Molecular and Physiological Effects on the Small Intestine of Weaner Pigs Following Feeding with Deoxynivalenol-Contaminated Feed.

We intended to assess how exposure of piglets to deoxynivalenol (DON)-contaminated feed impacted their growth, immune response and gut development. Piglets were fed traditional Phase I, Phase II and Phase III diets with the control group receiving 0.20-0.40 ppm DON (referred to as the Control group) and treatment group receiving much higher level of DON-contaminated wheat (3.30-3.80 ppm; referred to as DON-contaminated group). Feeding a DON-contaminated diet had no impact on average daily feed intake (ADFI) (p < 0.08) or average daily gain (ADG) (p > 0.10) but it did significantly reduce body weight over time relative to the control piglets (p < 0.05). Cytokine analysis after initial exposure to the DON-contaminated feed did not result in significant differences in serum interleukin (IL) IL1ß, IL-8, IL-13, tumor necrosis factor (TNF)-α or interferon (IFN)-γ. After day 24, no obvious changes in jejunum or ileum gut morphology, histology or changes in gene expression for IL-1ß, IL-6, IL-10, TNFα, or Toll-like receptor (TLR)-4 genes. IL-8 showed a trend towards increased expression in the ileum in DON-fed piglets. A significant increase in gene expression for claudin (CLDN) 7 gene expression and a trend towards increased CLDN 2-expression was observed in the ileum in piglets fed the highly DON-contaminated wheat. Because CLDN localization was not negatively affected, we believe that it is unlikely that gut permeability was affected. Exposure to DON-contaminated feed did not significantly impact weaner piglet performance or gut physiology.

Thyroid Hürthle cell tumors: research of potential markers of malignancy.

INTRODUCTION: Hurthle cell tumors (HCTs) are rare thyroid neoplasia. To date, capsular and/or vascular invasion are the only findings predicting malignancy. Recently, mutation of 19p13, encoding two proteins involved in cell proliferation and apoptosis (GRIM-19 and p19), has been described. The aim of our study is to evaluate the cellular proliferation index (Ki67), GRIM-19 and p19 expression as diagnostic markers of malignancy in HCT. MATERIALS AND METHODS: Eighty patients with HCT (32 carcinomas, 48 adenomas) whom underwent surgery in our center were included. Samples of both neoplastic lesions and adjacent normal thyroid tissue were analyzed by means of tissue micro-arrays. Correlations between expressions of Ki67, GRIM-19 and p19 and final histology were analyzed. RESULTS: Mean size of the lesion was higher in carcinomas than in adenomas (p = 0.01). GRIM-19 and p19 were significantly underexpressed in Hurthle cells tumors compared to normal tissue (p = 0.0004 and p = 0.0001, respectively). Ki67 and GRIM-19 were, respectively, higher and down-expressed in carcinomas compared to adenomas (p = 0.0004 and p = 0.005, respectively). On multivariate analysis, size correlates with carcinoma diagnosis. Neither GRIM-19 nor Ki67 index was related to size. The expression of p19 was reduced in both adenoma and carcinoma but differences were not statistically significant (p = 0.13). CONCLUSIONS: Our study suggest that Ki67 and GRIM-19 correlate with malignancy in HCT. The expression of p19 is down-regulated in HCT, but it is not diagnostic of carcinoma. Ki67 and GRIM-19 may potentially help as cytological markers of malignancy in HCT.

Effects of dietary soybean oil on pig growth performance, retention of protein, lipids, and energy, and the net energy of corn in diets fed to growing or finishing pigs.

The objectives of this experiment were 1) to determine if dietary soybean oil (SBO) affects the NE of corn when fed to growing or finishing pigs, 2) to determine if possible effects of dietary SBO on the NE of corn differ between growing and finishing pigs, and 3) to determine effects of SBO on pig growth performance and retention of energy, protein, and lipids. Forty-eight growing (initial BW: 27.3 ± 2.5 kg) and 48 finishing (initial BW: 86.0 ± 3.0 kg) barrows were used, and within each stage of growth, pigs were allotted to 1 of 6 groups. Two groups at each stage of growth served as an initial slaughter group. The remaining 4 groups were randomly assigned to 4 dietary treatments and pigs in these groups were harvested at the conclusion of the experiment. A low-lipid basal diet containing corn, soybean meal, and no added SBO and a high-lipid basal diet containing corn, soybean meal, and 8% SBO were formulated at each stage of growth. Two additional diets at each stage of growth were formulated by mixing 25% corn and 75% of the low-lipid basal diet or 25% corn and 75% of the high-lipid basal diet. Results indicated that addition of SBO had no effects on growth performance, carcass composition, or retention of energy, protein, and lipids but increased (P < 0.05) apparent total tract digestibility of acid hydrolyzed ether extract and GE. Addition of SBO also increased (P < 0.05) DE and NE of diets, but had no effect on the DE and NE of corn. Finishing pigs had greater (P < 0.05) growth performance and retention of energy, protein, and lipids than growing pigs. A greater (P < 0.05) DE and NE of diets was observed for finishing pigs than for growing pigs and the DE and NE of corn was also greater (P < 0.05) for finishing pigs than for growing pigs. In conclusion, addition of SBO increases the DE and NE of diets but has no impact on the DE and NE of corn. Diets fed to finishing pigs have greater DE and NE values than diets fed to growing pigs and the DE and NE of corn are greater for finishing pigs than for growing pigs.

Net energy of soybean oil and choice white grease in diets fed to growing and finishing pigs.

The objectives of this experiment were 1) to determine the NE of soybean oil (SBO) and choice white grease (CWG) fed to growing and finishing pigs, 2) to evaluate the effects of inclusion rate of SBO on the NE by growing and finishing pigs, and 3) to determine if there is a difference in the NE of SBO and CWG between growing and finishing pigs. Forty-eight growing (initial BW: 22.13 ± 1.78 kg) and 48 finishing (initial BW: 84.17 ± 5.80 kg) barrows were used, and they were housed and fed individually. Within each stage of growth, pigs were allotted to 8 outcome groups of 6 barrows based on BW. Within each outcome group, pigs were randomly allotted to 1 of 6 groups. Two groups at each stage of growth served as an initial slaughter group. Pigs in the remaining groups were assigned to 4 dietary treatments and slaughtered at the conclusion of the experiment. The basal diet contained corn, soybean meal, and no supplemental lipids. Three additional diets were formulated by mixing 95% of the basal diet and 5% SBO, 90% of the basal diet and 10% SBO, or 90% of the basal diet and 10% CWG. Average daily gain and G:F for finishing pigs and apparent total tract digestibility of energy for growing and finishing pigs increased (linear, P < 0.05) with lipid content, but was not affected by lipid source. The lipid gain:protein gain ratio and the energy retention also increased (linear, P ≤ 0.05) with lipid content in growing and finishing pigs. There were no interactive effects between lipid content and stage of growth or between lipid source and stage of growth on the NE of diets and the NE of dietary lipids. The NE of diets increased (linear, P < 0.01) with increasing SBO (2,056, 2,206, and 2,318 kcal/kg for diets containing 0, 5, or 10% SBO). The NE of the diet containing 10% CWG (2,440 kcal/kg) was greater (P < 0.05) than the NE of the diet containing 10% SBO. The NE of diets was greater (P < 0.05) for finishing pigs than for growing pigs regardless of lipid content or source. The NE of SBO included at 5% (5,073 kcal/kg) was not different from the NE of SBO included at 10% (4,679 kcal/kg), but the NE of CWG (5,900 kcal/kg) was greater (P < 0.05) than the NE of SBO. The stage of growth had no impact on the NE of SBO or CWG. In conclusion, the NE of lipids is not affected by the content of dietary lipids, but the NE of CWG is greater than the NE of SBO.

Management of acute malignant large-bowel obstruction with self-expanding metal stent.

BACKGROUND: Colorectal stents are being used for palliation and as a "bridge to surgery" in obstructing colorectal carcinoma. The purpose of this study was to review our experience with self-expanding metal stents (SEMS) as the initial interventional approach in the management of acute malignant large bowel obstruction. METHODS: Between February 2002 and May 2006, 67 patients underwent the insertion of a SEMS for an obstructing malignant lesion of the left-sided colon or rectum. RESULTS: In 55 patients, the stents were placed for palliation, whereas in 12 they were placed as a bridge to surgery. Stent placement was technically successful in 92.5% (n = 62), with a clinical success rate of 88% (n = 59). Two perforations that occurred during stent placement we retreated by an emergency Hartmann operation. In intention-to-treat by stent, the peri-interventional mortality was 6% (4/67). Stent migration was reported in 3 cases (5%), and stent obstruction occurred in 8 cases (13.5%). Of the nine patients with stents successfully placed as a bridge to surgery, all underwent elective single-stage operations with no death or anastomotic complication. CONCLUSIONS: Stent insertion provided an effective outcome in patients with malignant colonic obstruction as a palliative and preoperative therapy.

Factors affecting the concentration of sphingomyelin in bovine milk.

Sphingomyelin is a phospholipid located in the outer leaflet of the plasma membrane of most cells and is a component of the milk fat globule membrane. Sphingomyelin and its digestion products participate in several antiproliferative pathways that may suppress oncogenesis. Although milk and dairy products are important sources of sphingomyelin in the human diet, little is known about factors that influence sphingomyelin concentrations in milk fat or whether concentrations can be modified via the nutrition of cows. Sphingomyelin concentrations were determined in milk from Holstein and Jersey cows matched for parity and stage of lactation. Sphingomyelin was more concentrated in milk fat from Holstein cows than in milk fat from Jersey cows (1,044 vs. 839 microg/g of fat). Concentrations in whole milk did not differ because of greater milk fat content for milk from Jerseys. Differences between breeds may be related to the greater fat globule size in milk from Jerseys. Sphingomyelin content in whole milk increased with increasing days in milk because of associated increases in milk fat content. Regardless of breed, primiparous cows had greater amounts of stearic acid and less palmitic acid in sphingomyelin than did older cows. The sphingomyelin concentration in milk fat of cows in a commercial Jersey herd was lower for cows in their fourth or greater parity. Sphingomyelin content in whole milk was greater for cows in late lactation because of greater milk fat content. Feed restriction of multiparous Holstein cows to 37% of ad libitum dry matter intake increased milk fat content but did not affect milk sphingomyelin content or milk fat globule size. Supplementation of the diet with 4% soybean oil did not affect milk composition, sphingomyelin content, or milk fat globule size. Milk was sampled seasonally from 7 herds throughout Illinois during a 2-yr period. Sphingomyelin concentration in milk fat was greatest during summer and least during winter, but whole milk concentrations did not vary across seasons. We conclude that 1) sphingomyelin content of milk fat is greater in milk from Holsteins than that from Jerseys, 2) sphingomyelin content in whole milk increases with stage of lactation, and 3) sphingomyelin content of milk fat is greater during summer. However, efforts to produce milk with a greater sphingomyelin content through altering management and nutrition are unlikely to be successful.

Arthroscopic evaluation of potential structure-modifying drug in osteoarthritis of the knee. A multicenter, randomized, double-blind comparison of tenidap sodium vs piroxicam.

OBJECTIVE: To test the hypothesis that tenidap has a structure-modifying effect in human knee osteoarthritis. STUDY: multicenter, prospective, randomized, double blind, 1 year duration. PATIENTS: primary painful knee osteoarthritis (ACR criteria) of the medial tibiofemoral compartment, medial joint space width > or =2mm, at least 10% of one cartilage surface of the medial compartment affected by superficial fibrillation or worse at baseline arthroscopy. STUDY MEDICATION: once daily dosage of either tenidap 40 mg, tenidap 120 mg or piroxicam 20mg. STUDY ENDPOINTS: bilateral extended weight-bearing X-rays and knee arthroscopy under local anaesthesia were done at entry and after 1 year. Joint space width was measured in millimeters at the narrowest point of the medial compartment. Chondropathy was scored by using reader's overall assessment (VAS score, 100mm) and Société Française d'Arthroscopie (SFA) score (0-100). RESULTS: Patients (665) were randomized and 494 completed the study. After 1 year, intra-group radiological changes and radiological difference between both tenidap groups and the piroxicam group did not reach statistical significance. The intra-group arthroscopic deterioration of chondropathy was low, but statistically significant in the three study groups. However, there was no statistically significant difference between both tenidap groups and the piroxicam group. CONCLUSIONS: This study failed to demonstrate any difference between the treatment arms with regard to the structural progression of medial knee osteoarthritis as measured by radiography and arthroscopy. Arthroscopy did, however, appears to be more sensitive in detecting disease progression than the weight-bearing radiographs with fully extended knees. This study shows that it is possible to complete a large international trial using arthroscopy as an outcome measure of articular cartilage.

Concentrations of conjugated linoleic acid (cis-9, trans-11-octadecadienoic acid) are not increased in tissue lipids of cattle fed a high-concentrate diet supplemented with soybean oil.

Conjugated linoleic acid (CLA), a mixture of isomers of linoleic acid, has many beneficial effects, including decreased tumor growth in animal cancer models. The cis-9, trans-11 isomer of CLA (CLA9,11) can be formed in the rumen as an intermediate in biohydrogenation of linoleic acid. Recent data, however, indicate that tissue desaturation of trans-fatty acids is an important source of CLA9,11 in milk. Our objective was to determine whether supplementing a high-corn diet with soybean oil (SBO; a source of linoleic acid) would increase concentrations of CLA in ruminal contents and tissue lipids. Four ruminally cannulated steers were utilized in a Latin square design with 28-d periods. A control diet (80% cracked corn, 2.0% corn steep liquor, 8.0% ground corn cobs, and 10% supplement [soybean meal, ground shelled corn, minerals, and vitamins]) was supplemented with 2.5, 5.0, or 7.5% (DM basis) SBO. Supplemental SBO did not affect ruminal pH or concentrations of the major VFA. The proportion and amount (mg FA/g DM ruminal contents) of CLA9,11 were not increased by increasing dietary SBO. However, the proportion and amount of the trans-10, cis-12 CLA isomer (CLA10,12) in ruminal contents increased linearly (P < 0.006) as dietary SBO increased. Trans-18:1 isomers in ruminal contents increased linearly (P < 0.02) as dietary SBO increased. The proportion of CLA10,12 was correlated positively (P < 0.001) with proportions of trans-C 18:1 isomers in ruminal contents. Conversely, CLA9,11 was correlated negatively (P < 0.05) with the proportions of trans-18:1 in ruminal contents. The same high-corn diet, supplemented with 0 or 5% SBO, was fed to 20 Angus-Wagyu heifers for 102 d in a randomized complete block design to determine the effect of added SBO on tissue deposition of CLA. Supplemental SBO did not affect feed intake, gain:feed, or carcass quality. Tissue samples were obtained from the hindquarter, loin, forequarter, liver, large and small intestine, and subcutaneous, mesenteric, and perirenal adipose depots. The concentration of CLA9,11 was greatest in subcutaneous adipose tissue but was not affected in any tissue by SBO. Supplementing high-corn diets with SBO does not increase CLA9,11 concentrations in tissues of fattening heifers. Research is needed to identify regulatory factors for pathways of biohydrogenation that lead to increased concentrations of CLA10,12 in ruminal contents when high-oil, high-concentrate diets are fed.

Genetic diversity of Pseudomonas aeruginosa strains isolated from ventilated patients with nosocomial pneumonia, cancer patients with bacteremia, and environmental water.

Random amplified polymorphic DNA typing was used to study the genetic diversity of Pseudomonas aeruginosa strains from (i) ventilated patients with nosocomial pneumonia who were hospitalized in intensive care units, (ii) cases of bacteremia in cancer patients with severe neutropenia, and (iii) rivers and swimming pools. Genetic diversity was determined by three phylogenetic methods and by statistical analysis of population genetics. The population studied undergoes epidemic clonality with a high rate of genetic recombination. P. aeruginosa bacteremia and pneumonia are not caused by specific clones within this species.

Determinants of health status in fibromyalgia: a comparative study with systemic lupus erythematosus.

OBJECTIVE: To compare perceived health status in women with fibromyalgia (FM) and systemic lupus erythematosus (SLE) using the Medical Outcomes Study (MOS) Short Form Health Survey (SF-36); and to identify determinants of physical and mental health in each patient group. METHODS: A cross sectional study of 46 women with FM (mean age 48.13 yrs, SD 9.40) and 59 women with SLE (mean age 42.36 yrs, SD 11.31). Patients with FM were recruited from a rheumatology clinic and a rheumatology practice, while patients with SLE were recruited from 4 rheumatology clinics. Clinical examination determined disease activity (by Systemic Lupus Activity Measure) in SLE and a tender point count was used for FM. Patients completed questionnaires assessing health status (SF-36), stress (Hassles), social support (Social Support Questionnaire 6), and coping (Coping Inventory for Stressful Situations). RESULTS: Patients with FM reported more impairment on the following SF-36 subscales: physical function (p < 0.001), role physical (p < 0.001), bodily pain (p < 0.001), and vitality (p < 0.001). Physical component summary scores were also significantly lower (p < 0.001) for the FM group. Four hierarchical regression analyses were computed to determine factors related to physical and mental health in each patient group, with the following variables in the equation: age, income, disease activity (Step 1), hassles (Step 2), emotional and task coping, and social support (Step 3). Better physical health in FM was related to higher income (R2 = 0.17, p < 0.05). In the SLE group, better physical health was associated with younger age, less disease activity, and lower hassles (R2 = 0.37, p < 0.0001). Worse mental health among women with FM was associated with more hassles, more emotional coping, and less satisfaction with social support (R2 = 0.64, p < 0.0001), while lower income, higher hassles, and more emotional coping were linked to worse mental health in SLE (R2 = 0.46, p < 0.0001). CONCLUSION: Health related quality of life (HRQL) is impaired among women with FM and SLE, with FM patients reporting greater impairment along several dimensions. Enhancing the HRQL of patients with FM and SLE requires targeting specific modifiable psychosocial factors.

Enhanced reduction of fasting total homocysteine levels with supraphysiological versus standard multivitamin dose folic acid supplementation in renal transplant recipients.

The mild fasting hyperhomocysteinemia commonly observed in chronic (ie, >/=6 months posttransplantation) renal transplant recipients (RTRs) can be effectively treated with combined B-vitamin supplementation featuring supraphysiological doses of folic acid. There are no controlled data evaluating the comparative efficacy of supraphysiological versus standard multivitamin dose folic acid supplementation in reducing fasting total homocysteine (tHcy) levels among RTRs. We block-randomized 60 chronic, stable RTRs on the basis of their screening fasting tHcy level to 3 groups of 20 subjects treated for 12 weeks with folic acid at either 2.4 (group 1), 0.4 (ie, standard multivitamin dose) (group 2), or 0.0 (group 3) mg/d. All 60 study participants also received 50 mg/d vitamin B(6) and 0.4 mg/d vitamin B(12). The mean percent reductions (+/-SEM) in fasting tHcy were as follows: group 1, 32.3+/-2.4%; group 2, 23.4+/-2.3%; and group 3, 19.1+/-2.3%. ANCOVA accounting for the pretreatment matching and adjusted for pretreatment levels of fasting tHcy, folate, and albumin; change in creatinine during the study; and cyclosporine A use revealed significant overall group differences (P=0.005) and significant differences between groups 1 and 2 (P=0. 038) and groups 1 and 3 (P=0.001), but not between groups 2 and 3 (P=0.153). Moreover, a chi(2) analysis of participants with pretreatment tHcy levels >/=15 micromol/L (n=29) indicated that a significantly greater proportion of those in group 1 achieved posttreatment levels <12 micromol/L: group 1, 5 of 10 (50%); group 2, 1 of 11 (9%); and group 3, 0 of 8 (0%) (P=0.016; test of trend P=0. 007). We conclude that a supraphysiological dose of folic acid is superior to standard multivitamin dosing for the reduction of fasting tHcy levels in chronic RTRs.

Granulocyte-macrophage colony-stimulating factor modulates tapasin expression in human neutrophils.

Differential display-polymerase chain reaction (DD-PCR) was used to evaluate changes in mRNA expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) treated human neutrophils to better understand how this cytokine affects the functions of neutrophils at the molecular level. Although a variety of cDNA fragments were identified as modulated by GM-CSF with the use of DD-PCR, one fragment in particular, NGS-17 (neutrophil GM-CSF-stimulated fragment #17), was characterized. The NGS-17 fragment hybridized to a 3.8-kh mRNA that encodes for a protein of a predicted molecular mass of 47.6 kDa. After cloning and sequencing, this gene was found to code for the recently sequenced tapasin or TAP-A protein. Immunoprecipitation and immunoblotting studies using anti-tapasin antibodies showed that tapasin is expressed in neutrophils and is associated with the MHC class I-TAP complex. Moreover, tapasin expression was found to be induced by dimethyl sulfoxide and by retinoic acid in HL-60 cells. This is the first report on the expression of tapasin in human neutrophils. It provides novel information, at the molecular level, on how GM-CSF enhances the functions of these cells.

ABC50, a novel human ATP-binding cassette protein found in tumor necrosis factor-alpha-stimulated synoviocytes.

We have used the recently developed technique of differential display polymerase chain reaction to seek for new genes modulated by tumor necrosis factor-alpha (TNF-alpha) in cultured synoviocytes. One PCR fragment was shown to correspond to a new gene that was mapped by high-resolution fluorescence in situ hybridization to band 6p21.33. The cDNA of this gene was cloned, and the deduced amino acid sequence revealed consensus motifs for the nucleotide binding folds of the ATP-binding cassette (ABC) family of proteins. However, a hydropathy curve showed that the polypeptide does not contain the transmembrane domains that are typical of the subfamily of ABC transporters and are associated with transporter/channel functions. The new gene, called ABC50, is the first human and mammalian ABC protein found to lack transmembrane domains. Homology with some yeast ABC proteins suggests that ABC50 codes for a new human ribosomal protein involved in translation of mRNA. It could therefore play a role in the enhancement of protein synthesis that follows TNF-alpha treatment of synoviocytes and thus participate in the inflammatory processes mediated by this cytokine. Furthermore, since TNF-alpha also modulates the expression of MHC class I genes, and these genes are known to map to 6p21.33, it is hypothesized that ABC50 and MHC class I are part of the same chromatin expression domain.

The IL-1 and IL-1 receptor antagonist (IL-1Ra) response of human neutrophils to EBV stimulation. Preponderance of IL-Ra detection.

The present study investigated the effect of EBV on gene expression and protein synthesis of IL-1 and its natural IL-1R antagonist (IL-1Ra) in human peripheral blood neutrophils. EBV induced a rapid accumulation of IL-1 and IL-lRa mRNA in neutrophils that was associated with the later appearance of considerable amounts of IL-1alpha, IL-1beta, and IL-Ra proteins. Approximately 3200 and 610 times more IL-Ra than IL-1alpha a or IL-1beta, respectively, was secreted by neutrophils in response to EBV. The effect induced by EBV cannot reflect an overall metabolic activity of neutrophils, since EBV failed to induce granulocyte-macrophage OF synthesis. Heat-inactivated virus was unable to stimulate cytokine synthesis, whereas UV-irradiated virus retained the full IL-1- and IL-1Ra-inducing potential of the native particle. Furthermore, pretreatment of cells with cycloheximide or phosphonoacetic acid did not abrogate the effect of EBV, suggesting that EBV does not penetrate the cell, but that a virion's structural molecule is required to induce such an effect. In this respect, neutralization of the viral particles with the mAb 72A1, which is known to react with glycoprotein gp350 of the viral envelope, inhibits the production of IL-1 and IL-1Ra, suggesting that gp350 could be involved in this process. Thus, the elevated levels of IL-1Ra detected for EBV-stimulated neutrophils might be part of a mechanism used by the virus to evade the immune system.

Epstein-Barr virus modulates de novo protein synthesis in human neutrophils.

Neutrophils and macrophages represent the first line of defense against microbial invaders. However, the role of phagocytes in host response to viral infection is poorly understood. We have previously shown that Epstein-Barr virus (EBV) interacts with human monocytes and modulates cytokine production in this cell type, but its effects on neutrophils are still unknown. In the present study, we investigated the presence of EBV receptor (CR2 or CD21) on neutrophils by cytofluorometry using five different anti-CD21 monoclonal antibodies (MoAbs), as well as fluoroscein isothiocyanate-EBV (FITC-EBV). Whereas no significant amount of neutrophils reacted with anti-CD21 MoAbs, studies with FITC-EBV indicated that viral particles bind to 30% of cells (in some individuals, EBV binds to more than 50% of neutrophils). This interaction is specific as it was completely inhibited by nonconjugated virus or with labeled virus preincubated with neutralizing MoAbs. After EBV treatment, cellular aggregation was observed in neutrophil cultures, an indication that neutrophils were activated. Although EBV did not induce respiratory burst activity in neutrophils, pretreatment with infectious particles enhanced (priming effect) the fMLP-induced O2- release in neutrophils. Instead of restricting our analysis to specific cytokine genes, we investigated the effects of EBV on neutrophil transcriptional events in general. The effect of this virus on de novo synthesis of total cellular RNA was first investigated by measuring the incorporation of [5-3H] uridine into total RNA. The results showed that RNA synthesis in neutrophils was significantly increased (2.3- to 21.3-fold) by EBV compared with the unstimulated controls. Live and UV-inactivated virus markedly induced RNA synthesis, whereas heat-inactivated virus lost this ability. Induction of RNA transcription was EBV specific, as an EBV-neutralizing antiserum abolished this effect. Induction of protein synthesis was also studied by measuring the incorporation of [35S] methionine and [35S] cysteine into secreted and intracellular proteins in neutrophils incubated with EBV. The synthesis of both secreted and cytoplasmic proteins was induced by EBV. One- and two-dimensional gel electrophoresis analysis showed that EBV modulates protein synthesis, because activation of the synthesis of certain proteins was accompanied by the inhibition of others. Interleukin-1 beta (IL-1 beta) and IL-1 receptor antagonist (IL-1Ra) synthesis was found to be induced by EBV. Therefore, modulation of host-response proteins such as IL-1Ra could be one of the many mechanisms by which this virus avoids rejection.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of interleukin-2 on gene expression in human neutrophils.

Recently, the interleukin-2 receptor (IL-2R) was shown to be present on human neutrophils, and IL-2-neutrophil interactions are believed to be important in both tumor rejection and increased susceptibility to bacterial infections. Furthermore, neutrophils have been shown to synthesize host defense proteins, such as cytokines. In this study, we analyzed the effects of IL-2 on the induction of de novo RNA and protein synthesis in this cell type. When cells were stimulated with IL-2 alone, the level of incorporation of either [5-3H]-uridine or [35S]-methionine and [35S]-cysteine was similar to unstimulated cells. However, when cells were stimulated with the combination of a fixed concentration of granulocyte-macrophage colony-stimulating factor (GM-CSF), a dose-dependent effect of IL-2 was observed on the induction of both RNA and protein synthesis. In the presence of tumor necrosis factor-alpha or formyl-methionyl-leucyl-phenylalanine, however, IL-2 exerted no similar effect. Furthermore, the study of a large number of normal subjects (n = 55) showed reproducible categories of responders (low, intermediate, and high). The binding of IL-2 to the IL-2R complex on human neutrophils increased on GM-CSF-stimulated neutrophils compared with unstimulated cells. However, no increase in the level of expression of either the alpha or beta chains of this receptor complex was observed. This finding suggests that GM-CSF functionally activates the IL-2R, but does not regulate its level of expression. Finally, we found that human neutrophils constitutively express IL-2R gamma chain mRNA and thus have the potential to express the functional IL-2R complex. Our findings on IL-2-neutrophil interactions should lead to new avenues of research in understanding the responses of patients undergoing GM-CSF or IL-2 therapy.

Low molecular weight GTP-binding proteins in HL-60 granulocytes. Assessment of the role of ARF and of a 50-kDa cytosolic protein in phospholipase D activation.

Phospholipase D (PLD) activation by guanine nucleotides requires protein cofactors in both the plasma membrane and the cytosol. HL-60 cytosol was fractionated by ammonium sulfate and gel-permeation chromatography. Two cytosolic protein fractions were found to reconstitute the GTP gamma S (guanosine 5'-3-O-(thio)triphosphate)-stimulated PLD in a reconstitution assay consisting of 3H-labeled HL-60 membranes and eluted column fractions. The major peak of reconstituting activity was in the region of 50 kDa, and a second discrete peak of PLD reconstitution activity was observed in the region of 18 kDa. Rho GDP/GTP exchange inhibitor, Rho GDI, comigrated with Rac2 and RhoA, but not Rac1. RhoA and Rac2 were entirely complexed with Rho GDI and eluted with an apparent molecular mass of 43 kDa by gel filtration chromatography. The partial overlap between cytosolic Rac2 and RhoA with the 50-kDa peak of reconstituting activity was not consistent with the participation of cytosolic Rho-related GTPases in the activation of PLD by guanine nucleotides. However, recombinant Rho GDI, which inhibits nucleotide exchange on the Rho family of small GTP-binding proteins, reduced GTP gamma S-stimulated PLD activity in HL-60 homogenates. The stimulatory exchange factor, Smg GDS, which is active on Rho and Rac, could be partially separated from the PLD-stimulating factor(s) by gel-permeation chromatography. Moreover, recombinant Smg GDS failed to stimulate GTP-dependent PLD activity. Cytosolic ADP-ribosylation factor (ARF) was exclusively located in the 18-kDa peak of reconstitution activity. Faint amounts of membrane-bound ARF were also detected using the monoclonal antibody 1D9. The effects of the 50-kDa and 18-kDa PLD-inducing factors on the salt-extracted PLD activity were synergistic. The weak stimulatory effect of ARF alone suggested that the GTP gamma S-stimulated PLD activity is dependent on the presence of another protein(s), presumably ARF-regulatory proteins. We propose that a membrane-bound GTP-binding protein, possibly ARF, may be involved in the activation of PLD when combined with the component(s) of the 50-kDa fraction.

Human neutrophils produce high levels of the interleukin 1 receptor antagonist in response to granulocyte/macrophage colony-stimulating factor and tumor necrosis factor alpha.

Neutrophils, an abundant cell type at sites of inflammation, have the ability to produce a number of cytokines, including interleukin 1 (IL-1), IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-alpha). In this study, we have examined the ability of human neutrophils to produce the IL-1 receptor antagonist (IL-1Ra), a 17-23-kD protein recently isolated and cloned from macrophages. Since IL-1Ra has been shown to inhibit both the in vitro and in vivo effects of IL-1, its production by large numbers of tissue-invading neutrophils might provide a mechanism by which the effects of IL-1 are regulated in inflammation. Using antibodies that are specific for IL-1Ra and a cDNA probe encoding for this protein, we were able to show that neutrophils constitutively produce IL-1Ra. However, after activation by GM-CSF and TNF-alpha, IL-1Ra was secreted into the extracellular milieu where it constituted the major de novo synthesized product of activated neutrophils. None of a large array of other potent neutrophil agonists were found to affect the production of IL-1Ra by neutrophils. Quantitative measurements by enzyme-linked immunosorbent assay revealed that intracellular IL-1Ra is in eightfold excess of the amount secreted in supernatants when studying nonactivated neutrophils. However, in GM-CSF- and TNF-alpha-activated cells, this difference was reduced to values between four- and fivefold, as virtually all of the de novo synthesized IL-1Ra was secreted. In activated cells, the intracellular content of IL-1Ra was found to be in the 2-2.5-ng/ml range per 10(6) neutrophils, whereas levels reached the 0.5-ng/ml range in supernatants. This would imply that IL-1Ra is produced in excess of IL-1 by a factor of at least 100, an observation that is in agreement with the reported amounts of IL-1Ra needed to inhibit the proinflammatory effects of IL-1. Neutrophils isolated from an inflammatory milieu, the synovial fluid of patients with rheumatoid arthritis, were found to respond to GM-CSF and TNF-alpha in terms of IL-1Ra synthesis, indicating that the in vitro observations made in this study are likely to occur in an inflammatory setting in vivo.

Nuclear signaling in human neutrophils. Stimulation of RNA synthesis is a response to a limited number of proinflammatory agonists.

At inflammatory sites, neutrophils are stimulated by a range of proinflammatory molecules which elicit a number of cellular responses. Considerable information on the cytoplasmic events that occur following activation of neutrophils at the cell membrane level already exists. In this study, we have focused on the ability of neutrophil agonists to initiate nuclear signaling events by investigating the induction of de novo RNA synthesis. Of a total of 14 different known potent leukocyte agonists, only three had a significant effect on the induction of RNA synthesis in neutrophils; the formylated oligopeptide formyl-methionyl-leucylphenylalanine (fMet-Leu-Phe), granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha. All three agonists induced de novo RNA synthesis in neutrophils at concentrations known to be optimal for the activation of a number of other cellular responses occurring in inflammation. Of significance was the observation that activation of RNA synthesis in neutrophils is a G-protein-mediated event, is also dependent on tyrosine phosphorylation, but is not influenced by cAMP. Finally, we have demonstrated that all three agonists also induce de novo synthesis of a limited number of proteins, with granulocyte-macrophage colony-stimulating factor and fMet-Leu-Phe having the most potent effect. These studies define the effects of neutrophil agonists on de novo RNA and protein synthesis in a proinflammatory context and suggest that these events in neutrophils occur in a restricted fashion, highly dependent on the stimuli present at sites of inflammation.

Selective synthesis and secretion of a 23 kD protein by neutrophils following stimulation with granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha.

We tested a wide range of pro-inflammatory cytokines for their capacity to activate protein synthesis in neutrophils as analyzed b y [35S] methionine metabolic labelling experiments. Of all the cytokines tested, only GM-CSF and TNF alpha stimulated significant synthesis and secretion of a 23 kD protein which resolved into two bands on two dimensional gels. Under non-reducing conditions on one dimensional gels, its migration pattern remained the same indicating that the two bands most likely represent isoforms of the same protein. Immunoisolation studies using antibodies directed against size-relevant molecules did not lead to the identification of this molecule. The fact that this 23 kD molecule is induced in a highly specific and selective manner by GM-CSF and TNF alpha indicates that it may play a key role in some of the responses of neutrophils to these two cytokines. Therefore, full characterization of this 23 kD protein could provide important new knowledge on the mechanisms by which these two cytokines exert their biological effects on neutrophils.