Riccardo Ortolani

The F2-Isoprostane 8-epiprostaglandin F2α increases platelet adhesion and reduces the antiadhesive and antiaggregatory effects of NO

F2-isoprostanes are prostaglandin (PG) isomers produced in vivo through free radical-catalyzed peroxidation of arachidonic acid, which may affect platelet function. The current study investigated the effects of 8-epiprostaglandin F2alpha (8-epi-PGF2alpha) on critical events of platelet activation. A dose-dependent increase in platelet adhesion to fibrinogen- and plasma-coated microwells by 8-epi-PGF2alpha (1 to 1000 nmol/L) was observed when resting platelets (plasma from 1.3+/-0.2% to 5.5+/-0.2%, EC50 of 48 nmol/L; fibrinogen from 3.3+/-0.3% to 6.4+/-0.2%, EC50 of 35 nmol/L; mean+/-SEM, n=8, P<0.001) and thrombin-stimulated human platelets were used. The expression of the adhesion molecule glycoprotein IIb/IIIa was increased by 10 to 1000 nmol/L 8-epi-PGF2alpha in resting platelets (from 64.8+/-2.1% to 83.9+/-1.3%; n=5, P<0.01) and in stimulated platelets. The secretion of the glycoprotein GMP-140 increased only in the presence of both thrombin and 10 to 1000 nmol/L 8-epi-PGF2alpha (from 48.5+/-3.1% to 63.1+/-2.0%, P<0.05). The antiaggregatory effects of both the NO donor NOR-3 (basal, 21.4+/-4.6%; with 8-epi-PGF2alpha, 30.8+/-6.9%; n=14, P<0.05) and endothelial cells that release NO (basal, 18.5+/-4.6%; with 8-epi-PGF2alpha, 30.7+/-5.3%; n=15, P<0.001) were also reduced. All of these effects were prevented by the thromboxane receptor antagonist GR32191 but not affected by acetylsalicylic acid. An increase in free intracellular calcium concentration, measured with the use of fura 2, was observed with 8-epi-PGF2alpha. In conclusion, F2-isoprostanes may participate in oxidative injury by inducing platelet activation and by reducing the antiplatelet activity of NO: increased platelet adhesiveness and expression of the fibrinogen receptor are induced by nanomolar amounts of 8-epi-PG-F2alpha. Platelet secretion and aggregation can also be induced in the presence of platelet agonists.

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.

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.

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.

Expression of CD27 and CD23 on peripheral blood B lymphocytes in humans of different ages.

BACKGROUND Due to the fact that the coexpression of CD23 and CD27 has been reported to occur in B lymphocytic leukaemic clones and that there is debate about CD23 expression on memory B cells, we evaluated the behaviour of naive B cells (CD23-/CD27-) and memory B cells (CD27+) in the peripheral blood of a large number of humans of all ages. B cells were also distinguished into B2 (CD5-) and B1-a cells (CD5+). METHODS The cell surface expression of CD19, CD5, CD23 and CD27 was assessed on peripheral blood lymphocytes from 1,427 subjects of all ages undergoing peripheral blood immunophenotyping for a variety of reasons. RESULTS The absolute number of B lymphocytes and the percentage of naive cells (CD23-/CD27-) decreased with age whereas there was an increase in memory cells (CD27+). A small subset of B cells co-expressing CD23 and CD27 was present in humans of all ages, although the majority of CD27+ cells were CD23-. The percentages and rate of increase with age of B1-a CD23+/CD27+ were slightly higher than those of B2 cell counterparts. CONCLUSIONS On the basis of our data, age-associated changes in surface markers of B cells seem to be finely balanced and probably related to functional changes after antigen encounters, while the whole peripheral blood B-cell compartment undergoes a quantitative regression.

Immunology and Homeopathy. 4. Clinical Studies—Part 1

The evidence-based research of the effectiveness of homeopathic medicines in common immunologic disorders is reviewed. In part 1, we introduce methodological issues of clinical research in homeopathy, and criteria utilized to evaluate the literature. Then 24 studies (12 randomized and 12 non-randomized) on common upper respiratory tract infections and otorhinolaryngologic complaints are described. In part 2, the focus will be on allergic diseases and the effectiveness of homeopathy will be globally evaluated and discussed using the criteria of evidence-based medicine.

Circulating γδ T cells and the risk of acute‐phase response after zoledronic acid administration

The use of intravenous nitrogen‐containing bisphosphonates (N‐BPs) is associated with the appearance of an acute phase response (APR) in a proportion of the patients for reasons that are poorly understood. The APR was attributed to the indirect activation of γδ T cells with the release of interferon‐γ and tumor necrosis factor (TNF). Forty patients with postmenopausal or senile osteoporosis (age range = 53–91 years) never previously treated with intravenous (iv) bisphosphonate, received a single 5‐mg zoledronic acid (ZOL) iv infusion over 15 minutes. White blood cells were counted and analyzed with an automated hematology analyzer (ADVIA 2120i Siemens, New York, USA) and by flow cytometer (BD FACSCanto, Becton Dickinson). The occurrence of APR was defined by the occurrence of fever (>37 °C) during the next 2 days. Forty‐two percent of patients (17 of 40) receiving the infusion of ZOL experienced an APR. Compared with the others they were younger (69 ± 7 years versus 74 ± 8 years; p = 0.06), and both the proportion and absolute number of γδ T cells were significant higher (p = 0.02 and p = 0.013, respectively). Nonsignificant differences were found between the two groups for white blood cells and for the other circulating lymphocyte subpopulations. Age was inversely correlated with circulating γδ T cells (p = 0.003) but the difference between the two groups in circulating γδ T cells persisted for age‐adjusted values and vice versa. In conclusion, the results of this study indicate that the number of circulating γδ T cells, together with age, are important determinant of the occurrence of APR after intravenous infusion of ZOL and possibly of any other N‐BPs.