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.

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 (PBL). This study sought to determine the state of clock gene expression in human PBL, and leukocytes 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 (2ng/kg) or saline at either 09:00 or 21:00 h. Blood samples were collected at selected time points pre- and post-infusion. Measurements and Mains results Clock gene expression was determined in human PBL, neutrophils, and monocytes, by quantitative real-time polymerase chain reaction. The fold change for each gene was determined using the 2(-ΔΔCt) method. We show that endotoxin causes profound suppression of circadian clock gene expression, clearly manifested in human PBL, neutrophils, and monocytes. Clock, Cry1-2, Per3, CSNK1ε, Rora and Rev-erb gene expression were all reduced by 80-90% with the nadir between 3 to 6 hours post-infusion. Per1 and Per2 reached an expression nadir between 13 and 17 hours post-infusion. The levels of plasma interleukin-6 and tumor necrosis factor peaked and then returned to baseline within 6 hours. In contrast, clock gene expression remained suppressed for up to 17 hours, 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 PBL 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.

The lipopolysaccharide (LPS) receptor complex consists of two interacting receptors (CD14 and TLR4) and an associated protein (MD-2). When engaged by LPS, as in gram-negative infection, this complex transduces a signal detected by MyD88 and passed onward by a cascade of the IRAKs, TRAF6, and NIK, resulting in activation of NF-kappaB. A similar cascade, mediated by TLR2, occurs with ligands derived from gram-positive bacteria. In vitro studies of human monocytes have shown that TLR4 mRNA is paradoxically upregulated in response to "tolerizing" doses of LPS. This study evaluated changes in vivo of blood monocyte CD14, TLR4, TLR2, and MD-2 mRNA by reverse transcription followed by real-time polymerase chain reaction in surgical intensive care unit patients and in normal controls. In addition cell-surface receptor expression of TLR2, TLR4, and CD14 was assessed by flow cytometry in patients and normal controls. Inflammation-induced acute tolerance to LPS was evaluated by ex vivo whole blood tumor necrosis factor alpha production and was significantly reduced in patients compared with controls, confirming LPS hyporesponsiveness. Monocyte mRNA and cell-surface receptor expression of TLR4 were increased 2.4-fold (P < 0.05) and 1.7-fold (P <.002), respectively, in patients compared with normal controls. Monocyte TLR2 mRNA, MD-2 mRNA and CD14 and TLR2 cell-surface expression were not significantly changed compared with controls. The present study suggests that the acute inflammatory condition associated with peripheral cellular LPS hyporesponsiveness is neither specific to prior infectious challenge nor can be ascribed to significant alterations in expression of the cell-surface LPS binding complex proteins.

The E2F family of transcription factors can induce both cell proliferation and apoptosis. Whether they function as oncogenes or tumor suppressors appears to be tissue specific. Their role in breast carcinogenesis remains unclear. We found a decreased expression of E2F-1 and E2F-4 in 70% (7/10) of primary breast carcinomas and in all (10/10) metastatic nodal tissues when compared with the corresponding normal breast tissue. No tumor-specific mutation was detected, but polymorphisms were identified in E2F-1 exon 5 and in the polyserine tract of E2F-4. The presence of polymorphisms did not correlate with E2F expression. Among the 12 human breast cancer cell lines, one contained a missense mutation in E2F-1 exon 2. Five (42%) cell lines overexpressed E2F-1, while three (25%) expressed low levels of the protein. Our results suggest that not only are the E2Fs likely to function as tumor suppressors in breast cancer, but also that their down-regulation may be important in the development of metastases.