Immunoregulatory and anti-inflammatory effects of n-3 polyunsaturated fatty acids

1. Fish oils are rich in the long-chain n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids. Linseed oil and green plant tissues are rich in the precursor fatty acid, a-linolenic acid (18:3n-3). Most vegetable oils are rich in the n-6 PUFA linoleic acid (18:2n-6), the precursor of arachidonic acid (20:4n-6). 2. Arachidonic acid-derived eicosanoids such as prostaglandin E2 are pro-inflammatory and regulate the functions of cells of the immune system. Consumption of fish oils leads to replacement of arachidonic acid in cell membranes by eicosapentaenoic acid. This changes the amount and alters the balance of eicosanoids produced. 3. Consumption of fish oils diminishes lymphocyte proliferation, T-cell-mediated cytotoxicity, natural killer cell activity, macrophage-mediated cytotoxicity, monocyte and neutrophil chemotaxis, major histocompatibility class II expression and antigen presentation, production of pro-inflammatory cytokines (interleukins 1 and 6, tumour necrosis factor) and adhesion molecule expression. 4. Feeding laboratory animals fish oil reduces acute and chronic inflammatory responses, improves survival to endotoxin and in models of autoimmunity and prolongs the survival of grafted organs. 5. Feeding fish oil reduces cell-mediated immune responses. 6. Fish oil supplementation may be clinically useful in acute and chronic inflammatory conditions and following transplantation. 7. n-3 PUFAs may exert their effects by modulating signal transduction and/or gene expression within inflammatory and immune cells.

Endothelium-dependent vasodilation in response to Pseudomonas aeruginosa lipopolysaccharide: an in vitro study on canine arteries

Early systemic arterial hypotension is a common clinical feature of Pseudomonas septicemia. To determine if Pseudomonas aeruginosa endotoxin induces the release of endothelium-derived nitric oxide (EDNO), an endogenous nitrovasodilator, segments of canine femoral, renal, hepatic, superior mesenteric, and left circumflex coronary arteries were suspended in organ chambers (physiological salt solution, 95% O2/5% CO2, pH 7.4, 37oC) to measure isometric force. In arterial segments contracted with 2 µM prostaglandin F2a, Pseudomonas endotoxin (lipopolysaccharide (LPS) serotype 10(Habs) from Pseudomonas aeruginosa (0.05 to 0.50 mg/ml)) induced concentration-dependent relaxation of segments with endothelium (P<0.05) but no significant change in tension of arteries without endothelium. Endothelium-dependent relaxation in response to Pseudomonas LPS occurred in the presence of 1 µM indomethacin, but could be blocked in the coronary artery with 10 µM NG-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of nitric oxide synthesis from L-arginine. The inhibitory effect of L-NMMA on LPS-mediated vasorelaxation of the coronary artery could be reversed by exogenous 100 µM L-arginine but not by 100 µM D-arginine. These experiments indicate that Pseudomonas endotoxin induces synthesis of nitric oxide from L-arginine by the vascular endothelium. LPS-mediated production of EDNO by the endothelium, possibly through the action of constitutive nitric oxide synthase (NOSc), may decrease systemic vascular resistance and may be the mechanism of early hypotension characteristic of Pseudomonas septicemia.

Gap junctions in isolated rat aorta: evidence for contractile responses that exhibit a differential dependence on intercellular communication

Connexin43 (Cx43) is a major gap junction protein present in the Fischer-344 rat aorta. Previous studies have identified conditions under which selective disruption of intercellular communication with heptanol caused a significant, readily reversible and time-dependent diminution in the magnitude of a1-adrenergic contractions in isolated rat aorta. These observations have indentified a significant role for gap junctions in modulating vascular smooth muscle tone. The goal of these steady-state studies was to utilize isolated rat aortic rings to further evaluate the contribution of intercellular junctions to contractions elicited by cellular activation in response to several other vascular spasmogens. The effects of heptanol were examined (0.2-2.0 mM) on equivalent submaximal (»75% of the phenylephrine maximum) aortic contractions elicited by 5-hydroxytryptamine (5-HT; 1-2 µM), prostaglandin F2a (PGF2a; 1 µM) and endothelin-1 (ET-1; 20 nM). Statistical analysis revealed that 200 µM and 500 µM heptanol diminished the maximal amplitude of the steady-state contractile responses for 5-HT from a control response of 75 ± 6% (N = 26 rings) to 57 ± 7% (N = 26 rings) and 34.9 ± 6% (N = 13 rings), respectively (P<0.05), and for PGF2a from a control response of 75 ± 10% (N = 16 rings) to 52 ± 8% (N = 19 rings) and 25.9 ± 6% (N = 18 rings), respectively (P<0.05). In contrast, 200 µM and 500 µM heptanol had no detectable effect on the magnitude of ET-1-induced contractile responses, which were 76 ± 5.0% for the control response (N = 38 rings), 59 ± 6.0% in the presence of 200 µM heptanol (N = 17 rings), and 70 ± 6.0% in the presence of 500 µM heptanol (N = 23 rings) (P<0.13). Increasing the heptanol concentration to 1 mM was associated with a significant decrease in the magnitude of the steady-state ET-1-induced contractile response to 32 ± 5% (21 rings; P<0.01); further increasing the heptanol concentration to 2 mM had no additional effect. In rat aorta then, junctional modulation of tissue contractility appears to be agonist-dependent.

Studies on ATP-diphosphohydrolase nucleotide-binding sites by intrinsic fluorescence

Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10) and Desirée (ATPase/ADPase = 1) isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desirée and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desirée apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desirée isoapyrases.

Chronic intrathecal cannulation enhances nociceptive responses in rats

The influence of a chronically implanted spinal cannula on the nociceptive response induced by mechanical, chemical or thermal stimuli was evaluated. The hyperalgesia in response to mechanical stimulation induced by carrageenin or prostaglandin E2 (PGE2) was significantly increased in cannulated (Cn) rats, compared with naive (Nv) or sham-operated (Sh) rats. Only Cn animals presented an enhanced nociceptive response in the first phase of the formalin test when low doses were used (0.3 and 1%). The withdrawal latency to thermal stimulation of a paw inflamed by carrageenin was significantly reduced in Cn rats but not in Nv or Sh rats. In contrast to Nv and Sh rats, injection in Cn animals of a standard non-steroid anti-inflammatory drug, indomethacin, either intraperitoneally or into the spinal cord via an implanted cannula or by direct puncture of the intrathecal space significantly blocked the intensity of the hyperalgesia induced by PGE2. Cannulated animals treated with indomethacin also showed a significant inhibition of second phase formalin-induced paw flinches. Histopathological analysis of the spinal cord showed an increased frequency of mononuclear inflammatory cells in the Cn groups. Thus, the presence of a chronically implanted cannula seems to cause nociceptive spinal sensitization to mechanical, chemical and thermal stimulation, which can be blocked by indomethacin, thus suggesting that it may result from the spinal release of prostaglandins due to an ongoing mild inflammation.

Conventional and novel strategies in the treatment of adrenocortical cancer

Adrenocortical carcinoma is a highly malignant neoplasm with an incidence of two per million people per year. Several treatment strategies have resulted in temporary or partial tumor regression but very few cases have attained long survival. Surgical resection of the primary tumor and metastases is most effective. Several chemotherapeutic protocols have been employed with variable success. Mitotane (o,p'-DDD) is an adrenalytic drug effective in inducing a tumor response in 33% of patients treated. Mitotane requires metabolic transformation for therapeutic action. Tumors may vary in their ability to metabolize mitotane and the ability of tumors to transform mitotane may predict the clinical response to the drug. Preliminary data show a possible correlation between metabolic activity of neoplastic adrenocortical tissue and response to mitotane. We have attempted to develop mitotane analogs with enhanced adrenalytic effect. Compared to mitotane, a di-chloro compound, the bromo-chloro and di-bromo analogs appear to have a greater effect. Future approaches to the treatment of adrenocortical carcinoma are likely to be based on blocking or reversing the biological mechanisms of tumorigenesis. Angiogenic and chemotactic mechanisms may play a role in adrenal tumor growth and inhibition of these mechanisms may result in inhibition of tumor growth. New mitotane analogs with greater adrenalytic potential could be a promising approach to developing more effective and selective therapies for adrenal cancer. Alternative approaches should attempt to suppress tumor growth by means of compounds with anti-angiogenic and anti-chemotactic activity.

Bone marrow fibroblasts in patients with advanced lung cancer

In a previous study we demonstrated that the incidence of fibroblast colony-forming units (CFU-F) was very low in bone marrow primary cultures from the majority of untreated advanced non-small lung cancer patients (LCP) compared to normal controls (NC). For this reason, we studied the ability of bone marrow stromal cells to achieve confluence in primary cultures and their proliferative capacity following four continuous subcultures in consecutive untreated LCP and NC. We also evaluated the production of interleukin-1ß (IL-1ß) and prostaglandin E2 (PGE2) by pure fibroblasts. Bone marrow was obtained from 20 LCP and 20 NC. A CFU-F assay was used to investigate the proliferative and confluence capacity. Levels of IL-1ß and PGE2 in conditioned medium (CM) of pure fibroblast cultures were measured with an ELISA kit and RIA kit, respectively. Only fibroblasts from 6/13 (46%) LCP confluent primary cultures had the capacity to proliferate following four subcultures (NC = 100%). Levels of spontaneously released IL-1ß were below 10 pg/ml in the CM of LCP, while NC had a mean value of 1,217 ± 74 pg/ml. In contrast, levels of PGE2 in these CM of LCP were higher (77.5 ± 23.6 pg/ml) compared to NC (18.5 ± 0.9 pg/ml). In conclusion, bone marrow fibroblasts from LCP presented a defective proliferative and confluence capacity, and this deficiency may be associated with the alteration of IL-1ß and PGE2 production.

Antiproliferative effects of 1,25-dihydroxyvitamin D3 on breast cells: a mini review

The hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), the active form of vitamin D3, is an important regulator of calcium homeostasis, exerts antiproliferative effects on various cell systems and can induce differentiation in some kinds of hematopoietic cells. These effects are triggered by its receptor, vitamin D receptor (VDR), a phosphoprotein member of the nuclear receptor superfamily, which functions as a transcriptional factor. VDR binds as a heterodimer with retinoid X receptor (R X R) to hexameric repeats, characterized as vitamin D-responsive elements present in the regulatory region of target genes such as osteocalcin, osteopontin, calbindin-D28K, calbindin-D9K, p21WAF1/CIP1, TGF-ß2 and vitamin D 24-hydroxylase. Many factors such as glucocorticoids, estrogens, retinoids, proliferation rate and cell transformation can modulate VDR levels. VDR is expressed in mammary tissue and breast cancer cells, which are potential targets to hormone action. Besides having antiproliferative properties, vitamin D might also reduce the invasiveness of cancer cells and act as an anti-angiogenesis agent. All of these antitumoral features suggest that the properties of vitamin D could be explored for chemopreventive and therapeutic purposes in cancer. However, hypercalcemia is an undesirable side effect associated with pharmacological doses of 1,25-(OH)2D3. Some promising 1,25-(OH)2D3 analogs have been developed, which are less hypercalcemic in spite of being potent antiproliferative agents. They represent a new field of investigation.

Estrogen replacement therapy and cardioprotection: mechanisms and controversies

Epidemiological and case-controlled studies suggest that estrogen replacement therapy might be beneficial in terms of primary prevention of coronary heart disease (CHD). This beneficial effect of estrogens was initially considered to be due to the reduction of low density lipoproteins (LDL) and to increases in high density lipoproteins (HDL). Recent studies have shown that estrogens protect against oxidative stress and decrease LDL oxidation. Estrogens have direct effects on the arterial tissue and modulate vascular reactivity through nitric oxide and prostaglandin synthesis. While many of the effects of estrogen on vascular tissue are believed to be mediated by estrogen receptors alpha and ß, there is evidence for `immediate non-genomic' effects. The role of HDL in interacting with 17ß-estradiol including its esterification and transfer of esterified estrogens to LDL is beginning to be elucidated. Despite the suggested positive effects of estrogens, two recent placebo-controlled clinical trials in women with CHD did not detect any beneficial effects on overall coronary events with estrogen therapy. In fact, there was an increase in CHD events in some women. Mutations in thrombogenic genes (factor V Leiden, prothrombin mutation, etc.) in a subset of women may play a role in this unexpected finding. Thus, the cardioprotective effect of estrogens appears to be more complicated than originally thought and requires more research.

Design and applications of modified oligonucleotides

Oligonucleotides have a wide range of applications in fields such as biotechnology, molecular biology, diagnosis and therapy. However, the spectrum of uses can be broadened by introducing chemical modifications into their structures. The most prolific field in the search for new oligonucleotide analogs is the antisense strategy, where chemical modifications confer appropriate characteristics such as hybridization, resistance to nucleases, cellular uptake, selectivity and, basically, good pharmacokinetic and pharmacodynamic properties. Combinatorial technology is another research area where oligonucleotides and their analogs are extensively employed. Aptamers, new catalytic ribozymes and deoxyribozymes are RNA or DNA molecules individualized from a randomly synthesized library on the basis of a particular property. They are identified by repeated cycles of selection and amplification, using PCR technologies. Modified nucleotides can be introduced either during the amplification procedure or after selection.

Antagonism of the acute hemodynamic effects of captopril in decompensated congestive heart failure by aspirin administration

The concomitant use of angiotensin-converting enzyme inhibitors and aspirin may cause pharmacological antagonism. Hence we examined the effect of aspirin on the neurohormonal function and hemodynamic response to captopril in heart failure patients. Between April 1999 and August 2000, 40 patients were randomized into four equal groups: 1) captopril, 2) aspirin, 3) captopril-aspirin: captopril was given alone on the first day, followed by aspirin on the remaining days, and 4) aspirin-captopril: aspirin was given alone on the first day, followed by captopril on the remaining days. Hemodynamic, norepinephrine and prostaglandin measurements were performed pre- and post-medication for 4 days. Captopril (50 mg) was given orally every 8 h and 300 mg aspirin was given on the first day, and 100 mg/day thereafter. In the captopril group and only on the first day of captopril-aspirin, captopril produced increases in cardiac index (2.1 ± 0.6 to 2.5 ± 0.5 l min-1 m-2, P<0.0001), and reduced peripheral vascular resistance (1980 ± 580 to 1545 ± 506 dyn s-1 cm-5/m², P<0.0001) and pulmonary wedge pressure (20 ± 4 to 15 ± 4 mmHg, P<0.0001). In contrast, aspirin alone or associated with captopril showed no significant hemodynamic changes. Norepinephrine decreased (P<0.02) only in the captopril group. Prostaglandin levels did not differ significantly among groups. Thus, aspirin compromises the short-term hemodynamic and neurohormonal effects of captopril in patients with acute decompensated heart failure.

The L-arginine/nitric oxide/cyclic-GMP pathway apparently mediates the peripheral antihyperalgesic action of fentanyl in rats

There are only a few studies on the molecular mechanisms underlying the peripheral antihyperalgesic effect of opioids. The aim of this study was to investigate the molecular bases of the peripheral antihyperalgesic effect of fentanyl in a model of prostaglandin-induced chemical hyperalgesia. Prostaglandin E2 (1.4 nmol) injected into one hind paw of male Wistar rats (200-250 g, N = 6 in each experimental or control group) pretreated with indomethacin (2.5 mg/kg) potentiated the nocifensive response to formalin (1%) injection made 60 min later. Drugs applied locally 30 min after prostaglandin E2 induced the following effects: fentanyl (0.1-1.0 nmol) caused a dose-dependent reversal of the hyperalgesic state, naloxone (2 nmol) co-injected with fentanyl (1 nmol) completely reversed the antihyperalgesic effect, Nomega-nitro-L-arginine (NOARG, 0.05-0.2 µmol) in combination with fentanyl (1.0 nmol) caused a dose-dependent inhibition of the antihyperalgesic effect of fentanyl, co-administration of L-arginine (0.5 µmol) with NOARG (0.2 µmol) plus fentanyl (1.0 nmol) fully restored the antihyperalgesic effect, and the cyclic-GMP phosphodiesterase inhibitor UK-114,542-27 (5-[2-ethoxy-5-(morpholinylacetyl) phenyl]-1,6-dihydro-1-methyl-3-propyl-7H-pyrazolo [4,3-d]-pyrimidin-7-one methanesulfonate monohydrate; 0.5-2.0 µmol) potentiated a subeffective dose of fentanyl (0.1 nmol) in a dose-dependent manner. However, UK-114,542-27 (2.0 µmol) injected alone did not produce this antihyperalgesic effect. Systemically administered fentanyl (1.0 nmol, sc) did not cause antinociception. Taken together, these results support the view that fentanyl reverses prostaglandin E2-induced hyperalgesia, probably by activating an opioid receptor at the periphery, and furthermore the L-arginine/nitric oxide/cyclic-GMP pathway may mediate this peripheral effect of fentanyl.

Ionic radiocontrast inhibits endothelium-dependent vasodilation of the canine renal artery in vitro: possible mechanism of renal failure following contrast medium infusion

To determine if radiocontrast impairs vascular relaxation of the renal artery, segments (4-5 mm in length) of canine renal artery were suspended in vitro in organ chambers to measure isometric force (95% O2/5% CO2, at 37ºC). Arterial segments with and without endothelium were placed at the optimal point of their length-tension relation and incubated with 10 µM indomethacin to prevent synthesis of endogenous prostanoids. The presence of nonionic radiocontrast (iohexol, Omnipaque 350, 1 ml in 25 ml control solution, 4% (v/v)) did not alter endothelium-dependent relaxation to acetylcholine in rings precontracted with both norepinephrine and prostaglandin F2alpha (N = 6). When the rings were precontracted with prostaglandin F2alpha, the presence of ionic contrast did not inhibit the relaxation of the arteries. However, in canine renal arteries contracted with norepinephrine, the presence of ionic radiocontrast (diatrizoate meglumine and diatrizoate sodium, MD-76, 1 ml in 25 ml control solution, 4% (v/v)) inhibited relaxation in response to acetylcholine, sodium nitroprusside (N = 6 in each group), and isoproterenol (N = 5; P < 0.05). Rings were relaxed less than 50% of norepinephrine contraction. Following removal of the contrast, vascular relaxation in response to the agonists returned to normal. These results indicate that ionic radiocontrast nonspecifically inhibits vasodilation (both cAMP-mediated and cGMP-mediated) of canine renal arteries contracted with norepinephrine. This reversible impairment of vasodilation could inhibit normal renal perfusion and act as a mechanism of renal failure following radiocontrast infusion. In the adopted experimental protocol the isoproterenol-induced relaxation of renal arteries precontracted with norepinephrine was more affected, suggesting a pivotal role of the cAMP system.

An electronic pressure-meter nociception paw test for rats

The objective of the present investigation was to compare the sensitivity of an electronic nociceptive mechanical paw test with classical mechanical tests to quantify the intensity variation of inflammatory nociception. The electronic pressure-meter test consists of inducing the hindpaw flexion reflex by poking the plantar region with a polypropylene pipette tip adapted to a hand-held force transducer. This method was compared with the classical von Frey filaments test and with the rat paw constant pressure test, a modification of the Randall and Selitto test developed by our group. When comparing the three methods, the electronic pressure-meter and the rat paw constant pressure test, but not the von Frey filaments test, detected time vs treatment interactions in prostaglandin E2 (PGE2)-induced hypernociception. Both methods also detected the PGE2-induced hypernociception in dose- (50-400 ng/paw) and time- (1-4 h) dependent manners, and time vs treatment interactions induced by carrageenin (25-400 µg/paw). Furthermore, the electronic pressure-meter test was more sensitive at early times, whereas the constant pressure test was more sensitive at later times. Moreover, the electronic pressure-meter test detected the dose-dependent antinociceptive effect of local indomethacin (30-300 µg/paw) and dipyrone (80-320 µg/paw) on carrageenin- (200 µg/paw) and PGE2- (100 ng/paw) induced hypernociception, respectively, and also detected the ineffectiveness of indomethacin (300 µg) on the effect of PGE2. Our results show that the electronic pressure-meter provides a sensitive, objective and quantitative mechanical nociceptive test that could be useful to characterize new nociceptive inflammatory mediators and also to evaluate new peripheral analgesic substances.

An electronic pressure-meter nociception paw test for mice

The aim of the present investigation was to describe and validate an electronic mechanical test for quantification of the intensity of inflammatory nociception in mice. The electronic pressure-meter test consists of inducing the animal hindpaw flexion reflex by poking the plantar region with a polypropylene pipette tip adapted to a hand-held force transducer. This method was compared to the classical von Frey filaments test in which pressure intensity is automatically recorded after the nociceptive hindpaw flexion reflex. The electronic pressure-meter and the von Frey filaments were used to detect time versus treatment interactions of carrageenin-induced hypernociception. In two separate experiments, the electronic pressure-meter was more sensitive than the von Frey filaments for the detection of the increase in nociception (hypernociception) induced by small doses of carrageenin (30 µg). The electronic pressure-meter detected the antinociceptive effect of non-steroidal drugs in a dose-dependent manner. Indomethacin administered intraperitoneally (1.8-15 mg/kg) or intraplantarly (30-300 µg/paw) prevented the hypersensitive effect of carrageenin (100 µg/paw). The electronic pressure-meter also detected the hypernociceptive effect of prostaglandin E2 (PGE2; 10-100 ng) in a dose-dependent manner. The hypernociceptive effect of PGE2 (100 ng) was blocked by dipyrone (160 and 320 µg/paw) but not by intraplantar administration of indomethacin (300 µg/paw). The present results validate the use of the electronic pressure-meter as more sensitive than the von Frey filaments in mice. Furthermore, it is an objective and quantitative nociceptive test for the evaluation of the peripheral antinociceptive effect of anti-inflammatory analgesic drugs, which inhibit prostaglandin synthesis (indomethacin) or directly block the ongoing hypernociception (dipyrone).