P Martins

TLR2, TLR4, CD14, CD11B, and CD11c expressions on monocytes surface and cytokine production in patients with sepsis, severe sepsis, and septic shock

ABSTRACT 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-&agr;, IL-6, and IL-10 cytokine levels were measured in PBMC supernatants after LPS, IL-1&bgr;, and TNF-&agr; 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-1&bgr; and TNF-&agr; 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.

TLR signaling pathway in patients with sepsis.

The pathogenesis of sepsis involves complex interaction between the host and the infecting microorganism. Bacterial recognition and signaling are essential functions of the cells of innate immune systems and drive a coordinated immune response. One of the more intriguing aspects of sepsis is the fact that the protective and damaging host response are part of the same process, that is, the inflammatory response that is aimed to control the infectious process also underscores many of the pathophysiological events of sepsis. The discovery of Toll-like receptors (TLRs) in humans, and the early recognition of TLR-4 as the receptor that signals LPS bioactivity were major breakthroughs not only in the field of sepsis but also in immunology as a whole. In this article, we aimed to review TLR expression and signaling in the context of sepsis. The results obtained by our group show that TLR and other cellular surface receptors may be differently regulated on mononuclear cells and neutrophils, and that they are dynamically modulated across the stages of sepsis. Toll-like receptor signaling gene expression in mononuclear cells is decreased in more severe forms of the disease. In contrast, up-regulated genes are seen along the clinical spectrum of sepsis in neutrophils.

Expression of cell surface receptors and oxidative metabolism modulation in the clinical continuum of sepsis

BackgroundInfection 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.MethodsA 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.ResultsTLR2 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.ConclusionSurface 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.

Lipopolysaccharide-cell interaction and induced cellular activation in whole blood of septic patients.

We used biotinylatedLPS (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-α 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-α 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.

Bacterial Recognition and Induced Cell Activation in Sepsis

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.

Pathogenetic Aspects of Sepsis and Possible Targets for Adjunctive Therapy

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.

Glutamine in critically ill patients: is it a fundamental nutritional supplement?

Glutamine is the most abundant free amino acid in the human body and is necessary to modulate the inflammatory and oxidative stress responses in patients.(1,2) Systemic glutamine availability is determined by the balance of endogenous glutamine production (mainly in muscular tissue) and its use by glutamine consuming organs (gut, kidney, liver and the immune system). Several studies show that in catabolic intensive care unit (ICU) patients, the endogenous production of muscular glutamine is increased while the plasma levels of glutamine are decreased, indicating elevated glutamine needs.(3,4) We know also that low plasma glutamine values (< 420μmol/L) upon admission are related to increased mortality.(5) These findings are the rationale for the use of glutamine supplementation in the ICU population in order to replenish the muscle pool of glutamine, attenuate the efflux of this amino acid, and provide exogenous glutamine required to meet the elevated organ needs for improvement in protein synthesis, modulation of the immune system, reduction of oxidative stress, and preservation of the gut barrier. However, recent observations challenge this hypothesis. The plasma levels of glutamine are extremely variable in the ICU population(6) and are not always associated with increased mortality.(7) The glutamine supplementation does not stop the glutamine efflux from muscle because the endogenous muscular production and plasma levels of glutamine are related to the severity of the illness.(8) Intensive care unit patients are considered to be immunosuppressed as evidenced by reduced levels of and the presence of dysfunctional T lymphocytes, altered neutrophil activity and an imbalance in the production of cytokines.(9-13) The demonstration of the benefit of glutamine in increasing the number and functionality of effector cells of the immune response is evident in some studies,(14-19) while in others that answer is not so obvious.(20,21) We found (personal communication)(22) that in the ICU population, glutamine supplementation (0.40g/Kg/day) via parenteral nutrition improved cellular immune function with significant increases in CD14 and CD14 HLA-DR monocytes and significant decreases in T regulatory cells with a significant reduction in the appearance of nosocomial infection. Over the years, many studies examining glutamine in ICU populations have presented controversial results. We can differentiate between the small monocentric studies of the early years showing a significant reduction in Paulo Martins1

The Impact of Poor Prognostic Patients Admitted to the Intensive Care Unit on Family Member's Emotional Disorders

Background: Having a loved one admitted to the ICU is an extraordinarily stressful event, principally with poor prognosis and when death may occur. Aim: To evaluate the impact of poor prognosis patients admitted in the ICU on their family member’s emotional disorders. Design and setting: Prospective study conducted in a 22-bed mixed ICU in a tertiary hospital in Sao Paulo, Brazil. Family members completed the Hospital Anxiety and Depression Scale 48 hours post-admission. Family members answered the HADS, the Impact of Event Scale by phone at 30-days and 90-days after ICU discharge. Results: 95/575 patients admitted at the ICU were defined as poor prognostic patients. Poor prognostic patients required more mechanical ventilation (50.0% vs. 32.9%, p=0.002), tracheotomy (11.6% vs. 5.0%, p=0.014), vasopressors (54.7% vs. 36.8%, p=0.001), remained longer under mechanical ventilation (7 [3-15] vs. 3 [2-6] days, p=0.030) and stay longer at ICU (8 [5-18] vs. 4 [3-8], p<0.001) when compared with non-poor prognosis patients. They also had high mortality at ICU (32.6%), 30-days (60.0%) and 90-days (73.5%) and we also observed an intense emotional suffering among their respective family members during and after ICU discharge. Conclusion: Family members of poor prognosis patients admitted to the ICU were more likely to suffer with symptoms of anxiety, depression and post traumatic distress. Their loved ones needed more aggressive treatments during ICU and had higher mortality in a short time.

Characteristics and outcomes of palliative care patients in intensive care unit.

145 Background: Despite the growing palliative care movement, most admissions still occur in Intensive Care Units. The aim of this study was to determine the frequency of palliative care patients admitted in an ICU and assessed their outcomes. METHODS This prospective study was conducted in a tertiary private hospital, in an adult medical-surgical ICU with 22-bed in São Paulo, Brazil. Patients or their family member with ICU stay ≥ 48 hours were invited to participate. They were excluded if they had no conditions to answer the questionnaire or if they refuse to participate. During ICU stay we analyzed through the medical records and questionnaire their clinical condition and their oncologic status. We called them by telephonic assessment to assess their survival. RESULTS From March 2011 to March 2013 a total of 576 ICU patients were analyzed; of these, 280 were oncologic patients and 95 were palliative care. Of total, the majority was male gender (57.8%), median age was 67[54-79] years, SAPS III score was 54±18.4 points, SOFA was 3.1±3.0 and ICU Length of stay (LOS) was 9.0±11.3 days. ICU mortality was 16.5%, 1-month mortality was 22% and 3-months cumulative mortality was 28.6%. We could observe that palliative care patients were in majority cancer patients (75%vs 43.4%,p<0.001), with metastatic disease(81.7 vs 36.3, p<0.001), had greater mean time of initial diagnosis(3.21±3.7 vs 2.17±2.5, p=0.009), had greater ICU LOS (14.2±16.2 days vs 7.96±9.8, p<0.001) greater mean SAPS III (68.5±16.0 vs p<0.001) and SOFA (4.81±3.2 vs 2.81±2.8, p<0.001) when compared with non palliative patients care. They also needed more mechanical ventilation (50.0%vs32.6%, p=0.001), tracheotomy (11.6%vs 5.0%,p=0.014) and vasopressors (54.7% vs 36.8,p=0.001). The ICU mortality was greater (32.6% vs 6.8%, p<0.001), 1-month (60.0% vs 14.0%, p<0.001) and 3-months (73.5% vs 19.1%). CONCLUSIONS Palliative care suffers most in Intensive Care Unit and we observed a high mortality at 3-months after ICU discharge. We recommend more discussions before palliative care patients admissions in ICU to better provide them quality of life.