Association of autophagy-related 16-like 1 (ATG16L1) gene polymorphism with sepsis severity in patients with sepsis and ventilator-associated pneumonia.

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-α (TNF-α) 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-α 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.

Indication for a role of regulatory T cells for the advent of influenza A (H1N1)-related pneumonia.

Regulatory T cells (T(regs) ) have an anti-inflammatory role. A former study in a limited number of patients found that absolute counts of T(regs) 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. T(regs) 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 T(reg) 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 T(regs) equal to or above 16 mm(3) (P = 0·013). It is concluded that the advent of H1N1-related pneumonia is related to an early increase of the absolute T(reg) counts. This increase is probably not part of a hypo-inflammatory state of the host.

Immunomodulatory effect of three-day continuous administration of clarithromycin for experimental sepsis due to multidrug-resistant Pseudomonas aeruginosa.

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.

Early apoptosis of blood monocytes is a determinant of survival in experimental sepsis by multi-drug-resistant Pseudomonas aeruginosa.

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)-alpha 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-alpha 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 10(4) 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 10(4) 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-alpha.

Stimulation of innate immunity by susceptible and multidrug-resistant Pseudomonas aeruginosa: an in vitro and in vivo study.

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-alpha, IL-1beta, 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-alpha and of malondialdehyde. In vitro stimulation of human monocytes by the susceptible isolates elicited elevated production of malondiadeheyde, of IL-1beta and of IL-6 compared to stimulation by multidrug-resistant isolates. Similar differences were found for TNF-alpha 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-alpha 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.