Cytokine and nitric oxide levels in a rat model of immunologic protection from adjuvant-induced arthritis.

In the present study we investigated the correlation between the progression of adjuvant arthritis induced by Mycobacterium butyricum and the production of nitric oxide and some pro- and anti-inflammatory cytokines in arthritic rats and in rats treated with low intra-peritoneal doses of Mycobacterium 3 and 10 days after arthritis induction. The intra-peritoneal administration of Mycobacterium antigen significantly inhibited disease development. Compared to healthy rats, a rise in serum and peritoneal pro-inflammatory cytokines was observed in all arthritic rats already from the 14 day. The treatment with intra-peritoneal Mycobacterium was associated with a significant reduction in IL-6 serum concentrations and a slight decrease of IFN-gamma production by peritoneal macrophages. Nitrite/nitrate plasma and peritoneal levels were significantly higher in all arthritic rats. Intra-peritoneal administration of Mycobacterium caused a further increase in nitrite/nitrate plasma concentrations, while no differences were evident in nitric oxide production by peritoneal macrophages. From our data it is evident that among the variables here investigated, IL-6 seems to be the more representative marker of the disease and of the treatment effect. A possible role of nitric oxide as a modulator rather than a direct mediator in this model of inflammation is discussed.

Defective platelet response to arachidonic acid and thromboxane A(2) in subjects with Pl(A2) polymorphism of beta(3) subunit (glycoprotein IIIa).

The membrane complex alpha(IIb)beta(3) is the major receptor for fibrinogen and is involved in platelet adhesion and aggregation. Evidence has been presented that the Pl(A2) allele of the beta(3) Pl(A1/A2) gene polymorphism might be an independent risk factor for coronary thrombosis, but the matter is still controversial. We investigated the relationship between this polymorphism and possible alterations of platelet functions in vitro. The platelet adhesion to fibrinogen-coated microplate wells and the aggregation induced by several different agonists were tested in 63 healthy volunteers, among them, 49 subjects with Pl(A1/A1) polymorphism, 12 subjects with Pl(A1/A2) polymorphism and two subjects with (PlA2/A2) polymorphism. Subjects with PlA1/A2 polymorphism or with Pl(A2/A2) polymorphism showed significantly lower platelet responses as compared with Pl(A1/A1) subjects when either arachidonic acid or the thromboxane A(2) analogue, U46619, were used as agonists. In resting condition and after thrombin or ADP stimulation, platelet function was normal in all the subjects. An increased sensitivity to the anti-aggregatory effect of acetylsalicylic acid was observed in platelets from subjects with the Pl(A2) allele. Finally, using a flow-cytometric evaluation and determining the beta-thromboglobulin plasma levels, we did not find any evidence of a Pl(A2) platelet hyper-reactivity ex vivo. Our findings are not consistent with the hypothesis that the purported increase of cardiovascular risk in these subjects may be as a result of platelet hyperactivation. On the contrary, the Pl(A2) allele is associated with a platelet functional deficiency, specifically linked to the activation of the fibrinogen receptor by thromboxane A(2).

Influence of an acute exercise on neutrophil and platelet adhesion, nitric oxide plasma metabolites in inactive and active subjects.

In this work we studied the influence of an acute exercise either on nitrite/nitrate plasma levels or on neutrophil and platelet adhesion in inactive and active subjects. Twelve healthy subjects (6 inactives and 6 actives) exercised on a racing cycle ergometer performing stepwise increases in intensity until reaching, within 5 min, a heart rate of 150 beats x min(-1) which represents an oxygen consumption of about 75 % of the individual maximum rate of oxygen uptake. From peripheral venous blood samples (drawn from all subjects before, immediately after the end of exercise, and 1 hour later) neutrophils and platelets were isolated to test plate adhesion, and nitrite/nitrate concentrations were measured in the plasma. Immediately after the acute exercise, in active subjects we observed a significant decrease in the percentage of neutrophil adhesion (7.96+/-2.38 vs. 14.10+/-3.14), associated with an increase in nitrite/nitrate plasma levels (81.38+/-10.76 vs. 41.08+/-8.13 micromol x l(-1)), restored by a 40 min pre-incubation with NG-nitro-L-arginine methyl ester (L-NAME). In unstimulated platelets we observed a significant lower percentage of platelet adhesion in active subjects compared to inactives after exercise. With thrombin or adenosine 5'-diphosphate as agonists platelet adhesion did not result significantly different in active subjects compared to inactives. In conclusion, our data show that physical exercise can induce changes in some cell activities, even if transient, and favour the generation of nitric oxide. The lower adhesion of neutrophils and platelets induced by regular exercise could be an important goal in the prevention of vascular and inflammatory diseases.

Dual effects of a homeopathic mineral complex on carrageenan-induced oedema in rats.

Carrageenan oedema, a classical experimental model commonly used to test activity of anti-inflammatory drugs, was used to evaluate the therapeutic activity of a low-potency mineral complex (MC). The MC was administered in the right plantar surface of albino rats 60 min before, simultaneously and 30 min after injection of carrageenan, an irritant which causes a local, transitory increase of fluid volume. The administration of the MC 60 min before the injection of carrageenan primed the animal to enhanced inflammatory response to the irritant. The administration of MC contemporarily to carrageenan did not modify the kinetic and the extent of the oedema, while the administration of the MC 30 min after the induction of the oedema significantly reduced the early phase of the inflammatory reaction. This indicated that the therapeutic action of this MC is not due to conventional anti-inflammatory effect but to activation of endogenous regulatory mechanisms, a phenomenon which may be regarded as a simple application of the 'similia rule'.

Dual effects of a homeopathic mineral complex on carrageenan-induced oedema in rats

Carrageenan oedema, a classical experimental model commonly used to test activity of anti-inflammatory drugs, was used to evaluate the therapeutic activity of a low-potency mineral complex (MC). The MC was administered in... (AU)

A microplate-based colorimetric assay of the total peroxyl radical trapping capability of human plasma.

We developed a colorimetric assay estimating the radical-scavenging activity of human plasma. The test is based on a measure, in 96-well microplates at 450 nm, of the bleaching of carotenoid crocin by peroxyl radicals generated during thermal decomposition of 2, 2'-azobis-(2-amidinopopane) dihydrochloride (ABAP). The inhibition of this bleaching is a function of the antioxidant power of substances added to incubation mixture. We determined the optimal conditions for a sensitive, rapid, and reproducible assay of 50% inhibitory capacity (IC50) of a range of antioxidant substances and of plasma. Only a total of 200 microl of plasma is required in a complete dose-inhibition curve. The IC50 of normal human plasma resulted of 2.70 microl of plasma/250 microl assay volume. The total antioxidant capability (TAC) of plasma was defined as the reciprocal of IC50 and its value in a group of 19 healthy adults resulted in 0. 369 +/- 0.06. Intraassay and interassay coefficients of variation of plasma TAC were 6.13 and 4.80%, respectively. Measurement of samples with different uric acid concentration showed that antioxidant activity of uric acid accounts for approximately two-thirds of TAC.

Effect of Traumeel S, a homeopathic formulation, on blood-induced inflammation in rats.

OBJECTIVE: To evaluate the activity of Traumeel S (TRS), a homeopathic formulation containing Arnica montana and other plant extracts and minerals on an animal model of traumatic inflammation. DESIGN: TRS and individual components thereof were administered locally to rats 1 h before hind-paw injection with 0.1 ml of homologous blood and the development of oedema was measured over five hours. In each experiment, a control group was treated with saline. MAIN OUTCOME MEASURES: Paw volume of each rat was measured before oedema and 1, 3, and 5 h after oedema induction. Serum levels of IL-6 were determined at hour 5. RESULTS: The decrease of paw oedema, associated with the process of healing, was more rapid in rats treated with TRS (P < 0.05 after 3 h and P < 0.01 after 5 h). Similar effects were also induced by separate injection of most, but not all, TRS ingredients. The efficacy of complete mixture of TRS was higher than the combination of a selection of active components. TRS also reduced oedema development when administered after the oedema induction. The therapeutic effect of TRS was associated with a significant decrease of systemic interleukin-6 production. CONCLUSION: TRS seems to act by speeding up the healing process instead of blocking the development of oedema from the beginning. Moreover, its effect cannot be considered as the 'sum' of its active components and probably a synergistic interaction occurs to determine the final effect.

[Homeopathy in the perspective of scientific research]. / L'omeopatia nella prospettiva della ricerca scientifica.

A significant portion of the traditional concepts of homeopathy (similia principle, experimentation on healthy humans, the cure of whole person, the use of minimum doses or high dilution/potency of medicines) are amenable to investigations conducted according to criteria accepted by biomedical science. Even though many randomized and controlled clinical studies seem to demonstrate the efficacy of some homeopathic medicines and their superiority to placebo, other trials have given negative results. A definite clear answer is not yet possible due to the scarce quality of some published reports, to lack of reproduction by independent investigators and to the uncertainty regarding the methodologies to be used for testing the claims of homeopathy. As regards the possible physiopathological, biophysical and pharmacological explanations for the action of homeopathic remedies, there are models which tend to set the similia principle as a general expression of the action-reaction principle, within the context of dynamic systems theory. The clarification of the more controversial aspects regarding dilution/potency of medicines remains tied to several promising developments in physics of condensed matter, in chaos theory and in biophysics.

Study on paradoxical effects of NSAIDs on platelet activation.

We recently described a stimulatory effect of high doses (> 100 mumol/L) diclofenac on platelet adhesion. In this study we extend our research to the possible biochemical mechanisms of the observed effects, to other non steroidal anti-inflammatory drugs (NSAIDs) (flurbiprofen, indomethacin, acetylsalicylic acid, ibuprofen, nitrofenac and nitroflurbiprofen) and to the effect of high doses diclofenac and flurbiprofen on platelet aggregation. We observed that high doses of diclofenac and of flurbiprofen, but not of the other tested NSAIDs, increased platelet adhesion at doses ranging from 100 to 500 mumol/L, an effect completely removed by the 12-lipoxygenase-inhibitor nordihydroguaiaretic acid. Moreover, they had no pro-aggregating effect, inhibiting platelet aggregation induced by 10 mumol/L arachidonic acid and dose-dependently increasing the [Ca2+]i. Finally, whereas no basal nitric oxide release by washed platelets was detected, when platelets were incubated by 500 mumol/L diclofenac or flurbiprofen, the production of nitric oxide, as measured by amounts of nitrite released, was 4.4 +/- 0.5 and 3.8 +/- 0.4 pmol/5 x 10(8) platelets/min, respectively. Our data indicate that high doses diclofenac and flurbiprofen are promoters of the early phases of platelet activation, probably through the 12-lipoxygenase pathway.

A scientific reappraisal of the 'principle of similarity'.

In the history of therapeutics, the 'principle of similarity'--the treatment of 'same by same' or of 'like by like'--may be traced back to a number of medical traditions, including the systems of Hippocrates, Paracelsus and Hahnemann. Although in recent years we have witnessed a renaissance of interest in traditional medicines and 'holistic' medical practices, the reliability of the principle of similarity has still to be demonstrated on experimental grounds, and very few studies have been conducted to understand the underlying mechanism(s). Acceptance of this phenomenon requires supporting evidence of possible mechanisms and high-quality studies exploring its effectiveness in clinical medicine. The aim of this work is to provide a rational approach to the analysis of the various aspects of this historical yet also modern medical principle, in order to construct a plausible framework of ideas capable of facilitating further basic and clinical research into this field.

Studies of skin-window exudate human neutrophils: increased resistance to pentoxifylline of the respiratory burst in primed cells.

Human neutrophils were isolated both from peripheral blood (PB) and from aseptic inflammatory exudates obtained by the Senn's skin window technique (SW). The respiratory burst (O2- production) induced by in response to n-formyl-methionyl-lencyl-phenylalanine (fMLP) and by serum-treated zymosan (STZ) was investigated using a microplate assay. SW neutrophils were primed to enhanced fMLP-dependent O2- production in response to fMLP but not to STZ. Pentoxifylline, a cAMP-elevating drug, dose-dependently inhibited the respiratory burst in any experimental condition, but the dose-effect curves were markedly different according the stimulant used and the source of the cells. With fMLP as stimulant, a significant inhibition of the O2- production by PB neutrophils was obtained using doses of 10 micrograms/ml, while SW neutrophils were inhibited only by doses equal or higher than 100 micrograms/ml. With STZ as stimulant, the inhibition of the respiratory burst of PB neutrophils and of SW neutrophils was obtained only with doses higher than 400 micrograms/ml and 1 mg/ml respectively. Pentoxifylline dose-dependently (10 micrograms/ml to 1 mg/ml) increased the intracellular adenosine 3'-5'-cyclic monophosphate (cAMP) to the same extent in SW and in PB neutrophils. These data indicate that the priming of neutrophil oxidative metabolism by in vivo inflammation is associated with an increase in the resistance to the regulating effect of cAMP on the fMLP-dependent activation pathway of NADPH oxidase. The fact that therapeutic doses of pentoxifylline do not inhibit the respiratory burst of primed neutrophils may have relevance in the interpretation of the clinical effects of this drug.

Specific and long-lasting suppression of rat adjuvant arthritis by low-dose Mycobacterium butyricum.

We have tested the therapeutic effect of intraperitoneal injections of Mycobacterium butyricum on the development of adjuvant arthritis in rats and we have explored the specificity and the duration of effectivity of this treatment. Rats with induced arthritis were injected intraperitoneally with the causative antigen, Mycobacterium butyricum, at concentrations 10 times lower than the inducing one, on the 3rd and 10th day after arthritis induction. The severity of the disease was assessed on the basis of physical (arthritis index, paw swelling) and biochemical (serum interleukin-6) parameters. The treatment with Mycobacterium butyricum led to a significant suppression of adjuvant-induced arthritis. This therapeutic effect was both antigen-specific, because intraperitoneal aspecific inflammation did not prevent the disease, and long-lasting. The results obtained in this model confirm the possibility of modulating the autoimmune process even when the immunological response is already triggered, suggesting new therapeutic strategies, more suitable than preventive vaccination, in human autoimmune diseases.

Anti-inflammatory activity of polyphosphazene-based naproxen slow-release systems.

A biocompatible and biodegradable polyphosphazene bearing phenylalanine ethyl ester, imidazole and chlorine (10.7:1:2.5 molar ratio) as substituents of the phosphorus atoms of the polymer backbone was studied for the preparation of polymeric naproxen slow-release systems. Discs 2.5 cm in diameter and 0.5 mm (thin) or 0.65 mm (thick), loaded, respectively, with 20 and 13.5% naproxen, showed different drug release kinetics, the thin matrices releasing naproxen at a faster rate and for a shorter time. In-vivo studies in rats demonstrated the pharmacological efficacy of these two different delivery systems in the inhibition of acute or chronic inflammatory diseases. Subcutaneous implantation of the thin matrices in rats was found to reduce carrageenan oedema induced both 1 h and 7 days after implantation. Rats implanted with thick matrices showed a reduction in chronic inflammation caused by adjuvant arthritis. Approximately 78% inhibition of arthritic oedema was found 28 days after subcutaneous administration of the matrices whereas 28.7% inhibition was found after daily oral administration of naproxen. Blood levels of naproxen in arthritic rats after matrix implantation showed the presence of drug up to day 28. These positive results have encouraged us to study a controlled-release system suitable for use in man.

Dual effects of diclofenac on human platelet adhesion in vitro.

The effect of two non-steroidal anti-inflammatory drugs on the adhesion function of human platelets was evaluated. Platelets isolated from healthy human subjects were treated for 10 min with the indicated drugs and then incubated in fibrinogen-coated microwell plates in the absence or in the presence of ADP (10 microM) and thrombin (0.05 U/ml). After 1 h of incubation, adherent platelets were measured using an enzymatic assay. ADP- and thrombin-stimulated adhesion was significantly inhibited by high doses ( > 500 microM) of diclofenac, while doses ranging from 50 to 300 microM stimulated adhesion in the absence of agonists (resting platelets). A similar stimulatory effect on platelet adhesion was observed also with 200-500 microM flurbiprofen. Moreover, immunocytofluorimetry demonstrated that diclofenac dose-dependently (100-500 microM) induced the expression of GMP-140 and increased the expression of GPIIb/IIIa on the membrane of unstimulated platelets. High doses ( > 500 microM) of this drug inhibited thrombin-stimulated expression of GPIIb/IIIa and GMP-140.

Anti-inflammatory potency and gastrointestinal toxicity of a new compound, nitronaproxen.

Naproxen and its derivative nitronaproxen at the doses of 5 and 10 mg kg-1 were compared for their acute anti-inflammatory efficacy in a carrageenan oedema model and gastrointestinal toxicity in rats. Moreover, the effects of the two drugs were evaluated in the adjuvant arthritis, after chronic doses of 4 and 8 mg kg-1 administered orally for 18 days. The oedema reduction was maintained much longer (until 5 h) with nitronaproxen; the inhibition of arthritis was 50% or more with both doses of the examined drugs. From the histological examination of the stomachs, an extensive mucosal vasocongestion and haemorrhagic lesions have been observed in some rats treated with naproxen. The percentages of animals with ulcers were 50, 100 and 10 with naproxen 6 and 18 mg kg-1 and nitronaproxen 54 mg kg-1 respectively. A better gastrointestinal tolerability has been observed in arthritic and oedemic rats treated with nitronaproxen compared to naproxen: this could be due to the presence of nitric oxide that acts in maintaining the tissue perfusion and integrity.