Spatial distribution and temporal dynamics of invasive and native mosquitoes in a large Mediterranean city.

Mosquitoes, including invasive species like the Asian tiger mosquito Aedes albopictus, alongside native species Culex pipiens s.l., pose a significant nuisance to humans and serve as vectors for mosquito-borne diseases in urban areas. Understanding the impact of water infrastructure characteristics, climatic conditions, and management strategies on mosquito occurrence and effectiveness of control measures to assess their implications on mosquito occurrence is crucial for effective vector control. In this study, we examined data collected during the local vector control program in Barcelona, Spain, focusing on 234,225 visits to 31,334 different sewers, as well as 1817 visits to 152 fountains between 2015 and 2019. We investigated both the colonization and recolonization processes of mosquito larvae within these water infrastructures. Our findings revealed higher larval presence in sandbox-sewers compared to siphonic or direct sewers, and the presence of vegetation and the use of naturalized water positively influenced larval occurrence in fountains. The application of larvicidal treatment significantly reduced larvae presence; however, recolonization rates were negatively affected by the time elapsed since treatment. Climatic conditions played a critical role in the colonization and recolonization of sewers and urban fountains, with mosquito occurrence exhibiting non-linear patterns and, generally, increasing at intermediate temperatures and accumulated rainfall levels. This study emphasizes the importance of considering sewers and fountains characteristics and climatic conditions when implementing vector control programs to optimize resources and effectively reduce mosquito populations.

Mental impact of Covid-19 among Spanish healthcare workers. A large longitudinal survey.

AIMS: Longitudinal data on the mental health impact of the coronavirus disease 2019 (Covid-19) pandemic in healthcare workers is limited. We estimated prevalence, incidence and persistence of probable mental disorders in a cohort of Spanish healthcare workers (Covid-19 waves 1 and 2) -and identified associated risk factors. METHODS: 8996 healthcare workers evaluated on 5 May-7 September 2020 (baseline) were invited to a second web-based survey (October-December 2020). Major depressive disorder (PHQ-8 ≥ 10), generalised anxiety disorder (GAD-7 ≥ 10), panic attacks, post-traumatic stress disorder (PCL-5 ≥ 7), and alcohol use disorder (CAGE-AID ≥ 2) were assessed. Distal (pre-pandemic) and proximal (pandemic) risk factors were included. We estimated the incidence of probable mental disorders (among those without disorders at baseline) and persistence (among those with disorders at baseline). Logistic regression of individual-level [odds ratios (OR)] and population-level (population attributable risk proportions) associations were estimated, adjusting by all distal risk factors, health care centre and time of baseline interview. RESULTS: 4809 healthcare workers participated at four months follow-up (cooperation rate = 65.7%; mean = 120 days s.d. = 22 days from baseline assessment). Follow-up prevalence of any disorder was 41.5%, (v. 45.4% at baseline, p < 0.001); incidence, 19.7% (s.e. = 1.6) and persistence, 67.7% (s.e. = 2.3). Proximal factors showing significant bivariate-adjusted associations with incidence included: work-related factors [prioritising Covid-19 patients (OR = 1.62)], stress factors [personal health-related stress (OR = 1.61)], interpersonal stress (OR = 1.53) and financial factors [significant income loss (OR = 1.37)]. Risk factors associated with persistence were largely similar. CONCLUSIONS: Our study indicates that the prevalence of probable mental disorders among Spanish healthcare workers during the second wave of the Covid-19 pandemic was similarly high to that after the first wave. This was in good part due to the persistence of mental disorders detected at the baseline, but with a relevant incidence of about 1 in 5 of HCWs without mental disorders during the first wave of the Covid-19 pandemic. Health-related factors, work-related factors and interpersonal stress are important risks of persistence of mental disorders and of incidence of mental disorders. Adequately addressing these factors might have prevented a considerable amount of mental health impact of the pandemic among this vulnerable population. Addressing health-related stress, work-related factors and interpersonal stress might reduce the prevalence of these disorders substantially. Study registration number: NCT04556565.

Four-month incidence of suicidal thoughts and behaviors among healthcare workers after the first wave of the Spain COVID-19 pandemic.

Healthcare workers (HCW) are at high risk for suicide, yet little is known about the onset of suicidal thoughts and behaviors (STB) in this important segment of the population in conjunction with the COVID-19 pandemic. We conducted a multicenter, prospective cohort study of Spanish HCW active during the COVID-9 pandemic. A total of n = 4809 HCW participated at baseline (May-September 2020; i.e., just after the first wave of the pandemic) and at a four-month follow-up assessment (October-December 2020) using web-based surveys. Logistic regression assessed the individual- and population-level associations of separate proximal (pandemic) risk factors with four-month STB incidence (i.e., 30-day STB among HCW negative for 30-day STB at baseline), each time adjusting for distal (pre-pandemic) factors. STB incidence was estimated at 4.2% (SE = 0.5; n = 1 suicide attempt). Adjusted for distal factors, proximal risk factors most strongly associated with STB incidence were various sources of interpersonal stress (scaled 0-4; odds ratio [OR] range = 1.23-1.57) followed by personal health-related stress and stress related to the health of loved ones (scaled 0-4; OR range 1.30-1.32), and the perceived lack of healthcare center preparedness (scaled 0-4; OR = 1.34). Population-attributable risk proportions for these proximal risk factors were in the range 45.3-57.6%. Other significant risk factors were financial stressors (OR range 1.26-1.81), isolation/quarantine due to COVID-19 (OR = 1.53) and having changed to a specific COVID-19 related work location (OR = 1.72). Among other interventions, our findings call for healthcare systems to implement adequate conflict communication and resolution strategies and to improve family-work balance embedded in organizational justice strategies.

Hospital-acquired influenza infections detected by a surveillance system over six seasons, from 2010/2011 to 2015/2016.

BACKGROUND: In addition to outbreaks of nosocomial influenza, sporadic nosocomial influenza infections also occur but are generally not reported in the literature. This study aimed to determine the epidemiologic characteristics of cases of nosocomial influenza compared with the remaining severe cases of severe influenza in acute hospitals in Catalonia (Spain) which were identified by surveillance. METHODS: An observational case-case epidemiological study was carried out in patients aged ≥18 years from Catalan 12 hospitals between 2010 and 2016. For each laboratory-confirmed influenza case (nosocomial or not) we collected demographic, virological and clinical characteristics. We defined patients with nosocomial influenza as those admitted to a hospital for a reason other than acute respiratory infection in whom ILI symptoms developed ≥48 h after admission and influenza virus infection was confirmed using RT-PCR. Mixed-effects regression was used to estimate the crude and adjusted OR. RESULTS: One thousand seven hundred twenty-two hospitalized patients with severe laboratory-confirmed influenza virus infection were included: 96 (5.6%) were classified as nosocomial influenza and more frequently had > 14 days of hospital stay (42.7% vs. 27.7%, P < .001) and higher mortality (18.8% vs. 12.6%, P < .02). The variables associated with nosocomial influenza cases in acute-care hospital settings were chronic renal disease (aOR 2.44 95% CI 1.44-4.15) and immunodeficiency (aOR 1.79 95% CI 1.04-3.06). CONCLUSIONS: Nosocomial infections are a recurring problem associated with high rates of chronic diseases and death. These findings underline the need for adherence to infection control guidelines.

Effectiveness of antiviral treatment in preventing death in severe hospitalised influenza cases over six seasons.

We investigated the predictors of neuraminidase inhibitor (NAI) treatment in severe hospitalised influenza cases and the association between antiviral treatment and mortality. An observational epidemiological study was carried out in Catalonia (Spain) during 2010-2016 in patients aged ⩾18 years. Severe hospitalised cases of laboratory-confirmed influenza requiring hospitalisation were included. We collected demographic, virological and clinical characteristics. Mixed-effects logistic regression was used to estimate crude and adjusted odds ratio (aOR). We included 1727 hospitalised patients, of whom 1577 (91.3%) received NAI. Receiving NAI ⩽48 h after onset of clinical symptoms (aOR 0.37, 95% confidence interval (CI) 0.22-0.63), ⩽3 days (aOR 0.49, 95% CI 0.30-0.79) and ⩽5 days (aOR 0.50, 95% CI 0.32-0.79) was associated with a reduction in deaths. In patients admitted to the intensive care unit (ICU) (595; 34.5%), treatment ⩽48 h (aOR 0.32, 95% CI 0.14-0.74), ⩽3 days (aOR 0.44, 95% CI 0.20-0.97) and ⩽5 days (aOR 0.45, 95% CI 0.22-0.96) was associated with a reduction in deaths. Receiving treatment >5 days after onset of clinical symptoms was not associated with the reduction in deaths in hospitalised patients or those admitted to the ICU. NAI treatment of hospitalised patients with severe confirmed influenza was effective in avoiding death, mainly when administered ⩽48 h after symptom onset, but also when no more than 5 days had elapsed.

Trends and risk factors of hepatitis A in Catalonia after the introduction of a hepatitis A+B vaccination programme.

At the end of 1998, universal hepatitis A+B vaccination of 12 year olds was introduced in Catalonia. The aim was to examine trends in hepatitis A during 2005-2015 and assess risk factors by age group. We carried out an observational epidemiological study of the incidence and risk factors of hepatitis A reported to the surveillance system. Information on exposure was recorded for each case for the 2-6 weeks before symptom onset. Spearman's coefficient was used to evaluate the trends of rates. The chi-square test was used to compare categorical. We studied 2621 hepatitis A cases; the age mean was 26.6 years (SD=18.2), and >50% of cases were in the 20-49 years age group. The incidence decreased from 3.28/100 000 in 2005 to 1.50/100 000 in 2015. The rate for women decreased over time (P = .008), but the reduction was not significant in men (P = .234). Men consistently had higher rates than women with the biggest difference being in the 20-34 years age group (rate 8.8 vs 2.8). The greatest risk factor was travel to an endemic country (42.1%) in the 0-19 years age group and male-to-male sexual contact (18.6%) in the 20-49 years age group. The case fatality rate in adults aged >49 years was 0.4%. In conclusion, the vaccination programme of preadolescents resulted in a reduction in hepatitis A cases. However, a significant amount of cases still appear in immigrants and men who have sex with men. Hepatitis A in adults is an emerging health problem that will require new strategies.

Loss of p27 phosphorylation at Ser10 accelerates early atherogenesis by promoting leukocyte recruitment via RhoA/ROCK.

Reduced phosphorylation of the tumor suppressor p27(Kip1) (p27) at serine 10 (Ser10) is a hallmark of advanced human and mouse atherosclerosis. Apolipoprotein E-null mice defective for this posttranslational modification (apoE(-/-)p27Ser10Ala) exhibited increased atherosclerosis burden at late disease states. Here, we investigated the regulation of p27 phosphorylation in Ser10 at the very initial stages of atherosclerosis and its impact on endothelial-leukocyte interaction and early plaque formation. Hypercholesterolemia in fat-fed apoE(-/-) mice is associated with a rapid downregulation of p27-phospho-Ser10 in primary endothelial cells (ECs) and in aorta prior to the development of macroscopically-visible lesions. We find that lack of p27 phosphorylation at Ser10 enhances the expression of adhesion molecules in aorta of apoE(-/-) mice and ECs, and augments endothelial-leukocyte interactions and leukocyte recruitment in vivo. These effects correlated with increased RhoA/Rho-associated coiled-coil containing protein kinase (ROCK) signaling in ECs, and inhibition of this pathway with fasudil reduced leukocyte-EC interactions to control levels in the microvasculature of p27Ser10Ala mice. Moreover, apoE(-/-)p27Ser10Ala mice displayed increased leukocyte recruitment and homing to atherosusceptible arteries and augmented early plaque development, which could be blunted with fasudil. In conclusion, our studies demonstrate a very rapid reduction in p27-phospho-Ser10 levels at the onset of atherogenesis, which contributes to early plaque build-up through RhoA/ROCK-induced integrin expression in ECs and enhanced leukocyte recruitment.

Progestogens reduce thromboxane production by cultured human endothelial cells.

OBJECTIVES: Progestogens have been poorly studied concerning their roles in endothelial physiology. Prostanoids are vasoactive compounds, such as thromboxane A2, a potent vasoconstrictor, and prostacyclin, a vasodilator. We examined the effects of two progestogens used clinically, progesterone and medroxyprogesterone acetate, on thromboxane A2 production by cultured human umbilical vein endothelial cells (HUVEC) and investigated the role of progesterone receptors and the enzymes involved in production of thromboxane A2 and prostacyclin. METHODS: Cells were exposed to 1-100 nmol/l of either progesterone or medroxyprogesterone acetate, and thromboxane A2 production was measured in culture medium by enzyme immunoassay. Gene expression of prostacyclin synthase and thromboxane synthase was analyzed by quantitative real-time polymerase chain reaction. Expression of prostacyclin synthase protein was analyzed by Western blot. RESULTS: Both progestogens decreased thromboxane A2 release after 24 h. Protein and gene expression of prostacyclin synthase were increased after exposure to both progestogens, without changes in thromboxane synthase expression. These effects induced by progestogens were mediated through progesterone receptors, since they were decreased in the presence of the progesterone receptor antagonist RU486. The cyclo-oxygenase-1 selective inhibitor reduced thromboxane release. CONCLUSION: Progesterone and medroxyprogesterone acetate decreased HUVEC thromboxane release in a progesterone receptor-dependent manner, without changes in thromboxane synthase expression and enhanced prostacyclin synthase gene and protein expression.

Tuberculosis recurrence and its associated risk factors among successfully treated patients.

BACKGROUND: Little is known about recurrent tuberculosis (TB) in developed countries. The objective of this study was to determine the probability of TB recurrence and the associated risk factors among cured patients in a city with moderate TB incidence. METHODS: A population-based retrospective longitudinal study was carried out in Barcelona, Spain. All patients with culture-confirmed TB and drug susceptibility testing were included between 1995 and 1997 and followed until December 2005. The authors defined recurrence as a new TB event after a patient was considered cured and had remained free of the disease for a minimum of 1 year. Kaplan-Meier and Cox regression were used in the statistical analysis. HRs with 95% CIs were calculated. RESULTS: Among the 681 patients studied, the authors observed 29 recurrences (recurrence rate 0.53/100 person-years of follow-up). The mean incidence of TB in Barcelona from 1995 to 2005 was 36.25 cases per 100,000 inhabitants. The incidence of recurrence was 14.6 times higher in the cohort than the incidence of a first TB episode in the general population. The factors associated with recurrence at bivariate level were being male, being an immigrant, being an intravenous drug user (IDU), having human immunodeficiency virus, smoking, being an alcoholic, being in prison, and having both pulmonary and extrapulmonary TB. At multivariate level, only being an immigrant (HR 3.2, 95% CI 1.2 to 9), an IDU (HR 2.9, 95% CI 1.3 to 6.4) and male (HR 4.3, 95% CI 1.3 to 14.6) were associated. CONCLUSION: Having TB in the past is a risk factor for developing TB. Social policies must be implemented in populations at risk of recurrence, especially in immigrants and IDUs.

Identification of a critical Sp1 site within the endoglin promoter and its involvement in the transforming growth factor-beta stimulation.

Endoglin, a component of the transforming growth factor-beta (TGF-beta) receptor complex expressed on endothelial cells, is involved in cardiovascular morphogenesis and vascular remodeling, as exemplified by the fact that the endoglin gene is the target for the autosomal dominant disorder known as hereditary hemorrhagic telangiectasia type 1. Since haploinsufficiency is the underlying mechanism for hereditary hemorrhagic telangiectasia type 1, understanding the regulation of endoglin gene expression appears to be a crucial step to correct the disease. In this study we have identified an Sp1 site at -37 as a critical element for the basal transcription of the endoglin TATA-less promoter. Since endoglin promoter activity is stimulated by TGF-beta and this stimulation is located at the Sp1-containing proximal region, we have investigated the possible involvement of Sp1 in the TGF-beta-mediated induction. Mutation of the Sp1-binding sequence, or addition of the Sp1 inhibitor WP631, abolished both the basal transcription activity and the TGF-beta responsiveness of the endoglin promoter. Binding of Sp1 and Smad3 to the proximal promoter region -50/-29 was evidenced by electrophoretic mobility shift assays and DNA affinity precipitation studies. Furthermore, synergistic cooperation on the promoter activity between Sp1 and TGF-beta or Smad3 could be demonstrated by co-transfection experiments of reporter promoter constructs. The molecular mechanism underlying this cooperation appears to involve a direct physical interaction between Sp1 and Smad3/Smad4.

Cloning of the promoter region of human endoglin, the target gene for hereditary hemorrhagic telangiectasia type 1.

Endoglin (CD105) is a cell surface component of the transforming growth factor-beta (TGF-beta) receptor complex highly expressed by endothelial cells. Mutations in the endoglin gene are responsible for the hereditary hemorrhagic telangiectasia type 1 (HHT1), also known as Osler-Weber-Rendu syndrome (OMIM 187300). This is an autosomal dominant vascular disorder probably caused by a haploinsufficiency mechanism displaying low levels of the normal protein. To understand the mechanisms underlying the regulated expression of endoglin, a genomic DNA clone containing 3.3 kb of the 5'-flanking sequence of the human endoglin gene has been isolated. The 5'-flanking region of the endoglin gene lacks consensus TATA and CAAT boxes, but contains two GC-rich regions and consensus motifs for Sp1, ets, GATA, AP-2, NFkappaB, and Mad, as well as TGF-beta-, glucocorticoid-, vitamin D-, and estrogen-responsive elements. As determined by primer extension and 5' RACE experiments, a cluster of transcriptional start sites was found to be located 350 bp upstream from the translation initiation codon. To analyze the endoglin promoter activity, the upstream -400/+341 fragment was fused to the luciferase gene and transient transfections were conducted in several cell types. This construct displayed a tissue-specific activity in human and bovine endothelial cells. Analysis of various deletion constructs showed the existence of a basal promoter region within the -81/+350 fragment as well as major transcriptional regulatory elements within the -400/-141 fragment. Electrophoretic mobility shift assays demonstrated the specific interaction of a member of the ets family with a consensus motif located at position -68. A promoter construct mutated at this ets sequence showed a much reduced activity as compared with the wild-type construct, supporting the involvement of this ets motif in the basal activity of the promoter. The endoglin promoter exhibited inducibility in the presence of TGF-beta1, suggesting possible therapeutic treatments in HHT1 patients, in which the expression level of the normal endoglin allele might not reach the threshold required for its function. Isolation and characterization of the human endoglin promoter represents an initial step in elucidating the controlled expression of the endoglin gene.

Cloning of the human platelet endothelial cell adhesion molecule-1 promoter and its tissue-specific expression. Structural and functional characterization.

Platelet endothelial cell adhesion molecule-1 (PECAM-1; CD31) is a cell adhesion molecule involved in transendothelial migration and expressed by hemopoietic and endothelial cells. To understand the mechanisms underlying its regulated expression, a genomic clone containing 1555 bp of the 5'-flanking region and the first exon of the human PECAM-1 gene has been isolated. The 5'-flanking region of the PECAM-1 gene lacks a consensus TATA box, but contains consensus motifs for Sp1, EGR1, ets, helix-loop-helix (HLH) box, GATA, AP-2, C/EBP, YY1, CCACC, LyF-1, imperfect octamer, heptamer, high mobility group proteins (HMG) box, and nuclear factor-kappaB, as well as shear stress-, retinoic acid-, glucocorticoid-, and acute phase-responsive elements, and an Alu sequence. Successive 5' to 3' or 3' to 5' deletions revealed tissue-specific promoter activity within the two contiguous 0.22-kb NheI/BglII and 0.44-kb BglII/PstI fragments. The transcriptional activity displayed by the 0.22-kb NheI/BglII fragment was specific for the myeloid lineage, whereas the promoter activity of the 0.44-kb BglII/PstI fragment was apparently restricted to endothelial cells. The transcriptional activity of the 0.22-kb NheI/BglII fragment was confirmed by 5' RACE (rapid amplification of 5' cDNA ends) and S1 nuclease protection experiments that revealed previously unidentified transcription start sites. The 0.22-kb NheI/BglII promoter exhibited PMA inducibility in myeloid cells and contained a PMA-responsive element recognized by Sp1 and EGR-1 transcription factors. Isolation and characterization of the human PECAM-1 promoter represent an initial step in elucidating the controlled expression of the PECAM-1 gene.

Endoglin modulates cellular responses to TGF-beta 1.

Endoglin is a homodimeric membrane glycoprotein which can bind the beta 1 and beta 3 isoforms of transforming growth factor-beta (TGF-beta). We reported previously that endoglin is upregulated during monocyte differentiation. We have now observed that TGF-beta itself can stimulate the expression of endoglin in cultured human monocytes and in the U-937 monocytic line. To study the functional role of endoglin, stable transfectants of U-937 cells were generated which overexpress L- or S- endoglin isoforms, differing in their cytoplasmic domain. Inhibition of cellular proliferation and downregulation of c-myc mRNA which are normally induced by TGF-beta 1 in U-937 cells were totally abrogated in L-endoglin transfectants and much reduced in the S-endoglin transfectants. Inhibition of proliferation by TGF-beta 2 was not altered in the transfectants, in agreement with the isoform specificity of endoglin. Additional responses of U-937 cells to TGF-beta 1, including stimulation of fibronectin synthesis, cellular adhesion, platelet/endothelial cell adhesion molecule 1 (PECAM-1) phosphorylation, and homotypic aggregation were also inhibited in the endoglin transfectants. However, modulation of integrin and PECAM-1 levels and stimulation of mRNA levels for TGF-beta 1 and its receptors R-I, R-II, and betaglycan occurred normally in the endoglin transfectants. No changes in total ligand binding were observed in L-endoglin transfectants relative to mock, while a 1.5-fold increase was seen in S-endoglin transfectants. The degradation rate of the ligand was the same in all transfectants. Elucidating the mechanism by which endoglin modulates several cellular responses to TGF-beta 1 without interfering with ligand binding or degradation should increase our understanding of the complex pathways which mediate the effects of this factor.

Differential modulation of the expression of the intermediate filament proteins vimentin and nuclear lamins A and C by differentiation inducers in human myeloid leukemia (U-937, HL-60) cells.

Earlier reports seemed to indicate that the cytoplasmic intermediate filament protein vimentin and the nuclear intermediate-type filament proteins A and C lamins are expressed in a coordinate manner in human myeloid cells. We have comparatively studied the expression of the vimentin and the A/C lamin genes at the RNA and protein levels in human U-937 promonocytic and HL-60 promyelocytic cells treated with differentiation inducers. 12-O-Tetradecanoyl phorbol-13-acetate and cytosine arabinoside produced a coordinate and stable stimulation of both vimentin and A/C lamin expression in U-937 cells. A stable increase in vimentin expression was also produced by sodium butyrate and by dibutyryl cyclic AMP in U-937 cells and by dimethyl sulfoxide in HL-60 cells. In contrast, these agents produced only a transient increase in A/C lamin expression (maximum mRNA levels at 6-24 h), which later returned to expression levels similar to or even lower than those in untreated cells. Retinoic acid greatly inhibited vimentin expression in HL-60 cells, but it had little effect on A/C lamin expression. Taken together, the present results suggest that there are important differences in the mechanisms which regulate the expression of the vimentin and nuclear lamin genes, as well as in their implication in the differentiation of human myeloid cells.

Aggregated human immunoglobulins bind to modified proteins.

Human immunoglobulins treated at 55 degrees C in vitro are able to interact with maleylated bovine serum albumin (mBSA), but not with unmodified BSA. Gel filtration experiments demonstrated that the mBSA binding is associated with a high molecular weight complex of aggregated IgG. This aggregated IgG with binding capacity for mBSA could also be generated in vitro by treatment of human IgG at 37 degrees C or 40 degrees C and by incubation with human neutrophils. Furthermore, IgG aggregates with binding activity for mBSA could be detected in untreated synovial fluids from rheumatoid arthritis patients, indicating that these complexes occur in vivo. The phenomenon of binding to aggregated IgG was extended to other modified proteins such as maleylated human serum albumin (mHSA), acetyl low density lipoprotein (Ac-LDL) and BSA reacted with oxidized linolenic acid. Soluble forms of these modified proteins were able to compete for the interaction between aggregated IgG and surface-bound mBSA. We also found that aggregated IgG enhanced the Ac-LDL-dependent foam cell formation. These findings suggest a role for aggregated IgG in the metabolism of oxidized proteins.

Modulation by dexamethasone of insulin binding and insulin receptor mRNA levels in U-937 human promonocytic cells.

Treatment with 5 x 10(-6) M dexamethasone stimulated insulin binding in human promonocytic U-937 cells. When curvilinear Scatchard plots were examined according to the one-site model, only changes in affinity, but not in receptor numbers, were observed. However, when the two-site model was applied, an increase in both the affinity and the number of the high affinity-low capacity sites was observed, with maximum values at 15 h. By contrast, the low affinity-high capacity sites did not undergo significant alterations. Northern blot assays revealed two insulin receptor-related mRNAs of approximately 11 and 7 kb in size. Dexamethasone increased the levels of these RNAs, following similar kinetics to those of high affinity receptor expression. This suggests that the 11 and 7 kb species carry information for high affinity insulin receptors, and that in U-937 cells the expression of this receptor subclass is primarily regulated at the mRNA level.

Camptothecin induces differentiation and stimulates the expression of differentiation-related genes in U-937 human promonocytic leukemia cells.

We have studied the effect of the DNA topoisomerase I inhibitor camptothecin on growth, differentiation, and gene expression in U-937 human promonocytic leukemia cells. At a concentration of 20 nM, camptothecin caused significant DNA strand breakage and decreased the growth activity by accumulating cells preferentially at the G2 phase of the cycle. The growth arrest occurred concomitantly with an increase in cell size. Under those conditions, camptothecin induced differentiation, as demonstrated by (a) the capacity of the cells to generate reactive oxygen species, (b) the increase in the surface expression of the leukocyte integrins CD11b/CD18 and CD11c/CD18, (c) the increase in the cellular content of the intermediate filament protein vimentin, and (d) the decrease in the surface expression of the transferrin receptor. Camptothecin also induced the expression of differentiation markers in other human myeloid cells, namely, the promonocytic THP-1 and the myelomonocytic HL-60 cell lines. Northern blot assays revealed that camptothecin stimulated the expression of CD11b, CD11c, and vimentin at the mRNA level. Moreover, the drug increased the transcription rate of the vimentin gene, as shown by "run-on" transcription assays.

Vimentin expression as a late event in the in vitro differentiation of human promonocytic cells.

The administration of either 12-O-tetradecanoyl phorbol-13-acetate (TPA, 3 x 10(-8) M), sodium butyrate (SB, 10(-3) M), N6,2'-O-dibutyryladenosine-3':5'-cyclic monophosphate (dbcAMP, 10(-3) M), cytosine arabinoside (ara-C, 10(-7) M), amsacrine (mAMSA, 10(-7) M) or retinoic acid (RA, 10(-6) M) inhibits the growth activity of human promonocytic U-937 cells, by arresting them at G1 or at the G1/S border (SB, RA, ara-C), at G2 (mAMSA) or at G1 and G2 (dbcAMP). All these agents trigger cell differentiation, as proved by the increased expression of the maturation-associated CD11b and CD11c surface antigens, and induce the expression of the vimentin gene at both the protein and the mRNA levels. TPA, SB and dbcAMP behave as "early" inducers, in the sense that vimentin mRNA levels are rapidly increased (hour 6) upon drug administration. In contrast, mAMSA and RA behave as "late" inducers, since they do not increase vimentin mRNA levels until 48 to 72 hours, following the stimulation of surface antigen expression. The action of RA is characterized by an initial inhibition period, in which the basal level of vimentin mRNA is abolished (hour 24). Nevertheless, this RNA is later re-induced, to reach at 72 hours higher levels than in untreated cells. Moreover, RA is capable of delaying the early induction of vimentin expression caused by TPA and SB, without affecting the normal expression of differentiation markers. Taken together, these results strongly suggest that vimentin expression is not required at the initial stages of promonocytic cell differentiation, although it could play a role at an advanced stage.

Differentiation of human promonocytic leukemia U-937 cells with DNA topoisomerase II inhibitors: induction of vimentin gene expression.

The administration of the DNA topoisomerase II inhibitors 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) (10(-7) M), VP-16 (2 x 10(-7) M), or novobiocin (1.5 x 10(-4) M) reduces the growth activity of human promonocytic leukemia U-937 cells, by arresting them preferentially at the G2 (m-AMSA and VP-16) or at the G1 and G2 (novobiocin) phases of the cell cycle. Under these conditions, m-AMSA and VP-16 induce the differentiation of the cells efficiently, as proved both by an increase in the production of reactive oxygen species and by the activation of the surface expression of CD11b and CD11c, two differentiation-specific antigens. Novobiocin also induces the expression of those differentiation markers, but to a lesser extent. Analyses by Northern blot indicate that the topoisomerase II inhibitors reduce the levels of c-myc and beta-actin mRNA and increase the levels of vimentin mRNA. The expression of vimentin is also stimulated at the protein level, as indicated by immunofluorescence assays. This represents one of the few known instances in which topoisomerase inhibitors stimulate gene expression in eukaryotic cells.