Sialoglycan recognition is a common connection linking acidosis, zinc, and HMGB1 in sepsis.

Blood pH is tightly maintained between 7.35 and 7.45, and acidosis (pH <7.3) indicates poor prognosis in sepsis, wherein lactic acid from anoxic tissues overwhelms the buffering capacity of blood. Poor sepsis prognosis is also associated with low zinc levels and the release of High mobility group box 1 (HMGB1) from activated and/or necrotic cells. HMGB1 added to whole blood at physiological pH did not bind leukocyte receptors, but lowering pH with lactic acid to mimic sepsis conditions allowed binding, implying the presence of natural inhibitor(s) preventing binding at normal pH. Testing micromolar concentrations of divalent cations showed that zinc supported the robust binding of sialylated glycoproteins with HMGB1. Further characterizing HMGB1 as a sialic acid-binding lectin, we found that optimal binding takes place at normal blood pH and is markedly reduced when pH is adjusted with lactic acid to levels found in sepsis. Glycan array studies confirmed the binding of HMGB1 to sialylated glycan sequences typically found on plasma glycoproteins, with binding again being dependent on zinc and normal blood pH. Thus, HMGB1-mediated hyperactivation of innate immunity in sepsis requires acidosis, and micromolar zinc concentrations are protective. We suggest that the potent inflammatory effects of HMGB1 are kept in check via sequestration by plasma sialoglycoproteins at physiological pH and triggered when pH and zinc levels fall in late stages of sepsis. Current clinical trials independently studying zinc supplementation, HMGB1 inhibition, or pH normalization may be more successful if these approaches are combined and perhaps supplemented by infusions of heavily sialylated molecules.

Self-rated health and vital exhaustion, but not depression, is related to inflammation in women with coronary heart disease.

Poor subjective well-being has been associated with increased coronary heart disease (CHD) morbidity and mortality in population-based studies and with adverse outcomes in existing CHD. Little is known about the mechanisms responsible for this association, but immune activity appears to be a potential pathway. Despite the growing evidence linking immune activity to subjective feelings, very few studies have examined patients with CHD, and the results are conflicting. We examined consecutive women patients hospitalized for acute myocardial infarction, and/or underwent percutaneous transluminal coronary angioplasty or coronary artery bypass grafting. We assessed depression, vital exhaustion, and self-rated health by questionnaires. Circulating levels of high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), and interleukin-1 receptor antagonist (IL-1ra) concentrations were determined. After controlling for potential confounding factors there was a significant positive correlation between IL-6 levels and vital exhaustion and poor self-rated health. The association between hsCRP and vital exhaustion and self-rated health was borderline significant. In contrast, the correlations between psychological factors and IL-1ra levels were weak and non-significant, as were the correlations between inflammatory markers and depression. Similar relationships between the inflammatory markers and the measures of psychological well-being were obtained when the latter ones were categorized into tertiles. In conclusion, inflammatory activity, assessed by IL-6 and hsCRP levels, was associated with vital exhaustion and self-rated health in CHD women. These findings may provide further evidence for a possible psychoneuroimmune link between subjective well-being and CHD. Our observations also raise the possibility that a cytokine-induced sickness response in CHD may be better represented by constructs of vital exhaustion and self-rated health than of depression.

Increased expression of osteopontin is associated with long-term bee venom immunotherapy.

BACKGROUND: Venom allergen immunotherapy (VIT) is proven to be highly effective for insect allergy, but the mechanisms and the biomarkers associated with clinical efficacy remain elusive. OBJECTIVE: The aim of this study was to identify candidate biomarkers associated with successful VIT. METHODS: Gene chip array and clustering analyses of PBMCs from subjects with or without VIT were performed. RESULTS: From gene chip array and clustering analyses, an increased expression of osteopontin was found in patients who completed 5 to 6 years of VIT and discontinued therapy for 3 to 6 years (completed treatment group) compared with the untreated group. A significantly higher level of serum osteopontin was found in the completed treatment group compared with the untreated group (n = 16 in each group; P < .001). CONCLUSION: The upregulation of osteopontin after VIT suggests a role of osteopontin as a candidate biomarker for VIT.

The effect of vitamin K2 on bone metabolism in aged female rats.

Reactive oxygen species (ROS) may contribute to aging and osteoporosis resulting from marked decreases in plasma antioxidants in aged osteoporotic women. On the other hand, high-dose vitamin K2 (menaquinone-4: menatrenone, MK-4) supplementation has been reported to reduce ovariectomy-induced bone loss in rats and to decrease osteoporotic fracture in postmenopausal women. However, the mechanism by which vitamin K2 prevents osteoporosis is unclear. Recently, vitamin K2 has been suggested to preserve antioxidant activity as a novel function. Therefore, we investigated the effect of vitamin K2 on the osteoporosis of aged rats by evaluating the relationships between serum antioxidant levels and bone metabolism. Aged female rats exhibited significantly lower serum alkaline phosphatase activity and osteocalcin level, together with lower serum levels of antioxidants such as 17beta-estradiol, macrophage migration inhibitory factor (MIF) and glutathione peroxidase (GPx) activity, as compared with young female rats. On the other hand, vitamin K2 supplementation (500 mg/kg, food intake) for 98 days led to a significantly increased serum vitamin K2 level (3,045+/-915 ng/ml in the vitamin K2 supplemented group vs. 4.6+/-3.4 ng/ml in the control diet group; P<0.0001) with increased serum alkaline phosphatase activity and MIF level (P<0.05). Unexpectedly, however, it failed to increase the serum level of antioxidants such as GPx. Nor did it affect bone metabolism markers such as osteocalcin and osteopontin, which were significantly lower than in the young female rats (P<0.05). Finally, the histomorphometric properties of the proximal tibia in the femur were not altered by vitamin K2. These results suggest that high-dose vitamin K2 supplementation neither improves lowered antioxidant levels nor stimulates bone formation in aged rats.

Supplementation with vitamins C and E inhibits the release of interleukin-6 from contracting human skeletal muscle.

Contracting human skeletal muscle is a major contributor to the exercise-induced increase of plasma interleukin-6 (IL-6). Although antioxidants have been shown to attenuate the exercise-induced increase of plasma IL-6, it is unknown whether antioxidants inhibit transcription, translation or translocation of IL-6 within contracting human skeletal muscle. Using a single-blind placebo-controlled design with randomization, young healthy men received an oral supplementation with either a combination of ascorbic acid (500 mg day(-1)) and RRR-alpha-tocopherol (400 i.u. day(-1)) (Treatment, n= 7), or placebo (Control, n= 7). After 28 days of supplementation, the subjects performed 3 h of dynamic two-legged knee-extensor exercise at 50% of their individual maximal power output. Muscle biopsies from vastus lateralis were obtained at rest (0 h), immediately post exercise (3 h) and after 3 h of recovery (6 h). Leg blood flow was measured using Doppler ultrasonography. Plasma IL-6 concentration was measured in blood sampled from the femoral artery and vein. The net release of IL-6 was calculated using Fick's principle. Plasma vitamin C and E concentrations were elevated in Treatment compared to Control. Plasma 8-iso-prostaglandin F(2alpha), a marker of lipid peroxidation, increased in response to exercise in Control, but not in Treatment. In both Control and Treatment, skeletal muscle IL-6 mRNA and protein levels increased between 0 and 3 h. In contrast, the net release of IL-6 from the leg, which increased during exercise with a peak at 3.5 h in Control, was completely blunted during exercise in Treatment. The arterial plasma IL-6 concentration from 3 to 4 h, when the arterial IL-6 levels peaked in both groups, was approximately 50% lower in the Treatment group compared to Control (Treatment versus Control: 7.9 pg ml(-1), 95% confidence interval (CI) 6.0-10.7 pg ml(-1), versus 19.7 pg ml(-1), CI 13.8-29.4 pg ml(-1), at 3.5 h, P < 0.05 between groups). Moreover, plasma interleukin-1 receptor antagonist (IL-1ra), C-reactive protein and cortisol levels all increased after the exercise in Control, but not in Treatment. In conclusion, our results show that supplementation with vitamins C and E attenuated the systemic IL-6 response to exercise primarily via inhibition of the IL-6 protein release from the contracting skeletal muscle per se.

Mistletoe lectin I is a sialic acid-specific lectin with strict preference to gangliosides and glycoproteins with terminal Neu5Ac alpha 2-6Gal beta 1-4GlcNAc residues.

Mistletoe lectin I (ML-I) is a type II ribosome-inactivating protein, which inhibits the protein biosynthesis at the ribosomal level. ML-I is composed of a catalytically active A-chain with rRNA N-glycosidase activity and a B-chain with carbohydrate binding specificities. Using comparative solid-phase binding assays along with electrospray ionization tandem mass spectrometry, ML-I was shown to preferentially bind to terminally alpha2-6-sialylated neolacto series gangliosides from human granulocytes. IV(6)Neu5Ac-nLc4Cer, VI(6)Neu5Ac-nLc6Cer, and VIII(6)Neu5Ac-nLc8Cer were identified as ML-I receptors, whereas the isomeric alpha2-3-sialylated neolacto series gangliosides were not recognized. Only marginal binding of ML-I to terminal galactose residues of neutral glycosphingolipids with a Galbeta1-4Glc or Galbeta1-4GlcNAc sequence was determined, whereas a distal Galalpha1-4Gal, GalNAcbeta1-3Gal, or GalNAcbeta1-4Gal disaccharide did not bind at all. Among the glycoproteins investigated in Western blot and microwell adsorption assays, only those carrying Neu5Acalpha2-6Galbeta1-4GlcNAc residues, exclusively, predominantly, or even as less abundant constituents in an assembly with Neu5Acalpha2-3Galbeta1-4GlcNAc-terminated glycans, displayed high ML-I binding capacity. From our data we conclude that (i) ML-I has to be considered as a sialic acid- and not a galactose-specific lectin and (ii) neolacto series gangliosides and sialoglycoproteins with type II glycans, which share the Neu5Acalpha2-6Galbeta1-4GlcNAc terminus, are true ML-I receptors. This strict preference might help to explain the immunostimulatory potential of ML-I toward certain leukocyte subpopulations and its therapeutic success as a cytotoxic anticancer drug.

The effects of a herbal medicine (Mao-to) in patients with chronic hepatitis C after injection of IFN-beta.

We found that a herbal medicine (Mao-to) relieves the side effects of interferon (IFN)-beta and the combination therapy improves the biochemical response rate. However, the exact mechanism by which Mao-to is effective remains to be established. We conducted a controlled trial to clarify the effects of Mao-to. The study was carried out in 18 patients with chronic hepatitis C, and we examined subjective symptoms, body temperature and cytokines such as interleukin (IL)-beta, IL-1receptor antagonist (ra), IL-6 and TNF-alpha. Each patient received 6 million units of IFN-beta intravenously. Mao-to was given orally just before, just after, and 1 hour after IFN administration. The control study was carried out 6 months after the combination therapy of Mao-to and IFN-beta. The scores for general malaise, arthralgia and discomfort were significantly lower in the combination group than in control group. Body temperature did not significantly differ between the two groups. Plasma IL-6 level and IL-1ra were significantly elevated in the combination group compared to control (P = 0.0057 and 0.0003, respectively). Mao-to did not affect plasma concentrations of IL-1beta and TNF-alpha. We considered the increment of IL-1ra caused by Mao-to is to be one of the key factors involved in reducing the flu-like symptoms accompanying IFN-beta and improving the biochemical response rate.

A decrease in interleukin-1 receptor antagonist expression in the prefrontal cortex of schizophrenic patients.

Interleukin-1 (IL-1) mediates psychological stress responses by regulating monoamine metabolism and secretion of corticotropin-releasing factor, and is therefore, implicated in various psychiatric diseases. To evaluate the contribution of IL-1 signaling to the brain pathology of schizophrenia, we measured protein and/or mRNA levels for IL-1beta and endogenous IL-1 receptor antagonist (IL-1RA) in the postmortem brain tissues of prefrontal and parietal cortex, putamen, and hypothalamus. Both protein and mRNA levels of IL-1RA were specifically decreased in the prefrontal cortex of schizophrenic patients, whereas IL-1beta levels were not significantly altered in all the regions examined. The IL-1RA decrease was not correlated with the dose of antipsychotics given to patients. There was no influence of this illness on protein levels for IL-1 receptor type 1 in the prefrontal cortex, either. In contrast, IL-1RA serum levels were increased in schizophrenic patients, especially in drug-free patients, as reported previously. These findings suggest that chronic schizophrenia down-regulates IL-1RA production the prefrontal cortex, irrespective of its impact on the periphery. IL-1RA reduction might reflect an immunopathologic trait of the prefrontal region in schizophrenic patients.

Effects of glossopharyngeal nerve transection on central and peripheral cytokines and serum corticosterone induced by localized inflammation.

Bilateral transection of the glossopharyngeal nerves (GLOx) disrupts the immune-to-brain communication from the posterior oral cavity. The current report tested whether this effect is due to the afferent (sensory) or efferent (parasympathetic motor) components of the nerve. Injection of lipopolysaccharide (LPS) into the soft palate (ISP) of GLOx or sham-operated (SHAM) rats increased the circulating levels of interleukin-1beta (IL-1beta), interleukin-1 receptor antagonist (IL-1ra) and corticosterone (CORT), as well the hypothalamic content of IL-1beta; no difference in circulating levels and hypothalamic content was found between GLOx and SHAM at 2 and 4.5 h after LPS injection. These results indicate that glossopharyngeal neural efferents do not mediate the effects of GLOx on the immune-to-brain communication.

Effect of vitamin supplementation on cytokine response and on muscle damage after strenuous exercise.

The present double-blinded, placebo-controlled study investigated whether antioxidant vitamin supplementation was able to modulate the cytokine and lymphocyte responses after strenuous eccentric exercise. Furthermore, muscle enzyme release was examined to see whether antioxidant treatment could reduce muscle damage. Twenty male recreational runners randomly received either antioxidants (500 mg of vitamin C and 400 mg of vitamin E) or placebo for 14 days before and 7 days after a 5% downhill 90-min treadmill run at 75% .VO(2 max). Although the supplemented group differed significantly with regard to plasma vitamin concentration before and after exercise when compared with the placebo group, the two groups showed identical exercise-induced changes in cytokine, muscle enzyme, and lymphocyte subpopulations. The plasma level of interleukin (IL)-6 and IL-1 receptor antagonist increased 20- and 3-fold after exercise. The plasma level of creatine kinase was increased sixfold the day after exercise. The concentrations of CD4+ memory T cells, CD8+ memory and naïve T cells, and natural killer cells increased at the end of exercise. The total lymphocyte concentration was below prevalues in the postexercise period. In conclusion, the present study does not support the idea that exercise-induced inflammatory responses are induced by free oxygen radicals.

Inflammatory cytokines and their receptors in arterial and mixed venous blood before, during and after infusion of drained untreated blood.

Wound blood for postoperative autologous transfusion is drained through an area of damaged tissue, the surgical wound, and contains inflammatory mediators. The inflammatory cytokines interleukin-1-beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-alpha) and their modulators interleukin-1-receptor antagonist (IL-1Ra), interleukin 6 soluble receptor (IL-6sR), soluble tumour necrosis factor receptor 1 (sTNF-R1) and interleukin 10 (IL-10), together with white cell count (WCC) and white cell differential count were measured in arterial and mixed venous blood before, during and after infusion of postoperatively drained untreated blood in nine patients operated for thoracic scoliosis. We found a transient increase in IL-6, an increase in TNF-RI, an increase in IL-8 with granulocytosis and a decrease in IL-10 in the systemic circulation. The increase in IL-6 was higher in mixed venous than in arterial blood.

Serum bone sialoprotein in patients with primary breast cancer is a prognostic marker for subsequent bone metastasis.

Bone sialoprotein (BSP) is a noncoflagenous bone matrix protein that is important for both mineralization and cell-cell interactions. Tissue studies in primary breast cancers have shown that immunohistochemical expression of BSP is associated with a high incidence of bone metastases in the course of the disease. We used a RIA to investigate the importance of serum BSP as a marker for subsequent bone metastases. Between 1994 and 1996, preoperative blood samples were collected from 388 consecutive patients with nonmetastatic breast cancer and from 30 control patients with benign breast disease. Serum BSP concentrations were measured in a blinded fashion by RIA. The cutoff for elevated serum BSP values was 24 ng/ml, ie., two SDs above the normal mean value. Serum BSP was correlated with the risk of metastasis and analyzed with regard to its prognostic value. After a median follow-up period of only 20 months, 28 patients had developed metastases. Fourteen patients had bone metastases only, 9 visceral metastases only, and 5 a combination of osseous and visceral metastases. Of the 19 women with skeletal metastases, 17 had preoperative serum BSP values in excess of 24 ng/ml (median BSP values: 48.3 ng/ml for isolated metastatic bone disease, 30.6 ng/ml for combined metastases), whereas none of the women with visceral metastases only had elevated serum BSP concentrations (median BSP value: 12.3 ng/ml). The median serum BSP value in the control group (benign breast disease) was 8.8 ng/ml serum BSP; levels correlated with the size of the primary tumor, but not with any other prognostic factors. Using a multivariate regression analysis, serum BSP was found to be the most important independent prognostic factor for the development of skeletal metastasis (P < 0.001; relative risk, 94); its specificity was 96.7%, and its sensitivity was 89.5%. Our study shows that patients with preoperatively elevated serum BSP levels are at high risk of subsequent bone metastases in the first years after primary surgery. The mechanism of BSP in the pathogenesis of skeletal metastases is unclear. Because BSP contains an integrin recognition sequence, its expression in tumor cells may facilitate their adhesion to the bone surface. However, it is possible that a proportion of circulation BSP is derived from normal or tumor-induced bone turnover. Breast cancer patients with elevated serum BSP levels may benefit from osteoprotective adjuvant therapy with bisphosphonates.

Plasma levels of lipid and cholesterol oxidation products and cytokines in diabetes mellitus and cigarette smoking: effects of vitamin E treatment.

To evaluate the role of both oxidation and inflammation in atherosclerosis, we compared LDL oxidizability, in vivo lipid and cholesterol oxidation, and basal and lipopolysaccharide (LPS)-stimulated production of various cytokines in normolipidemic patients with diabetes mellitus (DM: n = 11), cigarettes smokers (n = 14). In addition, the effects of vitamin E (600 I.U./day for 4 weeks) on these parameters were evaluated. Initial LDL oxidation characteristics before and after vitamin E were identical in the 3 groups. Plasma thiobarbituric acid reactive substances were higher in DM and smokers versus controls (0.77 +/- 0.22, 0.74 +/- 0.14 versus 0.62 +/- 0.10 mumol malondialdehyde equivalents/l, respectively; P versus controls < 0.05) and normalized after vitamin E supplementation. Total plasma oxysterols were higher in smokers versus controls (354 +/- 104 versus 265 +/- 66 nmol/l, P < 0.05) and unaffected by vitamin E. The basal and LPS-stimulated levels of interleukin-1 beta and tumour necrosis factor alpha (TNF alpha) and the basal level of interleukin-1-receptor antagonist (IL-1RA) were identical for the 3 groups. LPS-stimulated IL-1RA was higher in DM versus controls (10.7 +/- 2.0 versus 8.1 +/- 1.7 pmol/l, P < 0.05). After vitamin E, TNF alpha dropped in controls and smokers, and IL-1RA in smokers only. Results suggest increased in vivo oxidative stress and inflammation in DM and smoking, which is partly overcome by vitamin E.

Pro- and anti-inflammatory cytokines in healthy volunteers fed various doses of fish oil for 1 year.

Dietary supplementation with n-3 fatty acids from fish oil alleviates inflammation in various chronic inflammatory disease states. Reductions in the production of pro-inflammatory cytokines interleukin 1 beta (IL-1 beta), tumour necrosis factor alpha (TNF-alpha), and interleukin 6 (IL-6) have been seen in humans after short-term n-3 fatty acid supplementation. We investigated long-term effects of dietary n-3 fatty acids on circulating cytokine concentrations and on ex vivo stimulated whole-blood production of IL-1 beta, TNF-alpha and interleukin 1 receptor antagonist (IL-1Ra), the naturally occurring antagonist of IL-1. A total of 58 monks with a mean age of 56 years were randomized into four groups and their diets were supplemented with 0, 3, 6, or 9 g of fish oil, providing 0, 1.06, 2.13 or 3.19 g of n-3 fatty acids per day. Subjects received equal amounts of saturated fatty acids, vitamin E and cholesterol. Compliance was excellent and erythrocyte fatty acid profiles closely reflected the amounts of n-3 fatty acids ingested. In the group receiving 9 g of fish oil per day, no influence of n-3 fatty acids on circulating cytokine concentrations was observed relative to placebo. Endotoxin-stimulated whole-blood cytokine production was measured at 26 and 52 weeks after the start and at 4, 8 and 26 weeks after cessation of supplementation. In all groups, the production of IL-1 beta and IL-1Ra was higher during supplementation than afterwards. However, no differences in cytokine production were noted between the placebo group and the various treatment groups at any point in time. Our results suggest that long-term supplementation of fish oil does not affect ex vivo cytokine production in man.

Identification, characterization, cloning, and expression of apolipoprotein C-IV, a novel sialoglycoprotein of rabbit plasma lipoproteins.

We have identified and characterized a novel proline- and arginine-rich protein component of lipoproteins, present in up to five sialylated isoforms, in rabbit blood plasma. The pI of the desialylated protein is 5.7. Based upon its N-terminal sequence, a complete cDNA sequence of 555 nucleotides was cloned from rabbit liver. The synthesized protein is predicted to contain 124 amino acids, including a typical signal peptide of 27 residues. The mature protein of 97 amino acids, designated apolipoprotein C-IV, is associated with the lipoproteins of blood plasma, primarily very low density and high density lipoproteins. It contains two potential amphipathic helices characteristic of plasma apolipoproteins and forms discoidal micelles with phosphatidylcholine. Northern analysis shows a single 0.6-kilobase apolipoprotein C-IV mRNA, detected only in the liver, and Southern analysis suggests a single copy gene. Sialylated apolipoprotein C-IV is secreted from transfected mammalian cells. Nucleotide sequence comparisons demonstrate a strong homology to portions of the upstream regions of the mouse and human apolipoprotein C2 genes, within each of which a distinct gene has recently been identified. The nucleotide sequences and the predicted amino acid sequences, as well as corresponding cDNA sequences in the rat and monkey, indicate that the apolipoprotein C4 gene has been highly conserved during mammalian evolution.

Increased serum interleukin-1-receptor-antagonist concentrations in major depression.

Recently, it has been shown that major depression may be accompanied by an increased production of interleukin-1 beta (IL-1 beta), an acute phase (AP) response and simultaneous signs of activation and suppression of cell-mediated immunity. The interleukin-1-receptor antagonist (IL-1-rA) is released in vivo during an AP response and serum levels are increased in many immune disorders. The release of IL-1-rA may limit the pro-inflammatory effects of IL-1. This study has been carried out to examine serum IL-1-Ra in 68 depressed subjects (21 minor, 25 simple major and 22 melancholic subjects) vs. 22 normal controls. Depressed subjects showed significantly higher serum IL-1-rA concentrations than healthy controls. 29% of all depressed subjects had serum IL-1-rA levels higher than the mean value +2 standard deviations of normal controls; 44% depressed subjects had IL-1-rA values greater than 0.215 ng/ml with a specificity of 90%. In depressed subjects, there was a significant and positive relationship between serum IL-1-rA and severity of illness. In depression, there were no significant relationships between serum IL-1-rA concentrations and indicants of hypothalamic-pituitary-adrenal (HPA)-axis activity, such as 24-h urinary cortisol and postdexamethasone cortisol values. Women had significantly higher serum IL-1-rA levels than men. The findings support the thesis that depression is accompanied by an immune-inflammatory response.

Altered red blood cell surface area in hereditary xerocytosis.

Hereditary xerocytes appear larger than normal red cells in scanning electron micrographs and exhibit a higher ghost packing volume. The major chemical components--protein, phosphorus, cholesterol and sialic acid--are increased uniformly, as are all polypeptides visible on gel electrophoresis patterns of xerocyte membranes. These data are consistent with a xerocyte surface area 15 to 25% above normal. Certain clinical anomalies common to this disorder, including unexpectedly low reticulocyte count and 2,3-diphosphoglycerate level, are discussed in the light of the present findings.