pre-Columbian peoples. Domesticated species are five times more likely than nondomesticated species to be hyperdominant. Across the basin, the relative abundance and richness of domesticated species increase in forests on and around archaeological sites. In southwestern and eastern Amazonia, distance to archaeological sites strongly influences the relative abundance and richness of domesticated species. Our analyses indicate that modern tree communities in Amazonia are structured to an important extent by a long history of plant domestication by Amazonian peoples.

Evaluation of Toll-like, chemokine, and integrin receptors on monocytes and neutrophils from peripheral blood of septic patients and their correlation with clinical outcomes

Recognition of pathogens is performed by specific receptors in cells of the innate immune system, which may undergo modulation during the continuum of clinical manifestations of sepsis. Monocytes and neutrophils play a key role in host defense by sensing and destroying microorganisms. This study aimed to evaluate the expression of CD14 receptors on monocytes; CD66b and CXCR2 receptors on neutrophils; and TLR2, TLR4, TLR5, TLR9, and CD11b receptors on both cell types of septic patients. Seventy-seven septic patients (SP) and 40 healthy volunteers (HV) were included in the study, and blood samples were collected on day zero (D0) and after 7 days of therapy (D7). Evaluation of the cellular receptors was carried out by flow cytometry. Expression of CD14 on monocytes and of CD11b and CXCR2 on neutrophils from SP was lower than that from HV. Conversely, expression of TLR5 on monocytes and neutrophils was higher in SP compared with HV. Expression of TLR2 on the surface of neutrophils and that of TLR5 on monocytes and neutrophils of SP was lower at D7 than at D0. In addition, SP who survived showed reduced expression of TLR2 and TLR4 on the surface of neutrophils at D7 compared to D0. Expression of CXCR2 for surviving patients was higher at follow-up compared to baseline. We conclude that expression of recognition and cell signaling receptors is differentially regulated between SP and HV depending on the receptor being evaluated.

Evaluation of Toll-like, chemokine, and integrin receptors on monocytes and neutrophils from peripheral blood of septic patients and their correlation with clinical outcomes.

Recognition of pathogens is performed by specific receptors in cells of the innate immune system, which may undergo modulation during the continuum of clinical manifestations of sepsis. Monocytes and neutrophils play a key role in host defense by sensing and destroying microorganisms. This study aimed to evaluate the expression of CD14 receptors on monocytes; CD66b and CXCR2 receptors on neutrophils; and TLR2, TLR4, TLR5, TLR9, and CD11b receptors on both cell types of septic patients. Seventy-seven septic patients (SP) and 40 healthy volunteers (HV) were included in the study, and blood samples were collected on day zero (D0) and after 7 days of therapy (D7). Evaluation of the cellular receptors was carried out by flow cytometry. Expression of CD14 on monocytes and of CD11b and CXCR2 on neutrophils from SP was lower than that from HV. Conversely, expression of TLR5 on monocytes and neutrophils was higher in SP compared with HV. Expression of TLR2 on the surface of neutrophils and that of TLR5 on monocytes and neutrophils of SP was lower at D7 than at D0. In addition, SP who survived showed reduced expression of TLR2 and TLR4 on the surface of neutrophils at D7 compared to D0. Expression of CXCR2 for surviving patients was higher at follow-up compared to baseline. We conclude that expression of recognition and cell signaling receptors is differentially regulated between SP and HV depending on the receptor being evaluated.

Increased neutrophil oxidative burst metabolism in systemic lupus erythematosus.

INTRODUCTION: There is increased frequency of discoid lesions (2.7%) and SLE (0.5%) in patients with chronic granulomatosus disease, but the literature is still controversial about phagocyte oxidative burst in SLE patients. MATERIALS AND METHODS: 300 SLE patients and 301 blood donors were evaluated for quantitation of the oxidative burst in phagocytes by flow cytometry based on the oxidation of 2,7-dichlorofluorescein-diacetate after stimuli with Staphylococcus aureus and Pseudomonas aeruginosa. RESULTS: Neutrophils from SLE patients displayed higher basal reactive oxygen species (ROS) production than healthy controls [Mean of fluorescence intensity (MFI) = 53.77 ± 11.38 vs 15.08 ± 2.63, p < 0.001] and after stimulation with S. aureus (MFI = 355.46 ± 58.55 vs 151.92 ± 28.25, p < 0.001) or P. aeruginosa (MFI = 82.53 ± 10.1 vs 48.99 ± 6.74, p < 0.001). There was stronger neutrophil response after bacterial stimuli (ΔMFI) in SLE patients than in healthy controls (S. aureus = 301.69 ± 54.42 vs 118.38 ± 26.03, p < 0.001; P. aeruginosa = 28.76 ± 12.3 vs 15.45 ± 5.15, p < 0.001), but no difference with respect to the oxidative burst profile according to disease activity (SLEDAI ≥ 6) or severity (SLICC-DI ≥2). Patients with kidney involvement presented higher basal and stimulated ROS production in neutrophils. DISCUSSION: The present findings corroborate the important role of innate immunity in SLE and implicate neutrophils in the pathophysiology of the disease.

Impact of a multidimensional infection control strategy on catheter-associated urinary tract infection rates in the adult intensive care units of 15 developing countries: findings of the International Nosocomial Infection Control Consortium (INICC).

PURPOSE: We aimed to evaluate the impact of a multidimensional infection control strategy for the reduction of the incidence of catheter-associated urinary tract infection (CAUTI) in patients hospitalized in adult intensive care units (AICUs) of hospitals which are members of the International Nosocomial Infection Control Consortium (INICC), from 40 cities of 15 developing countries: Argentina, Brazil, China, Colombia, Costa Rica, Cuba, India, Lebanon, Macedonia, Mexico, Morocco, Panama, Peru, Philippines, and Turkey. METHODS: We conducted a prospective before-after surveillance study of CAUTI rates on 56,429 patients hospitalized in 57 AICUs, during 360,667 bed-days. The study was divided into the baseline period (Phase 1) and the intervention period (Phase 2). In Phase 1, active surveillance was performed. In Phase 2, we implemented a multidimensional infection control approach that included: (1) a bundle of preventive measures, (2) education, (3) outcome surveillance, (4) process surveillance, (5) feedback of CAUTI rates, and (6) feedback of performance. The rates of CAUTI obtained in Phase 1 were compared with the rates obtained in Phase 2, after interventions were implemented. RESULTS: We recorded 253,122 urinary catheter (UC)-days: 30,390 in Phase 1 and 222,732 in Phase 2. In Phase 1, before the intervention, the CAUTI rate was 7.86 per 1,000 UC-days, and in Phase 2, after intervention, the rate of CAUTI decreased to 4.95 per 1,000 UC-days [relative risk (RR) 0.63 (95% confidence interval [CI] 0.55-0.72)], showing a 37% rate reduction. CONCLUSIONS: Our study showed that the implementation of a multidimensional infection control strategy is associated with a significant reduction in the CAUTI rate in AICUs from developing countries.

The status of conservation of urban forests in eastern Amazonia.

This study aims to identify the remnant tree flora in six forest fragments in the metropolitan area of Belém and to analyze these fragments in terms of biological conservation, species richness and diversity in the local urban landscape. The fragments and their respective sampling areas were as follows: Amafrutas reserve (15 ha), Trambioca Is. reserve (2 ha), Bosque Rodrigues Alves city park (15 ha), Combu Is. reserve (10 ha), Gunma Park reserve (10 ha) and Mocambo reserve (5 ha). Inventories were built from lineal plots of 250 m² and included trees with DBH equal to or greater than 10 cm at a height of 1.3 m above ground. Sixty-nine families and 759 species, of which eight were officially listed as endangered (Brazilian National Flora: Ministry of Environment, Normative Instruction of September, 2008; Pará State Flora: Decree Nº. 802 of February 2008) were recorded. These endangered species are: Aspidosperma desmanthum Benth. ex Müll. Arg. (Apocynaceae), Cedrela odorata L. (Meliaceae), Eschweilera piresii S.A Mori (Lecythidaceae), Euxylophora paraensis Huber (Rutaceae), Hymenolobium excelsum Ducke (Leguminosae), Manilkara huberi (Ducke) Chevalier (Sapotaceae), Tabebuia impetiginosa (Mart. ex DC.) Standl. (Bignoniaceae), Mezilaurus itauba (Meisn.) Taub. ex Mez (Lauraceae) and Qualea coerulea Aubl. (Vochysiaceae). Emergency actions such as implementing management plans for already existing Conservation Units, the creation of new such units in areas of primary forest fragments (as in the case of the Amafrutas reserve), as well as the intensification of actions of surveillance and monitoring, should be undertaken by Federal, State, and Municipal environmental agencies so as to ensure the conservation of these last primary forest remnants in the metropolitan area of Belém.

Vaccine antibodies and T- and B-cell interaction in juvenile systemic lupus erythematosus.

Both systemic lupus erythematosus (SLE) and its treatment can cause immunosuppression and a decreased response to vaccination. We evaluated 30 children and adolescents with SLE, and 14 age-matched healthy subjects (control group) regarding immunophenotyping and lymphocyte apoptosis by flow cytometry, while measles and tetanus antibodies were measured by enzyme-linked immunosorbent assay (ELISA). The SLE group was divided according to disease activity into inactive SLE and active SLE. Individuals with active SLE had lower CD4+ T and natural killer (NK) cells/mm(3) than the control group. Active and inactive SLE individuals had more CD38 molecules/CD8+ T cells and more CD4+ T, CD8+ T and B cells in apoptosis (as assessed by caspase-3 expression) than the control group. Patients with active SLE had a diminished CD28 expression on both CD4+ T and on CD8+ T cells and a higher CD86 expression on B cells than the control group. Measles antibody levels in the SLE groups were similar to the control group. In contrast, tetanus antibody levels were lower in the SLE groups than in the control group. The latter also directly correlated with the CD4+ T-cell and NK-cell counts from SLE patients (regression coefficient, 2.686 and 1.782; p = 0.010 and p = 0.039, respectively). We concluded that despite being up-to-date for tetanus vaccine, SLE patients presented with a poor immune response to tetanus vaccine.

Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood

Lipopolysaccharide (LPS) activates neutrophils and monocytes, inducing a wide array of biological activities. LPS rough (R) and smooth (S) forms signal through Toll-like receptor 4 (TLR4), but differ in their requirement for CD14. Since the R-form LPS can interact with TLR4 independent of CD14 and the differential expression of CD14 on neutrophils and monocytes, we used the S-form LPS from Salmonella abortus equi and the R-form LPS from Salmonella minnesota mutants to evaluate LPS-induced activation of human neutrophils and monocytes in whole blood from healthy volunteers. Expression of cell surface receptors and reactive oxygen species (ROS) and nitric oxide (NO) generation were measured by flow cytometry in whole blood monocytes and neutrophils. The oxidative burst was quantified by measuring the oxidation of 2',7'-dichlorofluorescein diacetate and the NO production was quantified by measuring the oxidation of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. A small increase of TLR4 expression by monocytes was observed after 6 h of LPS stimulation. Monocyte CD14 modulation by LPS was biphasic, with an initial 30 percent increase followed by a 40 percent decrease in expression after 6 h of incubation. Expression of CD11b was rapidly up-regulated, doubling after 5 min on monocytes, while down-regulation of CXCR2 was observed on neutrophils, reaching a 50 percent reduction after 6 h. LPS induced low production of ROS and NO. This study shows a complex LPS-induced cell surface receptor modulation on human monocytes and neutrophils, with up- and down-regulation depending on the receptor. R- and S-form LPS activate human neutrophils similarly, despite the low CD14 expression, if the stimulation occurs in whole blood.

Human monocytes tolerant to LPS retain the ability to phagocytose bacteria and generate reactive oxygen species

Tolerance to lipopolysaccharide (LPS) occurs when animals or cells exposed to LPS become hyporesponsive to a subsequent challenge with LPS. This mechanism is believed to be involved in the down-regulation of cellular responses observed in septic patients. The aim of this investigation was to evaluate LPS-induced monocyte tolerance of healthy volunteers using whole blood. The detection of intracellular IL-6, bacterial phagocytosis and reactive oxygen species (ROS) was determined by flow cytometry, using anti-IL-6-PE, heat-killed Staphylococcus aureus stained with propidium iodide and 2',7'-dichlorofluorescein diacetate, respectively. Monocytes were gated in whole blood by combining FSC and SSC parameters and CD14-positive staining. The exposure to increasing LPS concentrations resulted in lower intracellular concentration of IL-6 in monocytes after challenge. A similar effect was observed with challenge with MALP-2 (a Toll-like receptor (TLR)2/6 agonist) and killed Pseudomonas aeruginosa and S. aureus, but not with flagellin (a TLR5 agonist). LPS conditioning with 15 ng/mL resulted in a 40 percent reduction of IL-6 in monocytes. In contrast, phagocytosis of P. aeruginosa and S. aureus and induced ROS generation were preserved or increased in tolerant cells. The phenomenon of tolerance involves a complex regulation in which the production of IL-6 was diminished, whereas the bacterial phagocytosis and production of ROS was preserved. Decreased production of proinflammatory cytokines and preserved or increased production of ROS may be an adaptation to control the deleterious effects of inflammation while preserving antimicrobial activity.

Human monocytes tolerant to LPS retain the ability to phagocytose bacteria and generate reactive oxygen species.

Tolerance to lipopolysaccharide (LPS) occurs when animals or cells exposed to LPS become hyporesponsive to a subsequent challenge with LPS. This mechanism is believed to be involved in the down-regulation of cellular responses observed in septic patients. The aim of this investigation was to evaluate LPS-induced monocyte tolerance of healthy volunteers using whole blood. The detection of intracellular IL-6, bacterial phagocytosis and reactive oxygen species (ROS) was determined by flow cytometry, using anti-IL-6-PE, heat-killed Staphylococcus aureus stained with propidium iodide and 2',7'-dichlorofluorescein diacetate, respectively. Monocytes were gated in whole blood by combining FSC and SSC parameters and CD14-positive staining. The exposure to increasing LPS concentrations resulted in lower intracellular concentration of IL-6 in monocytes after challenge. A similar effect was observed with challenge with MALP-2 (a Toll-like receptor (TLR)2/6 agonist) and killed Pseudomonas aeruginosa and S. aureus, but not with flagellin (a TLR5 agonist). LPS conditioning with 15 ng/mL resulted in a 40% reduction of IL-6 in monocytes. In contrast, phagocytosis of P. aeruginosa and S. aureus and induced ROS generation were preserved or increased in tolerant cells. The phenomenon of tolerance involves a complex regulation in which the production of IL-6 was diminished, whereas the bacterial phagocytosis and production of ROS was preserved. Decreased production of proinflammatory cytokines and preserved or increased production of ROS may be an adaptation to control the deleterious effects of inflammation while preserving antimicrobial activity.

Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood.

Lipopolysaccharide (LPS) activates neutrophils and monocytes, inducing a wide array of biological activities. LPS rough (R) and smooth (S) forms signal through Toll-like receptor 4 (TLR4), but differ in their requirement for CD14. Since the R-form LPS can interact with TLR4 independent of CD14 and the differential expression of CD14 on neutrophils and monocytes, we used the S-form LPS from Salmonella abortus equi and the R-form LPS from Salmonella minnesota mutants to evaluate LPS-induced activation of human neutrophils and monocytes in whole blood from healthy volunteers. Expression of cell surface receptors and reactive oxygen species (ROS) and nitric oxide (NO) generation were measured by flow cytometry in whole blood monocytes and neutrophils. The oxidative burst was quantified by measuring the oxidation of 2',7'-dichlorofluorescein diacetate and the NO production was quantified by measuring the oxidation of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. A small increase of TLR4 expression by monocytes was observed after 6 h of LPS stimulation. Monocyte CD14 modulation by LPS was biphasic, with an initial 30% increase followed by a 40% decrease in expression after 6 h of incubation. Expression of CD11b was rapidly up-regulated, doubling after 5 min on monocytes, while down-regulation of CXCR2 was observed on neutrophils, reaching a 50% reduction after 6 h. LPS induced low production of ROS and NO. This study shows a complex LPS-induced cell surface receptor modulation on human monocytes and neutrophils, with up- and down-regulation depending on the receptor. R- and S-form LPS activate human neutrophils similarly, despite the low CD14 expression, if the stimulation occurs in whole blood.

Cytokine and hormonal profile in serum samples of patients undergoing controlled ovarian stimulation: interleukin-1beta predicts ongoing pregnancy.

BACKGROUND: Changes in the endometrium are not regulated exclusively by ovarian hormones; the immune system has also been implicated in normal endometrial function, similar to processes taking place during inflammatory and reparative path. Many cytokines are crucially important for reproductive processes, and the role of cytokines in the female reproductive system function has been broadly investigated during controlled ovarian stimulation (COS) for IVF attempts. The aim of this study was to evaluate the levels of serum cytokines and hormones, and the clinical outcomes of women who underwent COS and ICSI procedures. METHODS: The study prospectively included 96 patients (aged 22-43 years, unexplained or male infertility, n = 61; female infertility factors, n = 35) who underwent ICSI cycles. Serum levels of interleukin (IL-8, IL-6, IL-1beta, IL-10, IL-12), tumour necrosis factor and leukaemia-inhibitory factor (LIF) and the hormones FSH, estradiol, progesterone, anti-Mullerian hormone and Inhibin-B were measured on the day of oocyte retrieval. RESULTS: The ongoing pregnancy rate was 25.3%. The presence of serum IL-1beta positively affected the implantation rate (P = 0.004) and increased the chance of becoming pregnant by 15 fold. Furthermore, the percentage of patients with detectable serum IL-1beta levels who conceived (62.5%) was higher than those who failed to conceive (37.5%; P = 0.019). The LIF was undetectable in all serum samples, and no other factors influenced the clinical outcomes of patients undergoing ICSI cycles. CONCLUSIONS: Our findings revealed that detectable serum levels of IL-1beta on the day of oocyte retrieval in patients undergoing COS and ICSI are predictive of successful implantation and ongoing pregnancy.