A Genetic Modifier of the Gut Microbiome Influences the Risk of Graft-versus-Host Disease and Bacteremia After Hematopoietic Stem Cell Transplantation.

The human gut microbiome is involved in vital biological functions, such as maintenance of immune homeostasis and modulation of intestinal development and enhanced metabolic capabilities. Disturbances of the intestinal microbiota have been associated with development and progression of inflammatory conditions, including graft-versus-host disease (GVHD). The fucosyltransferase 2 (FUT2) gene produces an enzyme that is responsible for the synthesis of the H antigen in body fluids and on the intestinal mucosa. FUT2 genotype has been shown to modify the gut microbiome. We hypothesized that FUT2 genotype influences risk of GVHD and bacterial translocation after allogeneic hematopoietic stem cell transplantation (HSCT). FUT2 genotype was determined in 150 consecutive patients receiving allogeneic HSCT at our center. We abstracted clinical characteristics and outcomes from the transplantation database. Cumulative risk of any acute GVHD varied by FUT2 genotype, with decreased risk in those with A/A genotype and increased risk in those with G/G genotype. In contrast, the cumulative incidence of bacteremia was increased in those with A/A genotype. We conclude that the FUT2 genotype influences risk of acute GVHD and bacteremia after HSCT. We hypothesize that the mechanisms involve altered intestinal surface glycosylation and microbial composition but this requires additional study.

NADPH oxidase deficient mice develop colitis and bacteremia upon infection with normally avirulent, TTSS-1- and TTSS-2-deficient Salmonella Typhimurium.

Infections, microbe sampling and occasional leakage of commensal microbiota and their products across the intestinal epithelial cell layer represent a permanent challenge to the intestinal immune system. The production of reactive oxygen species by NADPH oxidase is thought to be a key element of defense. Patients suffering from chronic granulomatous disease are deficient in one of the subunits of NADPH oxidase. They display a high incidence of Crohn's disease-like intestinal inflammation and are hyper-susceptible to infection with fungi and bacteria, including a 10-fold increased risk of Salmonellosis. It is not completely understood which steps of the infection process are affected by the NADPH oxidase deficiency. We employed a mouse model for Salmonella diarrhea to study how NADPH oxidase deficiency (Cybb (-/-)) affects microbe handling by the large intestinal mucosa. In this animal model, wild type S. Typhimurium causes pronounced enteropathy in wild type mice. In contrast, an avirulent S. Typhimurium mutant (S.Tm(avir); invGsseD), which lacks virulence factors boosting trans-epithelial penetration and growth in the lamina propria, cannot cause enteropathy in wild type mice. We found that Cybb (-/-) mice are efficiently infected by S.Tm(avir) and develop enteropathy by day 4 post infection. Cell depletion experiments and infections in Cybb (-/-) Myd88 (-/-) mice indicated that the S.Tm(avir)-inflicted disease in Cybb (-/-) mice hinges on CD11c(+)CX3CR1(+) monocytic phagocytes mediating colonization of the cecal lamina propria and on Myd88-dependent proinflammatory immune responses. Interestingly, in mixed bone marrow chimeras a partial reconstitution of Cybb-proficiency in the bone marrow derived compartment was sufficient to ameliorate disease severity. Our data indicate that NADPH oxidase expression is of key importance for restricting the growth of S.Tm(avir) in the mucosal lamina propria. This provides important insights into microbe handling by the large intestinal mucosa and the role of NADPH oxidase in maintaining microbe-host mutualism at this exposed body surface.

Cyclooxygenase-2 deficiency leads to intestinal barrier dysfunction and increased mortality during polymicrobial sepsis.

Sepsis remains the leading cause of death in critically ill patients, despite modern advances in critical care. Intestinal barrier dysfunction may lead to secondary bacterial translocation and the development of the multiple organ dysfunction syndrome during sepsis. Cyclooxygenase (COX)-2 is highly upregulated in the intestine during sepsis, and we hypothesized that it may be critical in the maintenance of intestinal epithelial barrier function during peritonitis-induced polymicrobial sepsis. COX-2(-/-) and COX-2(+/+) BALB/c mice underwent cecal ligation and puncture (CLP) or sham surgery. Mice chimeric for COX-2 were derived by bone marrow transplantation and underwent CLP. C2BBe1 cells, an intestinal epithelial cell line, were treated with the COX-2 inhibitor NS-398, PGD(2), or vehicle and stimulated with cytokines. COX-2(-/-) mice developed exaggerated bacteremia and increased mortality compared with COX-2(+/+) mice following CLP. Mice chimeric for COX-2 exhibited the recipient phenotype, suggesting that epithelial COX-2 expression in the ileum attenuates bacteremia following CLP. Absence of COX-2 significantly increased epithelial permeability of the ileum and reduced expression of the tight junction proteins zonula occludens-1, occludin, and claudin-1 in the ileum following CLP. Furthermore, PGD(2) attenuated cytokine-induced hyperpermeability and zonula occludens-1 downregulation in NS-398-treated C2BBe1 cells. Our findings reveal that absence of COX-2 is associated with enhanced intestinal epithelial permeability and leads to exaggerated bacterial translocation and increased mortality during peritonitis-induced sepsis. Taken together, our results suggest that epithelial expression of COX-2 in the ileum is a critical modulator of tight junction protein expression and intestinal barrier function during sepsis.

Prevalence of type III secretion protein exoenzymes and antimicrobial susceptibility patterns from bloodstream isolates of patients with Pseudomonas aeruginosa bacteremia.

The purpose of this study was to determine the prevalence of two type III secretion effector proteins, exoU and exoS from bloodstream isolates of hospitalized patients with Pseudomonas aeruginosa (PSA) bacteremia, to characterize antimicrobial susceptibility patterns, and to compare mortality rates. PSA bloodstream isolates and antibiotic susceptibility profiles were collected from a university-affiliated hospital. ExoS and exoU genes were detected by polymerase chain reaction. Hospital mortality was assessed by medical chart review. 119 of 122 (97.5%) PSA bloodstream isolates contained either the exoS or exoU genes. ExoS was the most prevalent (n=86; 70.5%) followed by exoU (n=31; 25.4%), both genes (n=2; 1.6%) or neither gene (n=3; 2.5%). Isolates containing the exoU gene were significantly more likely to be resistant to cefepime, ceftazidime, piperacillintazobactam, carbapenems, fluoroquinolones, and gentamicin (p<0.05 for all). Mortality was high in patients with PSA bacteremia and did not differ among patients infected with the exoS isolates (n=37; 43%) or exoU isolates (n=11; 35%). One of two type III secretion effector proteins were almost universally present in PSA bloodstream isolates. Isolates containing the exoU gene were more likely to be resistant to multiple antibiotics.

Decreased alveolar macrophage apoptosis is associated with increased pulmonary inflammation in a murine model of pneumococcal pneumonia.

Regulation of the inflammatory infiltrate is critical to the successful outcome of pneumonia. Alveolar macrophage apoptosis is a feature of pneumococcal infection and aids disease resolution. The host benefits of macrophage apoptosis during the innate response to bacterial infection are incompletely defined. Because NO is required for optimal macrophage apoptosis during pneumococcal infection, we have explored the role of macrophage apoptosis in regulating inflammatory responses during pneumococcal pneumonia, using inducible NO synthase (iNOS)-deficient mice. iNOS(-/-) mice demonstrated decreased numbers of apoptotic macrophages as compared with wild-type C57BL/6 mice following pneumococcal challenge, greater recruitment of neutrophils to the lung and enhanced expression of TNF-alpha. Pharmacologic inhibition of iNOS produced similar results. Greater pulmonary inflammation was associated with greater levels of early bacteremia, IL-6 production, lung inflammation, and mortality within the first 48 h in iNOS(-/-) mice. Labeled apoptotic alveolar macrophages were phagocytosed by resident macrophages in the lung and intratracheal instillation of exogenous apoptotic macrophages decreased neutrophil recruitment in iNOS(-/-) mice and decreased TNF-alpha mRNA in lungs and protein in bronchial alveolar lavage, as well as chemokines and cytokines including IL-6. These changes were associated with a lower probability of mice becoming bacteremic. This demonstrates the potential of apoptotic macrophages to down-regulate the inflammatory response and for the first time in vivo demonstrates that clearance of apoptotic macrophages decreases neutrophil recruitment and invasive bacterial disease during pneumonia.

IdeS, a highly specific immunoglobulin G (IgG)-cleaving enzyme from Streptococcus pyogenes, is inhibited by specific IgG antibodies generated during infection.

IdeS, a recently discovered cysteine proteinase secreted by the important human pathogen Streptococcus pyogenes, interferes with phagocytic killing by specifically cleaving the heavy chain of immunoglobulin G. The fact that the enzyme targets one of the key molecules of the adapted immune response raised the question of whether an antibody response against IdeS could inhibit, i.e., neutralize, enzyme activity. Paired acute- and convalescent-phase serum samples from patients with pharyngotonsillitis (n = 10), bacteremia (n = 7), and erysipelas (n = 4) were analyzed. Antibodies with the ability to neutralize IdeS enzymatic activity were already found in two-thirds of acute-phase sera. However, patients who seroconverted to IdeS, in particular patients with pharyngotonsillitis and erysipelas, developed specific antibodies during convalescence with an increased capability to efficiently neutralize the enzymatic activity of IdeS. Also, the presence of neutralizing antibodies decreased the ability of IdeS to mediate bacterial survival in human immune blood. In patients with bacteremia, several acute-phase sera contained neutralizing antibodies, but no correlation was found to severity or outcome of invasive infections. Still, the fact that the human immune response targets the enzymatic activity of IdeS supports the view that the enzyme plays an important role during streptococcal infection.

Extremely high levels of alkaline phosphatase in adult patients as a manifestation of bacteremia.

BACKGROUND/AIMS: Jaundice resulting from severe bacterial infection is well known, particularly in pediatric literature. Extremely high levels of alkaline phosphatase (ALP) have rarely been emphasized as a manifestation of bacteremia in adults. The aim of this study was to evaluate the characteristics of extremely high levels of ALP in adult patients as a manifestation of bacteremia. METHODOLOGY: Extremely high levels of ALP were defined as being more than 1000 U/L. From April 1998 to May 1999, we retrospectively reviewed those patients' charts who had ALP above 1000 U/L. Sixteen patients that had bacteremia among 96 patients with extremely high levels of ALP at the emergency department of Taichung Veterans General Hospital were included in this study. RESULTS: Sixteen patients had bacteremia with extremely high levels of ALP, including 9 patients with malignant biliary obstruction (MBO), and 7 patients without MBO. The ALP levels ranged from 1002 to 2061 (1430.13+/-353.84) U/L. Ten patients were male, and 6 were female. Their ages ranged from 19 to 83 (56.13+/-16.51) years. A variety of gram-negative, and gram-positive organisms were identified, and Escherichia coli was the most common pathogen. Among the seven patients of bacteremia without MBO, 5 patients had underlying diabetes mellitus as the predisposing factor for development of the bacteremia. The ages of the bacteremia patients with MBO were older than those of patients without MBO (66.3+/-10.1 us. 43.0+/-13.7 years, P=0.0025). CONCLUSIONS: Bacteremia from a variety of organisms is a common cause for extreme elevation of ALP. Escherichia coli is the most common pathogen and presented more often in patients with MBO than those without MBO. In the setting o f extremely high levels of ALP as a manifestation of bacteremia, the patients with MBO are as common as those without MBO. We have demonstrated clinically that hepatic dysfunction during bacteremia may be manifested predominantly by extreme elevation of alkaline phosphatase with little abnormality in serum bilirubin.

Alveolar macrophage apoptosis contributes to pneumococcal clearance in a resolving model of pulmonary infection.

The role of alveolar macrophages (AM) in host defense against pulmonary infection has been difficult to establish using in vivo models. This may reflect a reliance on models of fulminant infection. To establish a unique model of resolving infection, with which to address the function of AM, C57BL/6 mice received low-dose intratracheal administration of pneumococci. Administration of low doses of pneumococci produced a resolving model of pulmonary infection characterized by clearance of bacteria without features of pneumonia. AM depletion in this model significantly increased bacterial outgrowth in the lung. Interestingly, a significant increase in the number of apoptotic AM was noted with the low-dose infection as compared with mock infection. Caspase inhibition in this model decreased AM apoptosis and increased the number of bacteremic mice, indicating a novel role for caspase activation in pulmonary innate defense against pneumococci. These results suggest that AM play a key role in clearance of bacteria from the lung during subclinical infection and that induction of AM apoptosis contributes to the microbiologic host defense against pneumococci.

Bioactive cytidine deaminase, an inhibitor of granulocyte-macrophage colony-forming cells, is massively released in fulminant meningococcal sepsis.

Cytidine deaminase (CDD) catalyzes the hydrolytic deamination of cytidine, which thereby is converted to uridine. CDD is found in serum and different tissues, with particularly high concentrations in polymorphonuclear neutrophils (PMN). We measured the CDD levels in plasma from patients with systemic meningococcal disease. Thirty-seven patients had significantly higher plasma levels of CDD than did 29 healthy control subjects (P=.0001). CDD levels in plasma or serum increased from a median of 96 ng/mL in healthy control subjects to medians of 168 ng/mL in patients without persistent shock (n=23; P=.001) and 422 ng/mL in patients with fulminant meningococcal septicemia (n=14; P=.0001). In most patients with fulminant septicemia, CDD levels in plasma increased during the first 3-53 h after the initiation of therapy (P=.003). CDD alone had no immediate harmful effect when injected into mice during a 4-day period. CDD may modulate the stimulatory effect of colony-stimulating factors on PMN in patients.

Tumor necrosis factor is a mediator of phospholipase release during bacteremia in baboons.

Phospholipase A (PLA) activity is elevated in plasma after polytrauma and in sepsis. During both situations, tumor necrosis factor (TNF) and free fatty acids (FFAs) are also increased. To determine whether there is a relationship between the changes in PLA and in TNF that occur during sepsis, we used a primate model of bacteremia. Live Escherichia coli (5 x 10(8) colony-forming units/kg) was injected into anesthetized baboons (n = 10). Four of these animals were pretreated with an anti-TNF antibody (anti-TNF Ab, 15 mg/kg). After the administration of bacteria (2 h), the plasma TNF was 10.2 +/- 3.2 ng/ml. PLA and FFA reached their maximum values of 191 +/- 14 U/l and 1.5 +/- 0.3 mM at 10 and 24 h after the injection of bacteria, respectively. In the animals treated with anti-TNF Ab, plasma TNF was undetectable; the highest values recorded for PLA and FFA were 71 +/- 23 U/l and 0.7 +/- 0.3 mM, respectively. Thus TNF appears to be an important mediator of PLA release during sepsis.

TNF-alpha and cyclooxygenase metabolites do not modulate C. albicans septic shock with disseminated candidiasis.

We analyzed differences in host regulation of tumor necrosis factor-alpha (TNF-alpha) production and pathophysiological responses in conscious rats after infection with two strains of pathogenic Candida albicans spp. (CA-1 and CA-2) compared with Escherichia coli serotype 055:B5 (EC). The hypothesis was tested that, in contrast to EC, hypotension, organ injury, and mortality after candidemia are not obligatorily dependent on TNF-alpha or TNF-alpha-induced cyclooxygenase pathway metabolites. Dose, viability, and strain-specific dependencies were established after intravenous 10(6) or 10(9) viable CA, as well as heat-killed (HK) or Formalin-inactivated (FI) CA blastospores, compared with live EC at the 24-h LD25 [10(9) colony-forming units (CFU)] and LD100 (10(10) CFU). Shock without endotoxemia developed 4-8 h after 10(9) live CA-1 or CA-2 (LD100 at 24 h) with disseminated yeast-mycelial transformation and increased microvascular permeability in multiple organs but not after HK or FI CA-1. Peak serum TNF-alpha after an LD100 of CA-1 or CA-2 was < 3% of LD25 EC values and was < 1% of peak values during lethal bacteremia. Similar pathogen-specific differences were found in liver- and lung-associated TNF. Production of functionally inactive TNF-alpha during candidemia was excluded by enzyme-linked immunosorbent assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Western blotting. Passive immunization against TNF-alpha 2 h before microbial challenge was not protective against CA but prevented otherwise lethal EC sepsis. Cyclooxygenase inhibition also failed to attenuate candidemic shock. We conclude that the magnitude and kinetics of TNF-alpha production and TNF-alpha-dependent immunophysiological responses are differentially regulated after lethal fungal vs. gram-negative bacterial infection. Thus TNF-alpha is not a pivotal mediator of the acute Candida septic shock syndrome with disseminated candidiasis.

Calcium-dependent and calcium-independent protease activities in skeletal muscle during sepsis.

Sepsis was produced in rats by implanting into their abdominal cavities fecal pellets containing Escherichia coli (10(2) colony-forming units [CFU]) and Bacteroides fragilis (10(4) CFU). Control rats were implanted with sterile pellets. A febrile response and hyperlactacidemia marked the onset of the septic injury. Control and septic rats were killed 24 and 48 hr after implantations, and posterior leg muscles were removed. Muscles were homogenized to prepare soluble fractions containing calcium-independent lysosomal (cathepsins B and L) and calcium-dependent cytosolic (calpain) proteases. Cathepsin and calpain activities were then assayed using standard procedures. There were no alterations in cathepsins B or L activities during sepsis. Calpain activity in septic muscle was significantly higher than that in control muscles. In vitro calpain sensitivity to Ca2+ was also higher in septic muscle than in controls. The cysteine protease inhibitor leupeptin caused a quantitatively greater inhibition of calpain activity in septic than in control muscles. These data indicate that whereas sepsis has no effect on Ca(2+)-insensitive lysosomal proteases, it is associated with an elevation of the Ca(2+)-dependent cytosolic protease activity.