Athina Savva

Effect of the Novel Influenza A (H1N1) Virus in the Human Immune System

Background The pandemic by the novel H1N1 virus has created the need to study any probable effects of that infection in the immune system of the host. Methodology/Principal Findings Blood was sampled within the first two days of the presentation of signs of infection from 10 healthy volunteers; from 18 cases of flu-like syndrome; and from 31 cases of infection by H1N1 confirmed by reverse RT-PCR. Absolute counts of subtypes of monocytes and of lymphocytes were determined after staining with monoclonal antibodies and analysis by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated from patients and stimulated with various bacterial stimuli. Concentrations of tumour necrosis factor-alpha, interleukin (IL)-1beta, IL-6, IL-18, interferon (FN)-alpha and of IFN-gamma were estimated in supernatants by an enzyme immunoassay. Infection by H1N1 was accompanied by an increase of monocytes. PBMCs of patients evoked strong cytokine production after stimulation with most of bacterial stimuli. Defective cytokine responses were shown in response to stimulation with phytohemagglutin and with heat-killed Streptococcus pneumoniae. Adaptive immune responses of H1N1-infected patients were characterized by decreases of CD4-lymphocytes and of B-lymphocytes and by increase of T-regulatory lymphocytes (Tregs). Conclusions/Significance Infection by the H1N1 virus is accompanied by a characteristic impairment of the innate immune responses characterized by defective cytokine responses to S.pneumoniae. Alterations of the adaptive immune responses are predominated by increase of Tregs. These findings signify a predisposition for pneumococcal infections after infection by H1N1 influenza.

Inhibition of caspase-1 activation in gram-negative sepsis and experimental endotoxemia

IntroductionDown-regulation of ex-vivo cytokine production is a specific feature in patients with sepsis. Cytokine downregulation was studied focusing on caspase-1 activation and conversion of pro-interleukin-1β into interleukin-1β (IL-1β).MethodsPeripheral blood mononuclear cells were isolated from a) 92 patients with sepsis mainly of Gram-negative etiology; b) 34 healthy volunteers; and c) 5 healthy individuals enrolled in an experimental endotoxemia study. Cytokine stimulation was assessed in vitro after stimulation with a variety of microbial stimuli.ResultsInhibition of IL-1β in sepsis was more profound than tumour necrosis factor (TNF). Down-regulation of IL-1β response could not be entirely explained by the moderate inhibition of transcription. We investigated inflammasome activation and found that in patients with sepsis, both pro-caspase-1 and activated caspase-1 were markedly decreased. Blocking caspase-1 inhibited the release of IL-1β in healthy volunteers, an effect that was lost in septic patients. Finally, urate crystals, which specifically induce the NLPR3 inflammasome activation, induced significant IL-1β production in healthy controls but not in patients with sepsis. These findings were complemented by inhibition of caspase-1 autocleavage as early as two hours after lipopolysaccharide exposure in volunteers.ConclusionsThese data demonstrate that the inhibition of caspase-1 and defective IL-1 β production is an important immunological feature in sepsis.

Risk assessment in sepsis: a new prognostication rule by APACHE II score and serum soluble urokinase plasminogen activator receptor.

IntroductionEarly risk assessment is the mainstay of management of patients with sepsis. APACHE II is the gold standard prognostic stratification system. A prediction rule that aimed to improve prognostication by APACHE II with the application of serum suPAR (soluble urokinase plasminogen activator receptor) is developed.MethodsA prospective study cohort enrolled 1914 patients with sepsis including 62.2% with sepsis and 37.8% with severe sepsis/septic shock. Serum suPAR was measured in samples drawn after diagnosis by an enzyme-immunoabsorbent assay; in 367 patients sequential measurements were performed. After ROC analysis and multivariate logistic regression analysis a prediction rule for risk was developed. The rule was validated in a double-blind fashion by an independent confirmation cohort of 196 sepsis patients, predominantly severe sepsis/septic shock patients, from Sweden.ResultsSerum suPAR remained stable within survivors and non-survivors for 10 days. Regression analysis showed that APACHE II ≥17 and suPAR ≥12 ng/ml were independently associated with unfavorable outcome. Four strata of risk were identified: i) APACHE II <17 and suPAR <12 ng/ml with mortality 5.5%; ii) APACHE II < 17 and suPAR ≥12 ng/ml with mortality 17.4%; iii) APACHE II ≥ 17 and suPAR <12 ng/ml with mortality 37.4%; and iv) APACHE II ≥17 and suPAR ≥12 ng/ml with mortality 51.7%. This prediction rule was confirmed by the Swedish cohort.ConclusionsA novel prediction rule with four levels of risk in sepsis based on APACHE II score and serum suPAR is proposed. Prognostication by this rule is confirmed by an independent cohort.

Soluble urokinase plasminogen activator receptor (suPAR) for assessment of disease severity in ventilator-associated pneumonia and sepsis

Urokinase plasminogen activator (uPAR) is a receptor mainly expressed on peripheral blood mononuclear cells and neutrophils. The role of its soluble form, namely suPAR, as a predictor of sepsis outcome in a homogenous cohort of 180 septic patients, was investigated. Blood from 180 patients with ventilator-associated pneumonia (VAP) and sepsis was collected for seven consecutive days. suPAR and PCT were measured in serum by an enzyme immunoassay and an immuno-time-resolved amplified cryptate assay respectively. Neutrophils and monocytes were isolated on day 1 and incubated. suPAR levels greater than 10.5 ng/ml had 80% specificity and 77.6% positive predictive value to discriminate between severe sepsis and sepsis. suPAR levels greater than 12.9 ng/ml had 80% specificity and 76.1% positive predictive value for prognosis of unfavorable outcome. suPAR levels were significantly lower among survivors than among non-survivors over follow-up. Step-wise Cox regression analysis found suPAR as an independent factor related with unfavorable outcome (p: 0.026). Concentrations of suPAR in supernatants of neutrophils of patients with sepsis were greater compared to controls. It is concluded that suPAR is a reliable marker of sepsis severity and a strong independent predictor of unfavorable outcome in VAP and sepsis. Neutrophils are involved in release.

Early changes of procalcitonin may advise about prognosis and appropriateness of antimicrobial therapy in sepsis

PURPOSE The objective of this study is to define if early changes of procalcitonin (PCT) may inform about prognosis and appropriateness of administered therapy in sepsis. METHODS A prospective multicenter observational study was conducted in 289 patients. Blood samples were drawn on day 1, that is, within less than 24 hours from advent of signs of sepsis, and on days 3, 7, and 10. Procalcitonin was estimated in serum by the ultrasensitive Kryptor assay (BRAHMS GmbH, Hennigsdorf, Germany). Patients were divided into the following 2 groups according to the type of change of PCT: group 1, where PCT on day 3 was decreased by more than 30% or was below 0.25 ng/mL, and group 2, where PCT on day 3 was either increased above 0.25 ng/mL or decreased less than 30%. RESULTS Death occurred in 12.3% of patients of group 1 and in 29.9% of those of group 2 (P < .0001). Odds ratio for death of patients of group 1 was 0.328. Odds ratio for the administration of inappropriate antimicrobials of patients of group 2 was 2.519 (P = .003). CONCLUSIONS Changes of serum PCT within the first 48 hours reflect the benefit or not of the administered antimicrobial therapy. Serial PCT measurements should be used in clinical practice to guide administration of appropriate antimicrobials.

Angiopoietin-2 is increased in septic shock: evidence for the existence of a circulating factor stimulating its release from human monocytes.

We aimed to investigate if angiopoietin-2 (Ang-2) participates in the septic process and what may be the role of monocytes as a site of release of Ang-2 in sepsis. Concentrations of Ang-2 were estimated in sera and in supernatants of monocytes derived form one already described cohort of 90 patients with septic syndrome due to ventilator-associated pneumonia (VAP). Mononuclear cells of 17 healthy volunteers were stimulated by serum of patients in the presence or absence of various intracellular pathway inhibitors. Ang-2 gene expression after stimulation was also tested. Ang-2 was higher in patients with septic shock compared to patients with sepsis, severe sepsis and controls. Ang-2 was significantly increased in non-survivors compared with survivors. Serum levels greater than 9700 pg/ml were accompanied by a 3.254 odds ratio for death (p: 0.033). Ang-2 release from monocytes of septic patients was slightly decreased after stimulation with lipopolysaccharide (LPS) of Escherichia coli O55:B5. Release of Ang-2 from healthy mononuclear cells was stimulated by serum of patients with shock but not by serum of non-shocked patients (p: 0.016). Release was decreased by LPS; increased in the presence of a TLR4 antagonist; and decreased by anti-TNF antibody. RNA transcripts of PBMCs after stimulation with serum of patients with septic shock were higher than those after LPS stimulation. It is concluded that Ang-2 is increased in serum in the event of septic shock and that its increase is related to unfavorable outcome. It seems that a circulating factor may exist in the serum of patients with septic shock that stimulates gene expression and subsequent release of Ang-2 from monocytes. TLR4 and TNFalpha modulate release of Ang-2.

Early Administration of Hydrocortisone Replacement After the Advent of Septic Shock: Impact on Survival and Immune Response*

Objectives:To investigate the impact of early initiation of hydrocortisone therapy on the clinical course of septic shock and on cytokine release. Design:Prospective study in patients with septic shock treated with low doses of hydrocortisone. Setting:ICUs and general wards. Patients:Over a 2-year period, 170 patients with septic shock treated with low doses of hydrocortisone were enrolled. Blood was sampled from 34 patients for isolation of peripheral blood mononuclear cells and cytokine stimulation before and 24 hours after the start of hydrocortisone. Interventions:None. Measurements and Main Results:After quartile analysis, patients were divided into those with early initiation of hydrocortisone (< 9 hr after vasopressors, n = 46) and those with late initiation of hydrocortisone (> 9 hr after vasopressors, n = 124). After adjusting for disease severity and type of infection, a protective effect of early hydrocortisone administration against unfavorable outcome was found (hazard ratio, 0.20; p = 0.012). Time of discontinuation of vasopressors was earlier among patients with initiation of hydrocortisone within 9 hours. Production of tumor necrosis factor-&agr; was lower among patients who had had hydrocortisone early. Conclusions:In patients receiving hydrocortisone for septic shock, early initiation of treatment was associated with improved survival. This treatment was also associated with attenuated stimulation of tumor necrosis factor-&agr;.

Role of tumor necrosis factor gene single nucleotide polymorphisms in the natural course of 2009 influenza A H1N1 virus infection

OBJECTIVES To identify the role of single nucleotide polymorphisms (SNPs) of the tumor necrosis factor (TNF) gene in the natural course of 2009 influenza A H1N1 virus infection. METHODS Genomic DNA was isolated from 109 patients with an H1N1 infection and from 108 healthy volunteers. SNPs of the TNF gene were assessed after electrophoresis of the digested PCR products by restriction enzymes. RESULTS The frequency of the -238 A allele was significantly greater among patients than among controls. Viral pneumonia developed in 20 of 96 non-carriers of at least one -238 A allele (20.8%) and in seven of 13 carriers of at least one -238 A allele (53.8%, p=0.016). Logistic regression analysis showed that the most important factors associated with the development of pneumonia were the presence of an underlying disease (p=0.021, odds ratio (OR) 3.08) and the carriage of at least one -238 A allele (p=0.041, OR 3.74). Gene transcripts of the TNF gene were greater among non-carriers of the -238 A allele than among carriers of the -238 A allele. CONCLUSIONS The -238 A SNP allele of the TNF gene imposes on the course of 2009 H1N1 virus infection and is an independent risk factor for pneumonia.

Long-term efficacy of etanercept in hidradenitis suppurativa: results from an open-label phase II prospective trial.

Please cite this paper as: Long‐term efficacy of etanercept in hidradenitis suppurativa: results from an open‐label phase II prospective trial. Experimental Dermatology 2010; 19: 538–540.

Ultrasound aids in diagnosis and severity assessment of hidradenitis suppurativa

cleaved caspase 3 protein. Under conditions of elevated growth factor signalling such as puberty, plasma insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) is decreased. On the other hand, ATRA treatment of human dermal papilla cells resulted in a fivefold elevation of IGFBP-3 levels. IGFBP-3 is FoxO-controlled at the promoter level and interacts in the nucleus with retinoid X receptor a (RXRa), which is essential for mediating the effects of IGFBP-3 on apoptosis (Fig. 2b). Thus, isotretinoin-induced FoxO1-activation of the IGFBP-3 promoter may facilitate sebocyte apoptosis. Overexpression of FoxO1 and IGFBP-3 may also mediate the anticomedogenic effect of isotretinoin as upregulated IGFBP-3 suppresses keratinocyte proliferation. The antiproliferative and apoptotic activity of nuclear IGFBP-3 may contribute to isotretinoin’s chemopreventive effect as well. In myeloid leukaemia cells and human breast cancer cells increased IGFBP-3 promoted apoptosis by enhancing the activity of RXRa. Oral isotretinoin treatment impairs insulin resistance. FoxO1 is a corepressor of PPARc, the most important mediator of insulin sensitization. Isotretinoin-induced upregulation of FoxO1 in adipose tissue may impair PPARc function, adipogenesis and insulin sensitivity. Severe acne and acneiform eruptions have been observed with high doses of tricyclic antidepressants and lithium therapy. In the brain of mice, lithium significantly decreased FoxO3 levels. In mice, elevated serotonergic activity increased Akt-mediated phosphorylation of FoxO1 and FoxO3a in various brain regions resulting in nuclear deficiency of FoxO1 and FoxO3a. FoxOs in the brain of rodents are intensely involved in the regulation of behavioural manifestations. Serotonin levels upregulated by antidepressants reduce nuclear concentrations of FoxO1 and FoxO3 in neuronal cells. FoxO1-deficient mice displayed reduced anxiety, whereas FoxO3-deficient mice presented with a significant antidepressant-like behaviour. Nuclear content of FoxO1 in the human brain elevated by isotretinoin treatment may explain the increased incidence of depression observed with isotretinoin therapy. Nuclear FoxO1 deficiency explains all the major pathogenetic factors of acne vulgaris. The upregulation of nuclear FoxO1 induced by isotretinoin is proposed to be the common underlying therapeutic mechanism of isotretinoin action. Furthermore, the enhanced nuclear levels of FoxO1 may explain the adverse effects mediated by isotretinoin on lipoprotein metabolism, insulin sensitivity and neuronal function. The indirect evidence presented from translational medicine should stimulate further research activities to understand the pathophysiology of acne and retinoid action at the level of transcriptional regulation.