Non-steroidal anti-inflammatory drugs increase MRP4 expression in an endometriotic epithelial cell line in a PPARa dependent manner.

OBJECTIVE: Endometriosis is a debilitating disease characterized by chronic inflammation. The transporter multidrug resistance-associated protein 4 (MRP4/ABCC4) is expressed in human endometrial tissue; it is overexpressed in ectopic endometrial tissue, and is modulated by the anti-inflammatory lipid Lipoxin A4 (LXA4). Recently, it was demonstrated that aspirin induces platelet MRP4 over-expression, through genomic modulation in megakaryocytes. Since patients with endometriosis frequently use aspirin or other non-aspirin Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), the aim of this study was to verify whether aspirin and other NSAIDs enhance MRP4 expression in 12Z human endometriotic epithelial cells and whether this was peroxisome proliferator-activated receptor alpha (PPARa) dependent. MATERIALS AND METHODS: MRP4 and PPARa expression was analyzed by Q-RT-PCR using TaqMan® Master Mix and TaqMan® Assay Reagents (Life Technologies, Monza, Italy) and Western blot. RESULTS: In 12Z cells, aspirin and other NSAIDs enhanced MRP4 mRNA and protein expression; these treatments also induced PPARa expression. Aspirin and diclofenac-induced increases in MRP4 expression were not observed in cells where PPARa was knocked down using siRNA. NSAIDs-induced MRP4 expression was correlated with augmented PGE2 secretion, indicating functional relevance. CONCLUSIONS: MRP4 expression was increased in cells treated with NSAIDs and the nuclear receptor PPARa is involved. Elevated PGE2 levels in cell supernatants correlate with its increased transport by MRP4 after NSAID treatment. More importantly, we provide evidence that in endometriotic epithelial cells aspirin and non-aspirin NSAIDs treatments alter gene expression.

Ferritin downregulation in HIV-infected cells.

Levels of serum ferritin are increased in AIDS patients in relation to the progression of the disease. To establish whether or not this in vivo increase could be due to a direct effect of the virus on the infected cells, three HIV-permissive cell lines, the CD4-positive HeLa-T4-6c and C8166 cells and the CD4-negative RD cells, were infected with HIV-1 strains. The expression of ferritin was followed during the course of acute infection, in parallel to other cellular components. Unexpectedly, all three cell lines showed a phase of decrease in their ferritin content after infection by HIV-1, not justified by the modest and late increase of ferritin in the fluids, due to disruption of infected cells. Since ferritin is involved in the control of cell growth and DNA synthesis, its downregulation may be implied both in cell toxicity and DNA abnormalities due to HIV infection.

Use of discriminant analysis to assess disease activity in pulmonary tuberculosis with a panel of specific and nonspecific serum markers.

Several cell activation markers, acute phase reactants, enzymes, and antituberculous antibody serum levels have been proposed as possible markers to monitor disease activity in patients with tuberculosis. They have all shown limited sensitivity or specificity. The authors therefore attempted to generate a canonical variable using discriminant analysis, including sensitive and specific parameters, to be a reliable marker in classifying patients correctly during the course of pulmonary tuberculosis. The following parameters were selected: two soluble cell activation markers (soluble interleukin-2 receptor and sCD8); the levels of immunoglobulin (Ig) G and IgM antibodies against the A60 antigen complex; and the presence of specific antibodies directed to eight different A60 components, revealed by Western blot analysis. The tests were performed on sera from three groups of patients with pulmonary tuberculosis. The first group comprised 25 patients with onset tuberculosis, clinically active (OTCA), evaluated at the time of admission. The second group included 28 patients with chemotherapy-treated tuberculosis, clinically active (CTCA), 2 months after therapy had begun. The third group included 20 patients with tuberculosis, nonclinically active (TNCA), who had had at least 1 year of effective therapy. The authors obtained an 80.9% rate of correct classification for the three groups and a rate of 100% when OTCA and TNCA were compared. The patients with CTCA were scarcely differentiated and tended to be distributed into the two other groups. To improve the separation between patients with CTCA and those with OTCA, a second canonical variable was generated with a 91.7% rate of correct classification, as compared with 71% obtained using the sCD8 as the best single variable. The mean values of the last canonical variable were statistically different (Mann-Whitney test, P = .049) when stratified for acid fast bacilli-positive or negative CTCA patients (microscopic detection). Three patients, followed during the entire course their disease, were, as expected, correctly positioned with respect to the subsequent disease phases.

Behaviour of several 'progression markers' during the HIV-Ab seroconversion period. Comparison with later stages.

Two acute phase reactants, four cytokines, five soluble factors and lymphocyte subpopulations have been simultaneously evaluated in 16 subjects before and closely after the HIV-Ab seroconversion time. The same variables have also been determined in 50 HIV-Ab-negative high risk subjects, in 36 CDC II-III and in 30 CDC IV patients, utilizing a mixed longitudinal epidemiological model. The results show significant variations of few parameters in the early phases (increase: sCD8, beta-2-Microglobulin, sIL-2R, sCD23, Neopterin, IFN-alpha; decrease: CD4+ lymphocytes). In the course of the disease, many others parameters progressively increase (IFN-tau, IL-4, IL-6, acid-alpha 1-glycoprotein, alpha 1-antitrypsin) or decrease (B- and T-lymphocytes). Ferritin, in particular, highly increases only in CDC IV stage. These data may be useful to monitor patients during the entire course of their disease and to suggest the time elapsed from seroconversion.

Soluble intercellular adhesion molecule-1 and procollagen III peptide are reliable markers of disease severity in psoriasis.

Levels of soluble intercellular adhesion molecule-1 (sICAM-1) and procollagen III peptide (PIIIP) were measured respectively by enzyme immunoassay (EIA) and radioimmunoassay (RIA) methods in sera from 14 patients affected with psoriasis. The same determinations were also performed on suction blister fluids (BFs) obtained from lesional and non-lesional skin. Fourteen normal subjects were used as controls. Significant correlations were found between the serum levels and psoriasis area and severity index (PASI), (R = 0.62 for sICAM-1 and R = 0.73 for PIIIp, respectively). Of the PASI components, infiltration and erythema represented the variables most closely related to PIIIP (R = 0.85; R = 0.72 respectively). Differently from PIIIP, whose levels were significantly lower in the sera than in skin BFs (serum: median value 1.05, range 0.7-2.3 vs. lesional skin fluid: 11.8, 4.8-30 U/ml), sICAM-1 molecules were found predominantly in the sera (serum: median 316, range 117-579 vs lesional skin fluid: median 70, range 31-252 ng/ml). These data cannot exclude that sICAM-1 molecules detected in suction BFs may derive from serum contamination.

Seroprevalence of anti-human parvovirus B19 antibodies in patients attending a centre for sexually transmitted diseases.

The aim of this study was to establish the serological prevalence of anti-human Parvovirus B19 (HP-B19) antibodies in a group of 321 patients attending a Centre for Sexually Transmitted Diseases (STDs) and epidemiologically examine whether this virus may also be sexually transmitted. For this purpose, the serum prevalence of anti-HP-B19 evaluated in STD patients (39%) was compared with that of 164 healthy blood donors (10%, p < 0.001), using commercially available ELISA methods detecting the anti-VP1 reactivity of the sera. The same STD patients were also analyzed for serum reactivities against 4 STD-causing microorganisms, namely T. pallidum (TPHA), HBV (HBcAb), HCV (HCV-Ab) and HIV (HIV-Ab), to observe possible associations with the serum anti-HP-B19 reactivity. These tests were also carried out with commercially available kits. The results suggest that the serum anti-HP-B19 antibody prevalence in patients with STDs is increased, also independently of their intravenous drug addition and varies with the reactivity pattern determined. In addition, as expected for a STD, the anti-HP-B19 prevalence is increased in homobisexual patients compared with heterosexuals.

[Electrophoretic mobility of lymphocytes in multiple myeloma and bronchial cancer]. / Mobilità elettroforetica dei linfociti nel mieloma multiplo e nel carcinoma bronchiale.

The electrophoretic mobility of peripheral lymphocytes was studied in normal subjects and patients with myeloma and bronchial carcinoma. Distinct differences in anodic mobility were noted between the sub-populations in all three groups. Total lymphocyte migration was significantly slower in the two tumour groups.

Increased doubled haploid plant regeneration from rice (Oryza sativa L.) anthers cultured on colchicine-supplemented media.

Plating rice anthers on a semisolid induction medium containing 250 or 500 mg/l colchicine for 24 or 48 h-incubations followed by transfer to colchicine-free medium and standard anther culture procedures resulted in overall 1.5- to 2.5- fold increases in doubled haploid green plant productions compared to control anther cultures. The addition of colchicine had no detrimental effects on the different anther culture efficiency parameters, but in some treatments led to significant enhancement of anther callusing frequency or callus green plant regenerating ability. The most efficient treatment raised doubled haploid plant recovery from 31% to 65.5%. These results suggest that post-plating colchicine treatment of anthers, since it was found to improve both anther culture efficiency and doubled haploid plant recovery frequency, could be integrated into rice doubled haploid plant production programmes.

Localization and identification of phenolic compounds in Theobroma cacao L. somatic embryogenesis.

Cocoa breeders and growers continue to face the problem of high heterogeneity between individuals derived from one progeny. Vegetative propagation by somatic embryogenesis could be a way to increase genetic gains in the field. Somatic embryogenesis in cocoa is difficult and this species is considered as recalcitrant. This study was conducted to investigate the phenolic composition of cocoa flowers (the explants used to achieve somatic embryogenesis) and how it changes during the process, by means of histochemistry and conventional chemical techniques. In flowers, all parts contained polyphenolics but their locations were specific to the organ considered. After placing floral explants in vitro, the polyphenolic content was qualitatively modified and maintained in the calli throughout the culture process. Among the new polyphenolics, the three most abundant were isolated and characterized by 1H- and 13C-NMR. They were hydroxycinnamic acid amides: N-trans-caffeoyl-l-DOPA or clovamide, N-trans-p-coumaroyl-l-tyrosine or deoxiclovamide, and N-trans-caffeoyl-l-tyrosine. The same compounds were found also in fresh, unfermented cocoa beans. The synthesis kinetics for these compounds in calli, under different somatic embryogenesis conditions, revealed a higher concentration under non-embryogenic conditions. Given the antioxidant nature of these compounds, they could reflect the stress status of the tissues.

Effect of data normalization for age on the correlations between corneometric values and serum molecule levels in plaque-type psoriatic patients.

Corneometry has been considered useful both to evaluate disease severity and to monitor psoriatic patients during treatment. However, a limitation of this technique is that the patient's age influences the corneometric determinations, thus reducing their clinical usefulness. The aim of this study was, therefore, to establish whether age normalization of the corneometric results may provide more reliable data for clinical use. Corneometric levels were determined in 10 plaque-type psoriatic patients, under standard conditions. Eight serum variables, including transforming growth factor-beta 1 and seven soluble membrane molecules, were assayed with commercially available immune-enzyme methods in the same patients, whose age and PASI scores were also recorded. The age normalization procedure improved all the correlation coefficients calculated on the lesional or non-lesional corneometric values versus the PASI scores as well as versus the other serum variables. This approach may render corneometric determinations more useful to evaluate disease status or treatment effect in patient groups with plaque-type psoriasis.

Minimal dose of aerosolized interferon-alpha in human subjects: biological consequences and side-effects.

Interferon-alpha (IFN-alpha) is a leucocyte-derived cytokine with pleiotropic effects on the cells of the immune system, including the ability to promote viral and microbial killing. This study was designed to evaluate the biologically active dosage of aerosolized lymphoblastoid IFN-alpha, in normal subjects and patients with chronic bronchitis, using serum 2'-5' oligoadenylate synthetase (OAS) as a marker of IFN-alpha activity. Three groups of subjects were included: two healthy groups and one of patients with chronic bronchitis. Group A (controls, n = 5) was studied in order to determine the minimal IFN-alpha dose able to induce biological effects without side-effects. IFN-alpha was given in a dose escalation trial including 0, 0.3 x 10(6), 1.0 x 10(6) and 3.0 x 10(6) IU.day-1 (5 day administration). Only the administration of 3.0 x 10(6) IU.day-1 of IFN-alpha induced a significant biological activity, increasing serum levels of OAS. Group B (controls, n = 5) and C (chronic bronchitis, n = 5) were given 3.0 x 10(6) IU.day-1 (10 day administration) in order to study serum, bronchoalveolar lavage fluid (BALF) and BALF cell modifications, after treatment. OAS serum levels and nitroblue tetrazolium (NBT) reduction tests, the latter used as a measure of phagocyte cell activity, increased both in normal subjects and in patients with chronic bronchitis. No significant change of serum IFN-alpha levels was found. It is concluded that aerosolized IFN-alpha administration to the lung is well-tolerated at biologically active doses. The activity can be monitored by quantifying OAS serum levels through a simple blood test.

Cytokine levels correlate with a radiologic score in active pulmonary tuberculosis.

Pulmonary tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. This microorganism is capable of inducing a delayed hypersensitivity reaction in the lung, with subsequent expression of the disease. This reaction depends on the presence of different cytokines that exert specific functions. The aim of this study was to evaluate the presence and the concentrations of nine different modulators in bronchoalveolar lavage fluid (BALF). For this purpose, 15 patients with active pulmonary tuberculosis were enrolled at the time of diagnosis, prior to institution of antituberculous therapy. All the patients demonstrated M. tuberculosis in the sputum, and their disease extention was defined by high-resolution computed tomography (HRCT) using a score which included the presence of six findings: miliary nodules, nodules < 10 mm, consolidation, ground glass, cavity and bronchial wall thickening. This score was more sensitive than an equivalent score calculated on the basis of chest radiology. HRCT score was calculated for each area of the two lungs in order to define the more and the less affected lung for each patient. The bronchoalveolar lavage (BAL) was performed in the more affected area for each lung. The HRCT total score for each washed area ranged between 1 and 15, and showed more significant differences between the more and less affected lungs (p = 0.0004) than those obtained with the individual radiologic findings (p ranged between 0.60 and 0. 004). The BAL concentrations of the nine cytokines evaluated for the more and less affected lungs were compared: interleukin-6 (IL-6), IL-8, IL-12, tumor necrosis factor-alpha (TNF-alpha), and interferon gamma (IFN-gamma) showed significant differences (p ranged between 0. 016 and 0.0007). In addition, each cytokine concentration was correlated with the HRCT score. Significant correlations were found with IL-12, IL-6, IL-8, IL-2, and TNF-alpha. The correlations between cytokines and HRCT total score were better than those observed with the individual radiologic findings. A correlation matrix for the different cytokines evaluated one against each other, has also been added to show common behavior of these modulators. A similar analysis was also performed for the radiologic abnormalities.

Effects of aerosolized interferon-alpha in patients with pulmonary tuberculosis.

Interferon-alpha (IFN-alpha) is a cytokine exerting pleiotropic activities, including antimicrobial effects, especially directed against intracellular infectious bacteria. It may be administered by aerosol to reach the lower respiratory tract without systemic side effects. The aim of the study reported here was the evaluation of aerosolized IFN-alpha treatment (3 MU/dose, given three times a week; total study dose: 72 MU/2 mo) in combination with conventional antimycobacterial therapy in patients with pulmonary tuberculosis. Two groups of 10 patients each were compared before and after 2 mo of conventional antituberculous chemotherapy with or without inhaled IFN-alpha. Several biologic (bronchoalveolar lavage fluid [BALF] cellularity, Mycobacterium tuberculosis [MT] number in sputum), biochemical (BALF concentrations of 10 cytokines, BALF IFN-alpha receptor levels), and clinical (fever, vital signs, high-resolution computed tomography [HRCT] images) measures were made in these patients at the time of their enrollment and at the end of the observation period of the study. Fever, MT number in sputum, and abnormalities in HRCT images showed significantly earlier resolution in the IFN-alpha-treated group, together with a more significant decrease in BALF interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha) concentrations and significantly greater pre- versus posttreatment variations in IL-2 and IFN-gamma. These data, taken together, suggest that IFN-alpha administration may favorably affect the evolution of pulmonary tuberculosis when combined with antimycobacterial therapy.

Mutual interactions between HIV-1 and cytokines in adherent cells during acute infection.

The production of cytokines by HIV-infected cells from adherent tissues as well as their effects on HIV replication in the same cells were investigated. CD4-transfected HeLa-T4-6c epithelial cells, CD4-positive normal lung fibroblasts and CD4-negative RD rhabdomyosarcoma cells were infected with HIV-1. All cultures were permissive for virus replication, which was completed within 48-72 h by Hela-T4-6c and RD cells and 2-3 weeks in normal fibroblasts. During the course of HIV replication, a series of cytokines (particularly IL-6 and TNF alpha) was produced and released in parallel to the peak of virus growth, in amounts varying with the cell system studied. Treatment of cultures with recombinant cytokines given at concentrations in the range of those induced by HIV-1 indicated that IL-6 and TNF alpha caused an increase of: i) CD4 expression, ii) HIV absorption to uninfected cells, and iii) release of infectious virions by infected cells. The fact that HIV-1 absorption and spread can be mediated by HIV-induced cytokines may be relevant in the pathogenesis of the in vivo disease, as it may constitute a possible self-enhancing model of HIV infection also in the solid tissues.