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Miniaturised methods have been used to construct dose-response curves for the effects of inhibitory drugs on prostaglandin synthesis using individual rectal biopsies obtained from patients with ulcerative colitis. The potency of different drugs has been compared. Sulphasalazine, 5 amino salicylic acid (5-ASA) and N-acetyl 5-ASA inhibited prostaglandin synthesis at high concentration, but sulphapyridine and prednisolone did not. Indomethacin and flurbiprofen were considerably more potent inhibitors. These data imply that sulphasalazine does not act by simple inhibition of prostaglandin synthesis but leave open the possibility that sulphasalazine or 5-ASA may be inhibitors of the synthesis of related lipoxygenase products. [ABSTRACT FROM PUBLISHER]

Introduction and Objectives: Chronic respiratory diseases are associated with an increased risk of cardiovascular diseases (CVD); however, it is unknown whether some respiratory diseases are at higher risk than others. In this perspective, head-to-head studies comparing bronchiectasis and chronic obstructive pulmonary disease (COPD) are encouraged. We explored whether the prevalence of cardiovascular risk factors (diabetes mellitus and hyperlipidemia) and cardiovascular comorbidity (systemic hypertension, ischemic heart diseases, cardiac arrhythmia, stroke) are different in these two diseases., Methods: The present retrospective case-control study aimed to compare patients with bronchiectasis with age and sex-matched individuals with COPD. A total of 63 patients with bronchiectasis and 63 with COPD were retained for analysis., Results: Patients with bronchiectasis had a lower risk of systemic hypertension (OR 0.42 (C.I. 0.20 to 0.87)) and diabetes mellitus (OR 0.28 (C.I. 0.09 to 0.81)). In contrast, ischemic heart diseases, cardiac arrhythmia, stroke, and hyperlipidemia did not differ between the two groups. Logistic regression analysis showed that age, male sex, and COPD remain independent risk factors for having at least one condition of a composite index including the above-mentioned CVD and CV risk factors. In detail, a patient with COPD has a risk of 4.648 times (C.I. 1.48 to 15.78) for having at least one CVD compared with a patient with bronchiectasis., Conclusions: The current findings suggest that subjects with bronchiectasis may experience lower cardiovascular risk than those with COPD. Larger studies are needed to confirm this preliminary observation and its clinical implications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Aims: Chemotherapy-induced dilated cardiomyopathy (CI-DCM) is a well-recognized phenotype of non-ischemic dilated cardiomyopathy (DCM), characterized by poor outcomes. However, a detailed comparison between idiopathic DCM (iDCM) and CI-DCM is still lacking., Methods and Results: All consecutive DCM patients enrolled in the Trieste Muscle Heart Disease Registry were analysed. CI-DCM and iDCM were defined according to current recommendations. The primary study outcome measure was all-mortality death and secondary outcomes were a) a composite of cardiovascular death/heart-transplantation/ventricular-assist-device implantation, and b) major ventricular arrhythmias. The study included 551 patients (499 iDCM and 52 CI-DCM). At enrolment, compared with iDCM, CI-DCM patients were older (51 ± 14 years vs. 58 ± 3 years, respectively, P < 0.001) and had a higher left ventricular ejection fraction (32% ± 9 vs. 35% ± 10, respectively, P = 0.03). Over a median follow-up of 90 months (IQR 54-140 months), CI-DCM patients had a higher incidence of all-cause mortality compared with iDCM (36.5% vs. 8.4% in CI-DCM and iDCM respectively, P < 0.001), while the incidence of major ventricular arrhythmias was higher in the iDCM group compared with CI-DCM (4% vs. 0%, in CI-DCM and iDCM respectively, P = 0.03). The risk of the composite outcome was comparable between the two groups (P = 0.91). At Cox multivariable analysis, the diagnosis of CI-DCM emerged as independently associated to primary outcome (HR 6.42, 95% C.I. 2.52-16.31, P < 0.001)., Conclusions: In a well-selected DCM cohort, patients with a chemotherapy-induced aetiology had a higher incidence of all-cause mortality compared with iDCM. Conversely, the incidence of life-threatening ventricular arrhythmic events was higher among patients with iDCM., (© 2022 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Background: Even in the era of highly active antiretroviral therapy (HAART), HIV-infected subjects are at higher risk of complications from vaccine-preventable diseases than those uninfected. The current international guidelines strongly recommend that these patients should receive all the routine childhood vaccinations. Although these children represent an appropriate target for immunization, the available data indicate suboptimal coverage rates., Methods: To evaluate seroprotection/seropositivity rates and vaccination coverage against the common vaccine-preventable diseases, all patients with vertically transmitted HIV-1 infection who attended San Martino Hospital were enrolled. Blood samples were collected for testing antibodies against diphtheria, tetanus, hepatitis A and B viruses by Enzyme-Linked ImmunoSorbent Assay and polioviruses by microneutralization test. In order to assess immunization coverage, retrospectively was recorded the vaccination history collecting data from Regional Immunization Database., Results: A total of 39 perinatally HIV-1 infected patients were included in the study. At the time of serum was obtained, the mean age was 18,1 years (range: 6-28). The median CD4+ T-lymphocyte count was 702 cells/mm(3) (2-1476 cells/mm(3)). Twenty-nine (74.4%) patients were found with HIV RNA load < 50 copies/mL. The proportion of subjects with protective anti-tetanus and anti-HBs were 43.6% and 30.8%, respectively. Seroprotection rates about 20% against rubella and measles were found, less than 20% against all the other antigens investigated. In particular, all patients resulted susceptible to mumps. High immunization rates were observed for polio and HBV (100% and 92.3%, respectively) and suboptimal for diphtheria-tetanus (84.6%). For the other recommended vaccines the rates were generally low. None of the patients received varicella vaccine doses., Conclusions: As in the HAART era the vertically acquired HIV infection has become a chronic treatable disease, the vaccine-induced long-term protection plays an increasingly significant role; despite good initial response to primary vaccination, subsequent decline and loss of detectable antibodies may be prevented by additional strategies for booster doses of vaccines in adolescents and young adults.

Altered EGFR activity is a causal factor for human tumor development, including malignant pleural mesotheliomas. The aim of the present study was the evaluation of the effects of Gefitinib on EGF-induced mesothelioma cell proliferation and the intracellular mechanisms involved. Cell proliferation, DNA synthesis and apoptosis were measured by MTT, thymidine incorporation and FACS analysis; EGFR, ERK1/2 and Akt expression and phosphorylation by Western blot, whereas receptor sites were analyzed by binding studies. Gefitinib inhibited EGF-induced proliferation in two mesothelioma cell lines, derived from pleural effusion (IST-Mes2) or tumor biopsy (ZL55). The treatment with Gefitinib induced cell cycle arrest in both cell lines, while apoptosis was observed only for high concentrations and prolonged drug exposure. EGF-dependent mesothelioma cell proliferation was mediated by EGFR and ERK1/2 phosphorylation, while Akt was not affected. Gefitinib inhibited both EGFR and ERK1/2 activation, being maximal at drug concentrations that induce cytostatic effects, suggesting that the proapoptotic activity of Gefitinib is independent from EGFR inhibition. Gefitinib treatment increased EGFR Bmax, possibly through membrane stabilization of inactive receptor dimers that we show to be induced by the drug also in the absence of EGF. EGFR activation of ERK1/2 represents a key pathway for pleural mesothelioma cell proliferation. Low concentrations of Gefitinib cause mesothelioma cell cycle arrest through the blockade of EGFR activity while high concentrations induce apoptosis. Finally, we propose that the formation of inactive EGFR dimers may contribute to the antitumoral activity of Gefitinib.

Because a subpopulation of cancer stem cells (tumor-initiating cells, TICs) is believed to be responsible for the development, progression, and recurrence of many tumors, we evaluated the in vitro sensitivity of human glioma TICs to epidermal growth factor receptor (EGFR) kinase inhibitors (erlotinib and gefitinib) and possible molecular determinants for their effects. Cells isolated from seven glioblastomas (GBM 1-7) and grown using neural stem cell permissive conditions were characterized for in vivo tumorigenicity, expression of tumor stem cell markers (CD133, nestin), and multilineage differentiation properties, confirming that these cultures are enriched in TICs. TIC cultures were challenged with increasing concentrations of erlotinib and gefitinib, and their survival was evaluated after 1-4 days. In most cases, a time- and concentration-dependent cell death was observed, although GBM 2 was completely insensitive to both drugs, and GBM 7 was responsive only to the highest concentrations tested. Using a radioligand binding assay, we show that all GBM TICs express EGFR. Erlotinib and gefitinib inhibited EGFR and ERK1/2 phosphorylation/activation in all GBMs, irrespective of the antiproliferative response observed. However, under basal conditions GBM 2 showed a high Akt phosphorylation that was completely insensitive to both drugs, whereas GBM 7 was completely insensitive to gefitinib, and Akt inactivation occurred only for the highest erlotinib concentration tested, showing a precise relationship with the antiproliferative effects of the drug. Interestingly, in GBM 2, phosphatase and tensin homolog expression was significantly down-regulated, possibly accounting for the insensitivity to the drugs. In conclusion, glioma TICs are responsive to anti-EGFR drugs, but phosphatase and tensin homolog expression and Akt inhibition seem to be necessary for such effect.

Background: The present study was aimed at investigating the effects of the co-administration of 4-OH Tamoxifen (4HT) and bicalutamide (BIC) on the human prostate cancer cell line LNCaP in vitro., Materials and Methods: The LNCaP FGC prostate cancer cell line was grown in vitro within a three-dimensional matrix formed by collagen gel under dihydrotestosterone (DHT) (0.1 nM) stimulation. Cells were incubated in the presence either of BIC at escalating concentrations (1 nM; 100 nM; 10 microM) or 4HT Tamoxifen (10 nM; 100 nM) or both compounds at the different concentrations studied. The cells were incubated for 144 hours, and growth was evaluated in non trypsinised cells by crystal violet vital dye staining assay., Results: BIC appeared to exert a dose-dependent inhibitory action, with the maximum inhibitory effect achieved by the 10 microM concentration. A comparable inhibitory effect was also observed after exposure to 4HT at both doses tested. No statistically significant interference was observed when BIC was combined with 4HT., Conclusion: 4HT, even at the higher concentration employed here, showed no major interference with the inhibitory effects of BIC.

This paper summarizes our most recent results of steroid enzyme studies on cultured breast and endometrial cancer cells. It deals mainly with estrogen 17 beta-hydroxysteroid oxidoreductase (17 beta HSOR) activity, which presides over estradiol (E2) and estrone (E1) interconversion, a major metabolic pathway of estrogens. Assessment of either the oxidative or reductive component of 17 beta HSOR was carried out on intact cells by means of an original approach based on reverse phase-high performance liquid chromatography and radioactive detection on line. This system allows the continuous monitoring of both precursor degradation and formation of several radiometabolites to assess rates and direction of steroid metabolism. Overall, hormone-responsive, estrogen receptor (ER)-positive cells, regardless of whether they were derived from breast (MCF7) or endometrial (Ishikawa) tumor tissues, showed a prevalence for reductive metabolism (E1-->E2), whilst oxidative pathways (E2-->E1) were largely dominant in non-responsive, ER-poor mammary (MDA-MB231) and endometrial (HEC-1A) cells. The above estimates of 17 beta HSOR activity were at variance with those obtained using the classical enzymology approach, not only in quantitative terms (being markedly lower using intact cell analysis), but also because the prevalent direction of estrogen metabolism was often reversed. Although striking methodological differences may well account for this discrepancy, intact cell analysis is undoubtedly more similar to the in vivo state than the artificial requirements of classical enzymology procedures.

Objective: To study the presence of estrogen-binding sites (EBS) in the synovial tissues of male and female patients with rheumatoid arthritis (RA) and in age- and sex-matched healthy controls., Methods: Both type 1 (high affinity, low binding capacity) and type 2 (reduced affinity, higher binding capacity) EBS were investigated in both soluble and nuclear fractions of homogenized synovial tissue samples by a dextran-coated charcoal method. To determine what type of synovial cell was positive for EBS, cryosections of synovial tissues were immunostained with a specific monoclonal anti-estrogen receptor antibody (anti-ER MAb) using both immunofluorescence and immunoperoxidase techniques. Double immunostaining with the anti-ER MAb and with specific MAb to detect different macrophage antigens (Ber-MAC3, MAC387, CD68) and CD8+ T cell subsets (CD29+, CD45RO+ and CD29-, CD45RO-) was performed., Results: Higher affinity EBS were found mostly in nuclear cell fractions of either RA or control synovial tissues (28 of the 33). These EBS were present to a lesser extent in soluble cell fractions (11 of the 33). Immunostaining showed the estrogen receptor-positive cells to be the macrophage-like synoviocytes and the CD8+, CD29+ T cells both in RA and in control synovial tissues. Higher nuclear content of EBS was consistent with more intense nuclear staining of synoviocytes and T cells., Conclusion: It is conceivable that the immunomodulatory activity exerted by estrogens is at least partly mediated through their interaction with EBS that are present on macrophage-like synoviocytes, functioning as antigen-processing and antigen-presenting cells, and on antigen-experienced (memory) CD8+ T lymphocytes (CD29+, CD45RO+).

A simple, rapid approach to the study of conversion rates and metabolic patterns of the steroids testosterone and estradiol is presented. It includes an optimized isocratic high-performance liquid chromatographic procedure in the reversed-phase mode and radioactive on-line detection. The purpose was to estimate the activity of key enzymes of steroid pathways, such as 17 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase, in in vivo conditions. Using this system, we obtained good efficiency and linearity of radio detection, under continuous flow conditions. Sensitivity limits were of the order of 50 and 70 cpm for [3H]estradiol and [14C]estrone, respectively, even though the efficiency was quite dissimilar (17.3% versus 56.2%). The applicability of this approach to studies of steroid metabolic pathways in growing cancer cells in culture is illustrated with examples of the conversion rates of both testosterone and estradiol. The high reproducibility (coefficients of variation of 2.7 and 5.1% for 3H and 14C, respectively) and good extraction efficiency (ranging from 86 to 94%) indicate the feasibility and reliability of this approach.

This review reports studies on long-term prostate cell lines using multiple experimental approaches. The main goal was to investigate the metabolism of testosterone (T) through in vitro conversion rates. Extensive studies were also carried out on growth curves, tritiated thymidine incorporation, and morphometry by either hormone-responsive or hormone-unresponsive, normal and neoplastic human (PC3 and DU-145) and canine (CAPE and CPA) cell lines. All of them were characterized for their content of both soluble and nuclear androgen receptors. Receptor studies at site I binding in both soluble and nuclear fractions were carried out to establish the hormone sensitivity status of cells. In two prostate epithelial cells, steroid metabolic conversions in vitro show predominantly an oxidative metabolism of T, forming mainly androstenedione. Conversion rates were greater than 50% in the first 24 hours and still higher after 72 hours. At the same time and under exactly the same experimental conditions, the other cells showed metabolic pathways in which reductive metabolism prevails, dihydrotestosterone (DHT) being the prevalent metabolite. Different metabolic patterns of steroids of several cell lines relate to the hormone sensitivity status of the cells; steroid receptor-endowed cells are maintaining higher levels of unconverted precursor than are receptor-empty cells. In fact, hormone-sensitive cells, such as cancer canine CPA and human DU-145, produced DHT early through slowly converting T. On the contrary, unresponsive cells such as human cancer cells PC3 and normal canine CAPE quickly metabolize T, but DHT formation was not observed. These significant differences between cells are highly reproducible provided the proportion between cell number and molar concentration of precursors is constant. Differences we observe cannot be attributed to different experimental conditions. Cell viability, extraction efficiency, and all other parameters used for monitoring cell growth kinetics do not substantiate these reported significant differences in metabolic abilities of cells. The divergent steroid metabolic pathway we observe in different prostate long-term cells appears to be an intrinsic, consistent, highly reproducible property of each cell line.

This paper presents a short review of the results obtained to date in our laboratory, with respect to the studies of steroid excretion profiles in both breast and endometrial cancer patients, by using gas-liquid chromatographic analysis. These data demonstrate the importance of minor estrogens, including catechol estrogens, and of their ratios with the classical ones, in studies of steroid metabolism in both breast and endometrial cancer. New data concerning postmenopausal endometrial cancer are consistent with our previous observations and demonstrate the necessity of measuring these steroids directly in tumors and examining patterns of metabolism in vitro. In order to analyze steroid metabolic patterns in vitro, however, high-performance liquid chromatography rather than gas-liquid chromatography methods are preferable on account of their selectivity, specificity, sensitivity and capacity to handle labile materials. With the aim of providing methods suitable for the complete resolution and analysis of these complex natural mixtures a method of computer-aided optimization of HPLC has been developed and its practical utility has been tested.

Studies on estrone metabolism by long term human endometrial cancer HEC-1A and Ishikawa cell lines are reported. These cell lines grow well in epithelial mono or plurilayer form, as previously reported. Ishikawa cells appear to be estrogen responsive whereas HEC-1A appear non responsive. In our experience Ishikawa cells show high affinity--low capacity estrogen binding sites in both soluble and nuclear fractions of the same range of ZR 75-1 and of MCF7, but HEC-1A cytosols gave Kd values in the order of 10(-8)-10(-9). These values are probably more representative of estrogen receptors of low affinity-high capacity (site II) and this is in agreement with previous results regarding their poor response to estrogens. These two different endometrial cancer cell lines exhibit at the same time, common and quite dissimilar metabolic patterns of estrogens. In fact, metabolic conversion studies carried out after 24th incubation at not far from physiological concentrations by using high pressure liquid chromatography in reverse phase mode plus "on line" radioactive detection showed: Both these well established cell lines are fast growing in culture with sufficient morphological or biochemical stability, at least during a limited number of passages and appear a useful material for studies on steroid metabolism. In both, estradiol (E2) and estrone (E1) were most part of converted products (more than 95%); negligible amounts of other radio-metabolites were observed. Quite different conversion rates of E1 to E2 have been shown by HEC-1A cells (6 times or more), with respect to Ishikawa.