Circulating CD40 ligand, Dickkopf-1 and P-selectin in HIV-infected patients.

OBJECTIVES: The aim of this study was to evaluate the circulating levels of CD40 ligand (CD40 L), Dickkopf-1 (DKK-1) and P-selectin, their relationships and their contributions to cardiovascular risk in subjects with HIV infection. METHODS: The study population included 80 HIV-infected patients, 14 (17.5%) of whom had diabetes mellitus (DM) and 32 (40.0%) of whom had arterial hypertension (AH). The HIV-infected patients were compared with a control group with similar demographic and clinical features. CD40L, DKK-1 and P-selectin levels were measured using an enzyme-linked immunosorbent assay. RESULTS: The HIV-infected patients showed higher levels of all the cardiovascular disease (CVD) markers. Both serum CD40L and DKK-1 were significantly higher in HIV-infected patients than in the HIV-negative controls (P < 0.001), while soluble P-selectin showed no significant between-group difference (P = 0.133), reflecting the role of HIV infection in CVD. In the HIV-infected group, patients with DM showed lower levels of CD40L and DKK-1 in comparison with the nondiabetic patients and patients with AH (P < 0.05, with Bonferroni correction). In contrast, patients with AH showed higher levels of CD40L and DKK-1 in comparison to patients without DM or AH (P < 0.05, with Bonferroni correction). Patients with AH showed higher levels of CD40L and DKK-1 than patients with DM (P < 0.05, with Bonferroni correction). CONCLUSIONS: In this study, we found that HIV-infected patients displayed significantly higher circulating levels of both CD40L and DKK-1, which were linearly and directly correlated, when compared to HIV-negative patients. The presence of diabetes was associated with lower levels of both CD40L and DKK-1, whereas the presence of hypertension was associated with higher levels of CD40L.

The novel HBx mutation F30V correlates with hepatocellular carcinoma in vivo, reduces hepatitis B virus replicative efficiency and enhances anti-apoptotic activity of HBx N terminus in vitro.

OBJECTIVE: We aimed to investigate HBx genetic elements correlated with hepatitis B virus (HBV) -related hepatocellular carcinoma (HCC) and their impact on (a) HBV replicative efficiency, (b) HBx binding to circular covalently closed DNA (cccDNA), (c) apoptosis and cell-cycle progression, and (d) HBx structural stability. METHODS: This study included 123 individuals chronically infected with HBV: 27 with HCC (77.9% (21/27) genotype D; 22.1% (6/27) genotype A) and 96 without HCC (75% (72/96) genotype D; 25.0% (24/96) genotype A). HepG2 cells were transfected by wild-type or mutated linear HBV genome to assess pre-genomic RNA (pgRNA) and core-associated HBV-DNA levels, HBx-binding onto cccDNA by chromatin immunoprecipitation-based quantitative assay, and rate of apoptosis and cell-cycle progression by cytofluorimetry. RESULTS: F30V was the only HBx mutation correlated with HCC (18.5% (5/27) in HCC patients versus 1.0% (1/96) in non-HCC patients, p 0.002); a result confirmed by multivariate analysis. In vitro, F30V determined a 40% and 60% reduction in pgRNA and core-associated HBV-DNA compared with wild-type (p <0.05), in parallel with a significant decrease of HBx binding to cccDNA and decreased HBx stability. F30V also decreased the percentage of apoptotic cells compared with wild-type (14.8 ± 6.8% versus 19.1 ± 10.1%, p <0.01, without affecting cell-cycle progression) and increased the probability of HBx-Ser-31 being phosphorylated by PI3K-Akt kinase (known to promote anti-apoptotic activity). CONCLUSIONS: F30V was closely correlated with HBV-induced HCC in vivo, reduced HBV replicative efficiency by affecting HBx-binding to cccDNA and increased anti-apoptotic HBx activity in vitro. This suggests that F30V (although hampering HBV's replicative capacity) may promote hepatocyte survival, so potentially allowing persistent production of viral progeny and initiating HBV-driven hepatocarcinogenesis. Investigation of viral genetic markers associated with HCC is crucial to identify those patients at higher risk of HCC, who hence deserve intensive liver monitoring and/or early anti-HBV therapy.

Transcription profile analysis of vastus lateralis muscle from patients with chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a disabling condition characterized by unexplained chronic fatigue that impairs normal activities. Many body systems are affected and etiology has not yet been identified. In addition to immunological and psychological aspects, skeletal muscle symptoms are prominent in CFS patients. In an effort to establish which pathways might be involved in the onset and development of muscle symptoms, we used global transcriptome analysis to identify genes that were differentially expressed in the vastus lateralis muscle of female and male CFS patients. We found that the expression of genes that play key roles in mitochondrial function and oxidative balance, including superoxide dismutase 2, were altered, as were genes involved in energy production, muscular trophism and fiber phenotype determination. Importantly, the expression of a gene encoding a component of the nicotinic cholinergic receptor binding site was reduced, suggesting impaired neuromuscular transmission. We argue that these major biological processes could be involved in and/or responsible for the muscle symptoms of CFS.

IL-32: a newly-discovered proinflammatory cytokine.

IL-32, a newly-discovered proinflammatory cytokine that activates the p38MAPK and NF-kappaB pathways, is an important player in innate and adaptive immune response. IL-32, a cytokine produced mainly by T, natural killer, and epithelial cells induces significant amounts of TNFalpha and MIP-2 and increases the production of both cytokines in a dose-dependent manner. IL-32 has been implicated in inflammatory disorders, mycobacterium tuberculosis infections, inflammatory bowel disease, and influenza A virus infection, as well as in some autoimmune diseases, such as rheumatoid arthritis, ulcerative colitis and Crohn?s disease and in human stomach cancer, human lung cancer and breast cancer tissues. Moreover, it has been reported that IL-32 has pro-inflammatory effects on myeloid cells and causes the differentiation of osteoclast precursors into multinucleated cells expressing specific osteoclast markers. We recently found that human IL-32 has the capacity to provoke histamine release in human-derived cord blood mast cells (HDCBMC), but not in LAD 2 cells nor in rat peritoneal mast cells (RPMC), showing that IL-32 may be specie specific and act more in mature human mast cells (HDCBMC) than in transformed mast cells (LAD 2 cells). Certainly, IL-32 is another potent proinflammatory cytokine, however, the specific role of this newly-discovered protein in the network of cytokine biology remains to be determined.

Acute urinary retention due to HSV-1: a case report.

Complications in urinary tract nervous routes due to herpes viruses as VZV and HSV-2 are well known. Acute urinary retention and chronic neuropathic pain are not rare when sacral dermatomes are involved by these viruses. However, an analogous condition has not yet been clearly ascribed to HSV-1 infection. We present a 32-year-old immunocompetent patient with fever, lumbar pain and acute urinary retention who had never had herpetic clinical manifestations. Urodynamic studies diagnosed a neurologic bladder with an absent filling sensation. Cystoscopic assessment revealed the presence of reddened and isolated small mucosal areas in the bladder walls. The search for herpes viruses in plasma and CSF by PCR assay were positive for HSV-1. After treatment with antiviral therapy the disease resolved. Intermittent catheterization was necessary and voiding dysfunction resolved after three weeks by its appearance. Neurological damage to the central nervous system (CNS) and/or PNS due to HSV-1 seems to be the most likely reason. The course of disease was benign and self-remitting.

Substance P upregulates LTB4 in rat adherent macrophages from granuloma induced by KMnO4.

Substance P (SP) is an important neuropeptide involved in neurogenic inflammation and most of its pathophysiological functions are mediated through binding to the neurokinin-1 receptor. SP exerts various proinflammatory actions on immune-cells, including macrophages. Several compounds such as cytokines have the capacity to activate and stimulate macrophages to produce arachidonic acid oxygenation and lipoxygenation products. Leukotriene B4 (LTB4) is one of the most important mediators of leukocyte activation in acute and chronic inflammatory reactions. LTB4 stimulates chemotaxis, lysosomal enzyme release, and cell aggregation. In this report, we studied the effect of SP on rat adherent granuloma macrophages (RAGMs). The chronic granuloma in rat was induced by dorsal injections of a potassium permanganate (KMnO4) saturated crystal solution (200 microl of a 1:40 dilution). After 7 days, all rats developed a subcutaneous granuloma in the injection site from which infiltrated macrophages were extracted, isolated, and cultured in vitro. We tested the hypothesis that SP stimulates the production of LTB4 in RAGMs and increases lipoxygenase expression. Here we show that the cell-free supernatant of RAGMs stimulated with SP (10 microM), resulted in statistically significant increases of LTB4 Preincubation of RAGMs with NDGA (nordihydroguaiaretic acid (10 microM), completely abolished the production of LTB(4) in the supernatants and lipoxygenase expression on RAGMs challenged with SP, or the cation ionophore A23187 (positive control). Similar effects were obtained when the cells were pretreated with dexamethasone (10 microM). Our results suggest that SP is able to stimulate the release of LTB4 and lipoxygenase expression in macrophages from chronic inflammatory granuloma and provide further evidence for a neuroinflammatory pathway.

RANTES (CCL5) potentiates calcium ionophore in the production of LTB4 in rat adherent macrophages from granuloma induced by KMnO4: inhibiton by NDGA.

The activation of monocytes/macrophages by several stimuli is an initial event in the inflammatory response. To ascertain the importance of LTB(4) and 5-lypoxigenase in the inflammatory site, we isolated and stimulated rat adherent granuloma macrophages (RAGMs) with calcium ionophore in the presence or absence of regulated on activation, normal T expressed and secreted (RANTES) [CCL5] at different concentrations. We tested the hypothesis that RANTES may influence the production of LTB(4) stimulated by calcium ionophore A23187 (2.5 microM/ml) in rat adherent granuloma macrophages derived from granuloma induced by potassium permanganate diluted 1:40 saturated solution. To test this hypothesis, we measured LTB(4) production, in rat granuloma macrophages stimulated with A23187 (2.5 microM) alone and in combination with RANTES at different concentrations. In these studies, the cell-free supernatant of stimulated RAGMs with the ionophore A23187, resulted in a drastic increase of LTB(4). However, when the cells were treated with the combination RANTES plus A23187 the stimulatory effect was more pronounced than A23187 alone. LTB(4) production was quantitated. The calcium ionophore A23187 directly induced LTB(4) in macrophages, this production was markedly enhanced when the cells were pretreated with RANTES. However, the addition of RANTES in the absence of calcium ionophore A23187 did not directly induce LTB(4) release, nor was lypoxigenase expression augmented. Preincubation of RAGMs with NDGA (nordihydroguiaretic acid) (10(-5)M) completely abolished the production of LTB4 on RAGMSs challenged with A23187 in combination with RANTES or A23187 alone in the supernatants. Similar effects were obtained when the cells were pretreated with dexamethasone. These data suggest, for the first time, that RANTES may stimulate the release of LTB(4), only when it is associated to other stimuli and for this reason we conclude that RANTES modulates inflammatory diseases, and may require other stimuli to be effective in amplifying its spectrum of action(s).

Localization and activity of iNOS in normal human lung tissue and lung cancer tissue.

Inducible nitric oxide synthase (iNOS) is one of three enzymes generating nitric oxide (NO) from the amino acid L-arginine. iNOS-derived NO plays an important role in several physiological and pathophysiological conditions. NO is a free radical which produces many reactive intermediates that account for its bioactivity. In the human lung, the alveolar macrophage is an important producer of cytokines and this production may be modified by NO. Moreover, high concentrations of NO have been shown to increase nuclear factor kappaB (NF-kB) activation. Recent investigations of NO expression in tumor tissue indicated that, at least for certain tumors, NO may mediate one or more roles during the growth of human cancer. We have studied iNOS in two tissue groups: normal human lung tissue and human lung cancer tissue. We localized iNOS in these tissues by immunohistochemistry and tested the mRNA expression by RT-PCR, the protein level by Western blot, and the protein activity by radiometric analysis. The results demonstrate different expression, localization and activity of iNOS in normal versus tumor tissue. This is suggestive of a role for NO production from iNOS in human lung cancer because high concentrations of this short molecule may transform to highly reactive compounds such as peroxynitrite (ONOO-); moreover, through the upregulator NF-kB, they can induce a chronic inflammatory state representing an elevated risk for cell transformation to cancer.

Expression and secretion of RANTES (CCL5) in granulomatous calcified tissue before and after lipopolysaccharide treatment in vivo.

RANTES (regulated on activation, normal T cell-expressed and secreted) is a CC chemokine appearing to be involved in the recruitment of leukocytes at inflammation sites. RANTES is produced by CD8(+) T cells, epithelial cells, fibroblasts, and platelets. It acts in vitro in leukocyte activation and human immunodeficiency virus suppression, but its role in vivo is still uncertain. In our study, we established the involvement of RANTES in an in vivo model of chronic inflammation induced by potassium permanganate, leading to calcified granulomas. In our rat model, RANTES expression (mRNA and protein) was significantly upregulated in granulomatous tissue; RANTES expression was further increased upon i.p. injection of lipopolysaccharide (LPS), while it was kept at basal levels by dexamethasone (Dex) given 18 hours before sacrifice. LPS and Dex increased and decreased, respectively, the recruitment of mononuclear cells in granulomatous tissue compared with control granulomas from phosphate-buffered saline (PBS)-treated animals. In granuloma tissue, levels of RANTES were higher in LPS-treated rats and lower in the Dex group compared to controls. RANTES was also found in the conditioned medium of granuloma tissue from treated (LPS or Dex) and untreated (PBS) rats. When LPS was added in vitro for 18 hours, RANTES was further increased, except in the Dex group (P > 0.05). On serum analysis, RANTES levels were higher in the LPS group and lower in the Dex group compared to controls. This study shows for the first time that RANTES is produced in vivo in chronic, experimental inflammatory states, an effect increased by LPS and inhibited by Dex.

Immunological activation of human umbilical cord blood mast cells induces tryptase secretion and interleukin-6, and histidine decarboxilase mRNA gene expression.

Allergy is the result of a complex immune cascade leading to the disregulated production of Th2 cytokines, the generation of allergen-specific IgE-producing B cells and the subsequent activation and degranulation of mast cells upon allergen challenge. Mast cell effector function significantly influences the quantity, duration and magnitude of most allergic reactions. Here, using isolated human umbilical cord blood mast cells (HUCBMC) from CD34+ cells, activated with anti-IgE (10 microg/ml) in culture, we found an augmented release of IL-6, tryptase and histamine (p < 0.01 compared with control). In addition, in these cells anti-IgE (10 microg/ml) activated the expression of histidine decarboxylase (HDC) and IL-6. In these studies we describe a new biological activity of anti-IgE in inducing histidine decarboxylase and IL-6, suggesting that this cytokine may have an important effect on allergic and inflammatory diseases mediated by mast cells. Moreover, with these data we confirm the immunoregulatory and inflammatory function of mast cells.

Erratum in Int J Biol Markers 2007; 22(3): 226-231.

Errata Corrige. In the article 'Localization and activity of iNOS in normal human lung tissue and lung cancer tissue' by Speranza L et al, which was published in the July-September issue of the International Journal of Biological Markers (Int J Biol Markers 2007; 22 (3): 226-231), the name of the 6th Author was misprinted. We reprint here with his correct name: S. Tet.

Inhibitory effect of quercetin on tryptase and interleukin-6 release, and histidine decarboxylase mRNA transcription by human mast cell-1 cell line.

Mast cells are involved in inflammatory processes and in allergic reactions where immunologic stimulation leads to degranulation and generation of numerous cytokines and inflammatory mediators. Mast cells have been proposed as an immune gate to the brain, as well as sensors of environmental and emotional stress, and are likely involved in neuropathologic processes such as multiple sclerosis. Among mast cell products, the protease tryptase could be associated with neurodegenerative processes through the activation of specific receptors (PARs) expressed in the brain, while interleukin (IL)-6 likely causes neurodegeneration and exacerbates dysfunction induced by other cytokines; or it could have a protective effect against demyelinisation. In this report we show that quercetin, a natural compound able to act as an inhibitor of mast cell secretion, causes a decrease in the release of tryptase and IL-6 and the down-regulation of histidine decarboxylase (HDC) mRNA from human mast cell (HMC)-1 cells. As quercetin dramatically inhibits mast cell tryptase and IL-6 release and HDC mRNA transcription by HMC-1 cell line, these results nominate quercetin as a therapeutical compound in association with other therapeutical molecules for neurological diseases mediated by mast cell degranulation.

Submaximal aerobic exercise with mechanical vibrations improves the functional status of patients with chronic fatigue syndrome.

AIM: Chronic fatigue syndrome (CFS) is an illness characterised by disabling fatigue of uncertain aetiology and other nonspecific symptoms. Typically CFS patients complain of a severe fatigue made worse by exercise, with a consistent reduction of working activity. A physical deconditioning could explain CFS features as well as a neuromuscular dysfunction, of central or peripheric origin. METHODS: Ten CFS patients were enrolled in a protocol of a rehabilitative treatment over a six-month period: they underwent a submaximal and predominantly aerobic exercise with a reduced O2 consumption using a Galileo 2000 system that provides mechanical vibrations characterised by sinusoid vertical sollecitations. Before and after such treatment, all patients underwent a pressure pain thresholds profile, an evaluation of physical and psychosocial parameters using the visual analogue scale (VAS) of Scott-Huskisson, and a muscle performance analysis by the CIBEX 6000 dynamometer. RESULTS: After the six-month period of study there was an overall improvement of the above described parameters as compared to the basal determinations. CONCLUSION: We conclude that the rehabilitative exertion provides an useful treatment for CFS patients particularly to realize an effective training of the explosive strength.

Rantes potentiates human macrophage aggregation and activation responses to calcium ionophore (A23187) and activates arachidonic acid pathways.

Regulated on activation, normal T-cell expressed and presumably secreted (RANTES), which generally mediates monocyte-macrophage (MO) activation and recruitment, is a protein of 8-10 kD that chemoattracts eosinophils, monocytes and certain T leukocyte subsets. RANTES is coded for by a gene cluster located on human chromosome 17 and is a human T-cell specific molecule. RANTES is a member of a beta intercrine subfamily reported to be a selective chemoattractant for human monocytes rather than neutrophils, and is also a chemoattractant for memory T lymphocytes, CD4+ cells. RANTES is a modulator of many important macrophage functions in addition to aggregation, such as chemotaxis and phagocytosis. Our investigations focussed on the ability to modulate the aggregation of macrophages induced by calcium ionophore A23187. The ionophore A23187 directly induced potent aggregation of MO which was markedly enhanced when the cells were pretreated with RANTES. However, the addition of RANTES in the absence of other co-stimuli did not directly induce aggregation. Additional cytokines examined for possible induction of macrophage aggregation were interleukin-1 (IL-1), tumor necrosis alpha (TNF-alpha), and IL-6; all proved to be incapable of inducing aggregation directly, nor did they enhance the effects of A23187 on macrophage aggregation. Additionally, we found that RANTES can directly stimulate MO to activate specific pathways of arachidonic acid cascade, inducing a synthesis and release of thromboxane (TxA2) and leukotriene B4 (LTB4). RANTES did not augment the potent ability of A23187 to induce increased production of LTB4 or TxA2 by human MO. These data suggest that RANTES can contribute directly to monocyte-leukocyte-activation during inflammatory responses, resulting in greater cell aggregation, activation, and specific pro-inflammatory arachidonic acid products release, such as TxA2 and LTB4.

Role of quercetin (a natural herbal compound) in allergy and inflammation.

An investigated flavonoid, quercetin, is reviewed in this article. Quercetin is a bioflavonoid found in red wine, grapefruit, onions, apples, black tea, and, in lesser amounts, in leafy green vegetables and beans. Quercetin has an antioxidant and anti-inflammatory activity and prevents cancer. Quercitin inhibits the growth of certain malignant cells in vitro, and histamine and most cyclin-dependent kinases and also displays unique anticancer properties. Quercetin is a natural compound that blocks substances involved in allergies and is able to act as an inhibitor of mast cell secretion, causes a decrease in the release of tryptase, MCP-1 and IL-6 and the down-regulation of histidine decarboxylase (HDC) mRNA from few mast cell lines. Quercetin is a safe, natural therapy that may be used as primary therapy or in conjunction with conventional methods.

IL-15 an immunoregulatory and anti-cancer cytokine. Recent advances.

Interleukins are mediators of inflammation, immunity and cancer. IL-15 is a cytokine produced by several leukocytes including phagocytes in response to infections and other signals that trigger innate immunity. IL-15 has many homologies to interleukin-2 (IL-2) and like IL-2, stimulates NK cells. This cytokine acts also on memory CD8+ T-cell. IL-15, therefore acts, probably through selective inhibition of tumor promoting molecules, as a new compound for the adjuvant treatment of solid tumors. In this review we propose a newly revised mechanism of interleukin 15 in inflammation and cancer.

Rat peritoneal mast cells release regulated upon activation normal T cell expressed and secreted (RANTES) after TNF-alpha activation.

Chemokines are a family consisting of at least ten distinct novel 8-10 kD cytokines. The cysteine-cysteine (C-C) chemokines are chemoattractant and activators for monocytes, T cells and mast cells. RANTES is the prototype of the C-C chemokine subfamily, purified from different sources with chemoattractant and activator properties. In this study we found that supernatants derived from TNF-alpha (scalar concentrations)-activated rat peritoneal mast cell cultures (5 x 10(5)/mL), incubated overnight, produced high levels of RANTES. This data describes an additional mode of generation of RANTES. Moreover, RANTES mRNA was not significantly produced in untreated cells, while it was dramatically increased by calcium ionophore A23187, LPS and TNF-alpha compared with the controls. These results underscore the importance of the presence of mast cells for the production of RANTES in the inflammatory process and contribute to an understanding of the mechanism by which RANTES profoundly affects inflammatory responses in vivo.