Biomedical subjects

David L Williams

Publications and source records attributed to David L Williams.

At least 19 recordsLinked to original sources

PubMed2025

In vitro protocol demonstrating five functional steps of trained immunity in mice: Implications on biomarker discovery and translational research.

We developed an in vitro methodology to study trained immunity using murine bone-marrow-derived macrophages stimulated with β-glucan and lipopolysaccharide (LPS). Longitudinal analysis of interleukin (IL)-6 and tumor necrosis factor (TNF) production demonstrates that trained macrophages secrete higher cytokine levels following primary stimulation with β-glucan compared to unstimulated macrophages (step 1). After a resting period, trained macrophages return to basal levels of cytokine production (step 2) but rapidly produce enhanced levels of IL-6 and TNF after secondary stimulation with LPS, compared to macrophages individually stimulated with either β-glucan (step 3) or LPS (step 4) alone. The combined cytokine production of macrophages after single stimulation with β-glucan (stimulus 1) and LPS (stimulus 2) is significantly lower than the cytokine levels produced by trained macrophages sequentially stimulated with both β-glucan and LPS (stimulus 1 + 2) (step 5). These results experimentally reproduce the distinctive functional stages that macrophages undergo during the training process.

Animals↗

PubMed2004

Dectin-1 uses novel mechanisms for yeast phagocytosis in macrophages.

The phagocytosis of pathogens is a critical event in host defense, not only for clearance of the invading microorganism, but also for the subsequent immune response. We have examined Dectin-1, a proinflammatory nonopsonic receptor for beta-glucans, and show that it mediates the internalization of beta-glucan-bearing ligands, including yeast particles. Although requiring tyrosine phosphorylation and the cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM)-like motif, uptake mediated by Dectin-1 was different from any previously reported phagocytic receptor and was not dependent on Syk-kinase in macrophages. Furthermore, intracellular trafficking of this receptor was influenced by the nature of the beta-glucan ligand, which has significance for the biologic activity of these immunomodulatory carbohydrates.

3T3 Cells↗

PubMed2004

Changes in plasma membrane properties and phosphatidylcholine subspecies of insect Sf9 cells due to expression of scavenger receptor class B, type I, and CD36.

In mammalian cells scavenger receptor class B, type I (SR-BI), mediates the selective uptake of high density lipoprotein (HDL) cholesteryl ester into hepatic and steroidogenic cells. In addition, SR-BI has a variety of effects on plasma membrane properties including stimulation of the bidirectional flux of free cholesterol (FC) between cells and HDL and changes in the organization of plasma membrane FC as indicated by increased susceptibility to exogenous cholesterol oxidase. Recent studies in SR-BI-deficient mice and in SR-BI-expressing Sf9 insect cells showed that SR-BI has significant effects on plasma membrane ultrastructure. The present study was designed to test the range of SR-BI effects in Sf9 insect cells that typically have very low cholesterol content and a different phospholipid profile compared with mammalian cells. The results showed that, as in mammalian cells, SR-BI expression increased HDL cholesteryl ester selective uptake, cellular cholesterol mass, FC efflux to HDL, and the sensitivity of membrane FC to cholesterol oxidase. These activities were diminished or absent upon expression of the related scavenger receptor CD36. Thus, SR-BI has fundamental effects on cholesterol flux and membrane properties that occur in cells of evolutionarily divergent origins. Profiling of phospholipid species by electrospray ionization mass spectrometry showed that scavenger receptor expression led to the accumulation of phosphatidylcholine species with longer mono- or polyunsaturated acyl chains. These changes would be expected to decrease phosphatidylcholine/cholesterol interactions and thereby enhance cholesterol desorption from the membrane. Scavenger receptor-mediated changes in membrane phosphatidylcholine may contribute to the increased flux of cholesterol and other lipids elicited by these receptors.

Animals↗

PubMed2004

Independent effects of APOE on cholesterol metabolism and brain Abeta levels in an Alzheimer disease mouse model.

The APOE epsilon4 allele is the most significant genetic risk factor associated with Alzheimer's disease to date. Epidemiological studies have demonstrated that inheritance of one or more epsilon4 alleles affects both the age of onset and the severity of pathology development. Dosage of APOE epsilon2 and epsilon3 alleles, however, appear to be protective against the effects of epsilon4. Although much of the biology of APOE in peripheral cholesterol metabolism is understood, its role in brain cholesterol metabolism and its impact on AD development is less defined. Several APOE transgenic models have been generated to study the effects of APOE alleles on APP processing and Abeta pathology. However, these models have potential limitations that confound our understanding of the effects of apolipoprotein E (APOE) levels and cholesterol metabolism on disease development. To circumvent these limitations, we have taken a genomic-based approach to better understand the relationship between APOE alleles, cholesterol and Abeta metabolism. We have characterized APOE knock-in mice, which express each human allele under the endogenous regulatory elements, on a defined C57BL6/J background. These mice have significantly different serum cholesterol levels and steady-state brain APOE levels, and yet have equivalent brain cholesterol levels. However, the presence of human APOE significantly increases brain Abeta levels in a genomic-based model of AD, irrespective of genotype. These data indicate an independent role for APOE in cholesterol metabolism in the periphery relative to the CNS, and that the altered levels of cholesterol and APOE in these mice are insufficient to influence Abeta metabolism in a mouse model of Alzheimer's disease.

Alleles↗

PubMed2004

SR-BI- and ABCA1-mediated cholesterol efflux to serum from patients with Alagille syndrome.

Alagille syndrome is associated with bile duct paucity resulting in liver disease. Patients can be divided into mildly and severely icteric groups, with both groups having altered lipoproteins. The incidence of ischemic heart disease is rare in severely cholestatic children despite increased total cholesterol and decreased high density lipoprotein cholesterol (HDL-C). The present studies examine the impact of altered lipid and lipoproteins on scavenger receptor class B type I (SR-BI)- and ABCA1-mediated efflux to serum from both groups. Efflux was compared with serum from 29 patients (15 with normal plasma cholesteryl ester, 14 with low cholesteryl ester). Efflux via SR-BI and ABCA1 was studied using cell systems having either low or high expression levels of these receptors. SR-BI efflux was lower (P = 0.04) with serum from severely icteric patients (3.9 +/- 1.4%) compared with serum from mildly icteric patients (5.1 +/- 1.4%) and was positively correlated with HDL-C and its apolipoproteins. SR-BI-mediated efflux was not correlated with any particular mature HDL but was negatively correlated with small lipid-poor prebeta-1 HDL. Consistent with severely icteric patients having high prebeta-1 HDL levels, the ABCA1 efflux was significantly higher with their serum (4.8 +/- 2.2%) compared with serum from mildly icteric patients (2.0 +/- 0.6%) and was positively correlated with prebeta-1 HDL. These studies demonstrated that prebeta-1 HDL is the preferred acceptor for ABCA1 efflux, whereas many particles mediate SR-BI efflux.

ATP Binding Cassette Transporter 1↗

PubMed2004

Differential regulation of cytokine and chemokine production in lipopolysaccharide-induced tolerance and priming.

LPS pretreatment of human pro-monocytic THP-1 cells induces tolerance to secondary LPS stimulation with reduced TNFalpha production. However, secondary stimulation with heat-killed Staphylococcus aureus (HKSa) induces priming as evidenced by augmented TNFalpha production. The pro-inflammatory cytokine, IFNgamma, also abolishes suppression of TNFalpha in LPS tolerance. The effect of LPS tolerance on HKSa and IFNgamma-induced inflammatory mediator production is not well defined. We hypothesized that LPS, HKSa and IFNgamma differentially regulate pro-inflammatory mediators and chemokine production in LPS-induced tolerance. THP-1 cells were pretreated for 24 h with LPS (100 ng/ml) or LPS (100 ng/ml) + IFNgamma (1 microg/ml). Cells were subsequently stimulated with LPS or HKSa (10 microg/ml) for 24 h. The production of the cytokines TNFalpha, IL-6, IL-1beta, and GMCSF and the chemokine IL-8 were measured in supernatants. LPS and HKSa stimulated TNFalpha (3070 +/- 711 pg/ml and 217 +/- 9 pg/ml, respectively) and IL-6 (237 +/- 8.9 pg/ml and 56.2 +/- 2.9 pg/ml, p < 0.05, n = 3, respectively) in control cells compared to basal levels (< 25 pg/ml). LPS induced tolerance to secondary LPS stimulation as evidenced by a 90% (p < 0.05, n = 3) reduction in TNFalpha. However, LPS pretreatment induced priming to HKSa as demonstrated by increased TNFalpha (2.7 fold, from 217 to 580 pg/ml, p < 0.05, n = 3 ). In contrast to suppressed TNFalpha, IL-6 production was augmented to secondary LPS stimulation (9 fold, from 237 to 2076 pg/ml, p < 0.01, n = 3) and also primed to HKSa stimulation (62 fold, from 56 to 3470 pg/ml, p < 0.01, n = 3). LPS induced IL-8 production and to a lesser extent IL-1beta and GMCSF. LPS pretreatment did not affect secondary LPS stimulated IL-8 or IL-1beta, although HKSa stimulation augmented both mediators. In addition, IFNgamma pretreatment reversed LPS tolerance as evidenced by increased TNFalpha levels while IL-6, IL-1beta, and GMCSF levels were further augmented. However, IL-8 production was not affected by IFNgamma. These data support our hypothesis of differential regulation of cytokines and chemokines in gram-negative- and gram-positive-induced inflammatory events. Such changes may have implications in the pathogenesis of polymicrobial sepsis.

Chemokines↗

PubMed2004

Subphysiologic apolipoprotein E (ApoE) plasma levels inhibit neointimal formation after arterial injury in ApoE-deficient mice.

OBJECTIVE: Apolipoprotein E (apoE) reduces mouse atherosclerosis progression independent of plasma cholesterol level effects. A mouse artery injury model was used to examine whether apoE exhibits beneficial lipid-independent effects on neointimal formation. METHODS AND RESULTS: ApoE-deficient (apoE-/-), wild-type (WT), and transgenic apoE-/- mice (secreting apoE at different levels from adrenal glands) underwent femoral artery injury. Mice with low expression of plasma apoE (0.1% of WT) had cholesterol levels approximately half those of apoE-/- littermates (but still approximately 6x >WT). Mice with higher expression (HE; 2% to 3% of WT) of plasma apoE had cholesterol levels approximately twice those of WT. Injured WT mouse (versus apoE-/-) arteries had a smaller mean intima-to-media (I/M) ratio (0.87 versus 1.96; P<0.05). HE mice tended to have lower mean I/M ratios (1.3; P>0.05 versus apoE-/- mice). Multiple regression analysis indicated that apoE levels were significantly associated with reduced I/M ratios, but plasma cholesterol levels were not, before or after adjusting for apoE. In addition, foam cell content of the neointima and media of injured arteries, a negative prognostic indicator in postangioplasty human lesions, was inversely related to plasma apoE levels. CONCLUSIONS: Similar to its effects on atherosclerosis progression, in a mouse model of restenosis, a subphysiological level of apoE was associated with beneficial effects on lesion size/composition.

Actins↗

PubMed2004

NF-kappaB activation is required for the development of cardiac hypertrophy in vivo.

In the present study, we examined whether NF-kappaB activation is required for cardiac hypertrophy in vivo. Cardiac hypertrophy in rats was induced by aortic banding for 1, 3, and 5 days and 1-6 wk, and age-matched sham-operated rats served as controls. In a separate group of rats, an IkappaB-alpha dominant negative mutant (IkappaB-alphaM), a superrepressor of NF-kappaB activation, or pyrrolidinedithiocarbamate (PDTC), an antioxidant that can inhibit NF-kappaB activation, was administered to aortic-banded rats for 3 wk. The heart weight-to-body weight ratio was significantly increased at 5 days after aortic banding, peaked at 4 wk, and remained elevated at 6 wk compared with age-matched sham controls. Atrial natriuretic peptide and brain natriuretic peptide mRNA expressions were significantly increased after 1 wk of aortic banding, reached a maximum between 2 and 3 wk, and remained increased at 6 wk compared with age-matched sham controls. NF-kappaB activity was significantly increased at 1 day, reached a peak at 3 wk, and remained elevated at 6 wk, and IKK-beta activity was significantly increased at 1 day, peaked at 5 days, and then decreased but remained elevated at 6 wk after aortic banding compared with age-matched sham controls. Inhibiting NF-kappaB activation in vivo by cardiac transfection of IkappaB-alphaM or by PDTC treatment significantly attenuated the development of cardiac hypertrophy in vivo with a concomitant decrease in NF-kappaB activity. Our results suggest that NF-kappaB activation is required for the development of cardiac hypertrophy in vivo and that NF-kappaB could be an important target for inhibiting the development of cardiac hypertrophy in vivo.

Adenoviridae↗

PubMed2004

Biochemical characterization of 2-Cys peroxiredoxins from Schistosoma mansoni.

Peroxiredoxins are a large family of peroxidases that have important antioxidant and cell signaling functions. Genes encoding two novel 2-cysteine peroxiredoxin proteins were identified in the expressed sequence tag data base of the helminth parasite Schistosoma mansoni, a causative agent of schistosomiasis. The recombinant proteins showed peroxidase activity in vitro with a variety of hydroperoxides and used both the thioredoxin and the glutathione systems as electron donors. Steady-state kinetic analysis indicated that the new peroxiredoxins had saturable kinetics, whereas a previously identified schistosome peroxiredoxin was found to function with more typical unsaturable (ping-pong) kinetics. The catalytic efficiencies S. mansoni peroxiredoxins were similar to those for other peroxiredoxins studied (10(4)-10(5) m(-1) s(-1)). Mutagenesis of S. mansoni peroxiredoxins indicated that glutathione dependence and kinetic differences were conferred by the C-terminal alpha-helix forming 22 amino acids. This is the first report of 2-cysteine peroxiredoxins efficiently utilizing reducing equivalents from both the thioredoxin and glutathione systems. Studies to determine the resistance to oxidative inactivation, important in regulating cell signaling pathways, showed that S. mansoni possess both bacterial-like resistant and mammalian-like sensitive peroxiredoxins. The susceptibility to oxidative inactivation was conferred by the C-terminal tail containing a tyrosine-phenylalanine motif. S. mansoni is the first organism shown to possess both robust and sensitive peroxiredoxins. The ability of schistosome peroxiredoxins to use alternative electron donors, and their variable resistance to overoxidation may reflect their presence in different cellular sites and emphasizes the significant differences in overall redox balance mechanisms between the parasite and its mammalian host.

Amino Acid Motifs↗

PubMed2004

Glycine 420 near the C-terminal transmembrane domain of SR-BI is critical for proper delivery and metabolism of high density lipoprotein cholesteryl ester.

Scavenger receptor BI, SR-BI, is a physiologically relevant receptor for high density lipoprotein (HDL) that mediates the uptake of cholesteryl esters and delivers them to a metabolically active membrane pool where they are subsequently hydrolyzed. A previously characterized SR-BI mutant, A-VI, with an epitope tag inserted into the extracellular domain near the C-terminal transmembrane segment, revealed a separation-of-function between SR-BI-mediated HDL cholesteryl ester uptake and cholesterol efflux to HDL, on one hand, and cholesterol release to small unilamellar phospholipid vesicle acceptors and an increased cholesterol oxidase-sensitive pool of membrane free cholesterol on the other. To further elucidate amino acid residues responsible for this separation-of-function phenotype, we engineered alanine substitutions and point mutations in and around the site of epitope tag insertion, and tested these for various cholesterol transport functions. We found that changing amino acid 420 from glycine to histidine had a profound effect on SR-BI function. Despite the ability to mediate selective HDL cholesteryl ester uptake, the G420H receptor had a greatly reduced ability to: 1) enlarge the cholesterol oxidase-sensitive pool of membrane free cholesterol, 2) mediate cholesterol efflux to HDL, even at low concentrations of HDL acceptor where binding-dependent cholesterol efflux predominates, and 3) accumulate cholesterol mass within the cell. Most importantly, the G420H mutant was unable to deliver the HDL cholesteryl ester to a metabolically active membrane compartment for efficient hydrolysis. These observations have important implications regarding SR-BI function as related to its structure near the C-terminal transmembrane domain.

Amino Acid Sequence↗

PubMed2004

Modulating Toll-like receptor mediated signaling by (1-->3)-beta-D-glucan rapidly induces cardioprotection.

OBJECTIVE: Immune and inflammatory signaling pathways, initiated by the innate response, are involved in myocardial ischemia/reperfusion (I/R) injury. Toll-like receptor (TLR) mediated MyD88-dependent NFkappaB pathways play a role in the induction of innate immunity. We have reported that glucan phosphate (GP) improved survival in experimental sepsis, which correlated with decreased tissue NFkappaB activation. In the present study, we report that GP rapidly induced cardioprotection against I/R injury in vivo. METHODS: Sprague-Dawley rats were pretreated with GP (40 mg/kg, i.p) 1 h before 45 min of ligation of the left anterior descending coronary followed by reperfusion for 4 and 24 h. Infarction size was examined by triphenyltetrazolium chloride (TTC) staining. NFkappaB activation was analyzed by electrophoretic mobility shift assay (EMSA). IkappaB kinase-beta (IKKbeta), IL-1 receptor-associated kinase (IRAK) and Phosphoinositide 3-kinase (PI3K) activities were determined by kinase assay with appropriate substrates. Association of TLR4 with MyD88 or with PI3K p85 was assessed by immunoprecipitation with anti-TLR4 followed by immunoblotting with anti-MyD88 or anti-p85. RESULTS: GP treatment reduced infarct size by 47% in rat hearts subjected to reperfusion for 4 h and by 50% following reperfusion for 24 h. The same protective effect was observed when GP was administrated 5 min after initiation of ischemia. The mechanisms of GP induced cardioprotection involve decreased association of TLR4 with MyD88, inhibition of I/R induced IRAK and IKKbeta activity and decreased NFkappaB activity. In addition, GP increased TLR4 phosphotyrosine, resulting in increasing PI3K/Akt activity in the myocardium, which correlated with decreased cardiac myocyte apoptosis following I/R. CONCLUSION: The results suggest that activation of the TLR mediated MyD88-dependent NFkappaB signaling pathway may play an important role in myocardial I/R injury, while stimulation of the PI3K/Akt signaling could serve a protective role. The data indicates that GP treatment shifts the TLR mediated activation signal in I/R from a predominantly NFkappaB pathway to a predominant PI3K/Akt signaling pathway.

Animals↗

PubMed2004

A novel selective allosteric modulator potentiates the activity of native metabotropic glutamate receptor subtype 5 in rat forebrain.

We found that N-[4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl]-2-hydroxybenzamide (CPPHA), is a potent and selective positive allosteric modulator of the metabotropic glutamate receptor subtype 5 (mGluR5). CPPHA alone had no agonist activity and acted as a selective positive allosteric modulator of human and rat mGluR5. CPPHA potentiated threshold responses to glutamate in fluorometric Ca(2+) assays 7- to 8-fold with EC(50) values in the 400 to 800 nM range, and at 10 microM shifted mGluR5 agonist concentration-response curves to glutamate, quisqualate, and (R,S)-3,5-dihydroxyphenylglycine (DHPG) 4- to 7-fold to the left. The only effect of CPPHA on other mGluRs was weak inhibition of mGluR4 and 8. Neither CPPHA nor the previously described 3,3'-difluorobenzaldazine (DFB) affected [(3)H]quisqualate binding to mGluR5, but although DFB partially competed for [(3)H]3-methoxy-5-(2-pyridinylethynyl)pyridine binding, CPPHA had no effect on the binding of this 2-methyl-6-(phenylethynyl)-pyridine analog to mGluR5. Although the binding sites for the two classes of allosteric modulators seem to be different, these different allosteric sites can modulate functionally and mechanistically similar allosteric effects. In electrophysiological studies of brain slice preparations, it had been previously shown that activation of mGluR5 receptors by agonists increased N-methyl-D-aspartate (NMDA) receptor currents in the CA1 region of hippocampal slices. We found that CPPHA (10 microM) potentiated NMDA receptor currents in hippocampal slices induced by threshold levels of DHPG, whereas having no effect on these currents by itself. Similarly, 10 microM CPPHA also potentiated mGluR5-mediated DHPG-induced depolarization of rat subthalamic nucleus neurons. These results demonstrate that allosteric potentiation of mGluR5 increases the effect of threshold agonist concentrations in native systems.

Allosteric Regulation↗

PubMed2004

Identification and characterization of a novel human myeloid inhibitory C-type lectin-like receptor (MICL) that is predominantly expressed on granulocytes and monocytes.

Inhibitory and activatory C-type lectin-like receptors play an important role in immunity through the regulation of leukocytes. Here, we report the identification and characterization of a novel myeloid inhibitory C-type lectin-like receptor (MICL) whose expression is primarily restricted to granulocytes and monocytes. This receptor, which contains a single C-type lectin-like domain and a cytoplasmic immunoreceptor tyrosine-based inhibitory motif, is related to LOX-1 (lectin-like receptor for oxidized low density lipoprotein-1) and the beta-glucan receptor (Dectin-1) and is variably spliced and highly N-glycosylated. We demonstrate that it preferentially associates with the signaling phosphatases SHP-1 and SHP-2, but not with SHIP. Novel chimeric analyses with a construct combining MICL and the beta-glucan receptor show that MICL can inhibit cellular activation through its cytoplasmic immunoreceptor tyrosine-based inhibitory motif. These data suggest that MICL is a negative regulator of granulocyte and monocyte function.

Alternative Splicing↗

PubMed2004

The role of SIGNR1 and the beta-glucan receptor (dectin-1) in the nonopsonic recognition of yeast by specific macrophages.

We recently demonstrated that the beta-glucan receptor Dectin-1 (betaGR) was the major nonopsonic beta-glucan receptor on macrophages (Mphi) for the yeast-derived particle zymosan. However, on resident peritoneal Mphi, we identified an additional mannan-inhibitable receptor for zymosan that was distinct from the Mphi mannose receptor (MR). In this study, we have studied the mannose-binding potential of murine Mphi and identified the dendritic cell-specific ICAM-3-grabbing nonintegrin homolog, SIGN-related 1 (SIGNR1), as a major MR on murine resident peritoneal Mphi. Both SIGNR1 and betaGR cooperated in the nonopsonic recognition of zymosan by these Mphi. When SIGNR1 was introduced into NIH3T3 fibroblasts or RAW 264.7 Mphi, it conferred marked zymosan-binding potential on these cells. However, in the nonprofessional phagocytes (NIH3T3), SIGNR1 was found to be poorly phagocytic, suggesting that other receptors such as betaGR may play a more dominant role in particle internalization on professional phagocytes. Binding of zymosan to RAW 264.7 Mphi expressing SIGNR1 resulted in TNF-alpha production. Treatment of RAW 264.7 Mphi expressing SIGNR1, which express low levels of betaGR, with beta-glucans had little effect on binding or TNF-alpha production, indicating that there was no absolute requirement for betaGR in this process. These studies have identified SIGNR1 as a major MR for fungal and other pathogens present on specific subsets of Mphi.

Animals↗

PubMed2004

Scavenger receptor class B type I-mediated cholesteryl ester-selective uptake and efflux of unesterified cholesterol. Influence of high density lipoprotein size and structure.

Scavenger receptor (SR)-BI catalyzes the selective uptake of cholesteryl ester (CE) from high density lipoprotein (HDL) by a two-step process that involves the following: 1) binding of HDL to the receptor and 2) diffusion of the CE molecules into the cell plasma membrane. We examined the effects of the size of discoidal HDL particles containing wild-type (WT) apoA-I on selective uptake of CE and efflux of cellular free (unesterified) cholesterol (FC) from COS-7 cells expressing SR-BI to determine the following: 1) the influence of apoA-I conformation on the lipid transfer process, and 2) the contribution of receptor binding-dependent processes to the overall efflux of cellular FC. Large (10 nm diameter) reconstituted HDL bound to SR-BI better (B(max) approximately 420 versus 220 ng of apoA-I/mg cell protein), delivered more CE, and promoted more FC efflux than small ( approximately 8 nm) particles. When normalized to the number of reconstituted HDL particles bound to the receptor, the efficiencies of either CE uptake or FC efflux with these particles were the same indicating that altering the conformation of WT apoA-I modulates binding to the receptor (step 1) but does not change the efficiency of the subsequent lipid transfer (step 2); this implies that binding induces an optimal alignment of the WT apoA-I.SR-BI complex so that the efficiency of lipid transfer is always the same. FC efflux to HDL is affected both by binding of HDL to SR-BI and by the ability of the receptor to perturb the packing of FC molecules in the cell plasma membrane.

Animals↗

PubMed2004

Modulation of the phosphoinositide 3-kinase pathway alters innate resistance to polymicrobial sepsis.

We examined the effect of modulating phosphoinositide 3-kinase (PI3K) activity in a murine model of cecal ligation and puncture-induced polymicrobial sepsis. Inhibition of PI3K activity with wortmannin increased serum cytokine levels and decreased survival time in septic mice. We have reported that an immunomodulator, glucan phosphate, induces protection in murine polymicrobial sepsis. We observed that glucan stimulated tissue PI3K activity, which positively correlated with increased survival in septic mice. We investigated the effect of PI3K inhibition on survival in septic mice treated with glucan. Treatment of mice with the PI3K inhibitors, wortmannin and LY294002, completely eliminated the protective effect of glucan, indicating that protection against septic mortality was mediated through PI3K. Inhibition of PI3K resulted in increased serum levels of IL1-beta, IL-2, IL-6, IL-10, IL-12, and TNF-alpha in septic mice. Apoptosis is thought to play a central role in the response to septic injury. We observed that inhibition of PI3K activity in septic mice resulted in increased splenocyte apoptosis and a change in the anatomic distribution of splenocyte apoptosis. We conclude that PI3K is a compensatory mechanism that suppresses proinflammatory and apoptotic processes in response to sepsis and/or inflammatory injury. Thus, PI3K may play a pivotal role in the maintenance of homeostasis and the integrity of the immune response during sepsis. We also observed that glucan phosphate decreased septic morbidity and mortality through a PI3K-dependent mechanism. This suggests that stimulation of the PI3K pathway may be an effective approach for preventing or treating sepsis and/or septic shock.

Androstadienes↗

PubMed2004

At low serum glucan concentrations there is an inverse correlation between serum glucan and serum cytokine levels in ICU patients with infections.

Glucans are fungal cell wall glucose polymers that are released into the blood of infected patients. The role of glucans in infection is unknown. We examined serum glucan and cytokine levels in intensive care unit (ICU) patients with infections. There was an inverse correlation (p<0.001) between serum glucan levels and interleukin (IL)-2), IL-4, tumor necrosis factoralpha (TNFalpha) and granulocyte macrophage-colony stimulating factor (GM-CSF) levels in infected ICU patients. The correlation between serum cytokines and serum glucan was only observed at glucan concentrations <40 pg/ml. No change was observed at serum glucan levels of >40 pg/ml. There was no correlation between serum glucan levels and systemic levels of IL-1beta, IL-5, IL-6, IL-8, IL-10 or IFNgamma. Interestingly, blood borne glucans did not suppress systemic cytokine levels in infected ICU patients, instead they were maintained at control levels. We conclude that circulating glucans may prevent cytokine upregulation in response to infection. This may represent an adaptive response to septic injury.

Adrenocorticotropic Hormone↗

PubMed2004

Pharmacokinetics of fungal (1-3)-beta-D-glucans following intravenous administration in rats.

Glucans are microbial cell wall carbohydrates that are shed into the circulation of patients with infections. Glucans are immunomodulatory and have structures that are influenced by bacterial or fungal species and growth conditions. We developed a method to covalently label carbohydrates with a fluorophore on the reducing terminus, and used the method to study the pharmacokinetics following intravenous administration of three highly purified and characterized glucans (glucan phosphate, laminarin and scleroglucan) that varied according to molecular size, branching frequency and solution conformation. Elimination half-life was longer (3.8+/-0.8 vs. 2.6+/-0.2 and 3.1+/-0.6 h) and volume of distribution lower (350+/-88 ml/kg vs. 540+/-146 and 612+/-154 ml/kg) for glucan phosphate than for laminarin and scleroglucan. Clearance was lower for glucan phosphate (42+/-6 ml/kg h) than for laminarin (103+/-17 ml/kg h) and scleroglucan (117+/-19 ml/kg h). Since plasma levels at steady state are inversely related to clearance, these differences suggest that pharmacokinetics could favor higher blood levels of glucans with certain physicochemical properties.

Animals↗