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Ana Ellen Queiroz Santiago, Plinio da Cunha Leal, Ed Carlos Rey Moura, Reinaldo Salomão, Milena Karina Coló Brunialti, Rioko Kimiko Sakata Department of Anesthesia, Universidade Federal de São Paulo, São Paulo, Brazil Background: Pregabalin is an anticonvulsant and has been used for postoperative analgesia. This study aimed to assess the effect of a single preoperative dose of pregabalin for analgesia after nephrectomy. Methods: The study was prospective, randomized, comparative, and double-blinded, conducted in 40 kidney transplant donors, between 18 and 60 years, American Society of Anesthesia physical status I or II. Epidural anesthesia was performed with 15 mL of 0.5% ropivacaine single shot and general anesthesia with 3 µg/kg of fentanyl, propofol, atracurium, and sevoflurane, and 50% of oxygen without nitrous oxide. Patients in group 1 were administered 300 mg of pregabalin and those in group 2 were administered placebo, in identical capsules, 1 hour prior to surgery. Postoperative analgesia was supplemented with tramadol. The following parameters were assessed: pain intensity after 6 and 24 hours; pain threshold, from the thenar and peri-incisional region, analgesic supplementation; ILs (IL6, IL8, and IL10) prior to surgery and after 6 and 24 hours. Results: The pain intensity was lower with pregabalin after 24 hours (G1: 2.5±2.4, G2: 3.0±2.6). There was no difference in the sensitivity of the thenar and peri-incisional region after 6 and 24 hours; in the number of patients requiring supplementation (G1=15%, G2=45%); concentrations of IL-6, IL-8, and IL-10; and side effects (nausea, vomiting, dizziness, and pruritus). Conclusion: Pregabalin in a single preoperative dose of 300 mg reduced pain intensity 24 hours after lumbotomy. Keywords: pregabalin, lumbotomy, postoperative analgesia, kidney transplant, nephrectomy

The clinical presentation of COVID-19 is highly variable, and understanding the underlying biological processes is crucial. This study utilized a proteomic analysis to investigate dysregulated processes in the peripheral blood mononuclear cells of patients with COVID-19 compared to healthy volunteers. Samples were collected at different stages of the disease, including hospital admission, after 7 days of hospitalization, and 30 days after discharge. Metabolic pathway alterations and increased abundance of neutrophil-related proteins were observed in patients. Patients progressing to critical illness had significantly low-abundance proteins in the pentose phosphate and glycolysis pathways compared with those presenting clinical recovery. Important biological processes, such as fatty acid concentration and glucose metabolism disorder, remained altered even after 30 days of hospital discharge. Temporal proteomic changes revealed distinct pathways in critically ill and non-critically ill patients. Our study emphasizes the significance of longitudinal cellular proteomic studies in identifying disease progression-related pathways and persistent protein changes post-hospitalization., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Background and Objectives: Sepsis is a complex disease that is characterized by activation and inhibition of different cell signaling pathways according to the disease stage. Here, we evaluated genes involved in the TLR signaling pathway, oxidative phosphorylation and oxidative metabolism, aiming to assess their interactions and resulting cell functions and pathways that are disturbed in septic patients., Materials and Methods: Blood samples were obtained from 16 patients with sepsis secondary to community acquired pneumonia at admission (D0), and after 7 days (D7, N = 10) of therapy. Samples were also collected from 8 healthy volunteers who were matched according to age and gender. Gene expression of 84 genes was performed by real-time polymerase chain reactions. Their expression was considered up- or down-regulated when the fold change was greater than 1.5 compared to the healthy volunteers. A p-value of ≤ 0.05 was considered significant., Results: Twenty-two genes were differently expressed in D0 samples; most of them were down-regulated. When gene expression was analyzed according to the outcomes, higher number of altered genes and a higher intensity in the disturbance was observed in non-survivor than in survivor patients. The canonical pathways altered in D0 samples included interferon and iNOS signaling; the role of JAK1, JAK2 and TYK2 in interferon signaling; mitochondrial dysfunction; and superoxide radical degradation pathways. When analyzed according to outcomes, different pathways were disturbed in surviving and non-surviving patients. Mitochondrial dysfunction, oxidative phosphorylation and superoxide radical degradation pathway were among the most altered in non-surviving patients., Conclusion: Our data show changes in the expression of genes belonging to the interacting TLR cascades, NADPH-oxidase and oxidative phosphorylation. Importantly, distinct patterns are clearly observed in surviving and non-surviving patients. Interferon signaling, marked by changes in JAK-STAT modulation, had prominent changes in both survivors and non-survivors, whereas the redox imbalance (iNOS signaling, oxidative phosphorylation and superoxide radical degradation) affecting mitochondrial functions was prominent in non-surviving patients., Competing Interests: The authors have declared that no competing interests exist.

Background: The nature of the inflammatory response underscoring the pathophysiology of sepsis has been extensively studied. We hypothesized that different cell functions would be differentially regulated in a patient with sepsis. We evaluated the modulation of monocyte functions during sepsis by simultaneously assessing their phagocytic activity, the generation of reactive oxygen species (ROS) and nitric oxide (NO), and the production of inflammatory cytokines (IL-6 and TNF-α)., Methods: Whole blood was obtained from patients with severe sepsis and septic shock both at admission (D0, n = 34) and after seven days of therapy (D7, n = 15); 19 healthy volunteers were included as a control group. The cells were stimulated with LPS, Pseudomonas aeruginosa, and Staphylococcus aureus. The ROS and NO levels were quantified in monocytes in whole blood by measuring the oxidation of 2,7-dichlorofluorescein diacetate and 4-amino-5-methylamino-2,7-difluorofluorescein diacetate, respectively. Intracellular IL-6 and TNF-α were detected using fluorochrome-conjugated specific antibodies. Monocyte functions were also evaluated in CD163+ and CD163- monocyte subsets., Results: The monocytes from septic patients presented with preserved phagocytosis, enhanced ROS and NO generation, and decreased production of inflammatory cytokines compared with the monocytes from healthy volunteers. TNF-α and IL-6 increased and ROS generation decreased in D7 compared with D0 samples. In general, CD163+ monocytes produced higher amounts of IL-6 and TNF-α and lower amounts of ROS and NO than did CD163- monocytes., Conclusions: We demonstrated that monocytes from septic patients, which are impaired to produce inflammatory cytokines, display potent phagocytic activity and increased ROS and NO generation.

Aim: Regarding the use of intravenous lidocaine in fibromyalgia, there are no well-controlled studies. This study aimed to evaluate the effect of intravenous lidocaine on pain intensity, clinical manifestations and plasma levels of interleukin (IL)-1, IL-6, and IL-8 in fibromyalgia patients., Methods: In a randomized double-blind study, group 1 patients received 240 mg of lidocaine in 125 mL of saline solution, while group 2 patients received 125 mL of saline, both once a week for 4 weeks (T1, T2, T3 and T4). All patients received amitriptyline. The following were assessed: pain intensity before treatment (T0) and at 1, 2, 3, 4 and 8 weeks after treatment; clinical manifestations; the fibromyalgia impact questionnaire (FIQ) before and at 4 and 8 weeks after; the levels of IL 1, 6 and 8 before and at 4 and 8 weeks after treatment., Results: Lower pain intensity was observed in the lidocaine group at T2, with no difference at the other time points. There was a reduction in pain intensity in both groups. The use of paracetamol and tramadol and plasma levels of IL-1, IL-6 and IL-8 did not differ between the groups. Clinical manifestations and side effects did not differ between groups., Conclusions: The combination of 240 mg of intravenous lidocaine (once a week for 4 weeks) with 25 mg of amitriptyline for 8 weeks had no meaningful impact in fibromyalgia patients., (© 2016 Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.)

Study Objective: Opioids are associated with hyperalgesia that can reduce their analgesic effect. The aim of this study was to determine whether the addition of ketamine reduces remifentanil-induced hyperalgesia; improves its analgesic effect; and alters interleukin 6 (IL-6), IL-8, and IL-10 levels., Design: This is a prospective, randomized, double-blind study., Setting: The setting is in a operating room and ward in a university hospital., Patients: There are 56 patients, aged ≥18 years, American Society of Anesthesiologists I or II, who underwent laparoscopic cholecystectomy., Interventions: Anesthesia was induced with remifentanil, 50% oxygen, and isoflurane. Patients randomized to group 1 received remifentanil (0.4 μg/kg per minute) and ketamine (5 μg/kg per minute), and patients randomized to group 2 received remifentanil (0.4 μg/kg per minute) and saline solution. Postoperative analgesia was achieved using morphine via patient-controlled analgesia., Measurements: The measurements were postoperative pain intensity during 24 hours; morphine consumption; time to first morphine supplementation; hyperalgesia (using monofilaments and an algometer) and allodynia (using a soft brush) in the thenar eminence of the nondominant hand and in the periumbilical region 24 hours after surgery; extent of hyperalgesia using a 300-g monofilament near the periumbilical region 24 hours after surgery; and serum levels of IL-6, IL-8, and IL-10., Main Results: Groups were similar for baseline characteristics. There were no differences in pain intensity, time to first request of morphine, and total 24 hours dose of morphine between groups. There was a difference in hyperalgesia using monofilaments 24 hours after the surgery in the thenar eminence of the nondominant hand, with a better profile for the experimental group. However, there were no differences in hyperalgesia using an algometer, in allodynia using a soft brush; in extent of hyperalgesia; or in levels of IL-6, IL-8, and IL-10., Conclusions: It was not possible to demonstrate that the addition of ketamine (5 μg/kg per minute) is effective in preventing or reducing remifentanil-induced postoperative hyperalgesia in laparoscopic cholecystectomy., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Mechanisms governing the inflammatory response during sepsis involve crosstalk between diverse signaling pathways, but current knowledge provides an incomplete picture of the syndrome. Microarray-based expression profiling is a powerful approach for the investigation of complex clinical conditions such as sepsis. In this study, we investigated whole-genome expression profiles in mononuclear cells from septic patients admitted in intensive care units with community-acquired pneumonia. Blood samples were collected at the time of sepsis diagnosis and seven days later since we aimed to evaluate the role of biological processes or genes possibly involved in patient recovery. Here we provide a detailed description of the study design, including clinical information, experimental methods and procedures regarding data analysis. Metadata corresponding to microarray results deposited in the database Gene Expression Omnibus (GEO) under the accession number GSE48080 are also described in this report. Our dataset allows the identification of genes possibly associated with host defense to infection as well as gene expression patterns associated with patient outcome.

Surfacen® is a clinical surfactant preparation of porcine origin. In the present study, we have evaluated the effect of Surfacen® in the modulation of oxidative burst in monocytes and neutrophils in human blood and pro-inflammatory cytokine production in peripheral blood mononuclear cells (PBMC). Reactive oxygen species (ROS) level was measured in monocytes and neutrophils by flow cytometry using 2,7-dichlorofluorescein diacetate (DCFH-DA) as substrate, while, tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels were estimated in PBMC supernatant by enzyme-linked immunosorbent assays (ELISA). Our results show that Staphylococcus aureus-induced ROS level was slightly affected by Surfacen® added to whole blood monocytes and neutrophils. The time course experiments of pre-incubation with Surfacen® showed no significant increase of ROS level at 2h; however, the ROS level decreased when pre incubated for 4h and 6h with Surfacen®. Pre-incubation of PBMC cells with Surfacen® at 0.125 and 0.5mg/mL showed a dose-dependent suppression of TNF-α levels measured after 4h of S. aureus stimulation, an effect less impressive when cells were stimulated for 24h. A similar behavior was observed in IL-6 release. In summary, the present study provides experimental evidence supporting an anti-inflammatory role of Surfacen® in human monocytes and neutrophils in vitro., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.

Mechanisms governing the inflammatory response during sepsis have been shown to be complex, involving cross-talk between diverse signaling pathways. Current knowledge regarding the mechanisms underlying sepsis provides an incomplete picture of the syndrome, justifying additional efforts to understand this condition. Microarray-based expression profiling is a powerful approach for the investigation of complex clinical conditions such as sepsis. In this study, we investigate whole-genome expression profiles in mononuclear cells from survivors (n = 5) and non-survivors (n = 5) of sepsis. To circumvent the heterogeneity of septic patients, only patients admitted with sepsis caused by community-acquired pneumonia were included. Blood samples were collected at the time of sepsis diagnosis and seven days later to evaluate the role of biological processes or genes possibly involved in patient recovery. Principal Components Analysis (PCA) profiling discriminated between patients with early sepsis and healthy individuals. Genes with differential expression were grouped according to Gene Ontology, and most genes related to immune defense were up-regulated in septic patients. Additionally, PCA in the early stage was able to distinguish survivors from non-survivors. Differences in oxidative phosphorylation seem to be associated with clinical outcome because significant differences in the expression profile of genes related to mitochondrial electron transport chain (ETC) I-V were observed between survivors and non-survivors at the time of patient enrollment. Global gene expression profiles after seven days of sepsis progression seem to reproduce, to a certain extent, patterns collected at the time of diagnosis. Gene expression profiles comparing admission and follow-up samples differed between survivors and non-survivors, with decreased expression of genes related to immune functions in non-survivors. In conclusion, genes related to host defense and inflammatory response ontology were up-regulated during sepsis, consistent with the need for a host response to infection, and the sustainability of their expression in follow-up samples was associated with outcomes.

Alzheimer's disease (AD) is a late-onset, progressive degenerative disorder that affects mainly the judgment, emotional stability, and memory domains. AD is the outcome of a complex interaction among several factors which are not fully understood yet; nevertheless, it is clear that oxidative stress and inflammatory pathways are among these factors. 65 elderly subjects (42 cognitively intact and 23 with probable Alzheimer's disease) were selected for this study. We evaluated erythrocyte activities of superoxide dismutase, catalase, and glutathione peroxidase as well as plasma levels of total glutathione, α-tocopherol, β-carotene, lycopene, and coenzyme Q10. These antioxidant parameters were confronted with plasmatic levels of protein and lipid oxidation products. Additionally, we measured basal expression of monocyte HLA-DR and CD-11b, as well as monocyte production of cytokines IL1-α, IL-6, and TNF-α. AD patients presented lower plasmatic levels of α-tocopherol when compared to control ones and also higher basal monocyte HLA-DR expression associated with higher IL-1α production when stimulated by LPS. These findings support the inflammatory theory of AD and point out that this disease is associated with a higher basal activation of circulating monocytes that may be a result of α-tocopherol stock depletion.

Since the definition of systemic inflammatory response syndrome/sepsis was originally proposed, a large amount of new information has been generated showing a much more complex scenario of inflammatory and counterinflammatory responses during sepsis. Moreover, some fundamental mechanisms of sensing and destroying invading microorganisms have been uncovered, which include the discovery of TLR4 as the lipopolysaccharide (LPS) gene, implications of innate immune cells as drivers of the adaptive response to infection, and the modulation of multiple accessory molecules that stimulate or inhibit monocyte/macrophage and lymphocyte interactions. The complexity of the infection/injury-induced immune response could be better appreciated with the application of genomics and proteomics studies, and LPS was a useful tool in many of these studies. In this review, we discuss aspects of bacterial recognition and induced cellular activation during sepsis. Because of the relevance of endotoxin (LPS) research in the field, we focus on LPS and host interactions as a clue to understand microorganisms sensing and cell signaling, then we discuss how this response is modulated in septic patients.

In this study, our aims were to evaluate the reactive oxygen species (ROS) and nitric oxide (NO) generation by monocytes and neutrophils from septic patients and to correlate their levels with clinical outcomes. Forty-nine septic patients and 19 healthy volunteers were enrolled in the study. The ROS and NO production was quantified in monocytes and neutrophils in whole blood by flow cytometry, constitutively, and after stimulation with Staphylococcus aureus and Pseudomonas aeruginosa. Nitric oxide production by monocytes was higher in septic patients compared with healthy volunteers for all conditions and by neutrophils at baseline, and ROS generation in monocytes and neutrophils was higher in septic patients than in healthy volunteers for all conditions. Nitric oxide production by monocytes and neutrophils was decreased at day 7 compared with that at admission (day 0) in survivors at baseline and after stimulation with S. aureus. Reactive oxygen species production by the monocytes and neutrophils was decreased in survivors at day 7 compared with day 0 under all conditions, except by neutrophils at baseline. No difference was found in NO and ROS generation by monocytes and neutrophils between day 7 and day 0 in nonsurvivors. Generation of NO and ROS by neutrophils and monocytes is increased in septic patients, and their persistence is associated with poor outcome.

Background: A shift from Th1 to Th2 as well as an increase in Treg CD4+T cell subsets has been reported in septic patients (SP). Furthermore, these patients display modulation of monocyte function, with reduced production of pro-inflammatory cytokines upon LPS stimulus, which resembles the phenotype of alternatively activated macrophages. In this study, we evaluated the percentages of T cells differentiated into Th1, Th17 and Treg subsets, as well as the percentage of monocytes expressing markers of alternatively activated monocytes/macrophages (AAM) in SP., Methodology/principal Findings: Peripheral blood mononuclear cells (PBMC) were obtained from 32 healthy volunteers (HV) and from SP at admission (D0, n = 67) and after 7 days of therapy (D7, n = 33). Th1 and Th17 (CD3+CD8-) lymphocytes were identified by the intracellular detection of IFN-γ and IL-17, respectively, spontaneously and after PMA/Io stimulation, and Treg cells were identified by Foxp3+CD127- expression. Monocytes were evaluated for CD206 and CD163 expression. Absolute numbers of CD4+T lymphocytes were measured in whole blood samples by flow cytometry. The Mann-Whitney or Wilcoxon test was applied, as appropriate. The percentage of Th1 cells was lower in SP than in HV at admission after PMA/Io stimulation, whereas the percentage of Th17 cells was higher. In patients' follow-up samples, a higher percentage of Th1 cells and a lower percentage of Th17 cells were observed on D7 compared with the D0 samples. Treg cells remained unchanged. Septic patients showed a markedly increased proportion of monocytes expressing CD163 and CD206., Conclusions/significance: Upon in vitro stimulus, the percentage of T helper lymphocytes producing IL-17 was higher in SP than in HV at admission, and the percentage producing IFN-γ was lower, a pattern that was reversed during follow-up. The increased expression of CD163 and CD206 indicates that monocytes may acquire the AAM phenotype during sepsis.

Background: Increased numbers of endothelial (EMP) and platelet (PMP) microparticles have been related to cardiovascular risk factors and coronary artery disease. Little is known about the early effects of statins and clopidogrel on these new biomarkers of vascular homeostasis. The aim of the present study was to evaluate pharmacokinetic interactions between atorvastatin and clopidogrel and their effects, alone or combined, on EMP, PMP, and endothelial progenitor cells (EPC)., Methods and Results: A prospective open-label study enrolled subjects with stable coronary disease (n=26). Drugs were given daily for 3 weeks (atorvastatin 80 mg, visits 1-3; clopidogrel 75 mg, visits 2-4). Counts of EPC (CD34+/CD133+/KDR+), EMP (CD51+) and PMP (CD42+/CD31+), and pharmacokinetic parameters over 24h were assessed at each visit. Atorvastatin plasma concentrations were increased by concomitant therapy with clopidogrel (maximum serum concentration [C(max)], P=0.002; area under the clopidogrel or atorvastatin plasma concentration vs. time curve from 0 to the last detectable concentration [AUC(last)], P=0.03). After atorvastatin withdrawal there was an increase in clopidogrel plasma concentrations (C(max), P=0.009; AUC(last), P=0.039). PMP were inversely correlated with clopidogrel C(max) on visit 3 (rho=-0.57, P=0.006) and on visit 4 (rho=-0.54, P=0.01), and with clopidogrel AUC(last) on visit 3 (rho=-0.44, P=0.04), and on visit 4 (rho=-0.57, P=0.005). In addition, clopidogrel C(max) was correlated with EPC (CD133+/KDR+) on visit 4 (rho=0.48, P=0.025). No correlations of atorvastatin and MP or EPC were found., Conclusions: The balance between platelet MP release and EPC mobilization seems influenced by clopidogrel plasma levels, suggesting a protective mechanism on coronary artery disease.

The pathogenetic mechanisms associated to the beneficial effects of mixed venous oxygen saturation (SvO(2))-guided resuscitation during sepsis are unclear. Our purpose was to evaluate the effects of an algorithm of SvO(2)-driven resuscitation including fluids, norepinephrine and dobutamine on hemodynamics, inflammatory response, and cardiovascular oxidative stress during a clinically resembling experimental model of septic shock. Eighteen anesthetized and catheterized pigs (35-45 kg) were submitted to peritonitis by fecal inoculation (0.75 g/kg). After hypotension, antibiotics were administered, and the animals were randomized to two groups: control (n = 9), with hemodynamic support aiming central venous pressure 8 to 12 mmHg, urinary output 0.5 mL/kg per hour, and mean arterial pressure greater than 65 mmHg; and SvO(2) (n = 9), with the goals above, plus SvO(2) greater than 65%. The interventions lasted 12 h, and lactated Ringer's and norepinephrine (both groups) and dobutamine (SvO(2) group) were administered. Inflammatory response was evaluated by plasma concentration of cytokines, neutrophil CD14 expression, oxidant generation, and apoptosis. Oxidative stress was evaluated by plasma and myocardial nitrate concentrations, myocardial and vascular NADP(H) oxidase activity, myocardial glutathione content, and nitrotyrosine expression. Mixed venous oxygen saturation-driven resuscitation was associated with improved systolic index, oxygen delivery, and diuresis. Sepsis induced in both groups a significant increase on IL-6 concentrations and plasma nitrate concentrations and a persistent decrease in neutrophil CD14 expression. Apoptosis rate and neutrophil oxidant generation were not different between groups. Treatment strategies did not significantly modify oxidative stress parameters. Thus, an approach aiming SvO(2) during sepsis improves hemodynamics, without any significant effect on inflammatory response and oxidative stress. The beneficial effects associated with this strategy may be related to other mechanisms.

This study was designed to assess the effect of GB virus (GBV)-C on the immune response to human immunodeficiency virus (HIV) in chronically HIV-infected and HIV- hepatitis C virus (HCV)-co-infected patients undergoing antiretroviral therapy. A cohort of 159 HIV-seropositive patients, of whom 52 were HCV-co-infected, was included. Epidemiological data were collected and virological and immunological markers, including the production of interferon gamma (IFN-γ) and interleukin (IL)-2 by CD4, CD8 and Tγδ cells and the expression of the activation marker, CD38, were assessed. A total of 65 patients (40.8%) presented markers of GBV-C infection. The presence of GBV-C did not influence HIV and HCV replication or TCD4 and TCD8 cell counts. Immune responses, defined by IFN-γ and IL-2 production and CD38 expression did not differ among the groups. Our results suggest that neither GBV-C viremia nor the presence of E2 antibodies influence HIV and HCV viral replication or CD4 T cell counts in chronically infected patients. Furthermore, GBV-C did not influence cytokine production or CD38-driven immune activation among these patients. Although our results do not exclude a protective effect of GBV-C in early HIV disease, they demonstrate that this effect may not be present in chronically infected patients, who represent the majority of patients in outpatient clinics.

Pre-exposure to low doses of LPS induces resistance to a lethal challenge, a phenomenon known as endotoxin tolerance. In this study, tolerance was induced in human PBMC by culturing cells with 1 ng/mL LPS for 48 h. Cells were subsequently challenged with 100 ng/mL LPS for 2, 6 and 24 h, and the expression of 84 genes encoding proteins involved in the TLR signaling pathway was evaluated at each time point by PCR array. LPS pretreatment did not modulate the expression of TLR4 and CD14 on the surface of monocytes. A gene was defined as tolerized when LPS pretreatment reversed the effect of LPS challenge on the expression of the gene or as non-tolerized when LPS pretreatment did not reverse the effects of LPS challenge. We observed impaired signal transduction through the NF-κB, JNK, ERK and TRIF pathways, whereas expression of p38 pathway-related genes was preserved in LPS-tolerant cells. These results show a distinct regulation of the TLR pathway cascades during tolerance; this may account for the differential gene expression of some inflammatory mediators, such as up-regulation of IL-10 and COX2 as well as down-regulation of TNF-α and IL-12. Depending on the effect of LPS-induced gene up-regulation or down-regulation, tolerance, as a reversion of such LPS effects, may result in repression or induction of gene expression., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Introduction: TLR-4 has also been identified as a receptor for endogenous alarmins, which are increased post transplantation. TLR-4 has also been associated with a polymorphism that could impact graft outcome., Objective: To assess the expression of TLR-4 in kidney transplant patients carrying or not a polymorphism., Methods: TLR-4 polymorphism (A299G/T399I) was studied in 200 renal transplant patients. Healthy volunteers were also enrolled as control group. The polymorphism analysis was performed using restriction enzymes technique (RFLP). Functionality of TLR-4 polymorphism was assessed in samples from controls by quantification of TNF-α after LPS stimulus. TLR-4 and -2 expressions were also analyzed by flow cytometry., Results: TLR-4 polymorphism was present in 8.5% of renal transplant patients. This polymorphism was associated with impairment in TNF-α secretion. In general, in renal transplant patients, TLR-4 expression in monocytes and in neutrophils was lower than in health volunteers. TLR-2 and TLR-4 expressions in healthy volunteers with A299G/T399I TLR-4 polymorphism was higher than in wild-type genotype healthy volunteers (p<0.01 and p<0.05, respectively), and also higher than A299G/T399I TLR-4 polymorphism renal transplant patients (p<0.05). TLR-2 expression on neutrophils in wild-type genotype renal transplant patients was higher compared to wild-type genotype healthy volunteers, and was also higher in relation to A299G/T399I kidney transplanted patients (p<0.01)., Conclusion: Stable renal transplant patients with TLR-4 polymorphism have a lower expression of TLR-4 and TLR-2 receptors in peripheral mononuclear cells, which ultimately indicate a less responsiveness for alarmins., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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.

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.

Acute respiratory distress syndrome secondary to sepsis is associated with high morbidity and mortality. The purpose of this study was to characterize the effects of ventilatory strategy and the modulating activity of pentoxifylline in a sepsis-induced lung dysfunction model. Male Wistar rats were randomly divided into six groups, undergoing two different ventilatory strategies. Rats received live Escherichia coli or saline intraperitoneally. After 6 hours, the septic animals were treated with either pentoxifylline (25 mg/kg for 20 minutes) or normal saline infusion and ventilated with low tidal volume (6 mL/kg; septic animals with E. coli intraperitoneal [IP] infusion, PTX-treated and ventilated with low tidal volume and septic animals with E. coli IP infusion and ventilated with low tidal volume, respectively) or high tidal volume (12 mL/kg; septic animals with E. coli IP infusion, PTX-treated and ventilated with high tidal volume and septic animals with E. coli IP infusion and ventilated with high tidal volume, respectively) for 3 hours. The control animals received normal saline infusion and, after 6 hours, were ventilated with low or high tidal volume (control animals with saline infusion and ventilated with low tidal volume and control animals with saline infusion and ventilated with high tidal volume, respectively). Lung dysfunctions were assessed by wet-to-dry lung ratios, total cell count, total protein, malondialdehyde, and tumor necrosis factor-alpha concentrations in bronchoalveolar lavage (BAL). Septic animals with E. coli IP infusion and ventilated with high tidal volume presented increased wet-to-dry lung ratios, total cell count, total protein, and malondialdehyde in BAL compared with the septic animals ventilated with low tidal volume. Septic animals treated with pentoxifylline presented higher arterial oxygenation and lower cellular influx, protein leakage, malondialdehyde concentration, and tumor necrosis factor-alpha levels in BAL compared with septic animals undergoing the same ventilatory support strategies (septic animals with E. coli IP infusion and ventilated with low tidal volume and septic animals with E. coli IP infusion and ventilated with high tidal volume). Ventilatory strategy modulated the inflammatory response and pulmonary alterations in a sepsis-induced acute lung injury model, and these effects are improved by pentoxifylline.

Objectives: Up- and down-regulation of inflammatory response was described in blood cells from septic patients, according to the stage of sepsis and the cells evaluated. This study aimed to evaluate the Toll-like receptor (TLR) signaling pathway gene expression in peripheral blood mononuclear cells (PBMC) and neutrophils in patients throughout the different stages of sepsis., Design: Prospective, observational study., Settings: Two emergency rooms and two intensive care units in one university and one teaching hospital., Patients and Controls: A total of 15 septic patients, five with sepsis, five with severe sepsis, and five with septic shock, in addition to five healthy volunteers were enrolled., Interventions: None., Measurements and Main Results: The Human-TLR Signaling Pathway, which comprises 84 genes related to TLR-mediated signal transduction, was evaluated by real time polymerase chain reaction in PBMC and neutrophils obtained from patients and controls. The fold change for each gene (2(-Delta DeltaCt)) was compared between the groups. Genes with fold changes greater than 2 and significant changes in DeltaCT are reported as differently expressed. The fold change ratios in PBMC gene expression between septic patients and healthy controls revealed a dynamic process according to the stage of sepsis, tending toward down-regulation of the TLR signaling pathway in PBMC in the more severe forms of the disease. However, the differential gene expression was restricted to five down-regulated genes in septic shock patients, which are found in the effector and downstream pathways. Neutrophils showed a different pattern of adaptation. Patients with sepsis, severe sepsis, and septic shock presented a broad gene up-regulation, which included all functional groups evaluated and persisted throughout the stages of the disease., Conclusions: TLR-signaling pathway genes are differently regulated in PBMC and neutrophils of septic patients, and are dynamically modulated throughout the different stages of sepsis.

We evaluated the recovery of cardiovascular function after transient cardiogenic shock. Cardiac tamponade was performed for 1 h and post-shock data were collected in 5 domestic large white female pigs (43 +/- 5 kg) for 6 h. The control group (N = 5) was observed for 6 h after 1 h of resting. During 1 h of cardiac tamponade, experimental animals evolved a low perfusion status with a higher lactate level (8.0 +/- 2.2 vs 1.9 +/- 0.9 mEq/L), lower standard base excess (-7.3 +/- 3.3 vs 2.0 +/- 0.9 mEq/L), lower urinary output (0.9 +/- 0.9 vs 3.0 +/- 1.4 mL x kg(-1) x h(-1)), lower mixed venous saturation, higher ileum partial pressure of CO2-end tidal CO2 (EtCO2) gap and a lower cardiac index than the control group. Throughout the 6-h recovery phase after cardiac tamponade, tamponade animals developed significant tachycardia with preserved cardiac index, resulting in a lower left ventricular stroke work, suggesting possible myocardial dysfunction. Vascular dysfunction was present with persistent systemic hypotension as well as persistent pulmonary hypertension. In contrast, oliguria, hyperlactatemia and metabolic acidosis were corrected by the 6th hour. The inflammatory characteristics were an elevated core temperature and increased plasma levels of interleukin-6 in the tamponade group compared to the control group. We conclude that cardiovascular recovery after a transient and severe low flow systemic state was incomplete. Vascular dysfunction persisted up to 6 h after release of tamponade. These inflammatory characteristics may also indicate that inflammatory activation is a possible pathway involved in the pathogenesis of cardiogenic shock.

Background: CD4+CD25high regulatory T (TReg) cells modulate antigen-specific T cell responses, and can suppress anti-viral immunity. In HTLV-1 infection, a selective decrease in the function of TReg cell mediated HTLV-1-tax inhibition of FOXP3 expression has been described. The purpose of this study was to assess the frequency and phenotype of TReg cells in HTLV-1 asymptomatic carriers and in HTLV-1-associated neurological disease (HAM/TSP) patients, and to correlate with measures of T cell activation., Results: We were able to confirm that HTLV-I drives activation, spontaneous IFNgamma production, and proliferation of CD4+ T cells. We also observed a significantly lower proportion of CTLA-4+ TReg cells (CD4+CD25high T cells) in subjects with HAM/TSP patients compared to healthy controls. Ki-67 expression was negatively correlated to the frequency of CTLA-4+ TReg cells in HAM/TSP only, although Ki-67 expression was inversely correlated with the percentage of CD127low TReg cells in healthy control subjects. Finally, the proportion of CD127low TReg cells correlated inversely with HTLV-1 proviral load., Conclusion: Taken together, the results suggest that TReg cells may be subverted in HAM/TSP patients, which could explain the marked cellular activation, spontaneous cytokine production, and proliferation of CD4+ T cells, in particular those expressing the CD25highCD127low phenotype. TReg cells represent a potential target for therapeutic intervention for patients with HTLV-1-related neurological diseases.

Objective: The aim of this study was to characterize using flow cytometry cultured human primary keratinocytes isolated from the epidermis and hair follicles by different methods., Methods: Human keratinocytes derived from discarded fragments of total skin and scalp hair follicles from patients who underwent plastic surgery in the Plastic Surgery Division at UNIFESP were used. The epidermal keratinocytes were isolated by using 3 different methods: the standard method, upon exposure to trypsin for 30 minutes; the second, by treatment with dispase for 18 hours and with trypsin for 10 minutes; and the third, by treatment with dispase for 18 hours and with trypsin for 30 minutes. Follicular keratinocytes were isolated using the standard method., Results: On comparing the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with dispase for 18 hours and with trypsin for 30 minutes, it was observed that the first group presented the largest number of viable cells, the smallest number of cells in late apoptosis and necrosis with statistical significance, and no difference in apoptosis. When we compared the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with trypsin, the first group presented the largest number of viable cells, the smallest number of cells in apoptosis with statistical significance, and no difference in late apoptosis and necrosis. When we compared the results of the group treated with dispase for 18 hours and with trypsin for 10 minutes with the results for follical isolation, there was a statistical difference in apoptosis and viable cells., Conclusion: The isolation method of treatment with dispase for 18 hours and with trypsin for 10 minutes produced the largest number of viable cells and the smallest number of cells in apoptosis/necrosis.

Background: Infection control depends on adequate microbe recognition and cell activation, yet inflammatory response may lead to organ dysfunction in sepsis. The aims of this study were to evaluate cell activation in the context of sepsis and its correlation with organ dysfunction., Methods: A total of 41 patients were prospectively enrolled: 14 with sepsis, 12 with severe sepsis and 15 with septic shock. A total of 17 healthy volunteers were included as a control group. Patients were admitted to the Intensive Care Units and Emergency Rooms of Hospital Sao Paulo (Federal University of Sao Paulo) and Hospital Santa Marcelina, Sao Paulo, Brazil. Toll-like receptor (TLR)2, TLR4, CD11b, CD11c and CD66b expression on neutrophil surfaces and oxidative metabolism measured by non-fluorescent dichlorofluorescein (DCFH) oxidation in neutrophils and monocytes, using whole blood, were evaluated using flow cytometry. Organ dysfunction was measured using the sepsis-associated organ failure assessment (SOFA) score., Results: TLR2 expression on neutrophils was found to be downregulated in septic shock patients compared to healthy volunteers (p = 0.05). No differences were found in CD11b and CD11c expression. CD66b expression was increased in the patient group compared to the control group (p = 0.01). Neutrophil and monocyte oxidative burst was increased in septic patients compared to the control group at baseline and after stimulation with phorbol myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (fMLP), lipopolysaccharide (LPS) and Staphylococcus aureus (p < 0.001 and p < 0.01, respectively, for neutrophils and monocytes in all tested conditions). A strong correlation was observed between neutrophil and monocyte oxidative metabolism. A SOFA score of 7 discriminated patients between survivors and non-survivors (area under the curve for reactive oxygen species (ROS) was 0.78; p = 0.02). ROS generation in patients with sepsis and septic shock with SOFA scores > 7 was higher than in patients with SOFA scores < 7, both in neutrophils and monocytes. However, oxidative burst in patients with sepsis was as high as in septic shock., Conclusion: Surface receptors expression on neutrophils may be modulated across the continuum of sepsis, and enhanced or decreased expression may be found depending on the receptor considered. ROS generation is upregulated both in neutrophils and monocytes in septic patients, and it is differently modulated depending on the stage of the disease and the stimuli used.

Purpose: Pathophysiological studies in humans regarding sepsis are difficult to perform due to ethical and methodological concerns. In this context, animal models of sepsis can be useful to better understand this condition and to test therapeutic strategies. The purpose of this study was to characterize a feasible and clinically relevant model of sepsis in pigs that could be useful for testing different therapeutic interventions., Methods: 5 White Large pigs were anesthetized, arterial and pulmonary catheters were introduced, and sepsis was induced by fecal peritonitis. Several biochemical indicators of organ dysfunction and infectious parameters were measured. The pigs were monitored until death, when fragments of organs were removed for pathology. Three animals without peritonitis served as controls and were sacrificed 24 hours after surgery without developing significant changes in organ function., Results: Septic pigs survived 17 hours on average (range, 16-18 h), and Escherichia coli was recovered from blood cultures. They developed a significant decrease in left ventricular work and a nonsignificant reduction in mixed venous oxygen saturation. Respiratory dysfunction was characterized by a decrease in the PaO2/FiO2 ratio and respiratory compliance. Pathology of the lungs revealed areas of pulmonary collapse, hemorrhage, pulmonary congestion, and discrete neutrophil infiltrate., Conclusions: Fecal peritonitis in pigs is a clinically relevant model of sepsis associated with acute lung injury without direct pulmonary insult. This model may prove to be useful for studying pathogenic aspects of secondary lung injury as well as for validating ventilatory or pharmacologic interventions.

Experimental models of sepsis-induced pulmonary alterations are important for the study of pathogenesis and for potential intervention therapies. The objective of the present study was to characterize lung dysfunction (low PaO2 and high PaCO2, and increased cellular infiltration, protein extravasation, and malondialdehyde (MDA) production assessed in bronchoalveolar lavage) in a sepsis model consisting of intraperitoneal (ip) injection of Escherichia coli and the protective effects of pentoxifylline (PTX). Male Wistar rats (weighing between 270 and 350 g) were injected ip with 10(7) or 10(9) CFU/100 g body weight or saline and samples were collected 2, 6, 12, and 24 h later (N = 5 each group). PaO2, PaCO2 and pH were measured in blood, and cellular influx, protein extravasation and MDA concentration were measured in bronchoalveolar lavage. In a second set of experiments either PTX or saline was administered 1 h prior to E. coli ip injection (N = 5 each group) and the animals were observed for 6 h. Injection of 10(7) or 10(9) CFU/100 g body weight of E. coli induced acidosis, hypoxemia, and hypercapnia. An increased (P < 0.05) cell influx was observed in bronchoalveolar lavage, with a predominance of neutrophils. Total protein and MDA concentrations were also higher (P < 0.05) in the septic groups compared to control. A higher tumor necrosis factor-alpha (P < 0.05) concentration was also found in these animals. Changes in all parameters were more pronounced with the higher bacterial inoculum. PTX administered prior to sepsis reduced (P < 0.05) most functional alterations. These data show that an E. coli ip inoculum is a good model for the induction of lung dysfunction in sepsis, and suitable for studies of therapeutic interventions.

The pathogenesis of sepsis involves complex interaction between the host and the infecting microorganism. Recognition and processing of microorganism antigens are essential functions of the cells of innate immune systems, and will ultimately, through the antigen presentation to the cells of adaptive immunity and the synthesis and secretions of mediators, such as cytokines, drive a coordinated immune response. Neutrophils and monocytes will therefore function as sensing and effectors cells. Fundamental in this process is the ability to discriminate self from non-self molecules. Of major interest in sepsis is that the protective and damaging host responses are part of the same process, that is, the inflammatory response that controls the infection process also underscores many of the pathophysiological events of sepsis. Moreover, this is a dynamic process according to the continuum of sepsis and its complications; up and down regulation of cellular activities may be differently regulated in different tissues, different cells and even in different functions of the same cell. This review will focus on microorganism recognition and signalization in sepsis, with emphasis on the neutrophils and monocytes adaptation during the ongoing disease.

Bacterial recognition and induced cellular activation are fundamental for the host control of infection, yet the limit between protective and harmful response is still inexact. Forty-one patients were enrolled in this study: 14 with sepsis, 12 with severe sepsis, and 15 with septic shock. Seventeen healthy volunteers (HV) were included as control. The expression of TLR2, TLR4, CD14, CD11b, and CD11c was analyzed on monocytes surface in whole blood. sCD14 was measured in serum, and TNF-alpha, IL-6, and IL-10 cytokine levels were measured in PBMC supernatants after LPS, IL-1beta, and TNF-alpha stimuli by ELISA. An increase in sCD14 and a decreased mCD14 were found in patients as compared with HV (P < 0.001). However, no differences in the expression of TLR2, TLR4, and CD11c were found among the groups. A trend toward differential expression of CD11b was observed, with higher values found in patients with sepsis as compared with HV. A negative regulation of the inflammatory cytokine production was observed in patients with severe sepsis and shock septic in relation to sepsis and HV, regardless of the stimulus. No significant difference in IL-10 production was found among the groups. In this study, we show that the inflammatory response is associated with the continuum of clinical manifestations of sepsis, with a strong inflammatory response in the early phase (sepsis) and a refractory picture in the late phases (severe sepsis and septic shock). Correlation between cell surface receptors and cytokine production after IL-1beta and TNF-alpha stimuli and the observation of a single and same standard response with the different stimulus suggest a pattern of immunology response that is not dependent only on the expression of the evaluated receptors and that is likely to have a regulation in the intracellular signaling pathways.

Glycolipoprotein (GLP) from pathogenic serovars of Leptospira has been implicated in the pathogenesis of leptospirosis by its presence in tissues of experimental animals with leptospirosis, the inhibition of the Na,K-ATPase pump activity, and induced production of cytokines. The aims of the present study were to investigate the induction of IL-6 by GLP in peripheral blood mononuclear cells (PBMC) and to demonstrate monocyte stimulation at the cellular level in whole blood from healthy volunteers. PBMC were stimulated with increasing concentrations (5 to 2500 ng/ml) of GLP extracted from the pathogenic L. interrogans serovar Copenhageni, lipopolysaccharide (positive control) or medium (negative control), and supernatants were collected after 6, 20/24, and 48 h, and kept at -80 degrees C until use. Whole blood was diluted 1:1 in RPMI medium and cultivated for 6 h, with medium, GLP and lipopolysaccharide as described above. Monensin was added after the first hour of culture. Supernatant cytokine levels from PBMC were measured by ELISA and intracellular IL-6 was detected in monocytes in whole blood cultures by flow-cytometry. Monocytes were identified in whole blood on the basis of forward versus side scatter parameters and positive reactions with CD45 and CD14 antibodies. GLP ( > or = 50 ng/ml)-induced IL-6 levels in supernatants were detected after 6-h incubation, reaching a peak after 20/24 h. The percentage of monocytes staining for IL-6 increased with increasing GLP concentration. Thus, our findings show a GLP-induced cellular activation by demonstrating the ability of GLP to induce IL-6 and the occurrence of monocyte activation in whole blood at the cellular level.

In this report we evaluated CD4(+) T, CD8(+) T and natural killer (NK) cell counts, the levels of naive/memory subsets within the CD4(+) T lymphocyte population, expression of CD38 on T lymphocytes, and CD4(+) and CD8(+) T cell cytokine production in two girls with hyper-IgM (HIM) syndrome. Both girls developed recurrent infections early in infancy, presenting a wide spectrum of clinical manifestations, with a strikingly different disease severity between them. CD4(+) T cell counts were low in both children (patient 1: 214 cells/mm(3) and patient 2: 392 cells/mm(3)), and the CD4/CD8 T cell ratio was 0.4 for patient 1, the patient with the more severe disease, and 1.4 for patient 2. NK cell numbers were low in patient 1 (60 cells/mm(3)) and borderline (286 cells/mm(3)) with regard to normal levels in patient 2. An imbalance of naive and memory/effector cell subsets was found in both girls, with the percentage of CD45RA(+) 27(+) (naive) CD4(+) T lymphocytes being 5.8 and 12.4 for patients 1 and 2, respectively. Expression of CD38 on the surface of T lymphocytes was low in patient 1. Detection of intracellular interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha in CD4(+) and CD8(+) T lymphocytes upon PMA-Io stimulus was preserved in both children. In conclusion, we found low numbers of CD4(+) T lymphocytes and a dramatic redistribution of naive and memory/effector CD4(+) T lymphocytes in two girls with non-X-linked HIM syndrome. Furthermore, we found low expression of CD38 on T lymphocytes and low numbers of NK cells in the patient with the more severe disease, indicating a possible role for these cells in the pathogenesis of this immunodeficiency.

Evaluation of HIV-induced IL-2 production by peripheral blood mononuclear cells (PBMC) and HIV-specific T helper and cytotoxic T lymphocyte (CTL) responses in health care workers (HCW) occupationally exposed to HIV reveals a high rate of response to HIV among non-seroconverters. IL-10 is also known to interfere with HIV infection in vitro. To evaluate the induction of IL-10 by HIV antigens in HCW occupationally exposed to HIV, 18 HCW with percutaneous injury were enrolled in this study, 9 of them exposed to HIV-contaminated blood, and 9 exposed to HIV-negative blood. PBMC were incubated on plates coated with HIV-1 antigens, and IL-10 was measured in supernatants by ELISA. Five of nine HCW exposed to HIV-contaminated blood presented HIV-induced IL-10. Two of nine HCW exposed to HIV-negative source patients also had detectable levels of HIV-induced IL-10, one of them in the sample obtained on the day of accidental exposure. There was a relationship between the type of device involved in injury and IL-10 production. Individuals exposed to hollow needles or scalpels presented HIV-induced IL-10, whereas those exposed to solid needles and to digital puncture did not, suggesting a relationship between infectious load and IL-10. Although occupational exposure to HIV leads to a low rate of seroconversion, these individuals can develop an antigen-specific immune response characterized in our study by induction of IL-10 in PBMC in vitro.

Leptospira interrogans glycolipoprotein (GLP) has been implicated in pathological and functional derangement seen in leptospirosis. The goal of this study was to evaluate GLP's ability to induce cellular activation, as assessed by cytokine production and expression of surface activation markers. GLP extracted from either pathogenic L. interrogans serovar Copenhageni or nonpathogenic Leptospira biflexa serovar Patoc (GLPp) was used to stimulate peripheral blood mononuclear cell cultures from healthy donors. Supernatant cytokine levels were measured by enzyme-linked immunosorbent assay. Expression of CD69 and HLA-DR on lymphocytes and monocytes, as well as lipopolysaccharide (LPS) binding, were measured by flow cytometry. At 6 h of incubation, GLP induced a significant rise in tumor necrosis factor alpha levels, which dropped progressively until 72 h of incubation. Interleukin-10 peak levels were obtained at between 24 and 48 h, with sustained levels until 72 h of incubation. The response magnitude was proportional to the GLP dose. CD69 expression on T lymphocytes and monocytes increased significantly, as did HLA-DR expression on monocytes. GLPp induced no CD69 or HLA-DR expression. GLP did not block biotinylated LPS binding to monocytes, suggesting that different pathways are used to induce cell activation. In conclusion, GLP induces cellular activation and may play a major role in the pathogenesis of leptospirosis.

Background: The use of whole blood (WB) in studying lipopolysaccharide (LPS)-induced cellular activation preserves the milieu in which LPS-cell interaction occurs in vivo. However, little information is available on using such a system at a single-cell level. We evaluated LPS binding and cell activation in WB by using flow cytometry. The influence of heparin or EDTA as anticoagulants was also addressed., Methods: Blood was obtained from healthy donors in EDTA and/or heparin tubes. Biotinylated LPS (LPSb) was used to evaluate cell binding of LPS in WB. Cells were surface stained with appropriate antibodies and LPSb was detected by adding streptavidin-allophycocyanin (APC). LPS-induced activation was evaluated by the expression of surface activation markers and by the detection of intracellular tumor necrosis factor-alpha (TNF-alpha)., Results: LPSb bound promptly to monocytes in EDTA- and heparin-treated blood. In EDTA-treated blood, membrane-bound LPSb decreased after 60 min of incubation, whereas it remained detectable in heparinized blood during the 6 h of incubation. LPS induced TNF-alpha and enhanced the expression of HLA-DR in monocytes, as well as the expression of CD69 in T and B lymphocytes. Induction of both TNF-alpha in monocytes and CD69 in lymphocytes was more efficient in heparinized blood., Conclusion: Detection of membrane-bound LPSb on monocytes differed in EDTA or heparin-treated blood, and cell activation was better obtained in heparinized blood., (Copyright 2002 Wiley‐Liss, Inc.)

We used biotinylated LPS (LPSb) and flow cytometry to study LPS-monocyte interaction and LPS-induced cellular activation in whole blood from septic patients (SP). Expression of surface activation markers was evaluated on monocytes (HLA-DR) and T lymphocytes (CD69 and CD95), and intracellular TNF-alpha on monocytes. Saturating curve and kinetics of LPSb detection on monocytes were similar in SP and healthy volunteers (HV). LPSb bound to monocytes was detected after 5 min of incubation in both groups, with a more pronounced decay in SP. Monocytes from SP had a lower expression of HLA-DR as compared to HV, both constitutive and upon LPS stimulation. The proportion of monocytes producing TNF-alpha after LPS stimulus was higher in HV than SP (mean +/- SD = 25.2 +/- 14.2% and 2.2 +/- 2.6%, respectively, P < 0.001). LPS-induced CD69 on T CD8+ and CD8- lymphocytes was similar for patients and controls. Expression of CD95 on T lymphocytes was higher in SP as compared to HV on T CD8+ cells (GMFI, mean +/- SD = 22.3 +/- 14.6 and 8.6 +/- 5.0, respectively, P = 0.01) and CD8- cells (GMFI, mean +/- SD = 28.3 +/- 7.7 and 14 +/- 4.3 respectively, P < 0.001). Thus, monocytes and lymphocytes seem to respond differently to LPS in septic patients. Monocyte hyporesponsiveness appears not to be related to a decreased binding capacity of LPS, but rather to an impaired signal transduction.

The outcome of patients with sepsis arises from multiple factors affecting both the host and the invading microorganisms. Age, presence of underlying disease, source of infection and some specific etiological agents have been related to prognosis. Appropriateness of antimicrobial therapy, considering the in vitro susceptibility tests for the infecting bacteria, has been strongly associated with the outcome. Therefore even after the cascade of sepsis has been triggered, the control of bacteria growth is still fundamental for the outcome of the infection. This is a major distinction point from experimental studies in which whole killed bacteria and their products are used as model of sepsis. However, even within the setting of adequate antimicrobial use, patients still die of sepsis. Thus, strategies focusing on further therapy targets are an important area of interest for basic and clinical research. Although such adjunctive sepsis therapy has failed to achieve consistent better survival rates so far, nevertheless, it improved our understanding of the pathophysiological events seen in sepsis that the possibility that a new and effective treatment may arise has been warmly considered. In this paper we aim to review some aspects of the pathogenesis of sepsis, focusing on recent advances and on possible targets for adjunctive therapy. Published clinical trials and experimental data supporting such trials are commented on.