Paolo Bellavite

The superoxide-forming enzymatic system of phagocytes

The formation of oxygen-derived free radicals by the phagocytes (neutrophils, eosinophils, monocytes and macrophages) is catalysed by a membrane-bound NADPH oxidase which is dormant in resting cells and becomes activated during phagocytosis or following interaction of the cells with suitable soluble stimulants. This enzyme is under investigation in many laboratories but its molecular structure remains to be clarified. Possible components such as flavoproteins, cytochrome b558, and quinones have been proposed on the basis of enzyme purification studies, effects of inhibitors, kinetic properties and analysis of genetic defects of the oxidase. An extensive discussion of the evidence for the participation of these constituents is reported. On the basis of the available information on the structure and the catalytic properties of the NADPH oxidase, a series of possible models of the electron-transport chain from NADPH to O2 is presented. Finally, the triggering mechanism of the respiratory burst is discussed, with particular reference to the stimulus-response coupling and the final modification(s) of the oxidase (phosphorylation, assembly, change of lipid environment, etc.) which are involved in its activation.

Defective platelet response to arachidonic acid and thromboxane A2 in subjects with PlA2 polymorphism of β3 subunit (glycoprotein IIIa)

The membrane complex αIIbβ3 is the major receptor for fibrinogen and is involved in platelet adhesion and aggregation. Evidence has been presented that the PlA2 allele of the β3 PlA1/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 PlA1/A1 polymorphism, 12 subjects with PlA1/A2 polymorphism and two subjects with PlA2/A2 polymorphism. Subjects with PlA1/A2 polymorphism or with PlA2/A2 polymorphism showed significantly lower platelet responses as compared with PlA1/A1 subjects when either arachidonic acid or the thromboxane A2 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 PlA2 allele. Finally, using a flow‐cytometric evaluation and determining the β‐thromboglobulin plasma levels, we did not find any evidence of a PlA2 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 PlA2 allele is associated with a platelet functional deficiency, specifically linked to the activation of the fibrinogen receptor by thromboxane A2.

Protein kinase C phosphorylates a component of NADPH oxidase of neutrophils

NADPH oxidase Cytochrome b−245 Phosphorylation Protein kinase Neutrophil activation Respiratory burst

Immunology and Homeopathy. 2. Cells of the Immune System and Inflammation

Here we describe the results of some experimental laboratory studies aimed at verifying the efficacy of high dilutions of substances and of homeopathic medicines in models of inflammation and immunity. Studies carried out on basophils, lymphocytes, granulocytes and fibroblasts are reviewed. This approach may help to test under controlled conditions the main principles of homeopathy such as ‘similarity’ of drug action at the cellular level and the effects of dilution/dynamization on the drug activity. The current situation is that few and rather small groups are working on laboratory models for homeopathy. Regarding the interpretation of data in view of the simile principle, we observe that there are different levels of similarity and that the laboratory data give support to this principle, but have not yet yielded the ultimate answer to the action mechanism of homeopathy. Evidence of the biological activity in vitro of highly diluted-dynamized solutions is slowly accumulating, with some conflicting reports. It is our hope that this review of literature unknown to most people will give an original and useful insight into the ‘state-of-the-art’ of homeopathy, without final conclusions ‘for’ or ‘against’ this modality. This kind of uncertainty may be difficult to accept, but is conceivably the most open-minded position now.

Study of platelet adhesion in patients with uncomplicated hypertension.

Objective To evaluate platelet function in patients with essential hypertension by sensitive methods investigating platelet adhesion and expression of some platelet glycoproteins (GP), namely GPIIb/llla (CD41/α2β3) and GMP- 140 (CD62/P-selectin/PADGEM). Other markers of platelet (β-thromboglobulin) and endothelium activation (von Willebrand factor) were also measured. Methods We studied 21 uncomplicated essential hypertensive patients and 20 healthy normotensive control subjects, non-smokers, matched for age and sex. Resting and stimulated platelet adhesion was performed with a colorimetric method using the activity of platelet acid phosphatase for the determination of the number of platelets adhering to human plasma- or fibrinogen-coated microwells. Platelet activation was characterized by flow cytometric measurement of GPIIb/llla and GMP-140 in whole blood and washed platelets suspensions, with antihuman fluorescent monoclonal antibodies. Results Thrombin-stimulated platelet adhesion to human plasma-coated microwells was significantly higher in hypertensive patients than in control subjects (0.05 U/ml thrombin: 13.4 ± 1.0 versus 7.7 ± 0.6% adhesion; 0.1 U/ml thrombin: 19.4 ±2.3 versus 12.6 ± 1.8%; means ± SEM), whereas platelet adhesion to fibrinogen-coated wells did not differ in the two groups. Flow-cytometry analysis of whole blood demonstrated a significantly increased expression of GMP-140 in hypertensive patients compared with normal subjects (percentage of CD62+ platelets: 7.3 ± 1.2 versus 3.7 ± 1; means ± SEM), whereas the expression of GPIIb/llla did not differ in the two groups (percentage of CD41a+ platelets: 72.5 ± 4.5 versus 70.4 ± 3.9). Moreover, flow cytometry showed an increased size of platelets in hypertensive patients compared with that in control subjects (forwards scattering: 46.5 ± 1.5 versus 38.9 ±1.1; means ± SEM). Flow-cytometric evaluation of washed platelet suspensions showed no statistically significant differences between the expression of GMP-140 and GPIIb/llla in the two groups. β-Thromboglobulin plasma levels were higher in hypertensive patients than they were in normal subjects (36.3 ± 2.0 versus 28.2 ± 1.3 ng/ml; means ± SEM). Von Willebrand factor plasma levels were not significantly different in the two groups (101.2 ± 10.3 versus 86.3 ± 5.6 U/dl). Conclusions These findings provide further evidence that there is a significant, albeit weak, platelet activation in hypertensive patients compared with normal subjects.

Differential response of human basophil activation markers: a multi-parameter flow cytometry approach.

BackgroundBasophils are circulating cells involved in hypersensitivity reactions and allergy but many aspects of their activation, including the sensitivity to external triggering factors and the molecular aspects of cell responses, are still to be focused. In this context, polychromatic flow cytometry (PFC) is a proper tool to investigate basophil function, as it allows to distinguish the expression of several membrane markers upon activation in multiple experimental conditions.MethodsCell suspensions were prepared from leukocyte buffy coat of K2-EDTA anticoagulated blood specimens; about 1500-2500 cellular events for each tested sample, gated in the lymphocyte CD45dim area and then electronically purified as HLADRnon expressing/CD123bright, were identified as basophilic cells. Basophil activation with fMLP, anti-IgE and calcium ionophore A23187 was evaluated by studying up-regulation of the indicated membrane markers with a two-laser six-color PFC protocol.ResultsFollowing stimulation, CD63, CD13, CD45 and the ectoenzyme CD203c up-regulated their membrane expression, while CD69 did not; CD63 expression occurred immediately (within 60 sec) but only in a minority of basophils, even at optimal agonist doses (in 33% and 14% of basophils, following fMLP and anti-IgE stimulation respectively). CD203c up-regulation occurred in the whole basophil population, even in CD63non expressing cells. Dose-dependence curves revealed CD203c as a more sensitive marker than CD63, in response to fMLP but not in response to anti-IgE and to calcium ionophore.ConclusionUse of polychromatic flow cytometry allowed efficient basophil electronic purification and identification of different behaviors of the major activation markers. The simultaneous use of two markers of activation and careful choice of activator are essential steps for reliable assessment of human basophil functions.

Neutrophil migration, oxidative metabolism, and adhesion in elderly and young subjects

Objective. To evaluate neutrophil functions in the elderly.Methods. We investigated the PMN migration in vivo and PMN superoxide production and adhesion in response to a variety of compounds; PMN have been isolated both from blood and from a skin experimental exudate (obtained by Senns skin window technique) of 25 normal elderly and of 25 normal young control subjects.Results. No difference was found in PMN migration in vivo (62.9±21.3×106 and 65.5±9.1×106 PMN/cm2/24 hours in elderly and young subjects respectively), neither were different the adhesion under basal condition and after some stimuli and the superoxide production in basal condition and in response to STZ and PMA in two groups. In elderly subjects superoxide production, in response to fMLP, markedly resulted lower than in young controls both by circulating PMNs (3.6±2.7 and 9.3±3.3 nMOLES O2−/106 PMN respectively, p<0.0001) and by exudate PMNs (13.6±4.3 and 19.4±6 nMOLES O2−106 PMNs respectively, p<0.005).Conclusion. Many PMN functions in the elderly do not differ from young people, suggesting that the overall defense function of these cells is not affected by aging. The only parameter that we have found to be different between the two groups is the poor superoxide production after fMLP stimulus of PMNs. The stimulus- and function-specificity of this defect in PMNs from elderly subjects indicates the existence of a dysregulation of the signal transduction pathway distal to fMLP receptor and proximal to NADPH oxidase activation.

Immunology and homeopathy. 3. Experimental studies on animal models.

A search of the literature and the experiments carried out by the authors of this review show that there are a number of animal models where the effect of homeopathic dilutions or the principles of homeopathic medicine have been tested. The results relate to the immunostimulation by ultralow doses of antigens, the immunological models of the ‘simile’, the regulation of acute or chronic inflammatory processes and the use of homeopathic medicines in farming. The models utilized by different research groups are extremely etherogeneous and differ as the test medicines, the dilutions and the outcomes are concerned. Some experimental lines, particularly those utilizing mice models of immunomodulation and anti-inflammatory effects of homeopathic complex formulations, give support to a real effect of homeopathic high dilutions in animals, but often these data are of preliminary nature and have not been independently replicated. The evidence emerging from animal models is supporting the traditional ‘simile’ rule, according to which ultralow doses of compounds, that in high doses are pathogenic, may have paradoxically a protective or curative effect. Despite a few encouraging observational studies, the effectiveness of the homeopathic prevention or therapy of infections in veterinary medicine is not sufficiently supported by randomized and controlled trials.

Immunology and homeopathy. 1. Historical background.

Homeopathy was born as an experimental discipline, as can be seen from the enormous amount of homeopathic data collected over more than two centuries. However, the medical tradition of homeopathy has been separated from that of conventional science for a long time. Conventional scientific wisdom dictates that homeopathy should have no effect above placebo but experiments on ultra-high dilutions of solutes together with some clinical data suggest the intriguing possibility that it might do in some circumstances. Today, an osmotic process between disciplines, previously seen as in conflict, is facilitated because over the last few decades homeopathy has initiated the methods of current medical science and a substantial number of experimental studies—at molecular, cellular and clinical levels—are available. One area of dialogue and of common progress is that of inflammation and immunity, probably because these are closely related to the traditional ‘vital force’ of the bodys self-healing power. In a series of papers we review the historical origins of homeopathy, the laboratory and animal models related to the field of immunopharmacology, the clinical evidence in favor and against the use of homeopathy in the inflammatory diseases and the hypotheses regarding its action mechanism(s). Finally, we will enlighten the specific characteristics of the homeopathic approach, which places great emphasis on identifying a cure for the whole organism.

Immunology and Homeopathy. 5. The Rationale of the ‘Simile’

The foundation of homeopathic medicine is the ‘Similia Principle’, also known as the ‘Principle of Similarity’ or also as the ‘Simile’, which reflects the inversion of pharmacological effects in healthy subjects as compared with sick ones. This article describes the inversion of effects, a widespread medical phenomenon, through three possible mechanisms: non-linearity of dose–response relationship, different initial pathophysiological states of the organism, and pharmacodynamics of body response to the medicine. Based on the systemic networks which play an important role in response to stress, a unitary and general model is designed: homeopathic medicines could interact with sensitive (primed) regulation systems through complex information, which simulate the disorders of natural disease. Reorganization of regulation systems, through a coherent response to the medicine, could pave the way to the healing of the cellular, tissue and neuro-immuno-endocrine homeodynamics. Preliminary evidence is suggesting that even ultra-low doses and high-dilutions of drugs may incorporate structural or frequency information and interact with chaotic dynamics and physical-electromagnetic levels of regulation. From the clinical standpoint, the ‘simile’ can be regarded as a heuristic principle, according to which the detailed knowledge of pathogenic effects of drugs, associated with careful analysis of signs and symptoms of the ill subject, could assist in identifying homeopathic remedies with high grade of specificity for the individual case.