Jacqueline E. Calvano

Human Toll-Like Receptor 4 Mutations but Not CD14 Polymorphisms Are Associated with an Increased Risk of Gram-Negative Infections

Human toll-like receptor 4 (hTLR4) and CD14 are known to be components of the lipopolysaccharide receptor complex. Our study investigated the association between TLR4 mutations (Asp299Gly and Thr399Ile) and CD14 polymorphism(s) with outcome in an intensive care unit (ICU) population at risk for sepsis. By use of a polymerase chain reaction-based restriction fragment-length polymorphism analysis technique, the hTLR4 gene was altered in 14 (18%) of 77 ICU patients (all positive for systemic inflammatory response syndrome) and in 5 (13%) of 39 volunteers. There was a significantly higher incidence of gram-negative infection among patients with the mutations (11 [79%] of 14), compared with that in the wild-type population (11 [17%] of 63; P=.004). No association between CD14 polymorphism(s) and the incidence of infection or outcome was observed. These findings indicate that hTLR4 mutations are associated with an increased incidence of gram-negative infections in critically ill patients in a surgical setting.

In vivo endotoxin synchronizes and suppresses clock gene expression in human peripheral blood leukocytes

Objectives:The intravenous administration of a bolus dose of endotoxin to healthy human subjects triggers acute systemic inflammatory responses that include cytokine production and dynamic changes in gene expression in peripheral blood leukocytes. This study sought to determine the state of clock gene expression in human peripheral blood leukocytes, and leukocyte subpopulations, challenged with in vivo endotoxin at two circadian/diurnal phases of the clock. Design:Clinical and laboratory investigation. Setting:University-based research laboratory and clinical research center. Subjects:Human volunteers. Interventions:Human subjects were administered a standard dose of endotoxin (2 ng/kg) or saline at either 0900 or 2100 hrs. Blood samples were collected at selected time points pre- and postinfusion. Measurements and Main Results:Clock gene expression was determined in human peripheral blood leukocytes, neutrophils, and monocytes by quantitative real-time polymerase chain reaction. The fold change for each gene was determined by use of the 2−&Dgr;&Dgr;CT method. We show that endotoxin causes profound suppression of circadian clock gene expression, clearly manifested in human peripheral blood leukocytes, neutrophils, and monocytes. Clock, Cry1-2, Per3, CSNK1 ϵ, Rora, and Rev-erb gene expression were all reduced by 80% to 90% with the nadir between 3 and 6 hrs postinfusion. Per1 and Per2 reached an expression nadir between 13 and 17 hrs postinfusion. The levels of plasma interleukin-6 and tumor necrosis factor peaked and then returned to baseline within 6 hrs. In contrast, clock gene expression remained suppressed for up to 17 hrs irrespective of the phase of the clock at the time of the endotoxin challenge. Endotoxin did not perturb the melatonin secretory rhythm. Conclusions:Circadian clock gene expression in peripheral blood leukocytes is dramatically altered and possibly uncoupled from the activity of the central clock during periods of acute systemic inflammation. The realignment of the central and peripheral clocks may constitute a previously unappreciated key factor affecting recovery from disease in humans.

Influence of the TNF-α and TNF-β Polymorphisms upon Infectious Risk and Outcome in Surgical Intensive Care Patients

Background: Tumor necrosis factor-alpha (TNF-α) is a well-documented central inflammatory mediator in sepsis. Specific polymorphisms of the TNF-α and TNF-β genes (TNF2 and LTA + 250, respectively) have been suggested to correlate with higher mortality in septic shock. This study sought to determine whether these polymorphisms of the TNF-α and -β genes are associated with an increased risk of infection in an at-risk surgical intensive care population. Materials and Methods: Forty-four consecutive patients with systemic inflammatory response syndrome were enrolled prospectively in the study. Genomic DNA was isolated from whole blood samples using standard phenol/chloroform extraction techniques. Specific fragments including the polymorphic sites of each gene were amplified by polymerase chain reaction, and restriction enzyme digestions were performed. Genotypes were determined by gel electrophoresis and confirmed by direct sequencing. Results: Eighty-six percent of the patients were TNF1 homozygotes (G:G at...

A novel model of common Toll-like receptor 4- and injury-induced transcriptional themes in human leukocytes

IntroductionAn endotoxin challenge, sepsis, and injury/trauma, trigger significant changes in human peripheral blood leukocytes (PBL) gene expression. In this study, we have sought to test the hypothesis that the Toll-like receptor 4 (TLR4) induced transcription patterns elicited in humans exposed to in vivo endotoxin would parallel gene expression patterns observed in trauma patients with initial non-infectious injury. In addition, we sought to identify functional modules that are commonly affected by these two insults of differing magnitude and duration.MethodsPBL were obtained from seven adult human subject experimental groups. The groups included a group of healthy, hospitalized volunteers (n = 15), that comprised four study groups of subjects challenged with intravenous endotoxin, without or with cortisol, and two serial samplings of trauma patients (n = 5). The PBL were analyzed for gene expression using a 8,793 probe microarray platform (Gene Chip® Focus, Affymetrix). The expression of a subset of genes was determined using qPCR.ResultsWe describe sequential selection criteria of gene expression data that identifies 445 genes that are significantly differentially expressed (both P ≤ 0.05 and >1.2 fold-change) in PBL derived from human subjects during the peak of systemic inflammatory responses induced by in vivo endotoxin, as well as in PBL obtained from trauma patients at 1 to 12 days after admission. We identified two functional modules that are commonly represented by this analysis. The first module includes more than 50 suppressed genes that encode ribosomal proteins or translation regulators. The second module includes up-regulated genes encoding key enzymes associated with glycolysis. Finally, we show that several circadian clock genes are also suppressed in PBL of surgical ICU patients.ConclusionsWe identified a group of >400 genes that exhibit similar expression trends in PBL derived from either endotoxin-challenged subjects or trauma patients. The suppressed translational and circadian clock modules, and the upregulated glycolytic module, constitute a robust and long lasting PBL gene expression signature that may provide a tool for monitoring systemic inflammation and injury.

Polymorphisms of heat shock protein-70 (HSPA1B and HSPA1L loci) do not influence infection or outcome risk in critically ill surgical patients

ABSTRACT Heat shock proteins (HSP) are induced in various stress conditions and have many cytoprotective effects, including formation of protein complexes for antigen presentation, stabilizing intracellular proteins, and facilitating protein folding. The HSP-70 gene exhibits polymorphisms at the HSPA1B and HSPA1L loci that reportedly influence cytokine levels and clinical outcomes in critically ill patients. These HSP variations also have been linked to TNF-&bgr; polymorphisms associated with poor outcomes. This study further evaluated outcomes and risk of infection of HSP polymorphisms in critically ill patients. Seventy-six consecutive surgical intensive care unit uninfected patients with established systemic inflammatory response features were prospectively enrolled. Genomic DNA was isolated from whole blood samples and specific fragments, including the relevant polymorphic sites, were amplified by PCR, and restriction digestions were performed. Genotypes were determined by electrophoresis and all were confirmed by direct sequencing. Plasma cytokine levels for TNF-&agr; were assayed in a subset of patients by enzyme-linked immunoabsorbant assay. None of the HSP alleles bore a significant relationship to nosocomial infection rates, organ specific dysfunctions, or mortality. No linkage of HSP genotype to common TNF-&agr; or TNF-&bgr; genotypes could be demonstrated, although the HSPA1L CT polymorphism was associated with higher levels of TNF-&agr; compared with the TT genotype. These data suggest that polymorphisms of the HSPA1L or HSPA1B loci do not influence infection or other highly morbid outcomes in surgical intensive care unit patients.

Cellular metabolic regulators: Novel indicators of low-grade inflammation in humans

Objective:The Toll-like receptor 4 (TLR4) ligand endotoxin triggers robust systemic inflammatory responses in humans at doses equal to or greater than 1 ng/kg. In this study, we tested the hypothesis that evidence of TLR4-induced responses would be detectable in leukocytes challenged with endotoxin doses that are below the threshold needed to trigger a characteristic systemic inflammatory phenotype in humans. Methods:Subjects were challenged with endotoxin at 1, 0.5, or 0.1 ng/kg (n = 5 per dose). Systemic responses were monitored for 24 hours. Blood samples, collected at designated intervals, were used to determine plasma cytokines levels, total and differential leukocyte counts, expression of leukocyte cell surface receptors, and changes in the leukocyte transcriptome. Western blotting was used to determine changes in leukocyte protein expression. Results:We found that in vivo endotoxin at doses below 1.0 ng/kg triggers weak and variable responses in humans. In marked contrast, we show that endotoxin at a concentration as low as 0.1 ng/kg triggers a transient decline in cellular ATP levels in leukocytes. This is associated with the appearance of a unique protein expression signature in leukocytes. The protein expression signature includes 3 prominent features: (i) AMP-activated protein kinase subunit &agr; (AMPK&agr;) degradation, (ii) increased hypoxia inducible factor-1 (HIF-1) &agr; expression, and (iii) autophagy, collectively indicative of a regulated metabolic response. An indistinguishable response phenotype was observed in human leukocytes treated with endotoxin in vitro. Conclusions:These data demonstrate for the first time in humans that a TLR4 ligand concentration that is below the threshold needed to trigger clinically evident systemic inflammatory manifestations initiates a transient decline in ATP levels, AMPK&agr; degradation, HIF-1&agr; expression, and autophagy in leukocytes. This establishes that low-grade TLR4 activation exerts control over leukocyte metabolism in the absence of systemic inflammatory indicators.

Insulin-like Growth Factor Binding Protein-3 Is Upregulated in LPS-treated THP-1 Cells

BACKGROUND Lipopolysaccharide (LPS) is a potent activator of human monocytic cells. We have determined that LPS stimulation of the human monocytic cell line, THP-1, results in an increased apoptotic rate. We hypothesized that cDNA expression array analysis could be used to identify target genes involved in the regulation of this process. METHODS THP-1 cells (1 x 10(6)/mL) were stimulated with LPS (1 microg/mL) or vehicle control. Apoptosis was measured at 0, 24, 48, 72 and 96 h using propidium iodide staining and flow cytometry to determine the percentage of cells with hypodiploid DNA. At 16 h, the Atlas Human cDNA expression array system, containing probes for 205 genes related to apoptosis, was used to survey and quantify transcript expression. The experiment was performed in duplicate and the membranes were normalized to cytoplasmic beta-actin. Standard Western blotting was performed on the conditioned medium to correlate secreted protein expression with RNA expression. Pretreatment with insulin-like growth factor I (IGF-I) was performed to determine whether the effects of insulin-like growth factor binding protein-3 (IGFBP-3) on apoptosis were IGF-dependent. RESULTS LPS stimulation of THP-1 cells resulted in a greater than 2-fold increase in the rate of apoptosis when compared to vehicle control. When the cDNA expression arrays were compared, there was a 500-fold increase in the expression of the IGFBP-3 transcript in the LPS-stimulated cells. Western blotting of culture medium verified an approximately 2-fold increase in secreted IGFBP-3. Pretreatment with IGF-I did not prevent the increase in apoptosis seen with LPS stimulation. CONCLUSIONS THP-1 cell apoptosis is increased in response to LPS stimulation and is associated with a significant induction of IGFBP-3 mRNA and protein. IGFBP-3, which reportedly promotes apoptosis and modulates the bioavailability of the pro-survival insulin-like growth factor 1, may serve to regulate apoptosis in monocytic cells in an IGF-independent manner. These data further support the investigation of the role of the IGF axis in programmed cell death of immune cells.

A single nucleotide polymorphism in the Mdm2 promoter and risk of sepsis.

BACKGROUND The Mdm2-SNP309(T/G) polymorphism has been shown to upregulate transcription of Mdm2 and subsequently attenuate the p53 pathway. Its role in regulating the human response to acute illness has not been reported. METHODS Patients from the surgical intensive care unit were prospectively enrolled. SNP309 genotype was determined, and a genotype-based comparison of clinical outcomes was performed. RESULTS Of the 85 enrolled patients, 41 had wild type (T/T) and 44 had mutant (32 T/G and 12 G/G) genotypes. The mutant-genotype group tended to have a longer LOS in both the surgical intensive care unit (P = .40) and the hospital (P = .08), but these trends did not reach significance. No observable genotype-based differences were noted in any other measured parameters. CONCLUSIONS The Mdm2-SNP309(G) allele may be associated with longer LOS. However, it does not appear to influence any other clinical characteristics, nor can it be used to predict clinical outcome.

DIFFERENTIAL CELL DEATH GENE EXPRESSION IN MONOCYTES FROM LPS-CHALLENGED HEALTHY HUMAN VOLUNTEERS PRE-TREATED WITH CORTISOL

GHRELIN INHIBITS SYMPATHETIC NERVOUS ACTIVITY IN SEPSIS. R. Wu, M. Zhou, P. Das*, W. Dong*, Y. Ji*, D. Yang*, M. Miksa, T.S. Ravikumar*, P. Wang. North Shore University Hospital-LIJ Medical Center, Manhasset, NY 11030. Our previous studies have shown that norepinephrine (NE) upregulates proinflammatory cytokines by activating !2A-adrenoceptor. Therefore, modulation of the sympathetic nervous system represents a novel treatment for sepsis. We have also shown that a novel stomach-derived peptide, ghrelin, is downregulated in sepsis and that its intravenous (IV) administration decreases proinflammatory cytokines and improves survival. This beneficial effect appears to be mediated through the central nervous system, as evidenced by the inhibitory effect of intracerebroventricular administration of ghrelin on LPS-induced TNF-! release in rats. However, it remains to be determined whether ghrelin inhibits sympathetic activity through central ghrelin receptors (i.e., GHSR-la) in sepsis. To study this, sepsis was induced in male rats by cecal ligation and puncture (CLP). First, ghrelin was given IV (4 nmol/rat) infused for 2.5h starting 0.5h prior to CLP) and plasma levels of NE was measured by ELISA. Ghrelin significantly reduced the elevated NE levels at 2h post-CLP. Second, the effect of ghrelin on c-fos expression (i.e., a measure of neuronal activations) in a sympathostimulatory nucleus in the brain [i.e., the periventricular hypothalamic nucleus (PHN)] was examined. A bolus IV injection of 2 nmol ghrelin at 5h after CLP was followed by a continuous infusion of 12 nmol ghrelin via an osmotic pump for 15h. The expression of c-fos increased markedly 20h after CLP. Ghrelin administration reduced c-fos expression in the PHN. Third, to determine whether the inhibitory effect of ghrelin on NE is mediated by ghrelin receptors, a GHSR-la antagonist [D-Arg D-Phe D-Trp Leu]-substance P was injected to normal animals (210 nmol/rat) for 1h and plasma NE and TNF-! levels were assessed. GHSR-la inhibition significantly elevated both NE and TNF-! levels even in normal animals. Finally, to determine whether ghrelin can directly affect NE production, a neuron catecholaminergic cell line, CAD cells, was incubated with various doses of ghrelin. Ghrelin reduced NE production from CAD cells in a dose-dependent manner. These results suggest that ghrelin has sympathoinhibitory properties which are likely mediated by central ghrelin receptors (NIH R01 GM053008).