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Background and objective The role of vascular endothelial growth factor (VEGF)-A in the resolution of ventilator-associated pneumonia (VAP) was investigated in clinical and mouse pneumonia models. Methods VEGF-A was measured for seven consecutive days by an immunosorbent assay in sera of 82 patients with VAP and changes from baseline were correlated with the resolution of VAP. Experimental animals were challenged intratracheally with Pseudomonas aeruginosa. Mouse bronchoalveolar lavage (BAL) samples and segments of lung tissue were obtained at 24, 48 and 124 h after bacterial challenge. Levels of VEGF-A, tumour Necrosis Factor alpha (TNF-α), interleukin (IL)-1β, interferon-gamma (IFNγ) and myeloperoxidase (MPO) activity were measured in these samples. Results VAP resolved in 36.1% of patients with a less than 45% increase of VEGF-A on day 5 compared to 65.2% of patients with a more than 45% increase (P = 0.014). This was also accompanied by an earlier resolution of VAP (log-rank: 7.99; P = 0.005) and it was not pathogen-specific. The increase of VEGF-A was an independent variable associated with VAP resolution in forward logistic regression analysis where Acute Physiology and Chronic Health Evaluation (APACHE) II and Sequential Organ Failure Assessment (SOFA) scores were included as independent variables. VEGF-A in mouse BAL and lung tissue increased significantly at 124 h but not with the other mediators. In mice pre-treated with bevacizumab, VEGF-A concentrations decreased while TNF-α and MPO significantly increased. Conclusion In patients, an association between increased levels of circulating VEGF-A and VAP resolution was observed. The mouse study suggests that elevated VEGF-A levels may be associated with lung inflammation resolution. Clinical trial registration NCT00297674 at www.clinicaltrials.gov.

The role of vascular endothelial growth factor (VEGF)-A in the resolution of ventilator-associated pneumonia (VAP) was investigated in clinical and mouse pneumonia models.VEGF-A was measured for seven consecutive days by an immunosorbent assay in sera of 82 patients with VAP and changes from baseline were correlated with the resolution of VAP. Experimental animals were challenged intratracheally with Pseudomonas aeruginosa. Mouse bronchoalveolar lavage (BAL) samples and segments of lung tissue were obtained at 24, 48 and 124 h after bacterial challenge. Levels of VEGF-A, tumour Necrosis Factor alpha (TNF-α), interleukin (IL)-1β, interferon-gamma (IFNγ) and myeloperoxidase (MPO) activity were measured in these samples.VAP resolved in 36.1% of patients with a less than 45% increase of VEGF-A on day 5 compared to 65.2% of patients with a more than 45% increase (P = 0.014). This was also accompanied by an earlier resolution of VAP (log-rank: 7.99; P = 0.005) and it was not pathogen-specific. The increase of VEGF-A was an independent variable associated with VAP resolution in forward logistic regression analysis where Acute Physiology and Chronic Health Evaluation (APACHE) II and Sequential Organ Failure Assessment (SOFA) scores were included as independent variables. VEGF-A in mouse BAL and lung tissue increased significantly at 124 h but not with the other mediators. In mice pre-treated with bevacizumab, VEGF-A concentrations decreased while TNF-α and MPO significantly increased.In patients, an association between increased levels of circulating VEGF-A and VAP resolution was observed. The mouse study suggests that elevated VEGF-A levels may be associated with lung inflammation resolution.NCT00297674 at www.clinicaltrials.gov.

Item does not contain fulltext Autophagy is a highly conserved mechanism of eukaryotic cells implicated in cell homeostasis and elimination of intracellular pathogens. Functional polymorphisms in genes encoding for autophagy have been associated with susceptibility to inflammatory and infectious diseases, but data on severe infections are missing. The aim of the present study was to assess whether polymorphisms in genes encoding proteins involved in autophagy influence susceptibility to ventilator-associated pneumonia (VAP). Mechanically ventilated patients with VAP were studied. Genotyping for autophagy-related 16-like 1 (ATG16L1, rs2241880) functional polymorphism was performed using the TaqMan single-nucleotide assay. Monocytes were isolated from patients and stimulated with lipopolysaccharide (LPS). Tumor necrosis factor-alpha (TNF-alpha) was measured in the supernatants of monocytes using an enzyme-linked immunosorbent assay. Procalcitonin (PCT) was also measured in the serum of patients by an immuno-time-resolved amplified cryptate technology assay. A total of 155 patients with VAP were enrolled in the study. Carriage of the minor A allele of ATG16L1 was associated with septic shock with at least one organ failure (odds ratio (OR): 2.40, p: 0.036). TNF-alpha production was significantly greater among the carriers of the polymorphism presenting with at least one organ failure (p: 0.040). PCT was increased upon worsening to septic shock and organ failure only among carriers of the minor frequency A alleles. In a homogeneous cohort of septic patients with VAP, the carriage of autophagy polymorphisms predisposes to VAP severity and septic shock development. This may be related with predisposition to immunoparalysis.

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.

Summary Regulatory T cells (Tregs) have an anti-inflammatory role. A former study in a limited number of patients found that absolute counts of Tregs increase when infection by the new influenza H1N1 virus is complicated with pneumonia. These results generate the question if H1N1-related pneumonia is associated with a state of hypo-inflammation. A total of 135 patients were enrolled with blood sampling within less than 24 h from diagnosis; 23 with flu-like syndrome; 69 with uncomplicated H1N1-infection; seven with bacterial pneumonia; and 36 with H1N1-related pneumonia. Tregs and CD14/HLA-DR co-expression were estimated by flow cytometry; concentrations of tumour necrosis factor-alpha (TNF-α), of interleukin (IL)-6 and of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) by an enzyme immunoassay; those of procalcitonin (PCT) by immuno-time-resolved amplified cryptate technology assay. Expression of human leucocyte antigen D-related (HLA-DR) on monocytes was similar between groups; absolute Treg counts were greater among patients with H1N1-related pneumonia than flu-like syndrome or H1N1-uncomplicated infection. Serum TNF-α of patients with bacterial pneumonia was greater than those of other groups, but IL-10 was similar between groups. Serum PCT was greater among patients with H1N1-related pneumonia and sTREM-1 among those with H1N1-related pneumonia. Regression analysis revealed that the most important factors related with the advent of pneumonia were the existence of underlying illnesses (P = 0·006) and of Tregs equal to or above 16 mm3 (P = 0·013). It is concluded that the advent of H1N1-related pneumonia is related to an early increase of the absolute Treg counts. This increase is probably not part of a hypo-inflammatory state of the host.

Background Because clarithromycin provided beneficiary nonantibiotic effects in experimental studies, its efficacy was tested in patients with sepsis and ventilator-associated pneumonia (VAP). Methods Two hundred patients with sepsis and VAP were enrolled in a double-blind, randomized, multicenter trial from June 2004 until November 2005. Clarithromycin (1 g) was administered intravenously once daily for 3 consecutive days in 100 patients; another 100 patients were treated with placebo. Main outcomes were resolution of VAP, duration of mechanical ventilation, and sepsis-related mortality within 28 days. Results The groups were well matched with regard to demographic characteristics, disease severity, pathogens, and adequacy of the administered antimicrobials. Analysis comprising 141 patients who survived revealed that the median time for resolution of VAP was 15.5 days and 10.0 days among placebo- and clarithromycin-treated patients, respectively (P = .011); median times for weaning from mechanical ventilation were 22.5 days and 16.0 days, respectively (p = .049). Analysis comprising all enrolled patients showed a more rapid decrease of the clinical pulmonary infection score and a delay for advent of multiple organ dysfunction in clarithromycin-treated patients, compared with those of placebo-treated patients (p = .047). Among the 45 patients who died of sepsis, time to death was significantly prolonged in clarithromycin-treated compared with placebo-treated patients (p = .004). Serious adverse events were observed in 0% and 3% of placebo- and clarithromycin-treated patients, respectively (P = .25). Conclusions Clarithromycin accelerated the resolution of VAP and weaning from mechanical ventilation in surviving patients and delayed death in those who died of sepsis. The mortality rate at day 28 was not altered. Results are encouraging and render new perspectives on the management of sepsis and VAP.

Based on former animal studies showing the effect of clarithromycin in experimental sepsis by multidrug-resistant Pseudomonas aeruginosa following administration of single doses, the significance of its administration for three consecutive days was evaluated. Acute pyelonephritis was induced in 20 rabbits after inoculation of the test isolate in the renal pelvis. Therapy was administered upon signs of sepsis in group B; A served as control. Survival was recorded; monocytes were isolated for determination of ex vivo TNFalpha secretion. Quantitative cultures of organs were performed after death. Mean survival of groups A and B was 2.65 and 7.95 days respectively. At 24 hours, serum malondialdehyde of group B, which is an index of the oxidant status in serum, was lower than A. Ex vivo release of TNFalpha by the isolated monocytes of group B was lower than A at 3.5 and 48 hours. Tissue bacterial load was similar in two groups after animal death. It is concluded that clarithromycin possessed considerable immunomodulatory effects restraining release of TNFalpha from blood monocytes.

To evaluate whether histological findings of skin in sepsis by Pseudomonas aeruginosa could be a predictive factor of progression to death, histological alterations after challenge by one multidrug-resistant isolate were studied in 24 rabbits. Acute pyelonephritis was induced after ligation of the right ureter and injection of 10 8 CFU per kg of body weight into the renal pelvis. Biopsy samples of skin were taken on necropsy. Mean survival of animals after bacterial challenge was 5.23 days. Main histological findings of skin were inflammation and swelling of dermis; thickening of endothelium; presence of thrombi in vessels; necrobiotic changes of the hair follicles. Serum TNFwas negatively correlated to histo- logy of dermis and follicles. Positive correlation was found between survival and swelling of dermis. It is concluded that prolongation of survival was accompanied by intense edema of the dermis. A punch skin biopsy might be a predictive fac- tor of sepsis outcome.

Summary Apoptosis of blood monocytes was studied in experimental sepsis by multi-drug-resistant Pseudomonas aeruginosa. Thirty-six rabbits were used, divided into the following groups: A (n = 6), sham; B (n = 6), administered anaesthetics; and C (n = 24), acute pyelonephritis induced after inoculation of the test isolate in the renal pelvis. Blood was sampled at standard time intervals for estimation of tumour necrosis factor (TNF)-α and isolation of monocytes. Half the monocytes were incubated and the other half was lysed for estimation of the cytoplasmic activity of caspase-3 by a kinetic chromogenic assay. No animal in groups A and B died; those in group C were divided into two subgroups, CI (n = 8) with present activity of caspase-3 of blood monocytes at 3·5 h and CII (n = 16) with absent activity. Their median survival was 2·0 and 3·5 days, respectively (P = 0·0089). Ex vivo secretion of TNF-α from monocytes was higher by monocytes of subgroup CII than subgroup CI at 3·5 h (P = 0·039) and of group A than CII at 48 h (P = 0·010). Median change of caspase-3 activity between 3·5 and 24 h of sampling was 56·1 and −5·8 pmol/min per 104 cells for subgroups CI and CII (P = 0·040), respectively. Respective changes between 3·5 and 48 h were 28 981·0 and 0 pmol/min per 104 cells (P = 0·036). Early induction of apoptosis in blood monocytes is of prime importance for the survival of the septic host and might be connected to changes of monocyte potential for the secretion of TNF-α.

Increasing numbers of admissions for sepsis impose a heavy burden on health care systems worldwide, while novel therapies have proven both expensive and ineffective. We explored the long-term mortality and hospitalization costs after adjunctive therapy with intravenous clarithromycin in ventilator-associated pneumonia (VAP). Two hundred patients with sepsis and VAP were enrolled in a published randomized clinical trial; 100 were allocated to blind treatment with a placebo and another 100 to clarithromycin at 1 g daily for three consecutive days. Long-term mortality was recorded. The hospitalization cost was calculated by direct quantitation of imaging tests, medical interventions, laboratory tests, nonantibiotic drugs and antibiotics, intravenous fluids, and parenteral and enteral nutrition. Quantities were priced by the respective prices defined by the Greek government in 2002. The primary endpoint was 90-day mortality; cumulative hospitalization cost was the secondary endpoint. All-cause mortality rates on day 90 were 60% in the placebo arm and 43% in the clarithromycin arm ( P = 0.023); 141 patients were alive on day 28, and mortality rates between days 29 and 90 were 44.4% and 17.4%, respectively ( P = 0.001). The mean cumulative costs on day 25 in the placebo group and in the clarithromycin group were €14,701.10 and €13,100.50 per patient staying alive, respectively ( P = 0.048). Respective values on day 45 were €26,249.50 and €19,303.10 per patient staying alive ( P = 0.011); this was associated with the savings from drugs other than antimicrobials. It is concluded that intravenous clarithromycin for three consecutive days as an adjunctive treatment in VAP and sepsis offers long-term survival benefit along with a considerable reduction in the hospitalization cost. (This study has been registered at ClinicalTrials.gov under registration no. NCT00297674.)

The objective of this study was to evaluate the efficacy of Norian skeletal repair system (SRS), a novel biodegradable and injectable form of calcium phosphate cement with a composition similar to that of cancellous bone, as a carrier for moxifloxacin, which is the most potent quinolone agent against staphylococci and Enterobacteriaceae. Norian SRS was mixed with moxifloxacin at a ratio of ca. 100:3 at room temperature and solidified in the bottom of a cylindrical vial. The same procedure was followed for acrylic bone cement. A total of five vials were prepared per system. Mueller-Hinton broth was placed over the free surface of both systems and the vials were transferred to a 37 degrees C incubator. The broth was replaced daily until visual degradation of both systems. Moxifloxacin was measured in aliquots of broth after passage through a high-performance liquid chromatography system. Optical degradation of both systems occurred after 450 days. Until Day 17, concentrations eluted from both systems were similar. After Day 18 until degradation, concentrations eluted by Norian SRS were statistically higher than those eluted by acrylic bone cement and ranged between 100 mg/L and 800 mg/L. The mean area under the concentration-time curve (AUC) over 450 days of sampling was 241 935.0 mg/L day for Norian SRS and 18 300.0 mg/L day for the acrylic bone cement system (P=0.043). Norian SRS is a novel biodegradable system providing excellent strength and mineralisation to bone. It was shown that this system allows in vitro elution of moxifloxacin at significant concentrations, making it a promising candidate for the therapy of chronic osteomyelitis.

To apply clarithromycin as an immunomodulatory treatment in experimental infection caused by pan-resistant Klebsiella pneumoniae.Acute pyelonephritis was induced in 80 rabbits after inoculation of the test isolate in the renal pelvis. Rabbits were divided into eight groups, with 10 animals in each group. In groups A-D, therapy was administered simultaneously with bacterial challenge as follows: A, controls; B, intravenous clarithromycin; C, amikacin; and D, both agents. In groups E-H, therapy was administered 24 h after bacterial challenge as follows: E, controls; F, intravenous clarithromycin; G, amikacin; and H, both agents. Blood was sampled for estimation of tumour necrosis factor-alpha (TNF-alpha) and malondialdehyde (MDA); monocytes were isolated for determination of intracellular activity of caspase-3 and ex vivo TNF-alpha secretion. Four days after bacterial challenge, animals were sacrificed for quantitative cultures and biopsies of organs.Serum TNF-alpha at 48 h was lower in groups B, C and D compared with group A. Activity of caspase-3 of monocytes was lower at 48 h in group D compared with group A. Bacterial loads of liver and spleen were decreased in group D compared with those of group A. The numbers of inflammatory cells of spleen of group B were lower compared with those of group A; those of kidney and mesenteric lymph nodes of group D were lower than those of group A. Serum MDA of group H was lower than that of group E and serum TNF-alpha of group F was lower compared with that of group E. TNF-alpha of monocyte supernatants and activity of caspase-3 of monocytes of group F were lower than those of group E. Bacterial tissue loads did not differ among groups E, F, G and H. The numbers of inflammatory cells of liver of groups F and H were lower compared with those of group E; those of kidney of groups F, G and H were lower compared with those of group E.Clarithromycin administered intravenously in experimental infection caused by pan-resistant K. pneumoniae attenuated systemic inflammatory response and local tissue damage. This effect is probably attributed to immunomodulatory intervention on blood monocytes.

To elucidate the role of innate immunity in the pathogenesis of meningitis, the effects of cerebrospinal fluid (CSF) and of sera from patients with bacterial and abacterial meningitis were studied on monocytes because of their resemblance in function with brain microglial cells. Monocytes were isolated from the mononuclear fraction of blood of healthy donors. They were incubated in growth medium single and in the presence of purified lipopolysaccharide or lipoteichoic acid of intact cells of Streptococcus pneumoniae and of samples of CSF and sera of patients. Samples were drawn from 10 patients with bacterial and 26 with abacterial meningitis. Concentrations of tumor necrosis factor α, interleukin (IL) 1β, IL-6, IL-10, IL-12, and interferon γ were estimated in cell supernatants by enzyme-linked immunosorbent assay and of procalcitonin by an immunochemiluminometric assay. It was found that the addition of CSF from patients with bacterial meningitis induced higher concentrations of IL-1β, IL-6, IL-10, IL-12, and procalcitonin than controls and than the addition of CSF from patients with abacterial meningitis. Intact cells of S. pneumoniae failed to elicit similar responses to CSF of patients. The presence of CSF from patients with abacterial meningitis induced higher levels of interferon γ and IL-6 than controls. It is concluded that cells of the innate immunity play a considerable role in the pathogenesis of meningitis. CSF triggering elicits cytokine responses that are higher in bacterial than abacterial meningitis; IL-1β, IL-6, IL-10, and procalcitonin are important mediators in bacterial meningitis and IL-6 and interferon γ in abacterial meningitis.

The potency of clarithromycin as immunomodulator was assessed in an experimental model of sepsis based on acute pyelonephritis by susceptible Escherichia coli. 55 rabbits were utilized; 5 for preliminary pharmacokinetic study and 50 for treatment. The latter were divided into 5 groups of treatment, A: controls; B: clarithromycin pretreatment; C: amikacin pretreatment; D: clarithromycin treatment on presentation of pulmonary oedema; and E; amikacin treatment on presentation of pulmonary oedema. Survival was recorded; tumour necrosis factor-alpha (TNFalpha), and malondialdehyde (MDA) were estimated in serum; activities of caspase-3 in monocyte cytosolic extracts were studied; and bacterial counts made in various organs. Median survival of animals of groups A, B, C, D and E was 1.0, 21.0, 12.5, 2.0 and 5.0 d, respectively. TNFalpha and MDA and monocyte caspase-3 activity of group A increased over time; no increases were detected in groups B and C. Concentrations of MDA and activities of monocytic caspase-3 were decreased after administration of clarithromycin in group D, an effect not occurring in group E. Bacterial load was decreased in renal tissue of group D compared to group A. It is concluded that intravenous clarithromycin might constitute a promising immunomodulator in sepsis even in the advent of pulmonary oedema.

Three multidrug-resistant strains of Pseudomonas aeruginosa were incubated ex vivo with sera sampled after a 10 min intravenous infusion of 25 mg/kg of arachidonic acid (AA) in 10 rabbits in the presence of ceftazidime and amikacin. Lipid peroxidation was assessed during bacterial growth. A statistically significant decrease in bacterial cells was found by the interaction of antimicrobials and serum sampled in the middle of infusion and 15 and 30 min after infusion of AA and was accompanied by elevated levels of malonodialdehyde. This effect of AA is probably attributed to lipid peroxidation and raises the possibility of its application in experimental infections.

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. Objective: To evaluate the long-term efficacy of etanercept for the management of hidradenitis suppurativa. Methods: Analysis was based on the long-term follow-up (weeks 24–144) of 10 patients enrolled in a prospective open-label phase II study; etanercept was initially administered subcutaneously 50 mg once weekly for 12 weeks in 10 patients. Disease recurrence and the need to restart etanercept were recorded. Results: Three patients did not report any disease recurrence. A second course of treatment with etanercept was needed in seven patients. Favourable responses were found in five; two patients failed treatment. Conclusions: The first treatment course achieved long-term disease remission in almost one-third of patients. The remaining needed a second treatment course but even in that case, their disease severity at restart was significantly lower compared with baseline.

One prospective, open-label, non-randomized study was conducted in 100 patients to define the antipyretic and analgesic effect of a new intravenous formulation of 1 g of paracetamol; 71 received paracetamol for the management of fever and 29 received paracetamol for pain relief after abdominal surgery or for neoplastic pain. Serial follow-up measurements of core temperature and of pain intensity were done for 6 h. Additional rescue medications were recorded for 5 days. Blood was sampled for the measurement of free paracetamol (APAP) and of glucuronide-APAP and N-sulfate-APAP by an HPLC assay. Defervescence, defined as core temperature below or equal to 37.1°C, was achieved in 52 patients (73.2%) within a median time of 3 h. Patients failing to become afebrile with the first dose of paracetamol became afebrile when administered other agents as rescue medications. Analgesia was achieved in 25 patients (86.4%) within a median time of 2 h. Serum levels of glucuronide-APAP were greater among non-responders to paracetamol. The presented results suggest that the intravenous formulation of paracetamol is clinically effective depending on drug metabolism. Keywords:: intravenous paracetamol, fever, pain, analgesia

Σκοπός Η εφαρμογή της κλαριθρομυκίνης ως ανοσοτροποποιητική θεραπεία στην πειραματική ουροσήψη από πολυανθεκτική Pseudomonas aeruginosa. Μέθοδοι Πρόκληση οξείας πυελονεφρίτιδας σε 40 κονίκλους μετά ενοφθαλμισμό του μικροβίου στη νεφρική πύελο. Η θεραπεία χορηγήθηκε κατά την εμφάνιση σημείων σήψης σε τέσσερις ομάδες: Α: μάρτυρες, B: ενδοφλέβιας κλαριθρομυκίνης, C: αμικασίνης και D: και των δύο φαρμάκων. Καταγράφηκε η επιβίωση και τα ζωτικά σημεία, έγινε συλλογή αίματος για καλλιέργεια και υπολογισμό προφλεγμονωδών μεσολαβητών, απομονώθηκαν μονοκύτταρα για καθορισμό του ρυθμού απόπτωσης και της ex vivo έκκρισης TNFα. Έγιναν ποσοτικές καλλιέργειες και βιοψίες οργάνων μετά τον θάνατο των πειραματικών προτύπων. Αποτελέσματα Βρέθηκαν αυξημένη θερμοκρασία ορθού και κορεσμός οξυγόνου στις ομάδες B και D συγκριτικά με τις A και C. Η μέση επιβίωση των ομάδων A, B, C και D ήταν 2.65, 7.15, 4.25 και 8.70 ημέρες αντίστοιχα. Δεν παρατηρήθηκαν διαφορές μεταξύ των ομάδων όσον αφορά το βακτηριακό φορτίο στο αίμα και τους ιστούς και τις ενδοτοξίνες στον ορό. Η MDA του ορού και η δραστικότητα της ολικής κασπάσης-3 των μονοκυττάρων της ομάδας D ελαττώθηκε μετά τη θεραπεία συγκριτικά με τις άλλες ομάδες. Διαπιστώθηκε αρνητική συσχέτιση μεταξύ της κυτταροπλασματικής κασπάσης-3 και της ex vivo έκκρισης TNFα των μονοκυττάρων του αίματος της ομάδας A, ενώ ανάλογη συσχέτιση δεν βρέθηκε για καμία άλλη ομάδα. Η ιστοπαθολογική ταξινόμηση του ήπατος και των πνευμόνων της ομάδας B ήταν χαμηλότερη από της ομάδας A. Συμπεράσματα Η όψιμη χορήγηση κλαριθρομυκίνης στην πειραματική ουροσήψη από πολυανθεκτική P.aeruginosa παρέτεινε την επιβίωση και βελτίωσε τα κλινικά ευρήματα. Η αποτελεσματικότητά της πιθανώς αποδίδεται σε ανοσοτροποποιητική παρέμβαση στα μονοκύτταρα του αίματος.

Background A previous randomized study showed that clarithromycin decreases the risk of death due to ventilator-associated pneumonia and shortens the time until infection resolution. The efficacy of clarithromycin was tested in a larger population with sepsis. Methods Six hundred patients with systemic inflammatory response syndrome due to acute pyelonephritis, acute intra-abdominal infections or primary Gram-negative bacteraemia were enrolled in a double-blind, randomized, multicentre trial. Clarithromycin (1 g) was administered intravenously once daily for 4 days consecutively in 302 patients; another 298 patients were treated with placebo. Mortality was the primary outcome; resolution of infection and hospitalization costs were the secondary outcomes. Results The groups were well matched for demographics, disease severity, microbiology and appropriateness of the administered antimicrobials. Overall 28 day mortality was 17.1% (51 deaths) in the placebo arm and 18.5% (56 deaths) in the clarithromycin arm (P = 0.671). Nineteen out of 26 placebo-treated patients with septic shock and multiple organ dysfunctions died (73.1%) compared with 15 out of 28 clarithromycin-treated patients (53.6%, P = 0.020). The median time until resolution of infection was 5 days in both arms. In the subgroup with severe sepsis/shock, this was 10 days in the placebo arm and 6 days in the clarithromycin arm (P = 0.037). The cost of hospitalization was lower after treatment with clarithromycin (P = 0.044). Serious adverse events were observed in 1.3% and 0.7% of placebo- and clarithromycin-treated patients, respectively (P = 0.502). Conclusions Intravenous clarithromycin did not affect overall mortality; however, administration shortened the time to resolution of infection and decreased the hospitalization costs.

The aim of this study was to investigate the impact of polymicrobial bloodstream infections (pBSIs) on the outcome of sepsis in an area where antimicrobial resistance is of concern. This was a retrospective analysis of data collected prospectively from patients developing BSI outside of an intensive care unit (non-ICU patients) or after ICU admission. Demographics and clinical characteristics were compared for patients with pBSI versus monomicrobial BSI (mBSI) and following stratification by ICU or non-ICU and severity of sepsis status. Possible risk factors for adverse outcome were explored by multivariate analysis, and outcomes were measured by Cox regression analysis. Among 412 patients with BSI, 47 patients (11.4%) with pBSI were recorded; compared with patients with mBSI, they had significantly higher APACHE II scores and presented more frequently with severe sepsis/septic shock. The all-cause 28-day mortality was significantly higher for pBSI versus mBSI (38.3% vs. 24.7%; P=0.033), whereas appropriateness of treatment was comparable (78.7% vs. 86.6%). Primary bacteraemia by combinations of Enterococcus faecalis, Klebsiella pneumoniae and Acinetobacter baumannii was predominant among pBSIs; in mBSIs, urinary tract infections by Escherichia coli, K. pneumoniae or Pseudomonas aeruginosa predominated. Multivariate analysis demonstrated pBSI as a significant contributor to 28-day mortality (HR=1.86; P=0.039), along with presence of two or more co-morbidities (HR=2.35; P=0.004). In conclusion, pBSIs differed epidemiologically from mBSIs, with the emergence of enterococcal species, and portended an almost two-fold increased risk of 28-day mortality. Prospective studies are warranted to elucidate possibly modifiable factors.

The immune response to a bacterial stimulus starts when pathogen-associated molecular patterns of the bacterial pathogens activate pattern recognition receptors of the innate immune system. This leads to production of proinflammatory and anti-inflammatory mediators aiming to contain infection and drive the clinical signs of sepsis. When sepsis and signs of failing organs are apparent, proinflammatory phenomena have ceased; a hypoinflammatory phase predominates, characterized by anergy of monocytes and apoptosis of T lymphocytes. The above sequence of events seems to differ from one patient to the next. The majority of therapies targeting the immune responses have failed to provide clinical benefit. Immunostimulation with IFN-γ and leukocyte growth factors, hemoperfusion with polymyxin B-embedded fiber column, and macrolides remain the most promising immunomodulators in clinical practice.

Debatable findings exist among various studies regarding the impact of single nucleotide polymorphisms (SNPs) within the promoter region of the tumor necrosis factor (TNF) gene for susceptibility to infections. Their impact was investigated in a cohort of mechanically ventilated patients who developed ventilator-associated pneumonia (VAP). Two-hundred and thirteen mechanically ventilated patients who developed VAP were enrolled. Genomic DNA was extracted and SNPs at the -376, -308 and -238 position of the promoter region of the TNF gene were assessed by restriction fragment length polymorphisms. Monocytes were isolated from 47 patients when they developed sepsis and stimulated by bacterial endotoxin for the production of TNFα and of interleukin-6 (IL-6). Patients were divided into two groups; 166 patients bearing only wild-type alleles of all three studied polymorphisms; and 47 patients carrying at least one A allele of the three studied SNPs. Time between start of mechanical ventilation and advent of VAP was significantly shorter in the second group than in the first group (log-rank: 4.416, p: 0.041). When VAP supervened, disease severity did not differ between groups. Stimulation of TNFα and of IL-6 was much greater by monocytes for patients carrying A alleles. Carriage of at least one A allele of the three studied SNPs at the promoter region of the TNF-gene is associated with shorter time to development of VAP but it is not associated with disease severity. Findings may be related with a role of the studied SNPs in the production of pro-inflammatory cytokines.

Objective: To evaluate the long-term efficacy of etanercept for the management of hidradenitis suppurativa. Methods: Analysis was based on the long-term follow-up (weeks 24-144) of 10 patients enrolled in a prospective open-label phase II study; etanercept was initially administered subcutaneously 50 mg once weekly for 12 weeks in 10 patients. Disease recurrence and the need to restart etanercept were recorded. Results: Three patients did not report any disease recurrence. A second course of treatment with etanercept was needed in seven patients. Favourable responses were found in five; two patients failed treatment. Conclusions: The first treatment course achieved long-term disease remission in almost one-third of patients. The remaining needed a second treatment course but even in that case, their disease severity at restart was significantly lower compared with baseline.

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.

Objective. To clarify whether time lapsing from advent of fever as a first sign of sepsis may be indicative of the potency of monocytes for the release of pro- and anti-inflammatory mediators.Methods. Monocytes were isolated from blood of 51 septic patients and 9 healthy donors. Monocytes were incubated in the absence and presence of patients' serum and concentrations of tumour necrosis factor-alpha (TNF), interleukin (IL)-6, IL-10, and malondialdehyde (MDA) were estimated in supernatants. Patients were divided into three groups: group A: 12 hours; group B: 12–24 hours, and group C: 24 hours between initiation of fever and blood sampling.Results. TNF of supernatants of groups B and C was higher than controls, as also were IL-6 of A and C, IL-10 of A and B, and MDA of A. IL-6 of group A was increased after addition of patients serum. A negative correlation was found between time from initiation of symptoms and IL-6 of monocyte supernatants incubated in the presence of patients serum. Median IL-6 of survivors was higher than nonsurvivors.Conclusion. Monocytes are potent for the release of pro- and anti-inflammatory mediators within the first 24 hours upon advent of fever related to sepsis; serum stimulates further release of IL-6 within the first 12 hours.

Based on the controversial findings of clinical studies regarding the influence of multidrug resistance on mortality, 10 susceptible and 10 multidrug-resistant (MDR) and extended-spectrum β-lactamase-producing isolates of Escherichia coli were applied to stimulate monocytes isolated from healthy donors. Immune mediators were estimated in supernatants. Four susceptible isolates (Group A) and four MDR isolates (Group B) were used to initiate acute pyelonephritis in 48 rabbits following inoculation of the pathogen into the right renal pelvis. Survival was recorded and blood monocytes were isolated and incubated to estimate the ex vivo release of tumour necrosis factor-alpha (TNFα). Release of TNFα, interleukin (IL)-6 and IL-8 was higher after 2 h and 4 h of stimulation by MDR isolates compared with susceptible isolates. The opposite occurred for the release of IL-12. Death occurred in 22 rabbits in Group A (91.7%) compared with 12 in Group B (50.0%) ( P = 0.003). Monocytes isolated at 24 h from Group A rabbits released significantly higher TNFα than monocytes from Group B. Tissue bacterial load after animal death was significantly higher in the kidneys of Group A rabbits. It is concluded that susceptible and MDR E. coli stimulate monocytes resulting in a different pattern of release of pro-inflammatory cytokines, which is accompanied by prolonged survival following experimental sepsis by MDR isolates.

Background To apply clarithromycin as an immunomodulatory treatment in experimental urosepsis by multidrug-resistant Pseudomonas aeruginosa. Methods Acute pyelonephritis was induced in 40 rabbits after inoculation of the test isolate in the renal pelvis. Therapy was administered upon signs of sepsis in four groups: A, controls; B, intravenous clarithromycin; C, amikacin; and D, both agents. Survival and vital signs were recorded; blood was sampled for culture and estimation of pro-inflammatory mediators; monocytes were isolated for determination of apoptotic rate and ex vivo TNFα secretion. Quantitative cultures and biopsies of organs were performed after death. Results Increased rectal temperature and oxygen saturation were found in groups B and D compared to A and C. Mean survival of groups A, B, C and D was 2.65, 7.15, 4.25 and 8.70 days respectively. No differences were noted between groups concerning bacterial load in blood and tissues and serum endotoxins. Serum MDA and total caspase-3 activity of monocytes of group D decreased following treatment compared to other groups. Negative correlation was detected between cytoplasmic caspase-3 and ex vivo secretion of TNFα of blood monocytes of group A; similar correlation was not found for any other group. Pathology scores of liver and lung of group B were lower than group A. Conclusion Clarithromycin administered late in experimental urosepsis by multidrug-resistant P. aeruginosa prolonged survival and ameliorated clinical findings. Its effect is probably attributed to immunomodulatory intervention on blood monocytes.

SUMMARY In attempt to investigate the stimulatory effect of Pseudomonas aeruginosa on innate immunity and to correlate it to its level of resistance to antimicrobials, 20 isolates were applied; 8 isolates were susceptible and 12 multidrug-resistant. Genetic diversity was defined by PFGE. Human monocytes of two healthy volunteers were in vitro stimulated by the isolates for the production of pro-inflammatory (TNF-α, IL-1β, IL-6, IL-8 and IL-12) and anti-inflammatory cytokines (IL-10), of malondialdehyde and of procalcitonin. Cytokines were estimated by EIA, malondialdehyde by the thiobarbiturate assay and procalcitonin by an immunochemiluminometric assay. Survival of 48 Wistar rats was recorded after induction of sepsis by the intraperitoneal injection of three susceptible and three multidrug-resistant isolates. To test whether comparative effect of the latter isolates on survival correlates with any difference of monocyte-mediated release of pro-inflammatory mediators, monocytes of two rats were in vitro stimulated for the production of TNF-α and of malondialdehyde. In vitro stimulation of human monocytes by the susceptible isolates elicited elevated production of malondiadeheyde, of IL-1β and of IL-6 compared to stimulation by multidrug-resistant isolates. Similar differences were found for TNF-α and IL-8, but they were not statistically significant. Production of IL-10 and IL-12 was not detected after stimulation with any isolate. Levels of procalcitonin were similar after induction with either susceptible or multidrug-resistant isolates. Mean survival of animals was 7·56, 21·80 and 55·20 h, respectively, after challenge by the susceptible isolates and 28·89, 61·8 and more than 120 h, respectively, after challenge by the multidrug-resistant isolates. Differences of survival were accompanied by greater rodent monocyte-release of TNF-α and malondialdehyde after stimulation by the susceptible isolates compared to multidrug-resistant ones. It is concluded that considerable differences are encountered on the stimulation of human monocytes by susceptible and resistant isolates of Pseudomonas aeruginosa. These results correlate with in vivo evidence and might influence decision on therapeutics.

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. J Crit Care

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.

In a recently published double-blind, randomized trial conducted by our study group, clarithromycin was intravenously administered in patients with ventilator-associated pneumonia (VAP) and sepsis for three consecutive days [1]. An earlier resolution of VAP and a fivefold decrease of the risk for death by septic shock and multiple organ failure (MODS) compared with placebo were shown.

Based on the central role of the triggering of monocytes for the initiation of the septic cascade, it was investigated whether apoptosis of blood monocytes in septic patients is connected to their final outcome. Blood monocytes were isolated from 90 patients with septic syndrome due to ventilator-associated pneumonia on days 1, 3, 5 and 7 from the initiation of symptoms. Apoptosis was defined after incubation with annexin-V-fluorescein isothiocyanate and propidium iodine and reading by a flow cytometer. The function of first-day monocytes was evaluated from the concentrations of tumour necrosis factor alpha (TNFα) and IL-6 in supernatants of cell cultures after triggering with endotoxins. TNFα, IL-6 and IL-8 were estimated in serum by an enzyme immunoassay. Mortality rates of patients with apoptosis ≤50% compared with patients with apoptosis >50% were 49.12% and 15.15%, respectively (P 50% compared with those patients with apoptosis ≤50% (P = 0.0032). Production of IL-6 by monocytes on the first day by patients with apoptosis ≤50% was similar compared with monocytes isolated from healthy controls. Serum concentrations of TNFα were higher in patients with monocyte apoptosis ≤50% and septic shock compared with patients with apoptosis >50% on day 7; similar findings occurred for serum IL-6 on days 1 and 7 and for serum IL-8 on days 1 and 5. Early apoptosis of monocytes upon presentation of clinical signs of sepsis is connected to a favourable outcome. These findings are of particular importance for the patient with septic shock, where they might constitute a mechanism of pathogenesis.

Background TREM-1 (triggering receptor expressed on myeloid cells), a receptor expressed on neutrophils and monocytes, is upregulated in sepsis and seems to tune the inflammatory response. We explored the expression of TREM-1 at the gene level and on cell membranes of monocytes and association with clinical outcome. Methods Peripheral venous blood was sampled from 75 septic patients (39 patients with sepsis, 25 with severe sepsis and 11 with septic shock) on sepsis days 1, 3 and 7. TREM-1 on monocytes was measured by flow cytometry; gene expression of TREM-1 in circulating mononuclear cells was assessed by real-time PCR. sTREM-1 was measured in serum by an enzyme immunoassay. Results Although surface TREM-1, sTREM-1 and TREM-1 gene expression did not differ between sepsis, severe sepsis and septic shock on day 1, survivors had greater expression of surface TREM-1 on days 3 and 7 compared to non-survivors. sTREM-1 on non-survivors decreased on day 3 compared to baseline. Patients with increase of monocyte gene expression of TREM-1 from day 1 to day 3 had prolonged survival compared to patients with decrease of gene expression of TREM-1 from day 1 to day 3 (p: 0.031). Conclusions Early decrease of gene expression of TREM-1 in monocytes is associated with poor outcome. A reciprocal decrease of the pro-inflammatory surface receptor TREM-1 linked with sepsis-induced immunosuppression may be part of the explanation. Electronic supplementary material The online version of this article (doi:10.1186/s12865-014-0063-y) contains supplementary material, which is available to authorized users.

Recent evidence suggests a link between excess lipid peroxidation and specific organ failures in sepsis. No study has been performed in sepsis by multidrug-resistant (MDR) Gram-negative bacteria. Lethal sepsis was induced in rats by the intraperitoneal injection of one MDR isolate of Pseudomonas aeruginosa. Produced malondialdehyde (MDA) was measured in tissues 5 hours after bacterial challenge with the thiobarbiturate assay followed by high-performance liquid chromatography (HPLC) analysis. Results were compared with those from a cohort of patients with ventilator-associated pneumonia (VAP) and sepsis by MDR Gram-negative bacteria. More precisely, serum MDA was measured on 7 consecutive days, and it was correlated with clinical characteristics. MDA of septic rats was greater in the liver, spleen, and aortic wall, and it was lower in the right kidney compared with sham operated-on animals. Findings were confirmed by the studied cohort. Circulating MDA was greater in patients with hepatic dysfunction and acute respiratory distress syndrome (ARDS) compared with patients without any organ failures. The opposite was found for patients with acute renal dysfunction. No differences were found between patients with ARDS without or with cardiovascular (CV) failure and patients without any organ failure. Serial measurements of MDA in serum of patients indicated that levels of MDA were greater in survivors of hepatic dysfunction and ARDS and lower in survivors of acute renal dysfunction. Animal findings and results of human sepsis are complementary, and they suggest a compartmentalization of lipid peroxidation in systemic infections by MDR gram-negative bacteria.

Introduction Early 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. Methods A 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. Results Serum 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