A Transcriptomic Approach to Understand Patient Susceptibility to Pneumonia After Abdominal Surgery.

OBJECTIVE: To describe immune pathways and gene networks altered following major abdominal surgery and to identify transcriptomic patterns associated with postoperative pneumonia. BACKGROUND: Nosocomial infections are a major healthcare challenge, developing in over 20% of patients aged 45 or over undergoing major abdominal surgery, with postoperative pneumonia associated with an almost 5-fold increase in 30-day mortality. METHODS: From a prospective consecutive cohort (n=150) undergoing major abdominal surgery, whole-blood RNA was collected preoperatively and at 3 time-points postoperatively (2-6, 24, and 48 h). Twelve patients diagnosed with postoperative pneumonia and 27 matched patients remaining infection-free were identified for analysis with RNA-sequencing. RESULTS: Compared to preoperative sampling, 3639 genes were upregulated and 5043 downregulated at 2 to 6 hours. Pathway analysis demonstrated innate-immune activation with neutrophil degranulation and Toll-like-receptor signaling upregulation alongside adaptive-immune suppression. Cell-type deconvolution of preoperative RNA-sequencing revealed elevated S100A8/9-high neutrophils alongside reduced naïve CD4 T-cells in those later developing pneumonia. Preoperatively, a gene-signature characteristic of neutrophil degranulation was associated with postoperative pneumonia acquisition ( P =0.00092). A previously reported Sepsis Response Signature (SRSq) score, reflecting neutrophil dysfunction and a more dysregulated host response, at 48 hours postoperatively, differed between patients subsequently developing pneumonia and those remaining infection-free ( P =0.045). Analysis of the novel neutrophil gene-signature and SRSq scores in independent major abdominal surgery and polytrauma cohorts indicated good predictive performance in identifying patients suffering later infection. CONCLUSIONS: Major abdominal surgery acutely upregulates innate-immune pathways while simultaneously suppressing adaptive-immune pathways. This is more prominent in patients developing postoperative pneumonia. Preoperative transcriptomic signatures characteristic of neutrophil degranulation and postoperative SRSq scores may be useful predictors of subsequent pneumonia risk.

Post-operative immune suppression is mediated via reversible, Interleukin-10 dependent pathways in circulating monocytes following major abdominal surgery.

INTRODUCTION: Post-operative infections occur frequently following major surgery. The magnitude of the post-operative immune response is associated with an increased risk of post-operative infections, although the mechanisms driving post-operative immune-dysfunction and the potential reversibility of this response with immune stimulants are not well understood. This study aims to describe the immediate immune response to major surgery and establish links to both post-operative infection and functional aspects of immune dysregulation. We also investigate the potential of clinically available immune stimulants to reverse features of post-operative immune-dysfunction. METHODS: Patients over 45 years old undergoing elective gastro-intestinal surgery with planned post-operative surgical ICU admission were recruited. The expression of selected genes was determined pre-operatively and at 2, 24 and 48 hours post-operatively using qRT-PCR. Circulating levels of Interleukin-10 protein were determined by ELISA. Peri-operative cell surface monocyte HLA-DR (mHLA-DR) expression was determined using flow cytometry. Gene expression and mHLA-DR levels were determined in healthy monocytes cultured in peri-operative serum with and without neutralising antibodies and immune stimulants. RESULTS: 119 patients were recruited; 44 developed a post-operative infection. Interleukin-10 mRNA and protein increased 4-fold post-operatively (P<0.0001), peaking within 2 hours of the procedure. Higher post-operative Interleukin-10 mRNA (P = 0.007) and protein (P = 0.001) levels were associated with an increased risk of infection. Cell surface mHLA-DR expression fell post-operatively (P<0.0001). Reduced production, rather than intracellular sequestration, accounted for the post-operative decline in cell surface mHLA-DR expression. Interleukin-10 antibody prevented the decrease in mHLA-DR expression observed when post-operative serum was added to healthy monocytes. GM-CSF and IFN-γ prevented the decline in mHLA-DR production through distinct pathways. CONCLUSIONS: Monocyte dysfunction and features of immune suppression occur frequently after major surgery. Greater post-operative Interleukin-10 production is associated with later infection. Interleukin-10 is an important mediator of post-operative reductions in mHLA-DR expression, while clinically available immune stimulants can restore mHLA-DR levels.

MicroRNAs in Acute Kidney Injury.

BACKGROUND: It is increasingly recognised that improved diagnosis, prognosis and treatment of acute kidney injury (AKI) requires an understanding of distinct underling cellular and molecular mechanisms (endotypes) that may distinguish overtly similar clinical AKI presentations. One important avenue of research is the post-transcriptional regulation of gene expression in response to kidney injury mediated by microRNAs. SUMMARY: This mini-review summarises the use of microRNAs as diagnostic and prognostic biomarkers in AKI. The contribution of microRNAs to the pathophysiology of AKI will be highlighted along with the potential for therapeutic applications. Key Messages: While there is great potential for a better understanding of AKI, microRNAs form a complex regulatory network. Understanding the role and significance of microRNAs in the context of AKI and critical illness is a major endeavour in translational medicine, requiring the integration of clinical and experimental data.

Acute Kidney Injury in Trauma Patients Admitted to Critical Care: Development and Validation of a Diagnostic Prediction Model.

Acute Kidney Injury (AKI) complicating major trauma is associated with increased mortality and morbidity. Traumatic AKI has specific risk factors and predictable time-course facilitating diagnostic modelling. In a single centre, retrospective observational study we developed risk prediction models for AKI after trauma based on data around intensive care admission. Models predicting AKI were developed using data from 830 patients, using data reduction followed by logistic regression, and were independently validated in a further 564 patients. AKI occurred in 163/830 (19.6%) with 42 (5.1%) receiving renal replacement therapy (RRT). First serum creatinine and phosphate, units of blood transfused in first 24 h, age and Charlson score discriminated need for RRT and AKI early after trauma. For RRT c-statistics were good to excellent: development: 0.92 (0.88-0.96), validation: 0.91 (0.86-0.97). Modelling AKI stage 2-3, c-statistics were also good, development: 0.81 (0.75-0.88) and validation: 0.83 (0.74-0.92). The model predicting AKI stage 1-3 performed moderately, development: c-statistic 0.77 (0.72-0.81), validation: 0.70 (0.64-0.77). Despite good discrimination of need for RRT, positive predictive values (PPV) at the optimal cut-off were only 23.0% (13.7-42.7) in development. However, PPV for the alternative endpoint of RRT and/or death improved to 41.2% (34.8-48.1) highlighting death as a clinically relevant endpoint to RRT.

Does major surgery induce immune suppression and increase the risk of postoperative infection?

PURPOSE OF REVIEW: Infection is the commonest cause of a postoperative complication. Following major surgery alterations in immune function are commonplace and these may contribute to an enhanced susceptibility to acquire nosocomial infections. This review will discuss postoperative infections in the context of an altered perioperative immune response and the factors influencing this response. RECENT FINDINGS: Up to 10% of patients undergoing elective in-patient surgery may develop a postoperative infection. Laboratory advances now permit systematic monitoring of single-cell immune signatures, which enable a clearer description of the interaction between tissue damage, immune modulation and clinical outcomes. Traditional candidate gene expression has identified pathways that define the detrimental immune modulating effects of perioperative allogeneic blood transfusion. Large clinical studies have demonstrated that the choice of anaesthetic technique may have an impact on postoperative infections through differential immune modulation. SUMMARY: Point of care tests are emerging that allow monitoring of the perioperative immune response. These could be further developed to introduce personalised care pathways. Consideration must also be given to anaesthesia techniques and perioperative treatments that may be associated with poor outcomes through immune modulation.

Features of Postoperative Immune Suppression Are Reversible With Interferon Gamma and Independent of Interleukin-6 Pathways.

OBJECTIVE: The aim of this study was to evaluate the role of interleukin (IL)-6 pathways in postoperative immune suppression and to assess the reversibility of this phenomenon. BACKGROUND: The postoperative period is characterized by increased IL-6 production and features of immune suppression. In vitro, IL-6 mediates anti-inflammatory effects through inhibition of interferon gamma (IFN-γ) pathways. The significance of the immunomodulatory effects of IL-6 in the clinical setting of postoperative immune suppression remains unclear. METHODS: Patients over 45 years old undergoing elective surgery, involving the gastrointestinal tract, were recruited. IL-6 levels were assayed using an enzyme linked immunosorbent assay preoperatively, and at 24 and 48 hours. Peripheral blood mononuclear cells from healthy volunteers were cultured in perioperative serum and CD14Human Leukocyte Antigen-DR (HLA-DR) [monocyte HLA-DR (mHLA-DR)] geometric mean florescent intensity was measured in the presence and absence of IL-6 neutralizing antibody and recombinant IFN-γ. RESULTS: Of the 108 patients, 41 developed a postoperative infection. The IL-6 levels increased 19-fold from the preoperative sample to 24 hours postoperatively (P < 0.0001). Higher IL-6 levels at 24 (P = 0.0002) and 48 hours (P = 0.003) were associated with subsequent postoperative infectious complications. mHLA-DR mean florescent intensity fell when healthy peripheral blood mononuclear cells were cultured with postoperative serum compared with preoperative serum (P = 0.008). This decrease was prevented by the presence of IFN-γ in the culture media, but not by the presence of IL-6-neutralizing antibody. CONCLUSIONS: IL-6 levels increase after a major surgery and are associated with an increased susceptibility to postoperative infections. Serum obtained from postoperative patients induces an immunosuppressive response, reflected in reduced mHLA-DR levels, mediated through IL-6 independent pathways and is reversible with IFN-γ. These data may have therapeutic implications for the prevention of infection in patients undergoing major surgery.

Systemic Inflammatory Response Syndrome After Major Abdominal Surgery Predicted by Early Upregulation of TLR4 and TLR5.

OBJECTIVES: To study innate immune pathways in patients undergoing hepatopancreaticobiliary surgery to understand mechanisms leading to enhanced inflammatory responses and identifying biomarkers of adverse clinical consequences. BACKGROUND: Patients undergoing major abdominal surgery are at risk of life-threatening systemic inflammatory response syndrome (SIRS) and sepsis. Early identification of at-risk patients would allow tailored postoperative care and improve survival. METHODS: Two separate cohorts of patients undergoing major hepatopancreaticobiliary surgery were studied (combined n = 69). Bloods were taken preoperatively, on day 1 and day 2 postoperatively. Peripheral blood mononuclear cells and serum were separated and immune phenotype and function assessed ex vivo. RESULTS: Early innate immune dysfunction was evident in 12 patients who subsequently developed SIRS (postoperative day 6) compared with 27 who did not, when no clinical evidence of SIRS was apparent (preoperatively or days 1 and 2). Serum interleukin (IL)-6 concentration and monocyte Toll-like receptor (TLR)/NF-κB/IL-6 functional pathways were significantly upregulated and overactive in patients who developed SIRS (P < 0.0001). Interferon α-mediated STAT1 phosphorylation was higher preoperatively in patients who developed SIRS. Increased TLR4 and TLR5 gene expression in whole blood was demonstrated in a separate validation cohort of 30 patients undergoing similar surgery. Expression of TLR4/5 on monocytes, particularly intermediate CD14CD16 monocytes, on day 1 or 2 predicted SIRS with accuracy 0.89 to 1.0 (areas under receiver operator curves). CONCLUSIONS: These data demonstrate the mechanism for IL-6 overproduction in patients who develop postoperative SIRS and identify markers that predict patients at risk of SIRS 5 days before the onset of clinical signs.

Epigenetic regulatory pathways involving microRNAs may modulate the host immune response following major trauma.

BACKGROUND: Posttraumatic nosocomial pneumonia is a common complication resulting in significant morbidity. Trauma-induced immunocompromise is associated with an enhanced susceptibility to pneumonia. In this study, we explore the hypothesis that posttranscriptional epigenetic regulation of gene expression may be an important factor in determining this immune phenotype. We describe the pattern of production of microRNAss (miRs) and their association with nosocomial pneumonia following severe trauma. METHODS: A convenience sample of 30 ventilated polytrauma patients ( UKCRN ID: 5637) and 16 healthy controls were recruited. Messenger RNA and protein levels of key cytokines were quantified within 2 hours of the injury and at 24 hours. Three miRs per cytokine were then selected based on miRBase target prediction scores and quantified using polymerase chain reaction. Nosocomial pneumonia was defined using the Centers for Disease Control and Prevention definitions. RESULTS: Median Injury Severity Score (ISS) was 29, and 47% of the patients developed nosocomial pneumonia. miR-125a and miR-202 decreased by 34% and 77%, respectively, immediately following injury, whereas their target, IL-10, increased messenger RNA levels 3-fold and protein levels 180-fold. Tumor necrosis factor α (TNF-α) and IL-12 gene expression decreased by 68% and 43%, respectively, following injury, and this was mirrored by a 10-fold increase in miR-181, an miR predicted to target TNF-α transcripts. Lower levels of miR-125a and miR-374b were associated with the later acquisition of hospital-acquired pneumonia. CONCLUSION: Alteration in the expression of miRs with highly predicted complementarity to IL-10 and TNF-α may be an important mechanism regulating the posttraumatic immunosuppressive phenotype in intensive care unit patients. LEVEL OF EVIDENCE: Retrospective observational study, level III.

The perioperative immune response.

PURPOSE OF REVIEW: A host of immune modulators are now available in clinical practice. The perioperative period is characterized by profound alterations in host immunity, which can result in poor outcomes, which include infection, cancer recurrence and organ failure. Manipulation of the perioperative immune response has the potential to improve outcomes. A complete understanding of the mechanisms and clinical consequences of altered immune function in this setting is therefore imperative. RECENT FINDINGS: Recent in-vivo data have emerged which further our understanding of the interaction between tissue damage, immune modulation and clinical outcomes by utilizing novel laboratory techniques capable of monitoring single-cell immune signatures. Traditional gene expression assays have continued to demonstrate their utility and have been instrumental in defining the host response to perioperative allogeneic blood transfusion. These mechanistic studies are complemented by large clinical studies describing associations between anaesthetic modalities and immune-related outcomes. SUMMARY: Laboratory techniques are now available that can monitor the perioperative immune response and could be further developed to introduce personalized care pathways. Consideration must also be given to anaesthesia techniques and perioperative treatments that, although not immediately harmful, may be associated with poor outcomes temporally distant from the treatment, secondary to induced immunosuppression.

Changes in gene expression following trauma are related to the age of transfused packed red blood cells.

BACKGROUND: Transfusion of packed red blood cells (PRBCs) is associated with an increased incidence of nosocomial infections and an increased risk of death. The duration of storage before transfusion may influence these outcomes. Here, we explore the association between the age of transfused PRBCs and specific patterns of inflammatory gene expression in severely injured trauma patients. METHODS: Severely injured trauma patients requiring intensive care unit treatment and receiving transfusion of PRBCs within 24 hours of the injury were recruited. Blood samples were obtained within 2 hours of the trauma, at 24 hours, and at 72 hours. Messenger RNA was extracted from whole blood, and gene expression was quantified using quantitative polymerase chain reaction. The median age of the units of PRBCs transfused to each patient was recorded. The primary outcome measure was the change in candidate gene expression over the initial 72 hours. RESULTS: Sixty-four patients were studied. Fifty-three patients (83%) were male, and the median age was 40.5 years (interquartile range [IQR], 31-59). Median Injury Severity Score (ISS) was 31.5 (IQR, 23-43), and 55 patients (86%) experienced a blunt injury. Forty-one patients (64%) developed a nosocomial infection, and 15 patients (23%) died before hospital discharge. Each patient received a median of 5 U of PRBCs (IQR, 4-9.8 U) during the first 24 hours of hospital admission. The median age of the units of PRBCs transfused in each patient was 20 days (IQR, 17-22 days). Older blood was associated with greater decreases in interleukin 12 (IL-12), IL-23, and RORγt (all p's < 0.05) gene expression over the initial 24 hours, greater decreases in IL-12 gene expression over 72 hours, and a rise in transforming growth factor ß gene expression over the first 72 hours. A multivariate analysis confirmed the independence of these associations. CONCLUSION: Increasing the duration of storage of PRBCs before transfusion is associated with a pattern of gene expression consistent with more severe immunosuppression. LEVEL OF EVIDENCE: Epidemiologic study, level III.

Perioperative blood transfusion is associated with a gene transcription profile characteristic of immunosuppression: a prospective cohort study.

INTRODUCTION: Blood transfusion in the perioperative period has frequently been associated with an excess of nosocomial infections. Whilst transfused whole blood induces specific host immune alteration that may predispose to nosocomial infections, the immunomodulating properties associated with leukodepleted blood remain incompletely understood. In this study, we explore the hypothesis that the transfusion of leukodepleted allogeneic blood during or following major gastrointestinal surgery is associated with an immunosuppressed phenotype, which may in turn predispose to postoperative infectious complications. METHODS: Patients aged over 45 years undergoing scheduled inpatient major gastrointestinal surgery were recruited. Gene expression profiles of specific inflammatory genes were assayed from blood collected preoperatively, at 24 and at 48 hours after surgery. Genes were selected based on their ability to represent specific immune pathways. Gene expression was quantified using quantitative real-time polymerase chain reaction (qRT-PCR) to measure messenger RNA (mRNA) levels. Postoperative infections were documented using predefined criteria. RESULTS: One hundred and nineteen patients were recruited. Fifteen (13%) patients required blood transfusion within 24 hours of surgery, 44 (37%) patients developed infections and 3 (2%) patients died prior to discharge. Patients receiving a blood transfusion were more likely to develop postoperative infections (P =0.02) and to have lower tumour necrosis factor alpha (TNFα), interleukin (IL)-12, IL-23 and RAR-related orphan receptor gamma T (RORγt) gene expression in the postoperative period (P <0.05). The TNFα/IL-10 mRNA ratio at 24 hours (P =0.0006) and at 48 hours (P =0.01) was lower in patients receiving a blood transfusion over this period. Multivariable analysis confirmed that these observations were independent of the severity of the surgical insult. CONCLUSIONS: An association between an immunosuppressive pattern of gene expression and blood transfusion following major elective gastrointestinal surgery is described. This gene expression profile includes a reduction in the activity of innate immunity and T helper cell type 1 (Th1) and T helper cell type 17 (Th17) pathways in those patients receiving a blood transfusion. Blood transfusion was also associated with an excess of infectious complications in this cohort. A mechanistic link is suggested but not proven.

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 ß-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 beta-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.

The Snow Snorkel: a proof of concept study.

OBJECTIVE: To demonstrate that the Snow Snorkel can be used safely by healthy volunteers buried in snow for up to 1 hour. METHODS: Nine healthy male volunteers were placed in a shoulder-width trench and buried with snow to a depth of 30 to 40 cm. The study was divided into 2 stages. The first stage (Stage 1) was performed with the Snow Snorkel in operation (60-minute duration) and was then followed by a second stage (Stage 2) (15-minute duration) when the device was removed. Arterial oxygen saturation (SaO2), heart rate (HR), respiratory rate (RR), axillary temperature (T), and 3-lead electrocardiography (ECG) were monitored throughout the study. RESULTS: Of the 9 volunteers who were enrolled, 7 were able to complete Stage 1, while only 3 were able to complete Stage 2. In those who completed Stage 1, the mean HR fell by 14.1 beats/min (P = .002), while RR (P = .5) and SaO2 (P = .7) remained unchanged compared to baseline measurements. There were no changes in T or ECG. CONCLUSIONS: Simple systems such as the Snow Snorkel are effective during snow burial and warrant further investigation.

The human response to infection is associated with distinct patterns of interleukin 23 and interleukin 27 expression.

OBJECTIVE: The development and progression of severe sepsis is related to a deficiency in pro-inflammatory cytokine production, characterised by lesser IFNgamma levels, which are not explained by variations in levels of the main putative regulator of IFNgamma, namely IL-12. As alternative regulators of IFNgamma may be of greater importance in human sepsis, we investigated the hypothesis that the development of severe sepsis is related to variations in IL-18, IL-23 and IL-27 gene expression. DESIGN AND SETTING: A prospective observational trial in a mixed intensive care unit (ICU) and hospital wards in a university teaching hospital. PATIENTS AND PARTICIPANTS: Sixty-two ICU patients with severe sepsis, 13 bacteraemic patients with no acute critical illness, and 10 healthy controls. MEASUREMENTS AND RESULTS: All subjects were assayed for IL-18, IL-23 and IL-27 mRNA levels in peripheral blood. IL-27 mRNA levels distinguished between the three groups, with levels highest in the ICU group, intermediate in the bacteraemic group and lowest in the control group. IL-23 distinguished between the groups, with levels lowest in the ICU group. In late sepsis IL-23 and TNFalpha mRNA levels were directly related. IL-18 mRNA levels did not distinguish between the patient groups. CONCLUSIONS: We conclude that the deficient pro-inflammatory response in patients with sepsis is expansive and includes deficient IL-23 and excessive IL-27 gene expression. This provides further evidence that upregulation of a cytokine-based immune response is beneficial in sepsis.

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

BACKGROUND: Hereditary 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. RESULTS: Vector 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-kappaB 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. CONCLUSION: Our novel observations suggest that both the ER overload response (EOR) and the unfolded protein response (UPR) are activated by mutant C282Y HFE protein.

Coagulopathy after cardiac surgery may be influenced by a functional plasminogen activator inhibitor polymorphism.

BACKGROUND: Cytokine-mediated inflammation and coagulopathy may occur after cardiac surgery. In this study we investigated the temporal pattern of plasminogen activator inhibitor-1 (PAI-1) gene expression after cardiac surgery and its relation with PAI genotype, and obtained preliminary data regarding its relation to perioperative morbidity. METHODS: The relative change in PAI-1 mRNA 1, 6, and 24 h after cardiopulmonary bypass (CPB) was measured from mononuclear cells in 82 patients undergoing elective cardiac surgery. DNA was analyzed for carriage of the 4G/5G PAI-1 polymorphism. RESULTS: PAI-1 gene expression decreased after CPB in all patients. A larger reduction in PAI-1 gene expression was observed in homozygous carriers of the 5G allele. Homozygous carriers of the 5G allele were also more likely to receive transfusion of coagulation blood products. There was no relation between change in PAI-1 gene expression and duration of CPB. CONCLUSIONS: PAI-1 gene expression decreased over time after CPB. We found a link between PAI-1 genotype, PAI gene expression, and transfusion of coagulation products after cardiac surgery.

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

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 alpha(TNFalpha), interferon gamma (IFNgamma), 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-alpha mRNA was detected in the ICU group when compared with the control group (P < 0.0001). However, TNF-alpha mRNA was most abundant in the bacteremic group (P = 0.0007). Lesser IFN-gamma 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-gamma mRNA (P = 0.0004) and lesser TNF-alpha mRNA (P = 0.001). Survivors had greater TNF-alpha 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.