The cause of Crohn's disease (CD) remains poorly understood. Counterintuitively, these patients possess an impaired acute inflammatory response, which could result in delayed clearance of bacteria penetrating the lining of the bowel and predispose to granuloma formation and chronicity. We tested this hypothesis in human subjects by monitoring responses to killed Escherichia coli injected subcutaneously into the forearm. Accumulation of 111In-labeled neutrophils at these sites and clearance of 32P-labeled bacteria from them were markedly impaired in CD. Locally increased blood flow and bacterial clearance were dependent on the numbers of bacteria injected. Secretion of proinflammatory cytokines by CD macrophages was grossly impaired in response to E. coli or specific Toll-like receptor agonists. Despite normal levels and stability of cytokine messenger RNA, intracellular levels of tumor necrosis factor (TNF) were abnormally low in CD macrophages. Coupled with reduced secretion, these findings indicate accelerated intracellular breakdown. Differential transcription profiles identified disease-specific genes, notably including those encoding proteins involved in vesicle trafficking. Intracellular destruction of TNF was decreased by inhibitors of lysosomal function. Together, our findings suggest that in CD macrophages, an abnormal proportion of cytokines are routed to lysosomes and degraded rather than being released through the normal secretory pathway.

Patients with advanced cirrhosis experience frequent infections leading to sepsis, which carries high mortality. While innate immune dysfunction underlies this vulnerability, the precise cause remains elusive. We found prostaglandin (PGE 2 ) elevated in acutely decompensated (AD) patients at immunosuppressive levels. Plasma from AD and end-stage liver disease (ESLD) patients suppressed macrophage cytokine secretion and bacteria killing in a PGE 2 receptor-dependent manner, effects not seen in stable cirrhosis. Mouse models (bile duct ligation and CCL4-liver injury) also demonstrated elevated PGE 2 , which when inhibited completely restored immune competence and survival following infection. Importantly, albumin binds/inactivates PGE 2 resulting in greater PGE 2 bioavailability. This results in enhanced immunosuppressive effects of AD plasma in patients with low albumin levels. Administering albumin to AD patients reversed immunosuppressive properties of their plasma; protective effects recapitulated in rodent survival studies. Thus, elevated PGE 2 combined with hypoalbuminemia mediates immunosuppression in AD and ESLD patients, which can be reversed with albumin. Cyclooxygenase (COX)-derived lipid mediators have broad immunosuppressive effects12-15 that could explain the aetiology of infection susceptibility in cirrhosis patients. Thus, we used a number of in vitro and in vivo assays using plasma from patients with AD and ESLD derived from clinical trials to investigate the role of bioactive lipid mediators in immunosuppression as well as animal models of liver injury for survival analyses. ResultsProstaglandin E 2 (PGE 2 ) is elevated in patients with acute decompensation at levels that are immunosuppressive via its effect on the EP2/3 receptor ESI/LC-MS/MS analysis of acutely decompensated patient plasma (day 1-2 of hospital admission) demonstrated significantly elevated PGE 2 , PGF 2 α, 5-and 15-HETE compared to HV ( Figure 1A and supplementary Figure 1E-G). However, only PGE 2 dampened TNFα release from LPS-stimulated human monocyte-derived macrophages when pre-treated with the mean concentrations observed in AD patients (0.1ng/ml) ( Figure 1B).Human monocyte-derived macrophages were incubated with culture media supplemented with 25% (vol./vol.) plasma from AD patients (see Table 1 for clinical characteristics).Compared to macrophages treated with media supplemented with HV plasma, AD plasma caused a significant decrease in LPS-stimulated TNFα that was reversed by pre-incubating Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts macrophages with the E-prostanoid (EP) 1-3/D-prostanoid (DP) 1 receptor antagonist, AH6809 ( Figure 1C). Additionally, macrophages were incubated with E. coli in the presence/absence of HV or AD plasma. Compared to macrophages treated with HV plasma, those with AD plasma exhibited reduced bacterial killing, an effect reversed by pretreatment with AH6809 (50μM) ( Figure 1D). AH6809 had no direct bactericidal effect while cell viability was unaffected b...

The gene which encodes alcohol dehydrogenase II (adhB) from Zymomonas mobilis was cloned in Escherichia coli as a 1.4-kilobase DNA fragment by using a novel indicator plate which directly detects the expression of this activity by recombinant colonies. The DNA sequence for this clone contained an open reading frame encoding a polypeptide of 383 amino acids, with a molecular weight of 40,141. Although this protein exhibited very little homology with other known alcohol dehydrogenases, the predicted amino acid composition was in excellent agreement with that reported for the purified alcohol dehydrogenase II protein from Z. mobilis. In Z. mobilis, the adhB gene was transcribed from tandem promoters which were separated by 100 base pairs and ended with a transcriptional terminator (13-base-pair palindrome). In Escherichia coli, only one of the Z. mobilis promoters was used, despite apparent similarity to the enteric consensus promoter. The adhB gene was transcribed at low levels in E. coli from the P2 promoter of Z. mobilis but was expressed well in E. coli under control of the lac promoter (approximately 0.25% of the total cell protein).

Single-nucleotide variations in C13orf31 (LACC1) that encode p.C284R and p.I254V in a protein of unknown function (called 'FAMIN' here) are associated with increased risk for systemic juvenile idiopathic arthritis, leprosy and Crohn's disease. Here we set out to identify the biological mechanism affected by these coding variations. FAMIN formed a complex with fatty acid synthase (FASN) on peroxisomes and promoted flux through de novo lipogenesis to concomitantly drive high levels of fatty-acid oxidation (FAO) and glycolysis and, consequently, ATP regeneration. FAMIN-dependent FAO controlled inflammasome activation, mitochondrial and NADPH-oxidase-dependent production of reactive oxygen species (ROS), and the bactericidal activity of macrophages. As p.I254V and p.C284R resulted in diminished function and loss of function, respectively, FAMIN determined resilience to endotoxin shock. Thus, we have identified a central regulator of the metabolic function and bioenergetic state of macrophages that is under evolutionary selection and determines the risk of inflammatory and infectious disease.

The genes encoding essential enzymes of the fermentative pathway for ethanol production in Zymomonas mobilis, an obligately ethanologenic bacterium, were inserted into Escherichia coli under the control of a common promoter. Alcohol dehydrogenase II and pyruvate decarboxylase from Z. mobilis were expressed at high levels in E. coli, resulting in increased cell growth and the production of ethanol as the principal fermentation product from glucose. These results demonstrate that it is possible to change the fermentation products of an organism, such as E. coli, by the addition of genes encoding appropriate enzymes which form an alternative system for the regeneration of NAD+.

No abstract

Glucose-6-phosphatase, an enzyme localized in the endoplasmic reticulum (ER), catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate. In humans, there are three differentially expressed glucose-6-phosphatase catabolic genes (G6PC1-3). Recently, it has been shown that mutations in the G6PC3 gene result in a syndrome associating congenital neutropenia and various organ malformations. The enzymatic function of G6PC3 is dependent on G6P transport into the ER, mediated by G6P translocase (G6PT). Mutations in the gene encoding G6PT result in glycogen storage disease type-1b (GSD-1b). Interestingly, GSD-1b patients exhibit a similar neutrophil dysfunction to that observed in G6PC3-deficient patients. To better understand the causes of neutrophil dysfunction in both diseases, we have studied the neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of patients with G6PC3 and G6PT syndromes. Unexpectedly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments indicated hypo-glycosylation of gp91(phox), the electron-transporting component of the NADPH oxidase, in all of these patients. Rigorous mass spectrometric glycomic profiling showed that most of the complex-type antennae which characterize the neutrophil N-glycome of healthy individuals were severely truncated in the patients' neutrophils. A comparable truncation of the core 2 antenna of the O-glycans was also observed. This aberrant neutrophil glycosylation is predicted to have profound effects on the neutrophil function and merit designation of both syndromes as a new class of congenital disorders of glycosylation.

Crohn's disease (CD) is a complex and highly heterogeneous chronic inflammatory disorder, primarily affecting the gastrointestinal tract. Genetic and functional studies have highlighted a key role for innate immunity in its pathogenesis. Profound systemic defects in innate immunity and acute inflammation are understood to result in markedly delayed clearance of bacteria from the tissues, leading to local chronic granulomatous inflammation and compensatory adaptive immunological changes. Macrophages, key orchestrators of acute inflammation, are likely to play an important role in the initial impaired innate immune response. Monocyte-derived macrophages from CD patients stimulated with Escherichia coli were shown to release attenuated levels of tumour necrosis factor and interferon-γ with normal secretion of interleukin-8 (IL-8), IL-10 and IL-6. In controls, the secretion of these cytokines was strongly positively correlated, which was not seen with CD macrophages. The transcriptomes of CD and control macrophages were examined in an attempt to understand the molecular basis of this defect. There were no differentially expressed genes identified between the two groups, consistent with genetic heterogeneity; however, a number of molecules were found to be under-expressed in subgroups of CD patients. The most common of these was optineurin (OPTN) which was under-expressed in approximately 10% of the CD patients. Reduced OPTN expression coincided with lower intracellular protein levels and diminished cytokine secretion after bacterial stimulation both in the patients and with small interfering RNA knockdown in THP-1 cells. Identifying and studying subgroups of patients with shared defective gene expression could aid our understanding of the mechanisms underlying highly heterogeneous diseases such as CD.