Mary White

The occurrence of severe sepsis and septic shock are related to distinct patterns of cytokine gene expression.

ABSTRACT Patient response to acute bacterial infection is highly variable. Differing outcomes in this setting may be related to variations in the immune response to an infectious insult. Using quantitative real-time polymerase chain reaction, we quantified gene expression of the tumor necrosis factor &agr;(TNF&agr;), interferon &ggr; (IFN&ggr;), and interleukin 10 (IL10), IL12p35, and IL4 genes in 3 patient groups. These groups consisted of an intensive care unit (ICU) cohort who presented with severe sepsis or septic shock, a group of noncritically ill ward patients with documented Gram-negative bacteremia, and a group of healthy controls. Greater interleukin 10 messenger RNA (mRNA) levels were detected in the ICU group in comparison with both the bacteremic and control groups (P < 0.0001). More TNF-&agr; mRNA was detected in the ICU group when compared with the control group (P < 0.0001). However, TNF-&agr; mRNA was most abundant in the bacteremic group (P = 0.0007). Lesser IFN-&ggr; mRNA levels were detected in the ICU group when compared with both the bacteremic and control groups (P < 0.0003). Cytokine mRNA levels were not associated with the occurrence of shock upon admission to ICU. On the seventh day of ICU stay, the presence of shock was associated with lesser IFN-&ggr; mRNA (P = 0.0004) and lesser TNF-&agr; mRNA (P = 0.001). Survivors had greater TNF-&agr; mRNA copy numbers on day 7 of ICU stay than nonsurvivors (P = 0.002). We conclude that a proinflammatory response is the appropriate response in the setting of infection and is associated with lesser requirements for inotropes and lesser mortality. Quantitative real-time polymerase chain reaction can be used to predict infection outcome in clinically relevant situations where enzyme-linked immunosorbent assay testing has proved disappointing.

Expression of hereditary hemochromatosis C282Y HFE protein in HEK293 cells activates specific endoplasmic reticulum stress responses

BackgroundHereditary Hemochromatosis (HH) is a genetic disease associated with iron overload, in which individuals homozygous for the mutant C282Y HFE associated allele are at risk for the development of a range of disorders particularly liver disease. Conformational diseases are a class of disorders associated with the expression of misfolded protein. HFE C282Y is a mutant protein that does not fold correctly and consequently is retained in the Endoplasmic Reticulum (ER). In this context, we sought to identify ER stress signals associated with mutant C282Y HFE protein expression, which may have a role in the molecular pathogenesis of HH.ResultsVector constructs of Wild type HFE and Mutant C282Y HFE were made and transfected into HEK293 cell lines. We have shown that expression of C282Y HFE protein triggers both an unfolded protein response (UPR), as revealed by the increased GRP78, ATF6 and CHOP expression, and an ER overload response (EOR), as indicated by NF-κB activation. Furthermore, C282Y HFE protein induced apoptotic responses associated with activation of ER stress. Inhibition studies demonstrated that tauroursodeoxycholic acid, an endogenous bile acid, downregulates these events. Finally, we found that the co-existence of both C282Y HFE and Z alpha 1-antitrypsin protein (the protein associated with the liver disease of Z alpha 1-antitrypsin deficiency) expression on ER stress responses acted as potential disease modifiers with respect to each other.ConclusionOur novel observations suggest that both the ER overload response (EOR) and the unfolded protein response (UPR) are activated by mutant C282Y HFE protein.

Post-operative infection and sepsis in humans is associated with deficient gene expression of γc cytokines and their apoptosis mediators

IntroductionLymphocyte homeostasis is dependent on the γc cytokines. We hypothesised that sepsis in humans is associated with differential gene expression of the γc cytokines and their associated apoptosis mediators.MethodsThe study population consisted of a total of 60 patients with severe sepsis, 15 with gram negative bacteraemia, 10 healthy controls and 60 patients undergoing elective lung resection surgery. Pneumonia was diagnosed by CDC NNIC criteria. Gene expression in peripheral blood leukocytes (PBLs) of interleukin (IL)-2, 7, 15 and interferon (IFN)-γ, Bax, Bim, Bcl-2 was determined by qRT-PCR and IL-2 and IL-7 serum protein levels by ELISA. Gene expression of IL-2, 7 and IFN-γ was measured in peripheral blood leukocytes (PBL), cultured in the presence of lipopolysacharide (LPS) and CD3 binding antibody (CD3ab)ResultsIL-2 gene expression was lower in the bacteraemia group compared with controls, and lower still in the sepsis group (P < 0.0001). IL-7 gene expression was similar in controls and bacteraemia, but lower in sepsis (P < 0.0001). IL-15 gene expression was similar in the three groups. Bcl-2 gene expression was less (P < 0.0001) and Bim gene expression was greater (P = 0.0003) in severe sepsis compared to bacteraemic and healthy controls. Bax gene expression was similar in the three groups.In lung resection surgery patients, post-operative pneumonia was associated with a perioperative decrease in IL-2 mRNA (P < 0.0001) and IL-7 mRNA (P = 0.003). IL-2 protein levels were reduced in sepsis and bacteraemia compared to controls (P = 0.02) but similar in pneumonia and non-pneumonia groups. IL-7 protein levels were similar in all groups.In cultured PBLs, IFN-γ gene expression was decreased in response to LPS and increased in response to CD3ab with sepsis: IL-7 gene expression increased in response to LPS in controls and to CD3ab with sepsis; Bcl-2 gene expression decreased in response to combined CD3ab and IL-2 with sepsis.ConclusionsPatients with infection and sepsis have deficient IL-2 and IL-7 gene expression in PBLs. Aberrant cytokine gene expression may precede the onset of infection.

Hospital-acquired pneumonia after lung resection surgery is associated with characteristic cytokine gene expression.

BACKGROUND Infection in humans has been linked with altered cytokine gene transcription. It is unclear whether this phenomenon is a consequence of an established disease process or precedes the infective process. The primary end point of this study was to determine whether hospital-acquired pneumonia (HAP) was associated with differential gene expression of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and IL-23p19. The secondary end point was to identify whether alteration in gene expression preceded the clinical onset of infection. METHODS Sixty consecutive patients undergoing elective thoracic surgery were recruited. HAP was diagnosed as per National Nosocomial Infection Surveillance guidelines. Messenger RNA (mRNA) and protein levels were analyzed preoperatively and 24 h and 5 days postoperatively. RESULTS Forty-one patients had an uncomplicated recovery. Nineteen patients developed HAP. IL-6, IL-10, IL-12p35, IL-23p19, IL-27p28, TNF-α, and IFN-γ mRNA and protein levels of IL-6, IL-23, and IFN-γ in peripheral blood leukocytes were analyzed before surgery and 24 h and 5 days postsurgery. IL-23p19 mRNA levels were reduced in the pneumonia group (median, 4.19; 10th-90th centile range, 3.90-4.71) compared with the nonpneumonia group (4.50; 3.85-5.32) day 1 postsurgery (P=02). IFN-γ mRNA levels were reduced in the pneumonia group (2.48; 1.20-3.20) compared with nonpneumonia group (2.81; 2.10-3.26) (P=03) day 5 postsurgery. Results are expressed as log to base 10 copy numbers of cytokine mRNA per 10 million β-actin mRNA copy numbers. All values are given as median and 10th to 90th centile range. CONCLUSIONS Cytokine gene expression is altered immediately following surgery in patients with postoperative HAP.

Transforming growth factor β-1 and interleukin-17 gene transcription in peripheral blood mononuclear cells and the human response to infection

INTRODUCTION The occurrence of severe sepsis may be associated with deficient pro-inflammatory cytokine production. Transforming growth factor beta-1 (TGFbeta-1) predominantly inhibits inflammation and may simultaneously promote IL-17 production. Interleukin-17 (IL-17) is a recently described pro-inflammatory cytokine, which may be important in auto-immunity and infection. We investigated the hypothesis that the onset of sepsis is related to differential TGFbeta-1 and IL-17 gene expression. METHODS A prospective observational study in a mixed intensive care unit (ICU) and hospital wards in a university hospital. Patients (59) with severe sepsis; 15 patients with gram-negative bacteraemia but without critical illness and 10 healthy controls were assayed for TGFbeta-1, IL-17a, IL-17f, IL-6 and IL-1beta mRNA in peripheral blood mononuclear cells (PBMC) by quantitative real-time PCR and serum protein levels by ELISA. RESULTS TGFbeta-1 mRNA levels are reduced in patients with bacteraemia and sepsis compared with controls (p=0.02). IL-6 mRNA levels were reduced in bacteraemic patients compared with septic patients and controls (p=0.008). IL-1beta mRNA levels were similar in all groups, IL-17a and IL-17f mRNA levels are not detectable in peripheral blood mononuclear cells. IL-6 protein levels were greater in patients with sepsis than bacteraemic and control patients (p<0.0001). Activated TGFbeta-1 and IL-17 protein levels were similar in all groups. IL-1beta protein was not detectable in the majority of patients. CONCLUSIONS Down regulation of TGFbeta-1 gene transcription was related to the occurrence of infection but not the onset of sepsis. Interleukin-17 production in PBMC may not be significant in the human host response to infection.

Highly sensitivity adhesion molecules detection in hereditary haemochromatosis patients reveals altered expression

Several abnormalities in the immune status of patients with hereditary haemochromatosis (HH) have been reported, suggesting an imbalance in their immune function. This may include persistent production of, or exposure to, altered immune signalling contributing to the pathogenesis of this disorder. Adhesion molecules L‐, E‐ and P‐Selectin, intercellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1) are some of the major regulators of the immune processes and altered levels of these proteins have been found in pathological states including cardiovascular diseases, arthritis and liver cancer. The aim of this study was to assess L‐, E‐ and P‐Selectin, ICAM‐1 and VCAM‐1 expression in patients with HH and correlate these results with HFE mutation status and iron indexes. A total of 139 subjects were diagnosed with HH (C282Y homozygotes = 87, C282Y/H63D = 26 heterozygotes, H63D homozygoyes = 26), 27 healthy control subjects with no HFE mutation (N/N), 18 normal subjects heterozygous for the H63D mutation served as age‐sex‐matched controls. We observed a significant decrease in L‐selectin (P = 0.0002) and increased E‐selectin and ICAM‐1 (P = 0.0006 and P = 0.0059) expression in HH patients compared with healthy controls. This study observes for the first time that an altered adhesion molecules profile occurs in patients with HH that is associated with specific HFE genetic component for iron overload, suggesting that differential expression of adhesion molecules may play a role in the pathogenesis of HH.