Malte Book

Differential expression of alpha- and beta-defensins in human peripheral blood.

Background  Human defensin peptides with broad‐spectrum antimicrobial activity have been implicated in the human defence response towards microbial invasion. Two families of defensins designated α‐ and β‐defensins, respectively, have been identified. Little is known about the expression of both defensin families in human peripheral blood. The purpose of this study was to examine the expression of α‐ and β‐defensin genes in human peripheral blood.

Induction of Bim and Bid gene expression during accelerated apoptosis in severe sepsis

IntroductionIn transgenic animal models of sepsis, members of the Bcl-2 family of proteins regulate lymphocyte apoptosis and survival of sepsis. This study investigates the gene regulation of pro-apoptotic and anti-apoptotic members of the Bcl-2 family of proteins in patients with early stage severe sepsis.MethodsIn this prospective case-control study, patients were recruited from three intensive care units (ICUs) in a university hospital. Sixteen patients were enrolled when they fulfilled the criteria of severe sepsis. Ten critically ill but non-septic patients and 11 healthy volunteers served as controls. Blood samples were immediately obtained at inclusion. To confirm the presence of accelerated apoptosis in the patient groups, caspase-3 activation and phosphatidylserine externalisation in CD4+, CD8+ and CD19+ lymphocyte subsets were assessed using flow cytometry. Specific mRNAs of Bcl-2 family members were quantified from whole blood by real-time PCR. To test for statistical significance, Kruskal-Wallis testing with Dunns multiple comparison test for post hoc analysis was performed.ResultsIn all lymphocyte populations caspase-3 (p < 0.05) was activated, which was reflected in an increased phosphatidylserine externalisation (p < 0.05). Accordingly, lymphocyte counts were decreased in early severe sepsis. In CD4+ T-cells (p < 0.05) and B-cells (p < 0.001) the Bcl-2 protein was decreased in severe sepsis. Gene expression of the BH3-only Bim was massively upregulated as compared with critically ill patients (p < 0.001) and 51.6-fold as compared with healthy controls (p < 0.05). Bid was increased 12.9-fold compared with critically ill patients (p < 0.001). In the group of mitochondrial apoptosis inducers, Bak was upregulated 5.6-fold, while the expression of Bax showed no significant variations. By contrast, the pro-survival members Bcl-2 and Bcl-xl were both downregulated in severe sepsis (p < 0.001 and p < 0.05, respectively).ConclusionsIn early severe sepsis a gene expression pattern with induction of the pro-apoptotic Bcl-2 family members Bim, Bid and Bak and a downregulation of the anti-apoptotic Bcl-2 and Bcl-xl proteins was observed in peripheral blood. This constellation may affect cellular susceptibility to apoptosis and complex immune dysfunction in sepsis.

CYP2D6 Genotype Dependent Oxycodone Metabolism in Postoperative Patients

Background The impact of polymorphic cytochrome P450 CYP2D6 enzyme on oxycodones metabolism and clinical efficacy is currently being discussed. However, there are only spare data from postoperative settings. The hypothesis of this study is that genotype dependent CYP2D6 activity influences plasma concentrations of oxycodone and its metabolites and impacts analgesic consumption. Methods Patients received oxycodone 0.05 mg/kg before emerging from anesthesia and patient-controlled analgesia (PCA) for the subsequent 48 postoperative hours. Blood samples were drawn at 30, 90 and 180 minutes after the initial oxycodone dose. Plasma concentrations of oxycodone and its metabolites oxymorphone, noroxycodone and noroxymorphone were analyzed by liquid chromatography-mass spectrometry with electrospray ionization. CYP2D6 genotyping was performed and 121 patients were allocated to the following genotype groups: PM (poor metabolizer: no functionally active CYP2D6 allele), HZ/IM (heterozygous subjects, intermediate metabolizers with decreased CYP2D6 activity), EM (extensive metabolizers, normal CYP2D6 activity) and UM (ultrarapid metabolizers, increased CYP2D6 activity). Primary endpoint was the genotype dependent metabolite ratio of plasma concentrations oxymorphone/oxycodone. Secondary endpoint was the genotype dependent analgesic consumption with calculation of equianalgesic doses compared to the standard non-CYP dependent opioid piritramide. Results Metabolism differed between CYP2D6 genotypes. Mean (95%-CI) oxymophone/oxycodone ratios were 0.10 (0.02/0.19), 0.13 (0.11/0.16), 0.18 (0.16/0.20) and 0.28 (0.07/0.49) in PM, HZ/IM, EM and UM, respectively (p = 0.005). Oxycodone consumption up to the 12th hour was highest in PM (p = 0.005), resulting in lowest equianalgesic doses of piritramide versus oxycodone for PM (1.6 (1.4/1.8); EM and UM 2.2 (2.1/2.3); p<0.001). Pain scores did not differ between genotypes. Conclusions In this postoperative setting, the number of functionally active CYP2D6 alleles had an impact on oxycodone metabolism. The genotype also impacted analgesic consumption, thereby causing variation of equianalgesic doses piritramide : oxycodone. Different analgesic needs by genotypes were met by PCA technology in this postoperative cohort.

Cytokine Promoter Polymorphisms in Severe Sepsis

The need to develop individualized risk profiles and drug therapy regimens motivates interest in genetic studies of critically ill patients. Gene promoter variants may predict interindividual variability in response to inflammatory stimuli, such as infection and trauma. Genomic variations also may affect gene expression profiles, as well as the structure and production of proteins. The genes involved in inflammation are numerous, as are genomic variations within most of those genes. Cytokine genes involved in inflammatory cascades are important candidate genes that may determine the extent of a persons response to injury. Understanding the genetic determination of the inflammatory process includes the possibility of developing valuable diagnostic tools and new therapeutic approaches in severe sepsis. To date, specific patterns of markers of genomic variation reliably indicating at-risk populations do not exist. Evaluation of possible genomic markers for risk stratification of patients with sepsis and persons at high risk of developing organ failure has begun at a level of well-powered genetic epidemiological research. Cytokine promoter variants may contribute substantially to studies of genetic predisposition of sepsis because they operate in a gene region of high regulatory activity.

Inducibility of the endogenous antibiotic peptide β-defensin 2 is impaired in patients with severe sepsis

IntroductionThe potent endogenous antimicrobial peptide human β-defensin 2 (hBD2) is a crucial mediator of innate immunity. In addition to direct antimicrobial properties, different effects on immune cells have been described. In contrast to the well-documented epithelial β-defensin actions in local infections, little is known about the leukocyte-released hBD2 in systemic infectious disorders. This study investigated the basic expression levels and the ex vivo inducibility of hBD2 mRNA in peripheral whole blood cells from patients with severe sepsis in comparison to non-septic critically ill patients and healthy individuals.MethodsThis investigation was a prospective case-control study performed at a surgical intensive care unit at a university hospital. A total of 34 individuals were tested: 16 patients with severe sepsis, 9 critically ill but non-septic patients, and 9 healthy individuals. Serial blood samples were drawn from septic patients, and singular samples were obtained from critically ill non-septic patients and healthy controls. hBD2 mRNA levels in peripheral white blood cells were quantified by real-time polymerase chain reaction in native peripheral blood cells and following ex vivo endotoxin stimulation. Defensin plasma levels were quantified by enzyme-linked immunosorbent assay.ResultsEndotoxin-inducible hBD2 mRNA expression was significantly decreased in patients with severe sepsis compared to healthy controls and non-septic critically ill patients (0.02 versus 0.95 versus 0.52, p < 0.05, arbitrary units). hBD2 plasma levels in septic patients were significantly higher compared to healthy controls and critically ill non-septic patients (541 versus 339 versus 295 pg/ml, p < 0.05).ConclusionIn contrast to healthy individuals and critically ill non-septic patients, ex vivo inducibility of hBD2 in peripheral blood cells from septic patients is reduced. Impaired hBD2 inducibility may contribute to the complex immunological dysfunction in patients with severe sepsis.

The PstI polymorphism of the endotoxin-inducible heat-shock protein 70-2 gene does not affect messenger RNA level in human whole-blood cultures

Objective: To determine whether the human leukocyte antigen linked biallelic heat-shock protein 70–2 (HSP70–2) gene polymorphism is associated with variable HSP70–2 messenger RNA expression. Design: Prospective observational study in consecutive healthy blood donors. Setting: Department of Anesthesiology, laboratory for molecular biology in a university hospital. Participants: 24 healthy blood donors. Interventions: None. Measurements and results: We studied the functional implication of the HSP70–2 (G/A) PstI gene polymorphism in 24 healthy, white blood donors with various HSP70–2 (G/A) genotypes by analyzing the endotoxin-inducible HSP70–2 mRNA expression by means of the reverse transcription–polymerase chain reaction. HSP70 expression was expressed semiquantitatively by calculating the ratio of HSP70–2 mRNA and the constitutively expressed glutaraldehyde 3-phosphate dehydrogenase mRNA. No significant differences in HSP70–2 mRNA expression after lipopolysaccharide (from Salmonella minnesota Re 595) stimulation were detected in individuals homozygous for the allele A (0.68, range 0.38–1, n = 10), in individuals homozygous for the allele G (0.79, range 0.42–1.1, n = 8), and in heterozygotes (HSP70–2 G/A; 0.52, range 0.4–0.67, n = 6; p > 0.05). Conclusions: The PstI polymorphism of the endotoxin-inducible HSP70–2 gene is not associated with variable HSP70–2 mRNA expression ex vivo. This finding is in accordance with the observation that HSP70–2 genotypes do not affect clinical outcome in human systemic inflammation.

Expression of the nociceptin precursor and nociceptin receptor is modulated in cancer and septic patients

BACKGROUND A role of nociceptin and its receptor (NOP) in pain and immune function has been suggested. The hypothesis was that mRNA expression of NOP and the nociceptin precursor pre-pronociceptin (pN/OFQ) in peripheral blood cells differs in end-stage cancer patients suffering from chronic pain and septic intensive care unit (ICU) patients compared with healthy controls. METHODS Blood samples were drawn from end-stage cancer patients and septic ICU patients. Additionally, postoperative patients representing individuals with surgical stress and healthy controls were enrolled as comparative groups. NOP and pN/OFQ mRNA expression, quantified by real-time polymerase chain reaction (RT-PCR), was compared between study groups, and associated to opioid medication, pain intensities, and the inflammatory markers procalcitonin (PCT) and interleukin-6. RESULTS NOP expression was significantly higher in cancer patients [normalized ratio, median (inter-quartile range): 10.2 (7.4/17.8)], postoperative patients [8.0 (5.3/10.2)], and ICU patients [6.6 (4.2/9.5)] compared with healthy controls [4.4 (2.7/7.0); P<0.001]. Expression of pN/OFQ was lower in cancer patients [3.8 (1.9/5.9)] and ICU patients [1.9 (1.0/2.7)] but not in postoperative patients compared with healthy controls [7.2 (6.1/9.4); P<0.001]. Increased plasma PCT was associated with decreased pN/OFQ in all patient groups. In cancer patients, no association was seen with pain scores, opioid medication or duration of analgesia, and NOP or pN/OFQ mRNA. CONCLUSIONS NOP and pN/OFQ expression in peripheral blood cells was modulated in end-stage cancer and septic patients compared with healthy controls, whereas changes in postoperative patients were minor. The involvement of the NOP-pN/OFQ system in inflammation, impaired immune function, and pain has to be further elucidated.

TNF-α Promoter Polymorphism in Relation to TNF-α Production and Clinical Status in Cystic Fibrosis

The severity of lung disease in cystic fibrosis may be related to the genetic propensity of the host to produce tumor necrosis fector α (TNF-α). A polymorphism in the promoter region of the TNF-α gene at nucleotide 308 relative to the transcription start site may be important in determing the host’s TNF-α response. The aim of this study was to assess the correlation between a TNF-308 promoter polymorphism, ex vivo TNF-α production (before and after lipopolysaccharide (LPS) stimulation), and clinical status [FEV1, weight (z-score), BMI, Shwachman score, incidence of diabetes mellitus, and Pseudomonas aeruginosa infection). Genotyping for the biallelic TNF-308 polymorphism was performed by using a real-time PCR cycler. Patients (homozygous for Delta F 508) were grouped according to genotype (TNF2 carriers, n = 16, median age = 15 yr, female/male = 5/11; TNF1 homozygotes, n = 37, median age = 21 yr, female/male = 18/19). TNF-α was measured using a chemiluminescent immunometric assay. There was a trend toward higher TNF-α values [median TNF2 carriers vs. TNF1 homozygotes: x = 56 vs. 43.5 pg/ml, n.s. (Mann–Whitney U-test] in those carrying the polymorphism and better lung function results [FEV1 (%) 81 vs. 65, n.s.]. These differences equalized [TNF2 carriers vs. TNF1 56 vs. 51 pg/ml, n.s.; FEV1 (%) 84 vs. 79, n.s.] after age adjustment (± 2 yr, n = 15, median age TNF2 vs. TNF1-17/18 yr). There were no significant differences for TNF values after LPS stimulation and the incidence of diabetes mellitus. The TNF-308 promoter polymorphism does not seem to influence TNF-α release in whole blood cells and clinical status.