Selective serotonin-reuptake inhibitor and norepinephrine dopamine reuptake inhibitor antidepressants do not affect natural killer cell activity in vitro

OBJECTIVE: This study aims to evaluate the citotoxic activity of two commonly used anti-depressants: paroxetine and bupropion. We also evaluated the in vitro natural killer activity (NKA) after incubating the blood samples with the antidepressants. METHODS: Peripheral blood samples from 15 healthy volunteers were collected and the mononuclear cells (PBMCs) were isolated and incubated for 24h with (or without = control cells) paroxetine and bupropion, in concentrations of 30, 100 and 1000 ng/ml. After the incubation period in both groups, the amount of dead cells was calculated using trypam blue technique. NKA was evaluated using the classic51Cr release assay. CONCLUSIONS: PBMCs dead cells occurred in both groups and in proportion to all pharmacological concentrations. Nevertheless, the NKA was not affected, even with the reduction in the number of effective cells.

Rabies virus infection of cultured adult mouse dorsal root ganglion neurons

An in vitro model of adult dorsal root ganglion neurons infection by rabies virus is described. Viral marked neurotropism is observed, and the percentage and the degree of infection of the neurons is higher than in non neuronal cells, even if neurons are the minority of the cells in the culture. The neuritic tree is also heavily infected by the virus.

The role of the eosinophil-selective chemokine, eotaxin, in allergic and non-allergic airways inflammation

Blood eosinophilia and tissue infiltration by eosinophils are frequently observed in allergic inflammation and parasitic infections. This selective accumulation of eosinophils suggested the existence of endogenous eosinophil-selective chemoattractants. We have recently discovered a novel eosinophil-selective chemoattractant which we called eotaxin in an animal model of allergic airways disease. Eotaxin is generated in both allergic and non-allergic bronchopulmonary inflammation. The early increase in eotaxin paralled eosinophil infiltration in the lung tissue in both models. An antibody to IL-5 suppressed lung eosinophilia, correlating with an inhibition of eosinophil release from bone marrow, without affecting eotaxin generation. This suggests that endogenous IL-5 is important for eosinophil migration but does not appear to be a stimulus for eotaxin production. Constitutive levels of eotaxin observed in guinea-pig lung may be responsible for the basal lung eosinophilia observed in this species. Allergen-induced eotaxin was present mainly in the epithelium and alveolar macrophages, as detected by immunostaining. In contrast there was no upregulation of eotaxin by the epithelial cells following the injection of Sephadex beads and the alveolar macrophage and mononuclear cells surrounding the granuloma were the predominant positive staining cells. Eotaxin and related chemokines acting through the CCR3 receptor may play a major role in eosinophil recruitment in allergic inflammation and parasitic diseases and thus offer an attractive target for therapeutic intervention.

A comparison of the inhibitory activity of selective PDE4 inhibitors on eosinophil recruitment in guinea pig skin

The elevation of intracellular cyclic AMP by phosphodiesterase (PDE)4 inhibitors in eosinophils is associated with inhibition of the activation and recruitment of these cells. We have previously shown that systemic treatment with the PDE4 inhibitor rolipram effectively inhibt eosinophil migration in guinea pig skin. In the present study we compare the oral potency and efficacy of the PDE4 inhibitors rolipram, RP 73401 and CDP 840 on allergic and PAF-induced eosinophil recruitment. Rolipram and RP 73401 were equally effective and potent when given by the oral route and much more active than the PDE4 inhibitor CDP 840. We suggest that this guinea pig model of allergic and mediator-induced eosinophil recruitment is both a sensitive and simple tool to test the efficacy and potency of PDE4 inhibitors in vivo.

Selective PDE4 inhibitors as potent anti-inflammatory drugs for the treatment of airway diseases

Phosphodiesterases (PDEs) are responsible for the breakdown of intracellular cyclic nucleotides, from which PDE4 are the major cyclic AMP metabolizing isoenzymes found in inflammatory and immune cells. This generated greatest interest on PDE4 as a potential target to treat lung inflammatory diseases. For example, cigarette smoke-induced neutrophilia in BAL was dose and time dependently reduced by cilomilast. Beside the undesired side effects associated with the first generation of PDE4 inhibitors, the second generation of selective inhibitors such as cilomilast and roflumilast showed clinical efficacy in asthma and chronic obstrutive pulmonary diseases trials, thus re-enhancing the interest on these classes of compounds. However, the ability of PDE4 inhibitors to prevent or modulate the airway remodelling remains relatively unexplored. We demonstrated that selective PDE4 inhibitor RP 73-401 reduced matrix metalloproteinase (MMP)-9 activity and TGF-beta1 release during LPS-induced lung injury in mice and that CI-1044 inhibited the production of MMP-1 and MMP-2 from human lung fibroblasts stimulated by pro-inflammatory cytokines. Since inflammatory diseases of the bronchial airways are associated with destruction of normal tissue structure, our data suggest a therapeutic benefit for PDE4 inhibitors in tissue remodelling associated with chronic lung diseases.

Selective suppression of leukocyte recruitment in allergic inflammation

Allergic diseases result in a considerable socioeconomic burden. The incidence of allergic diseases, notably allergic asthma, has risen to high levels for reasons that are not entirely understood. With an increasing knowledge of underlying mechanisms, there is now more potential to target the inflammatory process rather than the overt symptoms. This focuses attention on the role of leukocytes especially Th2 lymphocytes that regulate allergic inflammation and effector cells where eosinophils have received much attention. Eosinophils are thought to be important based on the high numbers that are recruited to sites of allergic inflammation and the potential of these cells to effect both tissue injury and remodelling. It is hoped that future therapy will be directed towards specific leukocyte types, without overtly compromising essential host defence responses. One obvious target is leukocyte recruitment. This necessitates a detailed understanding of underlying mechanisms, particularly those involving soluble che-moattractants signals and cell-cell adhesion molecules.

Sympathetic glial cells and macrophages develop different responses to Trypanosoma cruzi infection or lipopolysaccharide stimulation

Nitric oxide (NO) participates in neuronal lesions in the digestive form of Chagas disease and the proximity of parasitised glial cells and neurons in damaged myenteric ganglia is a frequent finding. Glial cells have crucial roles in many neuropathological situations and are potential sources of NO. Here, we investigate peripheral glial cell response to Trypanosoma cruzi infection to clarify the role of these cells in the neuronal lesion pathogenesis of Chagas disease. We used primary glial cell cultures from superior cervical ganglion to investigate cell activation and NO production after T. cruzi infection or lipopolysaccharide (LPS) exposure in comparison to peritoneal macrophages. T. cruzi infection was greater in glial cells, despite similar levels of NO production in both cell types. Glial cells responded similarly to T. cruzi and LPS, but were less responsive to LPS than macrophages were. Our observations contribute to the understanding of Chagas disease pathogenesis, as based on the high susceptibility of autonomic glial cells to T. cruzi infection with subsequent NO production. Moreover, our findings will facilitate future research into the immune responses and activation mechanisms of peripheral glial cells, which are important for understanding the paradoxical responses of this cell type in neuronal lesions and neuroprotection.

Frequency of B cells in normal mice which recognize self proteins

The mechanism whereby the immune system avoids self-aggression is one of the central issues of Immunology. The discovery of natural autoantibodies, mainly of IgM isotype, and of idiotypic interactions between antibodies indicates that elements of the immune system interact with self constituents and with themselves. Results of studies with soluble antibodies have indicated that the pool of circulating IgM represents the end result of a highly selective process of B cells activation and differentiation by self proteins resulting in the formation of a network. The objective of the present work was to determine the frequency of self-reacting B cells in normal mice. We were able to detect B cells that recognize self proteins present in extracts of different organs in normal adult, 2-3-month old, BALB/c and C57BL/6 mice with an ELISA spot assay. About 1% of total IgM-secreting cells among small, LPS-stimulated spleen cells reacted with organ extracts, whereas among large spleen cells the frequency was 5- to 10-fold lower. Immunization induced an increase in the frequency of IgM-secreting cells. The present results provide cellular evidence for the results of studies done at the serological level. The physiological role of these self-recognizing cells, as well as their participation in autoimmune processes, remain to be established

Role of Th1 and Th2 cells in autoimmune demyelinating disease

Evidence is accumulating that Th1 cells play an important role in the development of multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE), whereas Th2 cells contribute to recovery from disease. A major determinant in the development of Th1 and Th2 cells is the type of antigen-presenting cell (APC) involved and its functional characteristics, e.g., the production of interleukin-12. Therefore, modulation of APC might interfere with the development of Th1 type responses and as such be beneficial for MS and EAE. The potential of cytokines, in particular interleukin-10, and glucocorticoids to exert a selective effect on APC, and as a consequence to affect the Th1-Th2 balance in EAE, is discussed

The molecular basis of selective permeability of connexins is complex and includes both size and charge

Although gap junction channels are still widely viewed as large, non-specific pores connecting cells, the diversity in the connexin family has led more attention to be focused on their permeability characteristics. We summarize here the current status of these investigations, both published and on-going, that reveal both charge and size selectivity between gap junction channels composed of different connexins. In particular, this review will focus on quantitative approaches that monitor the expression level of the connexins, so that it is clear that differences that are seen can be attributed to channel properties. The degree of selectivity that is observed is modest compared to other channels, but is likely to be significant for biological molecules that are labile within the cell. Of particular relevance to the in vivo function of gap junctions, recent studies are summarized that demonstrate that the connexin phenotype can control the nature of the endogenous traffic between cells, with consequent effects on biological effects of gap junctions such as tumor suppression.

Activation of a P2Y4-like purinoceptor triggers an increase in cytosolic [Ca2+] in the red blood cells of the lizard Ameiva ameiva (Squamata, Teiidae)

An increasing number of pathophysiological roles for purinoceptors are emerging, some of which have therapeutic potential. Erythrocytes are an important source of purines, which can be released under physiological and physiopathological conditions, acting on purinergic receptors associated with the same cell or with neighboring cells. Few studies have been conducted on lizards, and have been limited to ATP agonist itself. We have previously shown that the red blood cells (RBCs) of the lizard Ameiva ameiva store Ca2+ in the endoplasmic reticulum (ER) and that the purinergic agonist ATP triggers a rapid and transient increase of [Ca2+]c by mobilization of the cation from internal stores. We also reported the ability of the second messenger IP3 to discharge the ER calcium pool of the ER. Here we characterize the purinoceptor present in the cytoplasmic membrane of the RBCs of the lizard Ameiva ameiva by the selective use of ATP analogues and pyrimidine nucleotides. The nucleotides UTP, UDP, GTP, and ATPgammaS triggered a dose-dependent response, while interestingly 2MeSATP, 2ClATP, alpha, ß-ATP, and ADP failed to do so in a 1- to 200-µm con- centration. The EC50 obtained for the compounds tested was 41.77 µM for UTP, 48.11 µM for GTP, 53.11 µM for UDP, and 30.78 µM for ATPgammaS. The present data indicate that the receptor within the RBCs of Ameiva ameiva is a P2Y4-like receptor due to its pharmacological similarity to the mammalian P2Y4 receptor.

Losses of immunoreactive parvalbumin amacrine and immunoreactive alphaprotein kinase C bipolar cells caused by methylmercury chloride intoxication in the retina of the tropical fish Hoplias malabaricus

To quantify the effects of methylmercury (MeHg) on amacrine and on ON-bipolar cells in the retina, experiments were performed in MeHg-exposed groups of adult trahiras (Hoplias malabaricus) at two dose levels (2 and 6 µg/g, ip). The retinas of test and control groups were processed by mouse anti-parvalbumin and rabbit anti-alphaprotein kinase C (alphaPKC) immunocytochemistry. Morphology and soma location in the inner nuclear layer were used to identify immunoreactive parvalbumin (PV-IR) and alphaPKC (alphaPKC-IR) in wholemount preparations. Cell density, topography and isodensity maps were estimated using confocal images. PV-IR was detected in amacrine cells in the inner nuclear layer and in displaced amacrine cells from the ganglion cell layer, and alphaPKC-IR was detected in ON-bipolar cells. The MeHg-treated group (6 µg/g) showed significant reduction of the ON-bipolar alphaPKC-IR cell density (mean density = 1306 ± 393 cells/mm²) compared to control (1886 ± 892 cells/mm²; P < 0.001). The mean densities found for amacrine PV-IR cells in MeHg-treated retinas were 1040 ± 56 cells/mm² (2 µg/g) and 845 ± 82 cells/mm² (6 µg/g), also lower than control (1312 ± 31 cells/mm²; P < 0.05), differently from the data observed in displaced PV-IR amacrine cells. These results show that MeHg changed the PV-IR amacrine cell density in a dose-dependent way, and reduced the density of alphaKC-IR bipolar cells at the dose of 6 µg/g. Further studies are needed to identify the physiological impact of these findings on visual function.

The blockade of cyclooxygenases-1 and -2 reduces the effects of hypoxia on endothelial cells

Hypoxia activates endothelial cells by the action of reactive oxygen species generated in part by cyclooxygenases (COX) production enhancing leukocyte transmigration. We investigated the effect of specific COX inhibition on the function of endothelial cells exposed to hypoxia. Mouse immortalized endothelial cells were subjected to 30 min of oxygen deprivation by gas exchange. Acridine orange/ethidium bromide dyes and lactate dehydrogenase activity were used to monitor cell viability. The mRNA of COX-1 and -2 was amplified and semi-quantified before and after hypoxia in cells treated or not with indomethacin, a non-selective COX inhibitor. Expression of RANTES (regulated upon activation, normal T cell expressed and secreted) protein and the protective role of heme oxygenase-1 (HO-1) were also investigated by PCR. Gas exchange decreased partial oxygen pressure (PaO2) by 45.12 ± 5.85% (from 162 ± 10 to 73 ± 7.4 mmHg). Thirty minutes of hypoxia decreased cell viability and enhanced lactate dehydrogenase levels compared to control (73.1 ± 2.7 vs 91.2 ± 0.9%, P < 0.02; 35.96 ± 11.64 vs 22.19 ± 9.65%, P = 0.002, respectively). COX-2 and HO-1 mRNA were up-regulated after hypoxia. Indomethacin (300 µM) decreased COX-2, HO-1, hypoxia-inducible factor-1alpha and RANTES mRNA and increased cell viability after hypoxia. We conclude that blockade of COX up-regulation can ameliorate endothelial injury, resulting in reduced production of chemokines.

Angiotensin II stimulates MCP-1 production in rat glomerular endothelial cells via NAD(P)H oxidase-dependent nuclear factor-kappa B signaling

Angiotensin II (Ang II) plays a crucial role in the pathogenesis of renal diseases. The objective of the present study was to investigate the possible inflammatory effect of Ang II on glomerular endothelial cells and the underlying mechanism. We isolated and characterized primary cultures of rat glomerular endothelial cells (GECs) and observed that Ang II induced the synthesis of monocyte chemoattractant protein-1 (MCP-1) in GECs as demonstrated by Western blot. Ang II stimulation, at concentrations ranging from 0.1 to 10 µm, of rat GECs induced a rapid increase in the generation of reactive oxygen species as indicated by laser fluoroscopy. The level of p47phox protein, an NAD(P)H oxidase subunit, was also increased by Ang II treatment. These effects of Ang II on GECs were all reduced by diphenyleneiodonium (1.0 µm), an NAD(P)H oxidase inhibitor. Ang II stimulation also promoted the activation of nuclear factor-kappa B (NF-κB). Telmisartan (1.0 µm), an AT1 receptor blocker, blocked all the effects of Ang II on rat GECs. These data suggest that the inhibition of NAD(P)H oxidase-dependent NF-κB signaling reduces the increase in MCP-1 production by GECs induced by Ang II. This may provide a mechanistic basis for the benefits of selective AT1 blockade in dealing with chronic renal disease.

Angiotensin II induces NF-κB, JNK and p38 MAPK activation in monocytic cells and increases matrix metalloproteinase-9 expression in a PKC- andRho kinase-dependent manner

Angiotensin II (ANG II), the main effector of the renin-angiotensin system, is implicated in endothelial permeability, recruitment and activation of the immune cells, and also vascular remodeling through induction of inflammatory genes. Matrix metalloproteinases (MMPs) are considered to be important inflammatory factors. Elucidation of ANG II signaling pathways and of possible cross-talks between their components is essential for the development of efficient inhibitory medications. The current study investigates the inflammatory signaling pathways activated by ANG II in cultures of human monocytic U-937 cells, and the effects of specific pharmacological inhibitors of signaling intermediates on MMP-9 gene (MMP-9) expression and activity. MMP-9 expression was determined by real-time PCR and supernatants were analyzed for MMP-9 activity by ELISA and zymography methods. A multi-target ELISA kit was employed to evaluate IκB, NF-κB, JNK, p38, and STAT3 activation following treatments. Stimulation with ANG II (100 nM) significantly increased MMP-9 expression and activity, and also activated NF-κB, JNK, and p38 by 3.8-, 2.8- and 2.2-fold, respectively (P < 0.01). ANG II-induced MMP-9 expression was significantly reduced by 75 and 67%, respectively, by co-incubation of the cells with a selective inhibitor of protein kinase C (GF109203X, 5 µM) or of rho kinase (Y-27632, 15 µM), but not with inhibitors of phosphoinositide 3-kinase (wortmannin, 200 nM), tyrosine kinases (genistein, 100 µM) or of reactive oxygen species (α-tocopherol, 100 µM). Thus, protein kinase C and Rho kinase are important components of the inflammatory signaling pathways activated by ANG II to increase MMP-9 expression in monocytic cells. Both signaling molecules may constitute potential targets for effective management of inflammation.