Immunomodulatory Protein Hydrolysates and Their Application.

Immunomodulatory protein hydrolysate consumption may delay or prevent western immune-related diseases. In order to purposively develop protein hydrolysates with an optimal and reproducible immunomodulatory effect, knowledge is needed on which components in protein hydrolysates are responsible for the immune effects. Important advances have been made on this aspect. Also, knowledge on mechanisms underlying the immune modulating effects is indispensable. In this review, we discuss the most promising application possibilities for immunomodulatory protein hydrolysates. In order to do so, an overview is provided on reported in vivo immune effects of protein hydrolysates in both local intestinal and systemic organs, and the current insights in the underlying mechanisms of these effects. Furthermore, we discuss current knowledge and physicochemical approaches to identify the immune active protein sequence(s). We conclude that multiple hydrolysate compositions show specific immune effects. This knowledge can improve the efficacy of existing hydrolysate-containing products such as sports nutrition, clinical nutrition, and infant formula. We also provide arguments for why immunomodulatory protein hydrolysates could be applied to manage the immune response in the increasing number of individuals with a higher risk of immune dysfunction due to, for example, increasing age or stress.

Expression and Sequence Variants of Inflammatory Genes; Effects on Plasma Inflammation Biomarkers Following a 6-Week Supplementation with Fish Oil.

(1) BACKGROUND: A growing body of literature suggest that polymorphisms (SNPs) from inflammation-related genes could possibly play a role in cytokine production and then interact with dietary n-3 fatty acids (FAs) to modulate inflammation. The aim of the present study was to test whether gene expression of selected inflammatory genes was altered following an n-3 PUFA supplementation and to test for gene-diet interactions modulating plasma inflammatory biomarker levels. (2) METHODS: 191 subjects completed a 6-week n-3 FA supplementation with 5 g/day of fish oil. Gene expression of TNF-α and IL6 was assessed in peripheral blood mononuclear cells (PBMCs) using the TaqMan technology. Genotyping of 20 SNPs from the TNF-LTA gene cluster, IL1ß, IL6 and CRP genes was performed. (3) RESULTS: There was no significant reduction of plasma IL-6, TNF-α and C-reactive protein (CRP) levels after the 6-week fish oil supplementation. TNF-α and IL6 were slightly overexpressed in PBMCs after the supplementation (fold changes of 1.05 ± 0.38 and 1.18 ± 0.49, respectively (n = 191)), but relative quantification (RQ) within the -0.5 to 2.0 fold are considered as nonbiologically significant. In a MIXED model for repeated measures adjusted for the effects of age, sex and BMI, gene by supplementation interaction effects were observed for rs1143627, rs16944, rs1800797, and rs2069840 on IL6 levels, for rs2229094 on TNF-α levels and for rs1800629 on CRP levels (p < 0.05 for all). (4) CONCLUSIONS: This study shows that a 6-week n-3 FA supplementation with 5 g/day of fish oil did not alter gene expression levels of TNF-α and IL6 in PBMCs and did not have an impact on inflammatory biomarker levels. However, gene-diet interactions were observed between SNPs within inflammation-related genes modulating plasma inflammatory biomarker levels.

Effect of tangweian jianji on upper gastrointestinal remodeling in streptozotocin-induced diabetic rats.

AIM: To investigate the effect of Tangweian Jianji (TWAJJ) on the biomechanical and morphometrical remodeling of the upper gastrointestinal tract in diabetic rats. METHODS: Diabetes was induced in 27 rats by injecting streptozotocin (40 mg/kg body weight), the animals were then divided into three groups (n = 9 in each group), i.e., diabetic control (DM); high dose (10 g/kg, T1) and low dose (5 g/kg, T2). Another 10 rats acted as normal controls (Control). TWAJJ was administered by gavage once daily. Blood glucose and serum insulin levels were measured. Circumferential length, wall thickness and opening angle were measured from esophageal, duodenal, jejunal and ileal ring segments. The residual strain was calculated from the morphometric data. Step-wise distension was carried out on esophageal and jejunal segments. The obtained data on the length, diameter and pressure changes were then used to calculate the circumferential and longitudinal stresses and strains. Real-time reverse transcription polymerase chain reaction was used to detect the receptor of advanced glycation end-products (RAGE) mRNA level in jejunal tissues. RESULTS: At the end of the experiment, the blood glucose level was significantly higher and the serum insulin level was significantly lower in DM, T1 and T2 groups than in the control group (Glucose: 30.23 ± 0.41 mmol/L, 27.48 ± 0.27 mmol/L and 27.84 ± 0.29 mmol/L vs 5.05 ± 0.04 mmol/L, P = 1.65 × 10(-16), P = 5.89 × 10(-19) and P = 1.63 × 10(-18), respectively; Insulin: 1.47 ± 0.32 µg/L, 2.66 ± 0.44 µg/L, 2.03 ± 0.29 µg/L and 4.17 ± 0.54 µg/L, P = 0.0001, P = 0.029 and P = 0.025, respectively). However, these levels did not differ among the DM, T1 and T2 groups. The wet weight per unit length, wall thickness and opening angle of esophageal and intestinal segments in the DM group were significantly higher than those in the control group (from P = 0.009 to P = 0.004). These parameters in the T1 group were significantly lower than those in the DM group (wet weight, duodenum: 0.147 ± 0.003 g/cm vs 0.158 ± 0.001 g/cm, P = 0.047; jejunum, 0.127 ± 0.003 g/cm vs 0.151 ± 0.002 g/cm, P = 0.017; ileum, 0.127 ± 0.004 g/cm vs 0.139 ± 0.003 g/cm, P = 0.046; wall thickness, esophagus: 0.84 ± 0.03 mm vs 0.94 ± 0.02 mm, P = 0.014; duodenum: 1.27 ± 0.06 mm vs 1.39 ± 0.05 mm, P = 0.031; jejunum: 1.19 ± 0.07 mm vs 1.34 ± 0.04 mm, P = 0.047; ileum: 1.09 ± 0.04 mm vs 1.15 ± 0.03 mm, P = 0.049; opening angle, esophagus: 112.2 ± 13.2˚ vs 134.7 ± 14.7˚, P = 0.027; duodenum: 105.9 ± 12.3˚ vs 123.1 ± 13.1˚, P = 0.046; jejunum: 90.1 ± 15.4˚ vs 115.5 ± 13.3˚, P = 0.044; ileum: 112.9 ± 13.4˚ vs 136.1 ± 17.1˚, P = 0.035). In the esophageal and jejunal segments, the inner residual stain was significantly smaller and the outer residual strain was larger in the DM group than in the control group (P = 0.022 and P = 0.035). T1 treatment significantly restored this biomechanical alteration (P = 0.011 and P = 0.019), but T2 treatment did not. Furthermore, the circumferential and longitudinal stiffness of the esophageal and jejunal wall increased in the DM group compared with those in the control group. T1, but not T2 treatment, significantly decreased the circumferential wall stiffness in the jejunal segment (P = 0.012) and longitudinal wall stiffness in the esophageal segment (P = 0.023). The mRNA level of RAGE was significantly decreased in the T1 group compared to that in the DM group (P = 0.0069). CONCLUSION: TWAJJ (high dose) treatment partly restored the morphometric and biomechanical remodeling of the upper gastrointestinal tract in diabetic rats.

Protective effects of grape seed proanthocyanidin extracts on cerebral cortex of streptozotocin-induced diabetic rats through modulating AGEs/RAGE/NF-kappaB pathway.

Diabetic encephalopathy is a severe complication in patients with long-term hyperglycemia. Oxidative stress is thought to be closely implicated in this disorder, so in this study, we examined whether grape seed proanthocyanidin extract (GSPE), a naturally occurring antioxidant derived from grape seeds, could reduce the injuries in the cerebral cortex of diabetic rats by modulating advanced glycation end products (AGEs)/the receptor for AGEs (RAGE)/nuclear factor-kappa B p65 (NF-kappaB p65) pathway, which is crucial in oxidative stress. Body weight and serum AGEs were tested; cerebral cortexes were isolated for morphological observations and the pyramidal cell layers were immunohistochemically stained for the detection of RAGE, NF-kappaB p65, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) as well. For RAGE and NF-kappaB p65, quantitative reverse transcriptase coupled to polymerase chain reaction (RT-PCR) was employed for determination of mRNA levels, and western blot was used to detect protein expression. Our results showed that long term hyperglycemia in diabetic rats caused the degeneration of neurons and the up-regulation of serum AGEs, and also the up-regulation of RAGE, NF-kappaB p65, VCAM-1 and ICAM-1 in the brain. We found that GSPE treatment improved the pathological changes of diabetic rats by modulating the AGEs/RAGE/NF-kappaB p65 pathway. This study enables us to further understand the key role that the AGEs/RAGE/NF-kappaB pathway plays in the pathogenesis of diabetic encephalopathy, and confirms that GSPE might be a therapeutical means to the prevention and treatment of this disorder.

Impact of cranberry on Escherichia coli cellular surface characteristics.

The anti-adhesive effects of cranberry have been attributed to both interactions of its components with the surface of bacterial cells and to inhibition of p-fimbriae expression. Previous reports also suggested that the presence of cranberry juice changed the Gram stain characteristics of Escherichia coli. Here, we show that the morphology of E. coli is changed when grown in the presence of juice or extract from Vaccinium macrocarpon (cranberry). Gene expression analysis indicates the down regulation of flagellar basal body rod and motor proteins. Consistent with this finding and previous reports, the SEM images indicate a decrease in the visible p-fimbriae. The iodine used in Gram-staining protocols was found to interact differently with the bacterial membrane when cells were cultured in spiked media. Slight alterations in the Gram stain protocol demonstrated that culturing in the presence of cranberry juice does not change the Gram stain characteristics contradicting other reports.

Effects of traditional chinese medicine on endotoxin and its receptors in rats with non-alcoholic steatohepatitis.

AIMS: The aim of this research is to study the effects of traditional Chinese medicine on endotoxin and its receptors in rats with nonalcoholic steatohepatitis (NASH). METHODS: Fifty-six SD rats were divided into seven groups. All the animals were fed high fatty diet for 12 weeks. Rats with non-alcoholic steatohepatitis (NASH) were treated with traditional Chinese medicine according to low-dose, middle-dose, high-dose and Lipitor from fifth week. All rats were killed at the end of 12th week. The liver pathology changes were observed under light microscope. The levels of serum lipoid, alanine aminotransferase (ALT), endotoxin (ET), tumor necrosis factor-alpha (TNF-alpha) and interleukine-1beta (IL-1beta) were determined. The expressions of CD14 and nuclear transcriptional factor kappaB (NF-kappaB) were observed by immunohistochemistry. The expressions of lipopolysaccharide binding protein (LBP), toll-like receptor-4 (TLR-4), myeloid differentiation-2 (MD-2) and induced nitric oxide synthase (iNOS) mRNA were detected by the reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The levels of serum endotoxin in the middle dose group (0.0225 +/- 0.0112 EU/l) were lower than those in high fatty diet model group (0.2249 +/- 0.0982 EU/l) at 12th week, the difference was significant (P < 0.01). In the middle dose group, mean values of serum TNF-alpha and IL-1beta levels decreased dramatically (1.604 +/- 0.302 ng/ml and 0.052 +/- 0.024 ng/ml) compared with those in the high fatty diet model group (4.029 +/- 1.180 ng/ml and 14.944 +/- 0.491 ng/ml; P < 0.01 and P < 0.01). The expressions of CD14 and NF-kappaB in the middle dose group decreased compared with those in the high fatty diet model group. The expressions of LBP mRNA (0.284 +/- 0.105) and TLR-4 mRNA (0.290 +/- 0.123) in the middle dose group down regulated compared with those in the high fatty diet model group (1.060 +/- 0.158 and 1.261 +/- 0.368; P < 0.01 and P < 0.01). In the middle dose group MD-2 and iNOS gene expressions were 0.132 +/- 0.058 and 0.164 +/- 0.061, respectively, which were significantly lower compared with the high fatty diet model group (0.795 +/- 0.294 and 1.029 +/- 0.388; P < 0.01 and P < 0.01). CONCLUSIONS: The mechanism of non-alcoholic steatohepatitis (NASH) maybe related to increasing the levels of serum endotoxin, upregulating endotoxin receptors of hepatic tissue and enhancing liver inflammatory injury. Traditional Chinese medicine is a good treatment for non-alcoholic steatohepatitis (NASH). It can produce a marked effect via relieving LPS-induced liver injury.

Inhibition of NADPH oxidase prevents advanced glycation end product-mediated damage in diabetic nephropathy through a protein kinase C-alpha-dependent pathway.

OBJECTIVE: Excessive production of reactive oxygen species (ROS) via NADPH oxidase has been implicated in the pathogenesis of diabetic nephropathy. Since NADPH oxidase activation is closely linked to other putative pathways, its interaction with changes in protein kinase C (PKC) and increased advanced glycation was examined. RESEARCH DESIGN AND METHODS: Streptozotocin-induced diabetic or nondiabetic Sprague Dawley rats were followed for 32 weeks, with groups randomized to no treatment or the NADPH oxidase assembly inhibitor apocynin (15 mg . kg(-1) . day(-1); weeks 16-32). Complementary in vitro studies were performed in which primary rat mesangial cells, in the presence and absence of advanced glycation end products (AGEs)-BSA, were treated with either apocynin or the PKC-alpha inhibitor Ro-32-0432. RESULTS; Apocynin attenuated diabetes-associated increases in albuminuria and glomerulosclerosis. Circulating, renal cytosolic, and skin collagen-associated AGE levels in diabetic rats were not reduced by apocynin. Diabetes-induced translocation of PKC, specifically PKC-alpha to renal membranes, was associated with increased NADPH-dependent superoxide production and elevated renal, serum, and urinary vascular endothelial growth factor (VEGF) concentrations. In both diabetic rodents and in AGE-treated mesangial cells, blockade of NADPH oxidase or PKC-alpha attenuated cytosolic superoxide and PKC activation and increased VEGF. Finally, renal extracellular matrix accumulation of fibronectin and collagen IV was decreased by apocynin. CONCLUSIONS: In the context of these and previous findings by our group, we conclude that activation of NADPH oxidase via phosphorylation of PKC-alpha is downstream of the AGE-receptor for AGE interaction in diabetic renal disease and may provide a novel therapeutic target for diabetic nephropathy.

Effect of the statin atorvastatin on intracellular signalling by the prostacyclin receptor in vitro and in vivo.

Prostacyclin plays a central role within the vasculature. We have previously established that the prostacyclin receptor (IP) undergoes isoprenylation, a lipid modification obligate for its function. The aim of the current study was to investigate the effect of the hydroxy methyl glutaryl co-enzyme A reductase inhibitor atorvastatin on signalling and function of the IP expressed in mammalian whole cells and in platelets isolated from patients undergoing therapeutic intervention with atorvastatin. Initially, the effect of atorvastatin on signalling by the human (h) and mouse (m) IP overexpressed in human embryonic kidney 293 cells and the hIP endogenously expressed in human erythroleukaemic 92.1.7 cells was investigated. Atorvastatin significantly reduced IP-mediated cAMP generation (IC(50) 6.6-11.1 microm) and [Ca(2+)](i) mobilization (IC(50) 7.2-16.4 microm) in a concentration-dependent manner, but had no effect on signalling by the nonisoprenylated beta(2) adrenergic receptor or the alpha or beta isoforms of the human thromboxane A(2) receptor (TP). Moreover, atorvastatin significantly reduced IP-mediated crossdesensitization of signalling by TP alpha (IC(50) 10.4 microm), but not by TP beta. In contrast to the whole-cell data, atorvastatin therapy did not interfere with IP-mediated cAMP generation or IP-induced inhibition of TP-mediated aggregation of platelets isolated from human volunteers undergoing therapeutic intervention with atorvastatin (10-80 mg per daily dose). In conclusion, while data generated in whole cells indicated that atorvastatin significantly impairs signalling by both the hIP and mP, the in vivo clinical data indicated that, at the administered therapeutic dose, atorvastatin does not significantly compromise IP signalling and function in humans.

Suppressive effects of tea polyphenol and conformational changes with receptor for advanced glycation end products (RAGE) expression in human hepatoma cells.

BACKGROUND/AIMS: It is reported that polyphenol is associated with low risk of hepatoma and that RAGE (receptor for advanced glycation end products) is important for cancer invasion. METHODOLOGY: Effects of teapolyphenol, EGCG (epigallocatechin-3-gallate) were studied. Proliferation of on human hepatoma cells, HLF, was measured with the use of WST-1 colorimetric assay. Cell invasion was analyzed by the Matrigel invasion assay. Morphology and immunohistological staining of expression of RAGE were also performed. RESULTS: Proliferation was inhibited with the addition of EGCG in a dose-dependent manner. EGCG (200 mumol/L) produced a profound growth suppression of HLF cells (24.5%). Cell invasion was also inhibited with preincubation of 100 mumol/L of EGCG (10.2%). In addition to the antitumor effects, neurite-like conformational changes of HLF cells were observed. Addition of EGCG (100 mumol/L) showed the expression of RAGE on cell surface in accordance to the morphological changes. CONCLUSIONS: The pathway associated to cell movement might be activated with RAGE expression. Although EGCG inhibits the growth and invasion, the cells which expressed RAGE seem to survive. Thus, the enrollment of RAGE should be analyzed to clarify the mechanisms of cancer resistance.

An olive oil-rich diet reduces scavenger receptor mRNA in murine macrophages.

During atherogenesis, a pathological accumulation of lipids occurs within aortic intimal macrophages through uptake of oxidised LDL via scavenger receptors. Here we investigated whether some of the anti-atherosclerotic effects ascribed to an olive oil rich-diet are mediated through effects on macrophage scavenger receptors (MSR). Male C57 Bl6 mice aged 6 weeks were fed for 12 weeks on a low-fat diet (containing 25 g corn oil/kg) or on high-fat diets containing 200 g coconut oil, olive oil or safflower oil/kg. Thioglycollate-elicited peritoneal macrophages were analysed for fatty acid composition by GC and the levels of mRNA coding for three MSR (MSRA type I, MSRA type II and CD36) were measured by reverse-transcription polymerase chain reaction. Feeding mice diets enriched with different fats resulted in significant differences in the fatty acid profile of macrophages, which reflected the fatty acid compositions of the diets. These differences were accompanied by a lower level of mRNA for MSRA type I, MSRA type II and CD36 in macrophages from mice fed an olive-oil-enriched diet compared with the mice fed on the low-fat diet. These data suggest that part of the protective effect of olive oil against atherosclerosis might be via reducing macrophage uptake of oxidised LDL. Whether this effect is due to the downregulation of gene transcription directly by unsaturated fatty acids or is the result of the effect of monounsaturated fatty acids or other components of olive oil on LDL composition and oxidation remains to be ascertained.

[Antioxidant and anti-AGE therapeutics: evaluation and perspectives]. / Thérapeutiques anti-oxydantes et anti-AGE: bilans et perspectives.

Diabetic patients exhibit an oxidative stress status, that is an imbalance between reactive oxygen species and antioxidant defences, in favour of the first ones. This oxidative stress, together with formation of advanced glycation endproducts (AGEs), is involved in diabetic complications. It could thus be of great interest to propose antioxidant and/or anti-AGE therapeutics as complementary treatment in these patients. Antioxidants can be classical molecules such as vitamin E, lipoic acid or N-acetylcysteine. Thus, vitamin E supplementation can improve insulin efficiency and glycemic equilibrium, as shown by the decrease of glycaemia, glycated haemoglobin and fructosamine values. In addition, this kind of supplementation lowers plasma lipid peroxidation and oxidizability of low density lipoproteins, which is involved in the atherogenesis process. Moreover, it allows to fight against complications such as retinopathy. A second category is represented by molecules able to fight against the effects of glycation end-products (AGEs). They can act: either by preventing cellular action of AGEs; this is obtained with soluble receptors of advanced glycation endproducts (sRAGE); or by inhibiting AGE formation (scavenging of reactive carbonyl intermediates). Nucleophilic compounds such as pyridoxamine, tenilsetam, 2,3-diaminophenazone, OPB-9195 or aminoguanidine can act in this way. Aminoguanidine is able to limit the development of the main diabetes-associated complications in animals. A double-blind clinical assay has been conducted in type 2 diabetic patients in the United States and the Canada, in order to determine if aminoguanidine is able to slow down the progression of diabetes-induced nephropathy. We will discuss about another guanidic molecule, i.e. metformin, which is also able to scavenge AGEs, in the last part of this review. A third category of molecules is constituted by oral antidiabetic molecules exhibiting antioxidant properties. They are thiazolidinediones (troglitazone) and sulfonylureas (gliclazide). Troglitazone and gliclazide can thus decrease LDL oxidizability and monocyte adhesion to endothelial cells, which is an early step in the atherogenesis process and which is stimulated by oxidised LDLs. Finally, a prospective way is devoted to oral antidiabetic drugs exhibiting both antioxidant and anti-AGE properties. A very used antidiabetic drug of interest is metformin (dimethylbiguanide), since it can prevent diabetes complications not only by lowering glycaemia, but also by inhibiting AGE formation and by stimulating antioxidant defences. The latter therapeutic approach constitutes a future way in the diabetes area, in order both to obtain a better glycemic control and a least development of diabetic complications.

Dietary fish oil reduces intercellular adhesion molecule 1 and scavenger receptor expression on murine macrophages.

During atherogenesis, a pathological accumulation of lipids occurs within aortic intimal macrophages through uptake of oxidised low-density lipoprotein (LDL) via scavenger receptors. Here we investigate whether some of the anti-atherosclerotic effects ascribed to dietary fish oil are mediated through effects on macrophage intercellular adhesion molecule 1 (ICAM-1) and scavenger receptor expression. Mice were fed on a low fat diet (containing 25 g/kg corn oil) or on high fat diets containing 200 g/kg coconut oil, safflower oil or fish oil. Thioglycollate-elicited peritoneal macrophages were analysed for fatty acid composition by gas chromatography. Macrophage scavenger receptor A (MSR-A) type I+type II and ICAM-1 expression were measured by flow cytometry and the levels of mRNA coding for MSR-A type I, MSR-A type II and ICAM-1 were measured by reverse-transcription polymerase chain reaction. Feeding mice diets enriched with different fats resulted in significant changes in the fatty acid profile of macrophages, which reflected the fatty acid compositions of the diets. Macrophages from the fish oil fed mice had the lowest expression of ICAM-1 and MSR-A at the level of both mRNA and cell surface expression. The reduced expression of ICAM-1 and MSR-A on macrophages from mice fed on a fish oil-rich diet supports our hypothesis that part of the protective effect of fish oil against atherosclerosis might be due to an altered macrophage phenotype and function ameliorating macrophage-induced plaque formation.

Solid-phase synthesis of chemotactic peptides using alpha-azido acids.

Four chemotactic peptides, For-Met-Xxx-Phe-OMe, with an alpha,alpha-disubstituted amino acid at position 2 have been synthesized by the azido acid method [Meldal M, Juliano MA, Jansson AM. 1997. Azido acids in a novel method of solid-phase peptide synthesis. Tetrahedron Lett. 38: 2531-2534] on solid-phase, and were tested for biological activity. Dipropylglycine in the central position (Xxx) was found to be as active as the natural chemotactic peptide for chemotactic activity toward human neutrophils. Higher yields were obtained than previously reported solution-phase syntheses of chemotactic peptides, and EEDQ was used successfully for the difficult solid-phase formylation of amino groups.

Effects of growth hormone and insulin-like growth factor I on opsonin receptor expression on local and systemic phagocytes in a lethal peritonitis model.

OBJECTIVE: To investigate the effects of pretreatment with growth hormone (GH) and insulin-like growth factor I (IGF-I) on phagocyte exudation and bacterial clearance, focusing on CD11b and CD32/CD16 expression on local and systemic phagocytes, in a lethal peritonitis model. DESIGN: Prospective randomized experimental study. SETTING: Research laboratory in a university hospital. SUBJECTS: Balb/c mice (n = 21). INTERVENTIONS: Mice were challenged intraperitoneally with 1 x 10(8) Escherichia coli, after 6 days of pretreatment with saline (control), GH (4.8 mg/kg/day), or IGF-I (24 mg/kg/day). Samples were harvested at 4 hrs after the challenge. MEASUREMENTS AND MAIN RESULTS: Viable bacterial counts in peritoneal lavage fluid (PLF) and blood were determined. Peritoneal exudative cells and peripheral blood leukocytes were counted and analyzed for receptor expressions by flow cytometry. GH reduced viable bacterial counts in PLF, as compared with the saline control. GH (three-fold) and IGF-I (two-fold) increased the number of peritoneal exudative neutrophils (PENs), as compared with the saline control. The number of PENs showed an inverse correlation with PLF viable bacterial counts. By contrast, there were no differences in peripheral blood neutrophil (PN) counts among the three groups, nor was there a correlation between PN and PEN counts. CD11b expression was greater on PENs than on PNs in all three groups. CD11b expression on PNs did not differ among the three groups. However, GH increased CD11b expression on PENs, as compared with saline and IGF-I, and this expression showed a positive correlation with PEN numbers and an inverse correlation with PLF viable bacterial counts. CD11b expression on peritoneal macrophages and peripheral blood monocytes did not differ among the three groups. There were no differences in phagocyte CD32/CD16 expression among the three groups. CONCLUSIONS: GH pretreatment enhanced CD11b expression on PENs, but not PNs, possibly in association with enhanced neutrophil recruitment, phagocytosis, and bacterial elimination by PENs, without activation of PNs. GH prophylaxis may be useful for reducing the frequency rate and severity of septic complications, via modulation of CD11b expression on local and systemic neutrophils.

Effect of precultivation conditions on colicin susceptibility in Escherichia coli.

The effect of 20 colicins and cloacin was studied after various precultivations. Nutrient agar supplemented with subinhibitory concentration of EDTA used for precultivation or elevating the growth-temperature of the inoculum from 37 degrees C to 42 degrees C increased the susceptibility of wild-type (smooth) Escherichia coli strains to the inhibitory action of some colicins. There were great differences among the colicins in respect to these effects. In case of rough mutants, their sensitivities did not change or eventually decrease after EDTA or heat pretreatment. The LPS pattern in SDS-PAGE of smooth cells grown in EDTA-containing nutrient medium changed in some degree towards the rough character. In case of precultivation at 42 degrees C this change was less considerable. It is supposed that both factors applied during precultivation have influence on colicin sensitivity by means of the change of receptor activity caused by LPS modification.

[The immunocorrective potentials of an antistaphylococcal immunosorbent in extracting staphylotoxin]. / Immunokorrigiruiushchie vozmozhnosti protivostafilokokkovogo immunosorbenta pri izvlechenii stafilotoksina.

The immunocorrective effect of antistaphylococcal adsorbent prepared on the basis of silochrome, a silica matrix, has been revealed in experiments on peripheral blood lymphocytes obtained from 18 donors. The expression of receptors on human immunoregulating cells has been suppressed by the addition of staphylotoxin at a toxic concentration into the system. The experiments have shown that immunosorption is capable of extracting the toxin from the solution to a considerable degree, thus preventing the suppression of the receptor apparatus of immunoregulating lymphocytes. Staphylotoxin has been found capable of forming complexes with plasma ingredients, which increases the adsorption capacity of the immunosorbent.

Polynucleotide binding to macrophage scavenger receptors depends on the formation of base-quartet-stabilized four-stranded helices.

Macrophage scavenger receptors exhibit unusually broad, but circumscribed, polyanionic ligand-binding specificity. For example, the polyribonucleotides poly(I) and poly(G) are ligands but poly(A) and poly(C) are not. To further investigate the molecular basis of this polynucleotide-binding specificity, we tested the capacity of various oligodeoxyribonucleic acids to inhibit the scavenger receptor-mediated degradation of 125I-labeled acetylated low density lipoprotein by Chinese hamster ovary cells expressing the type I bovine scavenger receptor. A series of short oligodeoxyriboguanines (dGn, where 5 < or = n < or = 37) were effective inhibitors. The dG6, dG12, and dA5G37 members of this series were shown by circular dichroism and UV spectroscopy to be assembled into four-stranded helices stabilized by G-quartets. [32P]dA5G37 bound directly to scavenger receptors. Partial or complete denaturation of the quadruplex structures of these oligonucleotides by boiling destroyed their inhibitory activity. Receptor activity was also inhibited by d(T4G4)4, a telomere-like oligonucleotide which forms an intramolecular quadruplex. In addition, conversion of the four-stranded potassium salt of poly(I) to the single-stranded lithium salt dramatically reduced its inhibitory activity. Addition of KCl to the Li+ salt resulted in the reformation of poly(I)'s quadruplex structure and restoration of its inhibitory activity. A variety of single-stranded and double-stranded oligo- and polydeoxyribonucleotides (e.g. dA37, HaeIII restriction fragments of phi X174) exhibited very little or no inhibitory activity. Thus, a base-quartet-stabilized four-stranded helix appears to be a necessary structural determinant for polynucleotide binding to and inhibition of scavenger receptors. This conformational requirement accounts for the previously unexplained polyribonucleotide-binding specificity of scavenger receptors. The spatial distribution of the negatively charged phosphates in polynucleotide quadruplexes may form a charged surface which is complementary to the positively charged surface of the collagenous ligand-binding domain of the scavenger receptor.

The modulation of the expression of IL-6 and its receptor in human osteoblasts in vitro.

Interleukin 6 (IL-6) probably plays a central role in the acute phase response and in haemopoiesis and may be involved in the control of bone turnover. We have studied the release of IL-6 from human trabecular bone cells treated with a variety of stimuli using a specific bioassay. In serum free medium, unstimulated human osteoblast-like cells produced IL-6 in the range of 1000-2050 pg/ml/24 h. Recombinant human interleukin 1 (IL-1 alpha) (10(-13)-10(-11) M), tumor necrosis factor alpha (TNF alpha) (10(-9)-10(-7) M) and lipopolysaccharide (5-500 ng/ml) all stimulated release of IL-6 from human bone cells. Maximal levels of 17,000 pg/ml were observed using the highest concentration of IL-1. 1,25(OH)2D3 and PTH did not stimulate IL-6 release. Using a specific sheep antihuman IL-6 antibody, all IL-6 activity could be neutralized. In parallel studies, ROS 17/2.8 rat osteosarcoma cells released around 50 pg/ml of IL-6 under basal conditions which were increased to a maximum of 900 pg/ml by treatment with PTH (10(-9) M). The cytokines were less effective and 1,25(OH)2D3 again had no effect. Modulation of expression of IL-6 mRNA in human osteoblast cells was examined using a human complementary deoxyribonucleic acid probe. The mRNA was constitutively expressed, and IL-1 (10(-11) M) and TNF (10(-7) M) induced further mRNA expression within 2 h, which was sustained over 24 h. 1,25(OH)2D3 (10(-7) M), IL-6 (2000 pg/ml), and PTH (10(-9) M) exerted no effects at any time point. Dexamethasone (10(-6) M) suppressed both basal and IL-1- and TNF-induced IL-6 mRNA expression. IL-6 receptor mRNA was constitutively expressed but was not regulated by any of the above agents. It is clear that rodent and human osteoblasts differ in their production of IL-6 and its modulation. These data support the hypothesis that IL-6 is produced locally in human bone by osteoblasts under the direction of other cytokines. This could have implications in bone remodeling, haemopoiesis, and systemic responses to local injury.

Cloning of complementary DNA encoding a functional human interleukin-8 receptor.

Interleukin-8 (IL-8) is an inflammatory cytokine that activates neutrophil chemotaxis, degranulation, and the respiratory burst. Neutrophils express receptors for IL-8 that are coupled to guanine nucleotide-binding proteins (G proteins); binding of IL-8 to its receptor induces the mobilization of intracellular calcium stores. A cDNA clone from HL-60 neutrophils, designated p2, has now been isolated that encodes a human IL-8 receptor. When p2 is expressed in oocytes from Xenopus laevis, the oocytes bind 125I-labeled IL-8 specifically and respond to IL-8 by mobilizing calcium stores with an EC50 of 20 nM. This IL-8 receptor has 77% amino acid identity with a second human neutrophil receptor isotype that binds IL-8 with higher affinity. It also exhibits 69% amino acid identity with a protein reported to be an N-formyl peptide receptor from rabbit neutrophils, but less than 30% identity with all other known G protein-coupled receptors, including the human N-formyl peptide receptor.