MT1a mRNA Expression in Human Lung Cells (BEAS 2B) After Airborne PM10 and Copper Exposure: A Possible Biomarker for Asthma

Puerto Ricans have the highest prevalence, morbidity and mortality of asthma than any other ethnic group in the U.S. and can be triggered by different stimuli such as airborne Particle pollution (PM). PM transfers transition metals, including cooper, to the human airways generating reactive oxygen species (ROS). Metallothionein protein (MT1A) is a free radical scavenger expected to be induced in lung cells by PM exposure; however, this has never been proven nor demonstrated. The hypothesis involves the amounts of PM10 in 2004, which would be significantly higher in the urban sites during months of March and June, thereby increasing PM metal exposure and inducing MT1A gene at the urban site. PM10 filters for Urban (Guaynabo) and rural (Fajardo) site were provided by the PREQB. Organic extracts were prepared from filters by means of Soxhlet Extraction and heavy metals were determined using Atomic Absorption Spectrometry. BEAS-2B cells were cultured and exposed to PM10 during 4 hrs. RNA was isolated and relative mRNA levels of MT1A were determined by RT-PCR. Urban PM10 concentrations were 35 ug/m3 and 60ug/m3 for the months of March and June, respectively. Concentrations of copper for this urban PM10 were 29.6 ppm and 50.3 ppm, for March and June, respectively. Exposure to Cu leads to an MT1A expression in lung cells demonstrating its response to metal exposure. Cellular response for the mRNA expression of MT1A was greater in relative high inflammation markers than in PM10 samples

A Novel Role of PM Extracts on the Post-Transcriptional Control of Pro-Inflammatory Mediators, IL-6 and CXCL8

Exposure to airborne particulate matter (PM) has been associated with the transcriptional up-regulation of pro-inflammatory mediators. However, the effect of PM on post-transcriptional regulation of pro-inflammatory mediators has not been fully explored. In this study, we examined the acute effect of organic extracts from urban PM, rural PM and diesel exhaust particles (DEP) on the post-transcriptional control of interleukin-6 (IL-6) and interleukin-8 (CXCL8) using a human bronchial epithelial cell line. Both PM and DEP extracts induced the release of IL-6 and CXCL8 after 24 h of exposure. Time-course experiments were conducted to examine changes in mRNA steady-state levels and half-lives. The steady-state levels of CXCL8 mRNA increase at 15 min on cells exposed to both PM and DEP extracts. Meanwhile only the urban extract induced significant increases of IL-6 mRNA levels at 15 min. Indirect measurements of IL-6 mRNA half-life showed a dramatic increase in cells exposed to the organic extracts. CXCL8 mRNA half-life increases in cells exposed to PM extracts and not DEP extract. Nuclear run-ons demonstrated that the urban PM and DEP extracts promoted an up-regulation in the transcription rate of CXCL8 at 15 min but not for IL-6. Urban and rural PM influences the post-transcriptional control of CXCL8

Selective ATP-Binding Cassette Subfamily C Gene Expression and Proinflammatory Mediators Released by BEAS-2B after PM2.5, Budesonide, and Cotreated Exposures

ATP-binding cassette subfamily C (ABCC) genes code for phase III metabolism proteins that translocate xenobiotic (e.g., particulate matter 2.5 (PM2.5)) and drug metabolites outside the cells. IL-6 secretion is related with the activation of the ABCC transporters. This study assesses ABCC1–4 gene expression changes and proinflammatory cytokine (IL-6, IL-8) release in human bronchial epithelial cells (BEAS-2B) exposed to PM2.5 organic extract, budesonide (BUD, used to control inflammation in asthmatic patients), and a cotreatment (Co-T: PM2.5 and BUD). A real-time PCR assay shows that ABCC1 was upregulated in BEAS-2B exposed after 6 and 7 hr to PM2.5 extract or BUD but downregulated after 6 hr of the Co-T. ABCC3 was downregulated after 6 hr of BUD and upregulated after 6 hr of the Co-T exposures. ABCC4 was upregulated after 5 hr of PM2.5 extract, BUD, and the Co-T exposures. The cytokine assay revealed an increase in IL-6 release by BEAS-2B exposed after 5 hr to PM2.5 extract, BUD, and the Co-T. At 7 hr, the Co-T decreases IL-6 release and IL-8 at 6 hr. In conclusion, the cotreatment showed an opposite effect on exposed BEAS-2B as compared with BUD. The results suggest an interference of the BUD therapeutic potential by PM2.5

Linking Endotoxins, African Dust PM10 and Asthma in an Urban and Rural Environment of Puerto Rico

African Dust Events (ADE) are a seasonal phenomenon that has been suggested to exacerbate respiratory and proinflammatory diseases in Puerto Rico (PR). Increases in PM10 concentration and the effects of biological endotoxins (ENX) are critical factors to consider during these storms. ENX promote proinflammatory responses in lungs of susceptible individuals through activation of the Toll-like receptors (TLR2/4) signaling pathways. The objective of the study was to evaluate the toxicological and proinflammatory responses stimulated by ADE PM10 ENX reaching PR using human bronchial epithelial cells. PM10 organic extracts from a rural and urban site in PR (March 2004) were obtained from ADE and non-ADE and compared. A retrospective data analysis (PM10 concentration, aerosol images, and pediatric asthma claims) was performed from 2000 to 2012 with particular emphasis in 2004 to classify PM samples. Urban extracts were highly toxic, proinflammatory (IL-6/IL-8 secretion), and induced higher TLR4 expression and NF-κB activation compared to rural extracts. ENX were found to contribute to cytotoxicity and inflammatory responses provoked by urban ADE PM10 exposure suggesting a synergistic potency of local and natural ENX incoming from ADE. The contribution of ADE PM10 ENX is valuable in order to understand interactions and action mechanisms of airborne pollutants as asthma triggers in PR

A comparative pharmacokinetic and pharmacodynamic study of two novel Cuban PEGylated rHuEPO versus MIRCERA® and ior®EPOCIM

Context: The recombinant human erythropoietin (rHuEPO) stimulates the erythropoiesis process. Because this glycoprotein has a short half-life, it needs to be administrated two to three times a week. One of the technics to solve this issue is the PEGgilation. Aims: To evaluate the pharmacokinetics (PK) and pharmacodynamics of two new branched PEGylated erythropoietins (i.e., an asymmetric 32 kDa-PEG2-rHuEPO and a symmetric 40 kDa-PEG2-rHuEPO molecule) compared to non-PEGylated ior®EPOCIM and MIRCERA®. Methods: Serum concentrations of both PEGylated and non-PEGylated erythropoietins were measured at various time points in order to determine PK parameters using non-compartmental analysis approach. The reticulocyte (%), erythrocyte count and hemoglobin levels were ascertained in order to compare the effect of these molecules after administrating a single intravenous dose (10 μg/kg) of each product in male New Zealand rabbits. Results: Both branched PEGylated erythropoietin forms exhibited half-lives that were significantly longer than ior®EPOCIM (p<0.05), but not statistically different to MIRCERA®. The mean elimination half-life increased from 4 h (ior®EPOCIM) to 131 h for the 32 kDa-PEG2-rHuEPO and 119 h for the 40 kDa-PEG2-rHuEPO. Conversely, MIRCERA® exhibits a half-life of 64 h. Both PEGylated erythropoietin products significantly enhanced the stimulating effect on reticulocytes and erythrocytes formation, as well as on hemoglobin levels, when compared to ior®EPOCIM treatment up to 42 days post-dose. Conclusions: The PEGylation strategy employed in this study is an effective method to modify the pharmacokinetics and pharmacodynamics of rHuEPO molecule achieving higher half-lives and, therefore, longer in vivo bioactivity. Both of the branched PEGylated-EPO forms tested are promising candidates for human testing