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The pharmacological profile of the lignan 7-hydroxymatairesinol (HMR/lignan, HMR) includes chemopreventive effects, antioxidant properties, and mild proestrogenic activity. The present study was devised to investigate the effects of HMR on THP-1 cells, an established model of human monocytes, and on human polymorphonuclear leukocytes (PMNs). In THP-1 cells, HMR concentration-dependently reduced LPS-stimulated tumor necrosis factor (TNF)-alpha secretion in the supernatant. HMR at low, sub-muM concentrations also reduced TNF-alpha mRNA, which was however enhanced by supra-muM concentrations of HMR. In human PMNs, HMR concentration-dependently reduced ROS production induced by either N-formyl-Met-Leu-Phe, phorbol myristate acetate or angiotensin II, as well as interleukin-8 production induced by either N-formyl-Met-Leu-Phe or angiotensin II. Results indicate that HMR is an effective inhibitor of both monocytic THP-1 cells and of human PMNs and warrant further studies to assess their relevance for the prevention and treatment of several conditions characterized by chronic systemic inflammation.

Long-term treatment of Parkinson disease (PD) is frequently associated with l-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesias (LIDs). L-DOPA-induced dyskinesias are likely due to changes in the signal transduction pathways, at the striatal level, related to pulsatile stimulation of dopamine receptors. We investigated whether markers of this phenomenon can also be detected peripherally. We analyzed mRNA expression for D5 (D1-like) and D3 (D2-like) receptors and levels of second messengers, such as cAMP and free intracellular Ca2+ ([Ca2+]i), in peripheral blood lymphocytes of PD patients with (LID+) or without LIDs (LID-). Patients with PD showed depressed [Ca2+]i rise in response to mitogen-induced activation. The defect was more pronounced in LID+ (-33% with respect to healthy controls) than in LID- patients (-20%). Peripheral blood lymphocyte levels of cAMP were decreased in both LID+ (3.8 +/- 2.9 pmol/10 cells) and LID- patients (4.2 +/- 2.4 pmol/10(6) cells), with respect to controls (6 +/- 2.6 pmol/10(6) cells). No differences were found in dopamine receptor mRNA expression. Our results demonstrate that second messenger levels are altered in the peripheral blood lymphocytes of PD patients treated with dopaminergic agents and that patients with LIDs show further alterations in the regulation of [Ca2+]i homeostasis. This may represent a distinctive trait of patients prone to develop dyskinetic movements.

Tourette syndrome (TS) is a neuropsychiatric disorder in which dopaminergic dysfunction and immune system abnormalities seem to coexist. Using real-time PCR, we determined mRNA expression of dopamine receptors (DRs) D1-5 in peripheral blood lymphocytes (PBLs) from 15 TS patients and 15 sex- and age-matched healthy controls (HCs). DRD5 mRNA levels in cells from TS were higher than in cells from HCs. In TS patients with obsessive-compulsive disorder, DRD5 mRNA levels in PBLs showed a highly positive correlation with the severity of compulsive symptoms. DRD5 mRNA upregulation in PBLs from TS patients may represent a peripheral marker of dopaminergic dysfunction and supports the involvement of the immune system in TS.

Objective Angiotensin II, through the activation of angiotensin II type 1 receptors, plays a crucial role in atherosclerosis. Statins may interfere with the effects of angiotensin II. Methods We have investigated the expression of angiotensin II type 1 receptor, angiotensin II type 2 receptor and angiotensinogen on circulating monocytes and T-lymphocytes from subjects at high risk for vascular events before and during simvastatin treatment, and healthy controls. In-vitro experiments were also performed to assess the ability of simvastatin to interfere with angiotensin II signalling. Results In comparison with controls, high-risk subjects had similar angiotensin II type 1 receptor expression on the cell membranes but significantly higher angiotensin II type 1 receptor mRNA levels at least in monocyte subsets whereas their expression on T cells was similar. Angiotensin II type 2 receptor mRNA expression was higher than controls in both monocytes and T lymphocytes. No differences were observed in angiotensinogen expression on monocytes while T lymphocytes of high-risk subjects show higher expression. One-month treatment of high-risk subjects with simvastatin resulted in a reduction of angiotensin II type 1 receptor mRNA without affecting angiotensin II type 2 receptor whereas angiotensinogen mRNA expression was reduced at least in monocytes. Incubation in vitro with simvastatin reduces the expression of angiotensin II type 1 receptor mRNA levels on monocytes from untreated subjects. Conclusion Simvastatin induces down-regulation of the angiotensin II type 1 receptor, interferes with angiotensin II activity in immune cells and contributes to the anti-inflammatory profile of statins that can explain the therapeutic effects of these drugs.

Background: Elevated blood pressure levels that are associated with hypalgesia and hypothyroidism have major influences on the cardiovascular system. The potential modulation of pain sensitivity by thyroid hormones is largely undetermined. Moreover, a few experimental studies show that peripheral benzodiazepine receptors (PBRs), which may be altered in hypothyroidism, seem to be related with pain perception. Methods: Dental pain threshold and tolerance were evaluated in 19 patients followed for differentiated thyroid carcinoma (1) in severe short-term hypothyroidism (phase 1) and (2) during thyroid stimulating hormone-suppressive LT 4 treatment (phase 2). PBR expression (cytofluorimetric evaluation) on peripheral blood mononuclear cells was also investigated in the 2 phases. Results: Pain perception differed throughout the study, the dental pain threshold was higher in phase 1 (P < 0.05) whereas pain tolerance was higher but not significantly (P = 0.07). Although the systolic blood pressure was higher during hypothyroidism (P < 0.01), no relationship was found between blood pressure changes and pain sensitivity variations. Moreover, the multiple regression analysis showed an independent association of the clinical phase with pain sensitivity (r = -2.61, P = 0.029), while accounting for systolic blood pressure. The intensity of PBRs was significantly higher in the first phase of the study (P = 0.047) whereas the ratio did not significantly differ. However, no relationship was observed between pain sensitivity and PBRs. Discussion: In conclusion, in athyreotic patients, the pain sensitivity is related to the thyroid status and is independent of the increase in blood pressure induced by thyroid hormone deprivation. The PBRs do not seem to have major influence on pain sensitivity changes in hypothyroidism.

CD4+CD25+ regulatory T lymphocytes (Tregs) are specialized T cells playing a key role in the control of immune homeostasis. Here, we show that human Tregs constitutively express tyrosine hydroxylase (TH, EC 1.14.16.2), the rate-limiting enzyme in the synthesis of catecholamines, and contain substantial amounts of dopamine, norepinephrine, and epinephrine, which are released upon treatment with reserpine. Catecholamine release results in reduced production of interleukin-10 and transforming growth factor-β by Tregs, and in down-regulation of Treg-dependent inhibition of effector T-lymphocyte (Teff) proliferation, which occurs without affecting the production of tumor necrosis factor-α or interferon-γ. Tregs and Teffs express on the cell membrane both D1-like and D2-like dopaminergic receptors to a similar extent (12%-29% of the cells). Catecholamine-dependent down-regulation of Tregs is, however, selectively reversed by pharmacological blockade of dopaminergic D1-like receptors, which in Tregs only (and not in Teffs) are also expressed at the level of mRNA and are functionally coupled to intracellular production of cAMP. These findings indicate that in human Tregs endogenous catecholamines subserve an autocrine/paracrine loop involving dopaminergic pathways and resulting in down-regulation of Treg function.

The renin-angiotensin system plays a key role in the regulation of cardiovascular functions and in particular angiotensin II type 1 receptor (AT1R)-operated pathways are involved in the modulation of inflammation in the vascular wall. In the present study we assessed the pattern of expression of AT1Rs on different human circulating leukocyte subsets. Venous blood was obtained from healthy male subjects. Leukocyte subsets were purified by immunomagnetic cell sorting or identified in whole blood using multiparametric cytometric analysis. RT-PCR analysis showed that AT1R mRNA was expressed in polymorphonuclear leukocytes (PMNs), monocytes, B-lymphocytes, and, to a lesser extent, T-lymphocytes. Flow cytometric analysis revealed that the frequency of expression of AT1Rs was: PMNs>monocytes>or=B-lymphocytes>>T-lymphocytes, while receptor density per positive cells was: PMNs>or=B-lymphocytes>T-lymphocytes>or=monocytes. AT1Rs are expressed on PMNs, monocytes, T- and B-lymphocytes, however the expression pattern is peculiar to each subset, possibly suggesting distinct roles in the various cell types. Investigating the expression and the functional role of AT1Rs on circulating leukocyte subsets, as well as their possible modifications in disease conditions before and after pharmacological treatments, is likely to provide novel clues to the comprehension of the mechanisms involved in the therapeutic efficacy of currently available agents.

Interferon (IFN)-gamma plays a pivotal role in the pathogenesis of multiple sclerosis (MS), while IFN-beta may be able to modify the clinical course of the disease, eventually also by counterbalancing IFN-gamma-mediated effects. Catecholamines (CA) exert important effects on the immune response, both as transmitters between the nervous and the immune system, as well as autocrine/paracrine mediators in immune cells, and several lines of evidence support their involvement in MS. In particular, dysregulated production of CA seems to occur in peripheral blood mononuclear cells (PBMCs) of MS patients. We assessed the effects of IFN-beta and IFN-gamma on endogenous CA in PBMCs. In cultured PBMCs stimulated with phytohaemagglutinin (PHA), IFN-beta increased CA production and induced CA release in the culture medium, while IFN-gamma decreased both CA production and the expression of mRNA for the CA-synthesizing enzyme tyrosine hydroxylase. Coincubation with both IFNs prevented the inhibitory effect of IFN-gamma, as well as the stimulatory effect of IFN-beta. IFNs are the first physiological compounds shown to affect endogenous CA in PBMCs: in view of the role of CA-dependent mechanisms in the immune response, these findings may help to better understand the mechanisms of action of IFN-beta as an immunomodulatory drug in MS.

Dopamine (DA) modulates apoptosis in neuronal and non-neuronal cells, and dopaminergic pathways contribute to neurodegenerative disease. Human lymphocytes express dopaminergic receptors and DA transporters, and synthesize endogenous catecholamines, which may modulate apoptosis in these cells. In the present study, dopaminergic modulation of apoptosis was investigated in human peripheral blood mononuclear cells (PBMCs) obtained from healthy donors. Twenty-four-hour DA reduced at 0.1-5 x 3 10(-6) M and enhanced at 1-5 x 310(-4) M spontaneous apoptosis. DA 1 x 310(-6) M was inhibited by the D1-like receptor antagonist SCH 23390 1 x 310(-6) M, but not by the D2-like receptor antagonists domperidone 1 x 3 10(-6) M or haloperidol 1 x 3 10(-6) M, while the effect of DA 5 x 3 10(-4) M was prevented by the antioxidants glutathione 5-10 mM or N-acetyl-l-cysteine 1-10 mM. Intracellular reactive oxygen species were respectively reduced and increased by 1-3 h incubation with DA 0.1-10 x 3 10(-6) M and 1-5x310(-4) M. Twenty-four-hour DA 1 x 3 10(-6) M or 5 x 3 10(-4) M had no effect on PBMC expression of Cu/Zn superoxide dismutase or Bcl-2; however, DA 5 x 3 10(-4) M decreased caspase-3 activity. In human PBMCs, DA seems to promote apoptosis through oxidative mechanisms but may also result in cell rescue from apoptotic death possibly through activation of D1-like receptors. The dual effect of DA on human PBMCs closely resembles that on striatal neurons. Lymphocytes of patients with Parkinson's disease may show reduced DA content and impaired DA transporter immunoreactivity. Human PBMCs may thus represent a simple and readily accessible model to study DA-related mechanisms relevant for neurodegenerative disease.

A rapid and simple HPLC-ED method is described to identify and measure catecholamines (CTs) and their major metabolites in immune cells. Using this method, intracellular CTs were quantified in human peripheral blood mononuclear cells (PBMCs), T and B lymphocytes, monocytes and granulocytes. Immune cell subsets were separated by density gradient centrifugation and immunomagnetic cell sorting. CTs were also found in the human hematopoietic cell lines NALM-6 (pre-B) and (in smaller amounts) in Jurkat (T lymphoblastoid) and U937 (promonocytic). In cultured PBMCs, intracellular CTs were reduced by both the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine and the chromaffin granule depletant reserpine. In NALM-6 cells, both alpha-methyl-p-tyrosine and the dopamine-beta-hydroxylase inhibitor disulfiram reduced intracellular CTs, supporting the presence of active synthetic pathways in these cells. Since sympathoadrenergic mechanisms play a key role in the interactions between the immune system and the nervous system, these findings may be relevant for a better understanding of the neuro-immune network.

Enteric ganglia can maintain integrated functions, such as the peristaltic reflex, in the absence of input from the central nervous system, which has a modulatory role. Several clinical and experimental observations suggest that homeostatic control of gut function in a changing environment may be achieved through adaptive changes occurring in the enteric ganglia. A distinctive feature of enteric ganglia, which may be crucial during the development of adaptive responses, is the vicinity of the final effector cells, which are an important source of mediators regulating cell growth. The aim of this review is to focus on the possible mechanisms underlying neuronal plasticity in the enteric nervous system and to consider approaches to the study of plasticity in this model. These include investigations of neuronal connectivity during development, adaptive mechanisms that maintain function after suppression of a specific neural input, and the possible occurrence of activity-dependent modifications of synaptic efficacy, which are thought to be important in storage of information in the brain. One of the applied aspects of the study of plasticity in the enteric nervous system is that knowledge of the underlying mechanisms may eventually enable us to develop strategies to correct neuronal alterations described in several diseases. GASTROENTEROLOGY 1999;117:1438-1458

We report that neutrophil function was impaired in former heroin addicts on chronic naltrexone maintenance. Of the subjects, 62.5% had elevated plasma ACTH, 25% had elevated plasma cortisol and one subject had increased urinary cortisol. All subjects showed enhanced expression of opioid receptors on monocytes, neutrophils and lymphocytes. In vitro, incubation with therapeutically relevant concentrations of naltrexone induced a slow increase of neutrophil cytoplasmatic free Ca(2+)concentrations ([Ca(2+)]()E2>i) and slowed down the [Ca(2+)]()E2>i rise induced by N-formyl-methionyl-leucyl-phenylalanine. Neither naltrexone nor its metabolite beta-naltrexol affected human neutrophil function in vitro. We conclude that impairment of neutrophil function during chronic naltrexone may be related to opioid receptor overexpression. With this regard, the possible role of naltrexone-induced [Ca(2+)]()E2>i changes deserves further investigation. 1999 Academic Press.

The aims of this study were to assess the possible role of peripheral benzodiazepine receptors (pBZrs)1 in mediating the in vitro effects of carbamazepine (CBZ) on some neutrophil functions in healthy volunteers and to investigate neutrophil function and pBZr expression in patients with epilepsia on CBZ monotherapy for at least 1 year. In vitro CBZ (42-168 microM) concentration-dependently inhibited chemotaxis induced by N-formyl-methionyl-leucyl-phenylalanine (FMLP) or lipopolysaccharide (LPS)-activated human serum. CBZ did not affect random migration, phagocytosis index, phagocytosis frequency, NBT reduction frequency, C. albicans lethality index and resting superoxide production. The pBZr antagonist PK 11195 (1 microM, per se ineffective) reversed the inhibitory effect of CBZ on chemotaxis induced by endotoxin-activated serum or FMLP. The pBZr agonist Ro 5-4864 (10-100 microM) mimicked the effect of CBZ on chemotaxis induced by endotoxin-activated serum or FMLP and had no effect on the other parameters. Neutrophils from epileptic patients on chronic CBZ monotherapy had impaired FMLP- and serum-induced chemotaxis and enhanced expression of pBZrs on neutrophils. These data strongly suggest an involvement of pBZrs in mediating the in vitro effects of CBZ on chemotaxis; furthermore, impairment of the same neutrophil function parameters and overexpression of pBZrs in patients are consistent with the hypothesis of an in vivo interaction of CBZ with pBZrs.

We investigated the relationships between common polymorphisms in CYP1A2 (CYP1A2(⁎)1C and (⁎)1F), CYP1A2-mRNA levels in circulating lymphocytes and clozapine(CLZ)-induced adverse drug reactions (ADRs) in 34 patients. Patients with ADRs had a higher frequency of CYP1A2 low activity allele combinations (8/12; 67%) and lower CYP1A2-mRNA levels than patients without ADRs (6/22; 27%, P=0.019).

We investigated dopaminergic inhibition of CD4+CD25(high) regulatory T lymphocytes (Treg) in relapsing-remitting multiple sclerosis (MS) patients treated with interferon (IFN)-β.MS patients were prospectively studied at baseline and during 1 year of IFN-β, and compared with healthy controls (HCs). Treg were separated by immunomagnetic sorting and the effect of dopamine (DA) on Treg was assessed in coculture experiments with homologous effector T lymphocytes (Teff). Tyrosine hydroxylase (TH), dopaminergic receptors (DR) D3 and D5, and forkhead box protein P3 (FoxP3) mRNA were assessed by real-time PCR. Circulating CD4+ T cell subsets were assessed by flow cytometry.In coculture experiments, Treg inhibition of Teff proliferation was reduced by DA in HCs and completely abolished in MS patients at baseline. However, in patients after 12 months of IFN-β, Teff proliferation was impaired and DA had no more effect on Treg. In comparison to cells from HCs, Treg from MS patients at baseline had increased mRNA for DR D5 and TH (but not for DR D3). During treatment with IFN-β, both DR D5 and TH mRNA decreased down to values lower than those of cells from HCs. In comparison to HCs, MS patients had a higher frequency of circulating Treg, both at baseline and after IFN-β, while FoxP3 mRNA levels in Treg were similar in patients and HCs and did not show major changes during IFN-β.Dopaminergic inhibition of Treg in MS patients is suppressed during IFN-β treatment. Treg play a key role in the suppression of autoimmunity, thus the effect may have a therapeutic repercussion.

Long term administration of anticonvulsants is sometimes associated with impairment of the humoral and/or cellular immune response. Furthermore, certain well known adverse reactions to antiepileptics may have an immunotoxicological origin e.g. lymphadenopathy, pseudolymphoma and systemic lupus erythematosus. However, two important questions remain unresolved. First, the possibility that epilepsy per se might be primarily associated with immune alterations makes it difficult to assess the pathogenetic role of a specific drug, especially in a patient population usually on multiple drug therapy. Secondly, the clinical relevance of some of the observed immunological abnormalities is still highly controversial. This review is intended to give an outline of the present state of knowledge on the effects of anticonvulsants on humoral, cellular and nonspecific immunity, with particular regard to some of the major clinical conditions that have been ascribed to drug-induced immune dysregulation, such as pseudolymphoma and systemic autoimmune diseases. The immunotoxic potential of anticonvulsants appears to be low, and immunological monitoring is not usually required except in patients with known immune defects.

CyP is a lipopolysaccharide (LPS)-like molecule extracted from the freshwater cyanobacterium Oscillatoria planktothrix FP1, which has been reported to be a potent competitive inhibitor of bacterial LPS. In the present study the ability of CyP to affect human polymorphonuclear leukocyte (PMN) function was investigated. PMNs were isolated from venous blood by standard density-gradient centrifugation. Cell migration was measured by use of the Boyden chamber assay. Interleukin (IL)-8 and tumor necrosis factor (TNF)-α production was measured using a sandwich-type enzyme-linked immunosorbent assay. PMN intracellular reactive oxygen species (ROS) levels were assessed by the use of a fluorescent probe coupled to spectrophotometry. CyP 10-100 μg/ml was chemotactic for PMNs without affecting the chemotactic response to either E. coli LPS or N-formyl-Met-Leu-Phe (fMLP). CyP per se did not affect PMN production of either IL-8 or TNF-α, but concentration-dependently reduced LPS-induced production of both cytokines. On the contrary, CyP had no effect either on fMLP-induced production of IL-8 or on PMN oxidative burst (at rest and after stimulation with fMLP), a response which is known to be independent from LPS-operated pathways. In human PMNs CyP behaves as a selective and effective LPS antagonist. These findings support the therapeutic potential of CyP in endotoxin-dependent disease.

Atherosclerotic calcifications are related to poor prognosis and all-cause mortality in large population studies.1 Moreover, vascular calcifications are inversely associated with the potential reduction of plaque volume in regression studies during statin treatment, which suggests that more calcified lesions are less likely to undergo positive remodeling.2 Recently, it has been suggested that calcification is a tightly regulated process of mineralization akin to bone formation.3 In plaques, the deposits consist of a nonhomogeneous composite that contains hydroxyapatite mineral nanocrystals embedded in a collagenous organic matrix, whereas at a nanoscale level, apatite crystals interact with cholesterol crystallites. In this context, mineralization may result from a basic template pattern generated by the organic …

The effect of the opioid agonist morphine and of the (-) and (+) stereoisomers of the antagonist naloxone were studied on superoxide generation from human granulocytes. Morphine or naloxone had no effect on basal or phorbol 12-myristate 13-acetate (PMA)-stimulated superoxide generation. Combined equimolar (-)naloxone and morphine concentrations (0.1 pM - 0.1 μ;M) inhibited PMA-stimulated superoxide generation, while combined (+)naloxone and morphine had no effect. The stereospecific effect of naloxone suggests the involvement of opioid receptors. Thus, inhibition of superoxide generation by combined (-)naloxone and morphine could result from the unmasking of non opioid effects of morphine. It is suggested that the opioid control of oxidative metabolism in human granulocytes could involve multiple receptors mediating opposite effects.

We investigated angiotensin type 1 receptor (AT1R) expression and interleukin-8 (IL-8) productions in polymorphonuclear leukocytes obtained from patients with peripheral arterial disease (PAD) undergoing femoral endarterectomy. Subjects at high cardiovascular risk (high-risk subjects, HRS) and healthy controls (HC) were also enrolled. To this end, patients with PAD were studied 1 month before surgery, at the time of surgery, and 3 and 6 months after surgery. Polymorphonuclear leukocytes were obtained from venous blood and evaluated for AT1R expression at messenger RNA (mRNA) and protein level and IL-8 production (by means of enzyme-linked immunosorbent assay). At baseline, AT1R membrane expression was similar in cells from patients with PAD, HRS, and HC, whereas AT1R mRNA was similar in patients with PAD and HC and higher in HRS. During the follow-up period, AT1R expression progressively decreased both on the cell membrane and at the mRNA level. Both resting and stimulated production of IL-8 was lower in patients with PAD in comparison to HC and HRS and did not change during the follow up period. In PAD patients, femoral endarterectomy is associated with reduction of AT1R expression however with no apparent effect on IL-8 production. The relevance of such effects for cardiovascular protection deserves consideration.

Aims Our study investigated reactive oxygen species (ROS) generation and angiotensin II type 1 receptor (AT1-R) expression in primed polymorphonuclear leukocytes (PMNs) of dyslipidaemic subjects over prolonged statin treatment. Methods and results Sixteen untreated dyslipidaemic subjects with moderately increased cardiovascular risk (National Cholesterol Education Program, Adult Treatment Panel III) were studied before and during long-term (1 year) simvastatin treatment. Neutrophils from dyslipidaemic subjects generated more ROS in comparison with cells from healthy control subjects. After 1 year of simvastatin treatment, ROS production (delta N -formyl-Met-Leu-Phe-induced generation and area under the curve) was significantly reduced. At baseline, AT1-R mRNA expression was also higher in dyslipidaemic subjects than in healthy controls and it was reduced after clinical treatment with simvastatin. In a subgroup of patients, a reduced angiotensin II-induced ROS generation was also observed upon clinical simvastatin treatment. Moreover, a direct effect of statin on the upregulated AT1-R expression was demonstrated in vitro in neutrophils of untreated dyslipidaemic subjects. Conclusion A consistent reversion of pro-inflammatory oxidative functional response and reduction of AT1-R expression in primed PMNs was observed in patients during long-term statin treatment. The AT1-R reduction over treatment may contribute to the normalization of dysregulated neutrophil activation which occurs in the pre-clinical phase of atherosclerosis.

Objective To investigate the endogenous dopaminergic/adrenergic system of lymphocytes in multiple sclerosis (MS) patients during treatment with interferon (IFN)-β. Methods Patients with relapsing-remitting MS undergoing IFN-β treatment were prospectively studied during the first year of treatment. Circulating lymphocytes were obtained at baseline and after 1, 3, 6 and 12 months of treatment and assayed for catecholamine (CA) production and mRNA expression of tyrosine hydroxylase (TH, the rate-limiting enzyme in the synthesis of CA), β 2 -adrenoceptors (AR) and D2, D3 and D5 dopaminergic receptors (DR). Results In cells from patients treated with IFN-β for 12 months the production of CA hugely increased and was less sensitive to IFN-γ-induced inhibition. Expression of mRNA for TH, β 2 -AR and DRD5 was already enhanced after 1 month and further increased up to 6–12 months of treatment. On the contrary, DRD2 mRNA progressively decreased and DRD3 mRNA did not significantly change over the whole study period. Conclusions In MS patients IFN-β treatment enhances the ability of lymphocytes to produce CA, and induces extensive modifications of both β 2 -AR and DR-operated pathways. The clinical relevance of these effects deserves consideration.

Aim of the study Achyrocline satureioides (Lam.) D.C. is a South American native medicinal herb known by the popular name of “Marcela”. Its infusion is widely utilized for the treatment of several digestive ailments, as an anti-inflammatory preparation, as a sedative and anti-atherosclerotic. Circumstantial evidence suggests that extracts of Achyrocline satureioides may have immunomodulatory properties. The present study was therefore devised to investigate the in vitro effects Achyrocline satureioides infusion on human peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs). Materials and methods Experiments were performed on cells isolated from venous blood obtained from healthy donors. PBMC proliferation and cytokine production were assessed by standard ELISA methods. Reactive oxygen species (ROS) production by PMNs was evaluated by spectrofluorimetry. Results In PBMCs, Achyrocline satureioides infusion in the 0.06–0.24 μg/ml quercetin equivalent (QE) concentration range concentration-dependently reduced PHA-induced proliferation and production of interferon (IFN)-γ and interleukin (IL)-4. Lower concentrations of the infusion (0.006–0.03 μg/ml QE), which were ineffective on cell proliferation, significantly increased the production of both IFN-γ and IL-4 and decreased the ratio IFN-γ/IL-4. In PMNs, Achyrocline satureioides infusion slightly increased the spontaneous generation of ROS only at concentrations ≥0.06 μg/ml QE. On the contrary, in the 0.0012–0.03 μg/ml QE concentration range the infusion profoundly inhibited fMLP-induced ROS generation as well as spontaneous and fMLP-induced IL-8 production. Conclusions The present results provide evidence that Achyrocline satureioides infusion may exert several immunomodulatory effects, in line with its traditional use as an anti-inflammatory agent in many disease conditions. Further studies are warranted to better characterize such effects and to assess their therapeutic relevance.

Morphine was found to inhibit human granulocyte aggregation and ATP, thromboxane B2 (TxB2), and leukotriene B4 (LTB4) secretion during cell aggregation. None of the opioid peptides tested [(D-Ala2, D-Leu5)-enkephalin (DADL), (D-Ala2, N-Me-Phe4, Gly-ol5)-enkephalin (DAGO) or dynorphin 1-9 (Dyn 1-9)] was capable of mimicking morphine effects, while Dyn 1-9 per se induced TxB2 and LTB4 secretion from granulocytes. Morphine inhibition of both cell aggregation and ATP, but not of arachidonic acid metabolism product secretion, was prevented by naloxone. The naloxone-sensitive impairment by morphine of CD11b-CD18 complex surface expression observed could play a role in opioid inhibition of granulocyte activation.

Lignans are plant polyphenols which may possess anticancer, antioxidant, antimicrobial, anti-inflammatory and immunomodulatory activities. In particular, the lignan 7-hydroxymatairesinol (HMR/lignan, HMR) is a novel precursor of the mammalian lignan enterolactone (EL). In the present study, we investigated the estrogenicity of HMR and of EL in comparison to estradiol (E2), by measuring their effects on growth and apoptotic markers in the human estrogen-sensitive cell line MCF-7. HMR, EL and E2 concentration-dependently increased the percentage of MCF-7 cells in the S phase of the cell cycle, with the following relative potencies: E2 congruent with EL>>HMR, and efficacies: E2>HMR>>EL. Treatment of MCF-7 cells with either HMR, EL or E2 also increased the Bcl-2/Bax mRNA ratio. The effects of HMR and EL were reduced in the presence of the estrogen receptor (ER) antagonist tamoxifene. We conclude that both HMR and its metabolite EL are endowed with estrogenic activity, which is likely to be exerted through the contribution of ER-dependent pathways and to target the same intracellular mechanisms acted upon by E2. The estrogenicity of HMR and EL is however milder than that of E2, as indicated by the lower potencies and efficacies of both lignans. The present results support the notion that dietary supplementation with HMR may result in a mild estrogenic activity, both directly and by providing a suitable source for endogenous EL.

Background Although extensive experimental evidence supports a primary role of polymorphonuclear leukocytes (PMNs) in atherosclerosis, few data exist concerning the functional properties of these cells and their pharmacological modulation in high-risk individuals. Objective The production of the proinflammatory chemokine interleukin-8 (IL-8), migration and chemotaxis, and reactive oxygen species (ROS) generation were investigated in a longitudinal study in PMNs obtained from high-risk individuals during statin treatment. As a secondary endpoint we compared PMN function of high-risk patients with that of controls. Methods and results PMNs were isolated from 21 high-risk individuals before treatment and 3 and 30 days after the beginning of simvastatin treatment, and from healthy controls. During treatment a significant reduction was observed both in resting (P = 0.009) and N-formyl-Met-Leu-Phe (fMLP)-stimulated (P = 0.008) IL-8 production, and in the chemotactic index (P = 0.038), whereas ROS generation did not significantly change. In comparison with cells from controls, PMNs obtained from patients before starting simvastatin treatment showed higher resting and fMLP-stimulated IL-8 release (P = 0.007 and P = 0.002, respectively) and ROS generation (resting, P = 0.009; and fMLP-stimulated, P = 0.046), whereas migration and the chemotactic index did not significantly differ. Conclusions An activation of neutrophils is present in high-risk individuals, shown by the enhanced production of IL-8, and increased ROS generation. The 4-week statin treatment is able to reduce the cell capability to produce IL-8, and to decrease chemotaxis, thus affecting the proinflammatory properties of PMNs.

Migration and superoxide anion (O2-) generation were studied in polymorphonuclear leukocytes (PMNs) from 14 athyreotic patients, previously treated by total thyroidectomy and radioiodine therapy for differentiated thyroid carcinoma, and from age- and sex-matched euthyroid healthy controls. Patients were studied twice: in hypothyroidism (visit 1) and after TSH-suppressive L-T4 replacement therapy (visit 2). Random migration and N-formyl-Met-Leu-Phe (fMLP) 0.1-microM induced chemotaxis were similar in cells from patients at both visit 1 and visit 2 and from healthy controls. On the contrary, resting O2- generation in cells from patients was significantly lower than control values, both at visit 1 and 2. At visit 1, fMLP 0.1 muM-induced O2- generation was significantly lower than control values, while phorbol-myristate acetate (PMA) 100-ng/ml induced O2- generation was similar in cells from patients and from controls. At visit 2 both responses increased, resulting in fMLP-induced O2- generation superimposable to control values and PMA-induced O2- generation significantly higher with respect to both visit 1 and cells from controls. In vitro exposure of PMNs from healthy subjects to L-T4 did not affect O2- generation in resting cells, and significantly increased that induced by fMLP or PMA only at high, supra-physiological concentrations. Neither TSH nor T3 had significant effects at any of the concentrations tested. The present results document the existence of a correlation between thyroid status and oxidative metabolism of human PMNs, which is however unlikely to depend upon a direct action of thyroid hormones on these cells.

The authors report two cases of catechol-O-methyltransferase (COMT) inhibitor-induced asymptomatic hepatic dysfunction in women with Parkinson disease. The patients were genotyped for the UDP-glucuronosyltransferase (UGT) 1A9 gene (which encodes the main COMT inhibitor-metabolizing enzyme), and found to carry mutations leading to defective glucuronidation activity. This suggests that UGT1A9 poor metabolizer genotype(s) may be a predisposing factor for COMT inhibitor-induced hepatotoxicity.

Activation of human peripheral blood mononuclear cells (PBMC) triggers endogenous production of catecholamines (CA) through protein kinase (PK) C-dependent induction of tyrosine hydroxylase (TH; EC 1.14.16.2), the first and rate-limiting enzyme in the synthesis of CA. Since CA themselves are major mediators of the neural input to the immune system, we have examined their ability to affect PKC-induced TH mRNA expression and CA production in human isolated PBMC. In T- and B-lymphocytes (but not in monocytes) the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) (but not its inactive analogue 4alpha-phorbol-12,13-didecanoate) induced TH mRNA expression which was followed by an increase in the amount of intracellular CA. Coincubation of human PBMC with dopamine (DA) (but not with norepinephrine or epinephrine) inhibited TPA-induced TH mRNA expression. The effect of DA was concentration-dependent and was mimicked by the dopaminergic D1-like receptor agonist SKF-38393 but not by the D2-like receptor agonist bromocriptine. The D1-like antagonist SCH-23390 shifted to the right the concentration-response curves of both DA and SKF-38393, while neither the D2-like antagonist domperidone, nor the alpha1-adrenoceptor antagonist prazosin, the alpha2-adrenoceptor antagonist yohimbine, or the beta-adrenoceptor antagonist propranolol affected to any significant extent the inhibitory effect of DA. SKF-38393 also significantly reduced TPA-induced increase of intracellular CA, an effect which was antagonized by SCH-23390. It is thus suggested that in human T- and B-lymphocytes PKC activation leads to TH mRNA expression and subsequent increase of intracellular CA, which can be inhibited by D1-like receptor activation. Inhibition of intracellular CA production in human PBMC promotes cell survival through reduction of activation-induced apoptosis, and dopaminergic modulation of TH expression and intracellular CA content may thus represent a novel mechanism in the cross-talk between the nervous and the immune system as well as among immune system cells.

Dopamine (DA) is a neurotransmitter in the central and peripheral nervous system, which can be either cytotoxic or cytoprotective under selected conditions. Such effects involve oxidative mechanisms and are likely to play a role in neurodegenerative disorders. Because increasing evidence points to peripheral blood lymphocytes (PBL) as a feasible model for studying DA-related mechanisms of cell death and survival, we have explored in these cells the effects of DA on oxidative metabolism and apoptosis. Our results show that, whereas DA 100-500 microM resulted in increased intracellular reactive oxygen species (ROS) levels and apoptotic cell death through oxidative stress, DA 0.1-5 microM decreased ROS levels and apoptosis. DA (both 1 and 500 microM) partially counteracted the decrease in Cu/Zn superoxide dismutase levels observed in untreated PBL. However, whereas the effect of the low dose lasted for the whole incubation period (24 h), the effect of DA 500 microM was transient. DA-dependent reduction of ROS levels and apoptosis was prevented by D1-like (but not D2-like) receptor antagonism. The present findings add knowledge about the sensitivity of PBL to DA and strengthen the rationale for exploiting these cells as an easily accessible peripheral model for the ex vivo investigation of oxidative stress-related dopaminergic mechanisms underlying human neurodegenerative diseases.

In this study, we investigated whether changes in the regulatory mechanisms of apoptosis and oxidative stress may be detected, peripherally, in patients with Parkinson's disease (PD). For this purpose, we measured caspase-3 activity, Bcl-2 concentrations, peripheral benzodiazepine receptor (PBR) expression and Cu/Zn superoxide dismutase (SOD) concentrations in lymphocytes of untreated PD patients, patients treated only with L-Dopa or with L-Dopa and dopamine agonists and healthy volunteers. Caspase-3 activity was significantly increased in all PD patient groups. Patients treated with L-Dopa and dopamine agonists showed the lowest values of Bcl-2, coupled with the highest density of PBRs, while increased levels of Cu/Zn SOD were found in the group under monotherapy with L-Dopa. We also found, in PD patients, clear, negative correlations between Bcl-2 levels and both duration and severity of the disease. Our findings point to the existence of changes in the regulatory mechanisms of apoptosis in PD patients -- observable outside the central nervous system -- which seem to be modulated by the pharmacological treatment with dopaminergic agents.

Several reports suggest that enteric cholinergic neurons are subject to a tonic inhibitory modulation, whereas few studies are available concerning the role of facilitatory pathways. Glutamate, the main excitatory neurotransmitter in the central nervous system (CNS), has recently been described as an excitatory neurotransmitter also in the guinea-pig enteric nervous system (ENS). The present study aimed at investigating the presence of glutamatergic neurons in the ENS of the human colon. At this level, the presence of ionotropic glutamate receptors of the NMDA type, and their possible interaction with the enteric cholinergic function was also studied. In the human colon, l -glutamate and NMDA concentration dependently enhance spontaneous endogenous acetylcholine overflow in Mg 2+ -free buffer, both effects being significantly reduced by the antagonists, (±)-2-amino-5-phosphonopentanoic acid (±AP5) and 5,7-diCl-kynurenic acid. In the presence of Mg 2+ , the facilitatory effect of l -glutamate changes to inhibition, while the effect of NMDA is significantly reduced. In addition, morphological investigations reveal that glutamate- and NR1-immunoreactivities are present in enteric cholinergic neurons and glial cells in both myenteric and submucosal plexus. These findings suggest that, as described for the guinea-pig ileum, glutamatergic neurons are present in enteric plexuses of the human colon. Modulation of the cholinergic function can be accomplished through NMDA receptors.

The endozepine triakontatetraneuropeptide (TTN) induces intracellular calcium ([Ca(2+)](i)) changes and is chemotactic for human neutrophils (PMNs). Because interleukin-8 (IL-8) production is Ca(2+) dependent and can be induced by chemotactic stimuli, we have investigated the ability of TTN to induce IL-8 production in PMNs, as well as the signal transduction mechanisms involved. Our results show that TTN increases IL-8 release and IL-8 mRNA expression in a concentration- and time-dependent fashion, and these effects are prevented by the Ca(2+) chelator BAPTA-AM. TTN-induced [Ca(2+)](i) changes and IL-8 mRNA expression are sensitive to pertussis toxin, to the phospholipase C (PLC) inhibitor U73122 (but not to its inactive analogue U73343) and to the protein kinase C (PKC) inhibitor calphostin C. It is therefore suggested that TTN-induced IL-8 production in human PMNs results from a G protein-operated, PLC-activated [Ca(2+)](i) rise, and PKC contributes to this effect. These findings further support the possible role of TTN in the modulation of the inflammatory processes.

Sympathoadrenergic mechanisms may play a role in multiple sclerosis (MS). We examined catecholamine (CA) levels and production and tyrosine hydroxylase (TH) expression in peripheral blood mononuclear cells (PBMCs) from MS patients, and the correlation between CA production and apoptosis in PBMCs. PBMCs from MS patients had increased norepinephrine (NE) levels. However, phytohaemagglutinin (PHA)-stimulated PBMCs from MS patients with active disease synthesized less dopamine (DA) than cells from both healthy controls and patients with inactive disease. PBMCs from patients with inactive disease showed lower expression of TH. Pharmacological inhibition of TH in cultured PBMCs stimulated with PHA reduced the percentage of apoptotic cells. Since a failure of activation-induced apoptosis in immune cells may be involved in MS, it is suggested that altered CA production by PBMCs may be implicated in such dysregulation.

Using immunocytochemistry coupled to fluorescence and electron microscopy, we investigated the expression and ultrastructural localization of tyrosine hydroxylase (TH, EC 1.14.16.2), the rate-limiting enzyme in the biosynthesis of catecholamines, in human peripheral blood mononuclear cells (PBMCs), with PC12 cells as positive controls. In unstimulated PBMCs, TH-specific immunoreactivity was localized to the plasma membrane. However, after stimulation with the polyclonal mitogen phytohaemagglutinin (PHA), TH immunoreactivity was almost completely localized to electron-dense cytoplasmic granules, which resembled those found in PC12. TH-positive granules, however, were larger (300–500 nm) than in PC12 cells (100–200 nm). Flow cytometry analysis of TH expression showed about 46–50% positive cells in unstimulated PBMCs and in PHA-stimulated PBMCs in the G0/G1 phase of the cell cycle, but more than 80% positive cells in PHA-stimulated PBMCs in the S+G2/M phase. In agreement with previous observations, PHA stimulation also induced de novo expression of TH mRNA as well as increased intracellular catecholamine content, suggesting the occurrence of TH upregulation at the level of both gene expression and enzyme activity. The ultrastructural localization of TH in human PBMCs seems therefore regulated by cell stimulation and related to the functional activity of the enzyme.

Although it is now established that immunocompetent cells produce catecholamines (CA), which in turn may act as autocrine/paracrine mediators, little is known about the mechanisms regulating CA production in these cells. In the present study, evidence is provided that stimulation of human cultured peripheral blood mononuclear cells (PBMCs) with phytohaemagglutinin (PHA) induces the expression of tyrosine hydroxylase (TH) mRNA and subsequently increases intracellular CA levels through protein kinase C (PKC) activation and the contribution of intracellular Ca(++)-dependent mechanisms. Increased production of CA in PHA-stimulated PBMCs suggests a preferential involvement of catecholaminergic pathways in the functional modulation of activated cells. These findings may help to better define the role of immunocompetent cell-derived CA in the neuroimmune network.

Purpose Drug treatment of idiopathic Parkinson's disease (IPD) is a difficult task, and comorbidity and comedication add to its complexity. Since in IPD there is little information about drug use, this study investigated drug prescribing and indications in IPD patients. Methods From June 1997 to April 1998, a cross-sectional survey of IPD outpatients was performed and demographic and clinical data and information about drug treatments was collected and analysed. Results In the 130 IPD patients included in the study, anti-Parkinson drug (APD) prescriptions increased with disease duration and severity. Levodopa was most frequently used, followed by dopamine agonists and anticholinergic agents. Levodopa with other APDs was given to older patients with later IPD onset. Prescriptions of drugs for other indications (non-APDs) were given to 80.8% of the patients and their number increased with patient age. Non-APD prescriptions concerned mainly the circulatory system, mental disorders, the musculo-skeletal system and the digestive system. Prescriptions for indications corresponding to secondary symptoms that often complicate IPD increased with patient age and also with IPD duration. Conclusion In IPD patients, disease duration and severity and patient age seem to be major determinants of drug use. Indications for drug prescription suggest that main comorbidity includes neuropsychiatric, circulatory, musculo-skeletal and digestive disorders. Analysis of prescribing patterns in IPD can provide a readily accessible indirect indicator of patient health status for both health services and epidemiologic research purposes. Copyright © 2002 John Wiley & Sons, Ltd.

In isolated human neutrophils, diazepam (10 nmol/l to 10 µmol/l) concentration-dependently increased migration and phagocytosis. Diazepam-induced migration and phagocytosis were inhibited by the peripheral benzodiazepine receptor (PBR) antagonist PK11195 (10 µmol/l). The PBR agonist Ro5-4864 (10 nmol/l to 10 µmol/l) did not affect migration but slightly enhanced phagocytosis, while clonazepam, which binds to the central-type benzodiazepine receptors but has no affinity for PBRs, was ineffective on both parameters up to 10 µmol/l. Phagocytosis induced by diazepam or Ro5-4864 was inhibited by the Ca2+ channel blocker L-verapamil (10 µmol/l), which however did not affect the action of diazepam on migration. Competition binding experiments performed by fluorescent staining of PBRs showed that diazepam directly interacts with PBRs on human neutrophils. Both diazepam and Ro5-4864 (10 nmol/l to 10 µmol/l) induced a rise of intracellular free Ca2+ concentrations ([Ca2+]i), which was inhibited by PK11195 (10 µmol/l) and L-verapamil (10 µmol/l) and prevented by extracellular Ca2+ chelation with EGTA (5 mmol/l). In conclusion, experimental evidence indicates that in human neutrophils diazepam stimulates both migration and phagocytosis through activation of PBRs. Diazepam-induced [Ca2+]i changes depend on a PBR-operated, L-verapamil-sensitive increase in the plasma membrane permeability and subsequent extracellular Ca2+ entry, and contribute to diazepam-induced phagocytosis. On the contrary, the effect of diazepam on migration seems to occur through Ca2+-independent mechanisms.

We studied the effects of two diazepambinding inhibitor (DBI)-derived peptides, triakonta-tetraneuropeptide (DBI 17–50, TTN) and eiksoneuropeptide (DBI 51–70, ENP), on cytosolic free Ca2+ concentrations ([Ca2+]i), chemotaxis, superoxide anion (O) generation, and phagocytosis in human neutrophils. Both TTN and ENP induced a rapid and transient rise of [Ca2+]i. The effect of TTN depended on the presence of extracellular Ca2+, whereas the effect of ENP also persisted after extracellular Ca2+ chelation. TTN induced neutrophil chemotaxis, stimulated O generation, and enhanced phagocytosis. ENP did not affect cell migration and oxidative metabolism but enhanced phagocytosis. Both peptides modulated N-formylmethionyl-leucyl-phenylalanine- and phorbol myristate acetate-induced O generation. Because neutrophils express benzodiazepine receptors of the peripheral type (pBRs) and DBI-derived peptides may interact with such receptors, we investigated the possible role of pBRs in TTN- or ENP-induced effects. The synthetic pBR ligand RO 5-4864 increased [Ca2+]i through extracellular Ca2+ influx and this effect was prevented by the pBR antagonist PK-11195. RO 5-4864, however, was ineffective on neutrophil migration and O generation and only slightly affected phagocytosis. Moreover, PK-11195 delayed the [Ca2+]i rise induced by TTN but did not significantly affect its extent, and had no effect on the [Ca2+]i rise induced by ENP. We conclude that DBI-derived peptides induce [Ca2+]i changes and modulate neutrophil function mainly through pBR-independent pathways. In view of the wide cell and tissue distribution of DBI in the brain and in peripheral organs, modulation of neutrophil function by DBI-derived peptides may be relevant for both the neuroimmune network and the development and regulation of the inflammatory processes. J. Leukoc. Biol. 67:637–643; 2000.

Evidence is presented that human neutrophils contain catecholamines and several of their metabolites. In vitro, incubation with alpha-methyl-p-tyrosine or pargyline affects intracellular dopamine, norepinephrine and their metabolites, suggesting catecholamine synthesis and degradation by these cells. Reserpine reduces intracellular dopamine and norepinephrine and desipramine reduces intracellular norepinephrine, suggesting the presence of storage and uptake mechanism. In view of the ability of catecholamines to affect neutrophil function, the present results support the hypothesis that autoregulatory adrenergic mechanisms may exist in these cells.

Evidence has been obtained that peripheral blood mononuclear cells contain dopamine, norepinephrine, epinephrine, and their metabolites. Pharmacologic inhibition of tyrosine hydroxylase or monoamine oxidase profoundly affected intracellular catecholamines (CTs) and their metabolites, indicating that these cells are able to synthesize and breakdown CTs. The sensitivity of intracellular CTs to reserpine and the presence of CTs in the extracellular medium suggest that CTs are stored and released. Moreover, the increase of extracellular CTs in the presence of monoamine uptake blockers point to the presence of functional uptake mechanisms. Altogether, these results indicate the existence of a CT lifecycle in human mononuclear cells and warrant further studies to investigate the role of adrenergic autoregulatory mechanisms in modulation of the immune response and in the pathogenesis of diseases involving the immune system.

Epileptic patients on long-term therapy with a single anticonvulsant showed enhanced expression of peripheral benzodiazepine receptors (pBZrs) on neutrophils, monocytes and lymphocytes. N-Formyl-methionyl-leucyl-phenylalanine-induced chemotaxis was significantly impaired in neutrophils from patients on carbamazepine (p < 0.01 vs. controls). Neutrophils from patients on phenytoin had enhanced phorbol myristate acetate-stimulated O–2 production (p < 0.01 vs. controls) and neutrophils from patients on valproic acid had impaired phagocytosis frequency and Staphylococcus aureus lethality index (p < 0.01 vs. controls). Overexpression of pBZrs on leukocytes may reflect the clinical response to anticonvulsants and may play a role in the immunological effects of some of these drugs.

Attitudes to adverse drug reaction (ADR) spontaneous reporting were investigated among all the National Health Service (NHS) doctors operating in the territory of the Area Health Authority n.1 of Varese (Italy), to assess their awareness of the reporting system and to identify reasons for under reporting. Three hundred and fifty doctors were sent questionnaires and 207 (59.1%) were returned completed. More than 77% of the responders stated to have noticed ADRs, which were mainly reported to the pharmaceutical manufacturers and, in a minority of cases, to the NHS. Fifty per cent did not report ADRs to anyone. Important factors for deciding to report were unusualness and severity of the reaction, and involvement of a new drug. The main reason for not reporting was the clinical negligibility of the reaction. There was little knowledge about the types of reactions to be preferentially reported and the purposes of ADR reporting systems. Nevertheless, nearly everyone asked for feed-back information about reported ADRs. NHS doctors in this district have little information concerning ADR reporting systems. Some effective measures to improve the situation could be: inclusion of pharmacovigilance into pre- and post-graduated continuing education programs, provision of guidelines for ADR spontaneous reporting and of feed-back information to reporters, implementation of regional pharmacovigilance units.

1. Lymphocyte subsets and neutrophil function were studied in chronic psychiatric patients on long-term multiple drug therapy, and the correlation with disease and drug treatments was also investigated. 2. Patients showed decreased CD4+ percentage, CD4+/CD8+ ratio, B lymphocyte percentage, increased absolute and percentage NK, and impaired neutrophil chemotaxis, phagocytosis, and oxidative metabolism. Correlations were found between: patient status and lower CD4+ percentage, CD4+/CD8+ ratio, neutrophil chemotaxis, phagocytosis, and oxidative metabolism; schizophrenia and higher absolute and percentage NK, and neutrophil random migration; benzodiazepines and higher CD8+ absolute number and lower NBT reduction frequency; carbamazepine and higher NK percentage, neutrophil chemotaxis, phagocytosis, stimulated O2-production, and lower microbicidal activity. 3. The present study provides direct evidence for the existence of immunologic abnormalities in a population of chronic psychiatric patients on long-term drug therapy. Although a possible role by other factors could not be excluded, such abnormalities are strongly associated with chronic administration of benzodiazepines and carbamazepine. Further investigations are warranted to confirm these findings and disclose the possible mechanism(s) involved.

A new HPLC assay method was used to investigate the pharmacokinetics of asiatic acid after oral administration of the total triterpenic fraction of Centella asiatica in single doses (30 or 60 mg) and after a 7-day treatment (30 or 60 mg twice daily). Twelve healthy volunteers received each treatment following a randomized cross-over design with trials separated by a 3-week interval. The time of peak plasma concentration was not affected by dosage difference or by treatment scheme. Differences in peak plasma concentration and area under the concentration vs. time curve from 0 to 24 h (AUC0-24) calculated after 30 or 60 mg administration (single dose) were accounted for by the different dose regimen. However, after chronic treatment with both 30 and 60 mg, peak plasma concentrations, AUC0-24 and half-life were significantly higher than those observed after the corresponding single dose administration. This phenomenon could be explained by a metabolic interaction between asiatic acid and asiaticoside, which is transformed into asiatic acid in vivo.

Peripheral benzodiazepine receptors (pBDZrs) are found in both brain and peripheral tissues and differ from the central BDZrs linked to the GABA-receptor-chloride channel complex in their different distribution, molecular structure and pharmacological sensitivity. The pBDZrs bind diazepam, but with low affinity, do not bind clonazepam, and bind the benzodiazepine (BDZ) Ro 5-4864 and the isoquinoline carboxamide PK 11195 with nanomolar affinity 1. Peripheral BDZrs have been shown on human neutrophils, monocytes and lymphocytes 2. Ligands for pBDZrs modulate a number of human leukocyte activities 3,4, however, no definite data are available yet concerning the intracellular signal transduction mechanisms of pBZr ligands in leukocytes. Ca++ is the most common signal transduction element modulating many crucial cellular processes. A role for BDZs as regulators of Ca++ channel activity has been suggested for both centrally and peripherally acting BDZ ligands5. However, the possible influence of BDZs on Ca++ movements in leukocytes has never been considered. The aim of the present study was to investigate the effect of BDZr ligands on [Ca]i in human neutrophils. In addition, the possible mechanisms responsible for such effect have also been examined.

Naloxone was found to prevent both opioid and non-opioid-induced migration of human granulocytes in a stereoselective way. Indeed, besides being able to inhibit morphine-induced migration, (-) but not (+), naloxone isomer proved to abolish either casein, serum of fMLP-induced chemotaxis. It is concluded that opioid-induced modulation of granulocyte migration is likely to be mediated through specific receptors, possibly of the μ type. Moreover, the antichemotactic effect of naloxone suggests an involvement of opioid receptors and/or endogenously released opioids in the mechanism of granulocyte activation by different chemoattractants.

Sixty-five patients undergoing long-term monotherapy for at least 3 months with phenytoin, carbamazepine or phenobarbital were screened for lymphocyte and immunoglobulin abnormalities. In 57% of patients the duration of therapy was longer than 12 months. The control subjects were matched for sex and age and none of them was taking drugs. The average serum immunoglobulin (IgG, IgA, IgM) values did not differ in control and patient groups. A significant decrease of OKT4+cells was seen with all drugs, while other lymphocyte subpopulations were differently affected depending on the drug used. It is concluded that long-term single-drug treatment with phenytoin, carbamazepine and phenobarbital exhibits immunosuppressant effects through a complex action which involves more than one lymphocyte subpopulation. Moreover, the possible interference of the disease state with the immune functions of epileptic patients is discussed.

Serum and CSF levels of penicillin have been determined in 13 syphilitic patients treated with relatively high doses of penicillin G by the intravenous route. Wide individual variability in the CSF/serum concentration ratio was observed. In all patients the concentration of the antibiotic in the CSF was equal to or greater than 0.03 IU/ml., which is considered to be the minimal therapeutic concentration. The potential clinical usefulness of different therapeutic regimens in the treatment of neurosyphilis is discussed.