Selective method for identification and quantification of Bifidobacterium animalis subspecies lactis BB-12 (BB-12) from the gastrointestinal tract of healthy volunteers ingesting a combination probiotic of BB-12 and Lactobacillus rhamnosus GG.

AIM: To develop a novel validated method for the isolation of Bifidobacterium animalis ssp. lactis BB-12 (BB-12) from faecal specimens and apply it to studies of BB-12 and Lactobacillus rhamnosus GG (LGG) recovered from the healthy human gastrointestinal (GI) tract. METHODS AND RESULTS: A novel method for isolating and enumerating BB-12 was developed based on its morphologic features of growth on tetracycline-containing agar. The method identified BB-12 correctly from spiked stool close to 100% of the time as validated by PCR confirmation of identity, and resulted in 97-104% recovery of BB-12. The method was then applied in a study of the recovery of BB-12 and LGG from the GI tract of healthy humans consuming ProNutrients® Probiotic powder sachet containing BB-12 and LGG. Viable BB-12 and LGG were recovered from stool after 21 days of probiotic ingestion compared to baseline. In contrast, no organisms were recovered 21 days after baseline in the nonsupplemented control group. CONCLUSIONS: We demonstrated recovery of viable BB-12, using a validated novel method specific for the isolation of BB-12, and LGG from the GI tract of healthy humans who consumed the probiotic supplement. SIGNIFICANCE AND IMPACT OF THE STUDY: This method will enable more detailed and specific studies of BB-12 in probiotic supplements, including when in combination with LGG.

Blood stream infection (BSI) and acute GVHD after hematopoietic SCT (HSCT) are associated.

Blood stream infection (BSI) and acute GVHD (aGVHD) are serious complications of hematopoietic SCT (HSCT). We hypothesized that the two events were not independent of one another. We studied (1) associations between BSI and aGVHD; and (2) the impact of BSI and/or aGVHD on death within 100 days after HSCT, using a retrospective cohort analysis. Risk factor analysis was carried out using multivariable Cox proportional hazards analyses. Of 211 patients who underwent allogeneic HSCT from January 2000 to December 2005 (58% of whom underwent reduced intensity transplantation), 82 (39%) developed BSI. In 49 patients (23%), grade (gr) 2-4 aGVHD occurred. Early BSI was independently associated with an increased occurrence of subsequent aGVHD gr 2-4. CMV seropositivity was independently associated with decreased occurrence of aGVHD. aGVHD gr 2-4 independently predicted subsequent first BSI. Both BSI and aGVHD gr 2-4 were significant independent predictors of death within 100 days after HSCT. There is a strong, independent association between BSI and aGVHD. Potential explanations include the elaboration of cytokines during BSI favoring the development of aGVHD and/or the immunosuppressive treatment of aGVHD favoring the development of BSI. Future studies should be directed at the mechanistic investigations of this association.

Blood stream infection after hematopoietic stem cell transplantation is associated with increased mortality.

Blood stream infection (BSI) is a serious complication of hematopoietic stem cell transplantation (HSCT). The aim of this retrospective cohort analysis was to describe BSI after HSCT, and to assess the predictors and outcomes of BSI after HSCT using multivariable modeling. Of the 243 subjects transplanted, 56% received allogeneic HSCT and 106 (43.6%) developed BSI. Of the 185 isolates, 68% were Gram-positive cocci, 21% were Gram-negative bacilli (GNR) and 11% were fungi. Type of allogeneic HSCT was an independent risk factor for BSI (hazard ratio (HR) 3.26, 95% confidence interval (CI) 1.50, 7.07, P = 0.01), as was the degree of HLA matching (HR 1.84, 95% CI 1.00, 3.37, P = 0.05). BSI was a significant independent predictor of mortality after HSCT (HR 1.79, 95% CI 1.18, 2.73, P = 0.007), after adjusting for acute graft-versus-host disease (GVHD) and allogeneic HSCT (both predicting death < or = 3 months after HSCT). In contrast to the effects of acute GVHD and allogeneic HSCT, the effect of BSI was evident throughout the post-HSCT period. GNR BSI and vancomycin-resistant enterococcal BSI also were significantly associated with death. We concluded that BSI is a common complication of HSCT associated with increased mortality throughout the post-HSCT period.

Late and atypical cytomegalovirus disease in solid-organ transplant recipients.

Posttransplantation cytomegalovirus (CMV) disease typically occurs 1-4 months after solid-organ transplantation. The case definition invariably includes unexplained fever for > or =3 days, often with leukopenia. Late and atypical presentation of CMV disease has been rarely reported. Five cases of late and atypical CMV disease in heart (n = 1), liver (n = 1), and kidney (n = 3) transplant recipients occurred within a 4-month period in early 1999. These patients presented at a median of 25 months after organ transplantation (range, 6 months to 22 years). Atypical findings included absence of fever in 3 patients, elevated white blood cell counts in 4 patients, and normal platelet counts in 4 patients. Four patients were at risk for primary CMV infection, and 3 received ganciclovir prophylaxis for 3 months. One patients was treated for rejection, and 2 patients had induction muromonab-CD3 (Orthclone; Orthobiotech). Two of the patients had pulmonary CMV disease, but neither of these patients had hypoxia. Two patients had enterocolitis, one of whom had chronic colitis for a year. These cases may represent a changing epidemiology and clinical presentation of CMV disease in solid-organ transplant recipients in an era of changing immunosuppression and improved CMV disease prevention in the early posttransplantation period.

Antimicrobial resistance in the chronically critically ill patient.

Infection caused by organisms resistant to conventional antimicrobial therapy is an emerging problem of global proportions. This article describes the epidemiology of infections caused by resistant organisms in chronically critically ill patients and explores factors and mechanisms that lead to the development of resistance. Specific organisms and strategies for the treatment and control of these resistance organisms are discussed.

Induction of circulating antagonists to IL-1 and TNF by IL-2 administration and their effects on IL-2 induced cytokine production in vitro.

The objectives of this study were to determine whether intensive immunotherapy with IL-2 results in detectable levels of circulating IL-1 and TNF antagonists and whether the levels achieved in vivo are sufficient to affect the generation of secondary proinflammatory cytokines such as IL-1 beta and TNF-alpha. We also sought to determine the extent to which endogenous TNF mediates the generation of an IL-1 antagonist by IL-2-activated PBMCs. In patients undergoing high dose IL-2 immunotherapy, plasma IL-1 receptor antagonist (IL-1ra) levels rose dramatically after the first IL-2 injection, reaching a plateau of 11.03 +/- 0.92 ng/ml within 4 h. TNF-soluble receptor p55 (TNFsRp55) was also detected in the plasma shortly after initiating treatment, and the levels progressively increased throughout the treatment course. PBMCs exposed to IL-2 expressed IL-1ra mRNA and secreted the IL-1ra protein, but neither PBMCs nor neutrophils shed TNFsRp55 in response to IL-2 or supernatants from IL-2-activated PBMCs. IL-1ra at concentrations achieved in the plasma during IL-2 immunotherapy (approximately 10 ng/ml) inhibited the in vitro production of IL-1 beta and TNF-alpha by IL-2-activated PBMCs by 65% and 30%, respectively. Although the monomeric receptor TNFsRp55 at concentrations achieved in the plasma had no effect on the in vitro production of IL-1ra, TNF-alpha, or IL-1 beta, the bivalent TNFsRp75-Fc chimera suppressed the generation of TNF and IL-1. IL-1ra synthesis was unaffected. These results suggest that the amount of IL-1ra generated in response to IL-2 is most likely sufficient to down-modulate the production of proinflammatory cytokines.

Cross-linking of the beta-glucan receptor on human monocytes results in interleukin-1 receptor antagonist but not interleukin-1 production.

The beta-glucan receptor, found on monocytes and neutrophils, binds glucose polymers derived from fungi. Ligands for the receptor have various immunomodulatory effects, including increased microbicidal killing activity. We have investigated the effect of beta-glucans on the production of interleukin-1 (IL-1) and its naturally occurring inhibitor, the IL-1 receptor antagonist (IL-1Ra). Particulate beta-glucan induced IL-1Ra production from human peripheral blood mononuclear cells (PBMC) but did not stimulate IL-1 beta synthesis or gene expression in these same cells. Monomeric (soluble) beta-glucan did not induce IL-1Ra production. However, when preincubated with PBMC, monomeric beta-glucan significantly (P < .01) reduced particulate beta-glucan induction of IL-1Ra by 40%, suggesting that crosslinking of beta-glucan receptors is required for induction of IL-1Ra. In support of this, monomeric beta-glucan immobilized on plastic surfaces stimulated IL-1Ra production. Vitamin D3, which increases the functional capacity of beta-glucan receptors, increased IL-1Ra production induced by particulate beta-glucan, whereas dexamethasone suppressed IL-1Ra synthesis. Because of their differential effects on cytokine production, beta-glucans may be used to therapeutic advantage in the diseases in which IL-1 is implicated.

Hematologic and immunomodulatory effects of an interleukin-1 receptor antagonist coinfusion during low-dose endotoxemia in healthy humans.

Endotoxin is a component of gram-negative bacteria that causes hematologic and immunologic changes through its induction of cytokines. Interleukin-1 receptor antagonist (IL-1Ra) is a naturally occurring inhibitor of IL-1 that competes with IL-1 for occupancy of cell-surface receptors but possesses no agonist activity. We investigated the ability of human recombinant IL-1Ra to block the effects of low-dose endotoxin. Fourteen healthy male volunteers between 18 and 30 years old were injected intravenously with 3 ng/kg Escherichia coli endotoxin. Concurrent with the injections, nine volunteers received a 3-hour continuous intravenous infusion of IL-1Ra. The other five subjects were given a 3-hour infusion of saline. Volunteers injected with endotoxin experienced a threefold increase in circulating neutrophils over baseline. This neutrophilia was significantly reduced by 48% in subjects administered endotoxin plus IL-1Ra (P = .0253). Ex vivo mitogen-induced peripheral blood mononuclear cell proliferation decreased by greater than 60% at 3 and 6 hours after endotoxin injection (P = .0053). This endotoxin-induced reduction in mitogen response was reversed in subjects coinjected with IL-1Ra (P = .0253). Endotoxin-induced symptoms, fever, and tachycardia were unaffected by IL-1Ra. IL-1 appears to be an important mediator in endotoxemia because some of its hematologic and immunomodulatory effects can be blocked by IL-1Ra.

Measuring circulating cytokines.

Cytokines, i.e., regulatory proteins derived primarily (but not exclusively) from cells of the immune system, are receiving increasing attention for their influences on physiological processes. This paper outlines several of the unique characteristics of cytokines and discusses the pitfalls encountered when measuring them in biological fluids. At present, each available assay has a combination of advantages and drawbacks; therefore, investigators must be aware of the trade-offs and choose the assay that best addresses their needs. The factors that affect cytokine measurement also influence cytokine activity in vivo; thus they are important from a physiological as well as methodological standpoint. Moreover, the absolute concentration of a single cytokine is probably less important than the balance between that cytokine and its natural antagonists.

Comparison of first use and reuse of cuprophan membranes on interleukin-1 receptor antagonist and interleukin-1 beta production by blood mononuclear cells.

The magnitude of the changes in a variety of blood constituents on exposure to the dialysis membrane has been used as an index of "biocompatibility," and dialyzer reuse has been postulated to improve biocompatibility by attenuating these changes. We studied the hemodialysis-induced changes in the in vitro production of interleukin-1 receptor antagonist (IL-1Ra) and interleukin-1 beta (IL-1 beta) by peripheral blood mononuclear cells (PBMCs), and compared the effect of first use and reuse of cuprophan membranes on these changes. Studies were performed during dialysis with first use and third reuse of the same kidney. The cell content and production of IL-1Ra and IL-1 beta by unstimulated and endotoxin- or IgG-stimulated PBMCs were studied just prior to dialysis, and from the afferent and efferent limbs of the blood circuit 15 minutes after the start of dialysis. Interleukin-1 receptor antagonist and IL-1 beta were measured by specific radioimmunoassay and are expressed as picograms per 2.5 x 10(6) PBMCs. Fifteen minutes after the start of dialysis, the number of PBMCs harvested from 10 mL of blood decreased from 19.8 +/- 4.7 x 10(6) predialysis to 14 +/- 3 x 10(6) (P = 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of tumor necrosis factor soluble receptor p55 in blood samples from healthy and endotoxemic humans.

The tumor necrosis factor (TNF) soluble receptor derived from the cell surface p55 TNF receptor (TNFsRp55) is a naturally occurring substance generated during infection and inflammation. TNFsRp55 inhibits biologic effects of TNF. An RIA was developed to quantitate TNFsRp55 in human blood. Recovery of TNFsRp55 from blood anticoagulated with EDTA was optimal compared with recovery from serum or heparinized plasma. TNF did not interfere with the assay. With the RIA based on radiolabeled nonglycosylated Escherichia coli-derived recombinant TNFsRp55, a mean concentration of 198 +/- 15 pg/mL was found in 14 volunteers. When glycosylated CHO cell-derived TNFsRp55 was used, the mean level was 1656 +/- 95 pg/mL. Infusion of endotoxin into volunteers induced TNFsRp55, which peaked at 517 +/- 99 pg/mL for the E. coli-based RIA and 7300 +/- 1810 pg/mL for the CHO cell-based RIA. These findings demonstrate that blood collected in EDTA is optimal for measuring circulating TNFsRp55 and that this soluble receptor is present in health but elevated during endotoxemia.

In vitro production of interleukin-1 receptor antagonist in chronic renal failure, CAPD and HD.

Dialysis-related symptoms are believed to be mediated, at least in part, by monocyte/macrophage-derived pro-inflammatory cytokines including interleukin-1 (IL-1) and tumor necrosis factor (TNF). Measuring the production of interleukin-1 receptor antagonist (IL-Ra), a naturally occurring inhibitor of IL-1, opens avenues to study the balance between these two cytokines in patients. We studied the cell content and production of IL-1 beta and IL-Ra by unstimulated and endotoxin- or IgG-stimulated peripheral blood mononuclear cells (PBMC) in undialyzed patients with chronic renal failure (CRF), patients on continuous ambulatory peritoneal dialysis (CAPD) and patients on chronic hemodialysis with reuse cuprophan membranes (HD), and compared them to healthy controls. IL-1 beta and IL-Ra were measured by specific radioimmunoassay. IL-1 beta was undetectable in freshly harvested PBMC from healthy controls, CRF, CAPD or HD. In contrast, the content of IL-Ra in HD patients (2828 +/- 466 pg/ml) was significantly higher than that in healthy controls (643 +/- 53 pg/ml, P < 0.01), CRF (1097 +/- 320 pg/ml, P < 0.01) or CAPD (1398 +/- 390 pg/ml, P < 0.05). In endotoxin-stimulated PBMC, IL-1 beta production by HD patients (9375 +/- 1687 pg/ml) was not significantly different from healthy controls (8429 +/- 1621 pg/ml). However, endotoxin-stimulated IL-Ra production by HD patients (32,350 +/- 8276 pg/ml) was greater than that from healthy controls (11,284 +/- 1250 pg/ml, P < 0.001), CRF (12,263 +/- 2680 pg/ml, P < 0.01) or CAPD patients (11,822 +/- 1797 pg/ml, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1 beta (IL-1 beta), IL-1 receptor antagonist, and TNF alpha production in whole blood.

The ability of an individual to mount defense responses to infection depend in part on the capacity to produce cytokines such as interleukin 1 (IL-1) and tumor necrosis factor (TNF). The specialized equipment, labor intensity, and sterile practice required for the standard in vitro evaluation of cytokine production can make such evaluation impractical in some clinical situations. We report a method for stimulating whole blood to produce cytokines that can be implemented in laboratories without tissue culture facilities and requires minimal sample preparation. IL-1 beta and TNF alpha production in whole blood samples was stimulated with endotoxin and/or phytohemagglutinin in standard EDTA-containing vacuum collection tubes. After incubation, plasma was removed and frozen for later assay. Comparison of this whole blood method with isolated mononuclear cell cultures indicated a significant correlation for IL-1 beta production (r = 0.746, P = 0.005). This technique also produced the newly described cytokine, IL-1 receptor antagonist. We conclude that the whole blood method is an acceptable alternative to isolated cell culture methods for measuring IL-1 beta in situations that preclude the standard in vitro approach.

Interleukin-1 receptor antagonist circulates in experimental inflammation and in human disease.

Interleukin-1 receptor antagonist (IL-1ra) is a 22-Kd protein that shares homology with IL-1 beta, binds to the IL-1 receptor, but has no known agonist properties. This inhibitor appears to be the first cytokine whose sole function is to block the actions of another cytokine. Exogenous IL-1ra administration has been shown to reduce mortality in experimental septic shock. We now report that IL-1ra is endogenously produced and circulates in experimental inflammation and in clinical disease. After experimental endotoxemia in human volunteers, IL-1ra concentrations increase from a baseline concentration of 460 +/- 200 pg mL-1 to 14,870 +/- 290 pg mL-1 at 3 hours (P less than .05). IL-1ra is also detectable in all plasma samples from critically ill patients with a mean concentration of 8,680 +/- 2,060 pg mL-1 (range 320 to 55,370 pgs mL-1). In nonhuman primates, Escherichia coli septic shock induces elevated plasma levels of IL-4ra (P less than .05). However, in animals that eventually succumb to septic shock, Il-1ra appears in quantities presumed inadequate to block the pathologic sequelae associated with high IL-1 beta levels. The findings suggest that IL-1ra may play a role in modulating the systemic host responses to a variety of nonlethal disease states by altering the balance between cytokines and their antagonists.

Effect of interleukin-1 (IL-1) blockade on cytokine synthesis: II. IL-1 receptor antagonist inhibits lipopolysaccharide-induced cytokine synthesis by human monocytes.

Lipopolysaccharide (LPS) stimulates interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) gene expression and synthesis in human peripheral blood mononuclear cells (PBMC). IL-1 can also induce PBMC to synthesize IL-1 and TNF alpha. In the present study, we used IL-1 receptor antagonist (IL-1ra) to determine the relative contribution of an IL-1-positive feedback loop to the total amount of LPS-induced cytokine synthesis. Pretreatment of PBMC with human recombinant IL-1ra reduced LPS-induced cytokine synthesis in a dose-dependent manner (P less than .001). Maximal inhibition was 33% for IL-1 alpha (P less than .01), 43% for IL-1 beta (P = .001), and 20% for TNF alpha (P less than .05). We consistently observed IL-1ra suppression of LPS-induced cytokines in PBMC of 38 volunteers. However, this phenomenon was not specific for LPS; 1 microgram/mL IL-1ra inhibited IL-1 beta synthesized in response to human recombinant IL-2 by 44% (P less than .001), toxic shock syndrome toxin-1 by 26% (P less than .05), and phorbol 12-myristate 13-acetate by 76% (P less than .001). IL-1ra added to PBMC 4 or 8 hours after stimulation with LPS still inhibited IL-1 beta synthesis by 44% (P less than .001) or 25% (P = .01), respectively. The steady state messenger RNA levels of IL-1 beta were reduced in PBMC stimulated by LPS in the presence of IL-1ra. In monocytes isolated by elutriation, IL-1ra reduced LPS-induced IL-1 alpha by 16% (P less than .001), IL-1 beta by 14% (P less than .05), and TNF alpha by 24% (P = .01). We conclude that IL-1-induced IL-1 significantly contributes to LPS-induced cytokine synthesis.

Interleukin-1 (IL-1) receptor blockade reduces endotoxin and Borrelia burgdorferi-stimulated IL-8 synthesis in human mononuclear cells.

Interleukin-1 (IL-1) is a potent stimulator of IL-8 production by fibroblasts and monocytes. In the present study, we asked how much of endotoxin (LPS)-induced IL-8 production by human peripheral blood mononuclear cells was due to IL-1 induced by LPS. Cells were stimulated with either IL-1 beta, LPS, or Borrelia burgdorferi, and total IL-8 was determined by a specific radioimmunoassay. The addition of saturating concentrations of IL-1 receptor antagonist protein (IRAP) reduced the IL-1 beta-, LPS-, and B. burgdorferi-induced IL-8 synthesis by 85, 50, and 40%, respectively. Increasing the concentration of LPS did not affect the reduction in IL-8 synthesis observed in the presence of IRAP. Significant inhibition of the IL-1 beta-induced IL-8 synthesis was observed when IRAP was added 60 or 90 min after IL-1 beta; similarly, IL-8 synthesis after LPS was also reduced by delayed addition of IRAP. These data suggest that the ameliorative effects of IL-1 receptor blockade in models of inflammation and infection may be due, in part, to suppression of IL-1-induced IL-8.

Production of interleukin-1-receptor antagonist during experimental endotoxaemia.

Interleukin-1 (IL-1) has been implicated in the pathogenesis of sepsis. IL-1-receptor antagonist (IL-1ra) is a naturally occurring inhibitor of IL-1 activity that competes with IL-1 for occupancy of cell-surface receptors but possesses no agonist activity. We induced endotoxaemia in 9 healthy human volunteers by injection of Escherichia coli endotoxin, and measured plasma concentrations of IL-1 and IL-1ra by radioimmunoassay during the next 24 h. Peak plasma concentrations of IL-1ra were about a hundred-fold greater than those of IL-1 beta. No IL-1 or IL-1ra were detectable in the plasma of 4 volunteers injected with saline. Our results suggest that the predominant natural response to endotoxin in man is the production of antagonist rather than agonist.

Production of interleukin-1 receptor antagonist and interleukin-1 beta by peripheral blood mononuclear cells is differentially regulated.

We studied the relationship between the production of the 23-Kd interleukin-1 receptor antagonist (IL-1ra) and IL-1 beta in cultures of human peripheral blood mononuclear cells (PBMC) using a specific radioimmunoassay for IL-1ra that had a sensitivity of 166 +/- 11 pg/mL. PBMC cultured without human serum made little IL-1ra or IL-1 beta. In the presence of 1% AB serum, there was no increase in IL-1 beta (0.25 +/- 0.13 ng/mL) but IL-1ra production increased sevenfold to 3.4 +/- 0.5 ng/mL. IgG (2.5 to 100 micrograms/mL IgG) or granulocyte-macrophage colony-stimulating factor (GM-CSF) (1 to 100 ng/mL) had no significant effect on IL-1 beta production but increased IL-1ra production up to 18-fold (18.2 +/- 3.9 ng/mL). Using endotoxin as a stimulant, 82% +/- 2% of IL-1ra was secreted in comparison with 52% +/- 9% of IL-1 beta. Culture conditions of PBMC influenced the production of IL-1ra but not IL-1 beta. Rocking endotoxin-stimulated PBMC produced 75% less IL-1ra but the same amount of IL-1 beta when compared with PBMC cultured in stationary plastic tubes. Rocking IgG-or GM-CSF-stimulated PBMC also produced 75% to 80% less IL-1ra. GM-CSF or IL-1 beta at concentrations that elicited submaximal production of IL-1ra potentiated IgG-induced IL-1ra production. The production of IL-1ra and IL-1 beta are under differential regulation because serum, IgG, and GM-CSF were potent stimuli for the production of IL-1ra but not IL-1 beta, and the prevention of cell-cell contact of PBMC reduced IL-1ra but not IL-1 beta production.

Turnover and fate of I-Ak antigen on the murine macrophage cell surface.

The macrophage plasma membrane is a major site of the cell's activities, including phagocytosis, antibody-dependent cellular cytotoxicity, and antigen presentation. To present antigen, the expression by the macrophage of immune region-associated (Ia) antigen is required. The turnover and fate of this cell surface constituent was studied in macrophages cultured with lymphokine or recombinant interferon-gamma. Surface-labeled subregion I-Ak antigen was lost from the cell surface at a rapid rate, with a half-life of approximately 24 hours. However, the shedding of I-A antigen into the culture fluid was not detected. Therefore, the loss of I-A antigen from the macrophage surface is most likely by its degradation. Upon removal of lymphokine or interferon from macrophage cultures, I-A antigen expression declined, with an apparent half-life of 2 days.