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Ethnopharmacological Relevance: The use of "Mexican calea" (Calea zacatechichi Schltdl.) in ritualistic ceremonies, due to its dream-inducing effects, was until recently limited to indigenous communities in Mexico. However, the plant has recently gained popularity in Western societies being commonly used in recreational settings. Despite the traditional and recreational uses, mechanisms underlying its reported oneirogenic effects remain unknown, with no data available on its neurotoxic profile., Aim of the Study: The scarcity of toxicological data and the unknown role of major neurotransmitter systems in the dream-inducing properties of the plant prompted us to investigate which neurotransmitters might be affected upon its consumption, as well as the potential cytotoxic effects on neurons and microglial cells. Furthermore, we aimed to explore a relationship between the recorded effects and specific constituents., Materials and Methods: Effects on cholinergic and monoaminergic pathways were investigated using enzymatic assays, with the latter also being conducted in neuronal SH-SY5Y cells along with the impact on glutamate-induced excitotoxicity. Investigation of the neurotoxic profile was approached in neuronal SH-SY5Y and microglial BV-2 cells, evaluating effects on metabolic performance and membrane integrity using MTT and LDH leakage assays, respectively. Potential interference with oxidative stress was monitored by assessing free radical's levels, as well as 5-lipoxygenase mediated lipid peroxidation. Phenolic constituents were identified through HPLC-DAD-ESI(Ion Trap)MS n analysis., Results: Based on the significant inhibition upon acetylcholinesterase (p < 0.05) and tyrosinase (IC 50  = 60.87 ± 7.3 μg/mL; p < 0.05), the aqueous extract obtained from the aerial parts of C. zacatechichi interferes with the cholinergic and dopaminergic systems, but has no impact against monoamine oxidase A. Additionally, a notable cytotoxic effect was observed in SH-SY5Y and BV-2 cells at concentrations as low as 125 and 500 μg/mL (p < 0.05), respectively, LDH leakage suggesting apoptosis may occur at these concentrations, with necroptosis observed at higher ones. Despite the neurocytotoxic profile, these effects appear to be independent of radical stress, as the C. zacatechichi extract scavenged nitric oxide and superoxide radicals at concentrations as low as 62.5 μg/mL, significantly inhibiting also 5-lipoxygenase (IC 50  = 72.60 ± 7.3 μg/mL; p < 0.05). Qualitative and quantitative analysis using HPLC-DAD-ESI(Ion Trap)MS n enabled the identification of 28 constituents, with 24 of them being previously unreported in this species. These include a series of dicaffeoylquinic, caffeoylpentoside, and feruloylquinic acids, along with 8 flavonols not previously known to occur in the species, mainly 3-O-monoglycosylated derivatives of quercetin, kaempferol, and isorhamnetin., Conclusions: Our findings regarding the neuroglial toxicity elicited by C. zacatechichi emphasize the necessity for a thorough elucidation of the plant's toxicity profile. Additionally, evidence is provided that the aerial parts of the plant inhibit both acetylcholinesterase and tyrosinase, potentially linking its psychopharmacological effects to the cholinergic and dopaminergic systems, with an apparent contribution from specific phenolic constituents previously unknown to occur in the species. Collectively, our results lay the groundwork for a regulatory framework on the consumption of C. zacatechichi in recreational settings and contribute to elucidating previous contradictory findings regarding the mechanisms underlying the dream-inducing effects of the plant., 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. Published by Elsevier B.V.)

Mitoxantrone (MTX) is a pharmaceutical drug used in the treatment of several cancers and refractory multiple sclerosis (MS). Despite its therapeutic value, adverse effects may be severe, namely the frequently reported cardiotoxicity, whose mechanisms need further research. This work aimed to assess if inflammation or oxidative stress-related pathways participate in the cardiotoxicity of MTX, using the mouse as an animal model, at two different age periods (infant or adult mice) using two therapeutic relevant cumulative doses. Histopathology findings showed that MTX caused higher cardiac toxicity in adults. In MTX-treated adults, at the highest dose, noradrenaline cardiac levels decreased, whereas at the lowest cumulative dose, protein carbonylation increased and the expression of nuclear factor kappa B (NF-κB) p65 subunit and of M1 macrophage marker increased. Moreover, MTX-treated adult mice had enhanced expression of NF-κB p52 and tumour necrosis factor (TNF-α), while decreasing interleukin-6 (IL-6). Moreover, while catalase expression significantly increased in both adult and infant mice treated with the lowest MTX cumulative dose, the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glutathione peroxidase only significantly increased in infant animals. Nevertheless, the ratio of GAPDH to ATP synthase subunit beta decreased in adult animals. In conclusion, clinically relevant doses of MTX caused dissimilar responses in adult and infant mice, being that inflammation may be an important trigger to MTX-induced cardiotoxicity.

Limited fitness for practice may result from a mismatch between education and practice. Aiming to meet the common interests of academics and practitioners, the Portuguese Pharmaceutical Society (PPS) developed the Education and Practice Platform (EPP). The EPP includes one representative from each pharmacy faculty, and all Councils of Speciality Boards of Practice. Brainstorming with involved parties enabled sharing of interests, concerns and identifying a common path. Aims, mission, vision and values were set. The EPP's mission is to: act as an enabler to foster the quality and adequacy of education through sharing best practices, ultimately leading to facilitate professional integration, and to foster quality development in teaching practices with recognition for autonomy in freedom to teach and to learn. Its vision is an alignment of education and practice with the PPS' statutes to ensure validation of the competences defined for each practice area, and compliance with international guidance. Key performance indicators (KPIs) were set. Activities developed include the creation of a national forum to discuss education and practice, development of workshops on teaching methods and pharmacy internships, enhanced representation in international events and response to global and national requests. Ongoing work focuses on the creation of a common training framework in hospital and community pharmacy practice adapted to Portugal. The EPP is a worldwide case study, encouraging the development of discussion contributing to an open climate of sharing best practices, indirectly leading to foster a better alignment between education and practice. Many of these results are so far intangible in scientific terms but worth describing., Competing Interests: The authors declare no conflict of interest.

Predictive in vitro models are valuable alternatives to animal experiments for evaluating the transport of molecules and (nano)particles across biological barriers. In this work, an improved triple co-culture of air-blood barrier was set-up, being exclusively constituted by human cell lines that allowed to perform experiments at air-liquid interface. Epithelial NCI-H441 cells and endothelial HPMEC-ST1.6R cells were seeded at the apical and basolateral sides of a Transwell® membrane, respectively. Differentiated THP-1 cells were also added on the top of the epithelial layer to mimetize alveolar macrophages. Translocation and permeability studies were also performed. It was observed that around 14-18% of 50-nm Fluorospheres®, but less than 1% of 1.0 µm-Fluorospheres® could pass through the triple co-culture as well as the epithelial monoculture and bi-cultures, leading to the conclusion that both in vitro models represented a significant biological barrier and could differentiate the translocation of different sized systems. The permeability of isoniazid was similar between the epithelial monoculture and bi-cultures when compared with the triple co-culture. However, when in vitro models were challenged with lipopolysaccharide, the release of interleukin-8 increased in the bi-cultures and triple co-culture, whereas the NCI-H441 monoculture did not show any proinflammatory response. Overall, this new in vitro model is a potential tool to assess the translocation of nanoparticles across the air-blood barrier both in healthy state and proinflammatory state. STATEMENT OF SIGNIFICANCE: The use of in vitro models for drug screening as an alternative to animal experiments is increasing over the last years, in particular, models to assess the permeation through biological membranes. Cell culture models are mainly constituted by one type of cells forming a confluent monolayer, but due to its oversimplicity they are being replaced by three-dimensional (3D) in vitro models, that present a higher complexity and reflect more the in vivo-like conditions. Being the pulmonary route one of the most studied approaches for drug administration, several in vitro models of alveolar epithelium have been used to assess the drug permeability and translocation and toxicity of nanocarriers. Nevertheless, there is still a lack of 3D in vitro models that mimic the morphology and the physiological behavior of the alveolar-capillary membrane. In this study, a 3D in vitro model of the air-blood barrier constituted by three different relevant cell lines was established and morphologically characterized. Different permeability/translocation studies were performed to achieve differences/similarities comparatively to each monoculture (epithelium, endothelium, and macrophages) and bi-cultures (epithelial cells either cultured with endothelial cells or macrophages). The release of pro-inflammatory cytokines (namely interleukin-8) after incubation of lipopolysaccharide, a pro-inflammatory inductor, was also evaluated in this work., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Mannose receptor is highly expressed on alveolar macrophages, being a potential target to promote the specific local drug delivery of anti-tuberculosis agents through the use of functionalized nanocarriers. In this work, isoniazid (Isn)-loaded solid lipid nanoparticles (SLN), reinforced with stearylamine (SA) were produced by double emulsion technique and further surface-functionalized with mannose in a straightforward chemical approach. Upon pre-formulation assessment, SLN close to 500 nm average size, positively charged and with association efficiency of ISN close to 50% were obtained. Functionalization with mannose was performed after SLN production and confirmed by Fourier transform infrared spectroscopy (FTIR). Both functionalized and non-functionalized SLN demonstrated to devoid of toxicity when tested in human lung epithelial cell line (NCI-H441) and differentiated THP-1 (dTHP-1), reducing the intrinsic cytotoxicity of Isn when incorporated into SLN. Uptake studies were conducted on same macrophage-like cells and the results showed that fluorescent mannosylated SLN (M-SLN) were more efficient in be internalized comparatively to SLN. Moreover, the uptake of M-SLN was reduced when cells were pre-incubated with mannose, demonstrating the receptor-dependence internalization of functionalized SLN. These functionalized nanocarriers may represent a useful platform to target alveolar macrophages for delivering anti-infective drugs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Cashew gum (CG) was grafted with N-isopropylacrylamide (NIPA) by radical polymerization to originate a stimuli-sensitive copolymer for drug delivery purposes. NMR and IR spectroscopy confirmed the insertion of NIPA onto the cashew gum chains. The graft copolymer (CG:NIPA) demonstrated thermal responsiveness. The critical aggregation concentration of the copolymers at 25°C was higher than at 50°C. At temperatures lower than the LCST, the nanoparticle size ranged from 12 to 21nm, depending on the CG:NIPA ratio, but above the LCST the particles aggregated, increasing the particle size. Regarding the potential for future oral application, the nanoparticles showed no cytotoxic activity against the Caco-2 and HT29-MTX intestine cell lines. Epirubicin was encapsulated into nanoparticles of CG-NIPA (1:1), resulting in a 64% association efficiency and 22% loading capacity. Thus, the CG:NIPA graft copolymer demonstrates good potential for used in controlled drug delivery systems., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Combining two or more antiretroviral drugs in one medical product is an interesting but challenging strategy for developing topical anti-HIV microbicides. We developed a new vaginal delivery system comprising the incorporation of nanoparticles (NPs) into a polymeric film base - NPs-in-film - and tested its ability to deliver tenofovir (TFV) and efavirenz (EFV). EFV-loaded poly(lactic-co-glycolic acid) NPs were incorporated alongside free TFV into fast dissolving films during film manufacturing. The delivery system was characterized for physicochemical properties, as well as genital distribution, local and systemic 24h pharmacokinetics (PK), and safety upon intravaginal administration to mice. NPs-in-film presented suitable technological, mechanical and cytotoxicity features for vaginal use. Retention of NPs in vivo was enhanced both in vaginal lavages and tissue when associated to film. PK data evidenced that vaginal drug levels rapidly decreased after administration but NPs-in-film were still able to enhance drug concentrations of EFV. Obtained values for area-under-the-curve for EFV were around one log10 higher than those for the free drugs in aqueous vehicle (phosphate buffered saline). Film alone also contributed to higher and more prolonged local drug levels as compared to the administration of TFV and EFV in aqueous vehicle. Systemic exposure to both drugs was low. NPs-in-film was found to be safe upon once daily vaginal administration to mice, with no significant genital histological changes or major alterations in cytokine/chemokine profiles being observed. Overall, the proposed NPs-in-film system seems to be an interesting delivery platform for developing combination vaginal anti-HIV microbicides., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Purpose: The freezing step in lyophilization is the most determinant for the quality of biopharmaceutics. Using insulin as model of therapeutic protein, our aim was to evaluate the freezing effect in the stability and bioactivity of insulin-loaded PLGA nanoparticles. The performance of trehalose, sucrose and sorbitol as cryoprotectants was evaluated., Methods: Cryoprotectants were co-encapsulated with insulin into PLGA nanoparticles and lyophilized using an optimized cycle with freezing at -80°C, in liquid nitrogen, or ramped cooling at -40°C. Upon lyophilization, the stability of protein structure and in vivo bioactivity were assessed., Results: Insulin was co-encapsulated with cryoprotectants resulting in particles of 243-394 nm, zeta potential of -32 to -35 mV, and an association efficiency above 90%. The cryoprotectants were crucial to mitigate the freezing stresses and better stabilize the protein. The insulin structure maintenance was evident and close to 90%. Trehalose co-encapsulated insulin-loaded PLGA nanoparticles demonstrated enhanced hypoglycemic effect, comparatively to nanoparticles without cryoprotectant and added with trehalose, due to a superior insulin stabilization and bioactivity., Conclusions: The freezing process may be detrimental to the structure of protein loaded into nanoparticles, with negative consequences to bioactivity. The co-encapsulation of cryoprotectants mitigated the freezing stresses with benefits to protein bioactivity.

Unlabelled: Topical pre-exposure prophylaxis (PrEP) with antiretroviral drugs holds promise in preventing vaginal transmission of HIV. However, significant biomedical and social issues found in multiple past clinical trials still need to be addressed in order to optimize protection and users' adherence. One approach may be the development of improved microbicide products. A novel delivery platform comprising drug-loaded nanoparticles (NPs) incorporated into a thin polymeric film base (NPs-in-film) was developed in order to allow the vaginal administration of the microbicide drug candidate tenofovir. The system was optimized for relevant physicochemical features and characterized for biological properties, namely cytotoxicity and safety in a mouse model. Tenofovir-loaded poly(lactic-co-glycolic acid) (PLGA)/stearylamine (SA) composite NPs with mean diameter of 127nm were obtained with drug association efficiency above 50%, and further incorporated into an approximately 115μm thick, hydroxypropyl methylcellulose/poly(vinyl alcohol)-based film. The system was shown to possess suitable mechanical properties for vaginal administration and to quickly disintegrate in approximately 9min upon contact with a simulated vaginal fluid (SVF). The original osmolarity and pH of SVF was not affected by the film. Tenofovir was also released in a biphasic fashion (around 30% of the drug in 15min, followed by sustained release up to 24h). The incorporation of NPs further improved the adhesive potential of the film to ex vivo pig vaginal mucosa. Cytotoxicity of NPs and film was significantly increased by the incorporation of SA, but remained at levels considered tolerable for vaginal delivery of tenofovir. Moreover, histological analysis of genital tissues and cytokine/chemokine levels in vaginal lavages upon 14days of daily vaginal administration to mice confirmed that tenofovir-loaded NPs-in-film was safe and did not induce any apparent histological changes or pro-inflammatory response. Overall, obtained data support that the proposed delivery system combining the use of polymeric NPs and a film base may constitute an exciting alternative for the vaginal administration of microbicide drugs in the context of topical PrEP., Statement of Significance: The development of nanotechnology-based microbicides is a recent but promising research field seeking for new strategies to circumvent HIV sexual transmission. Different reports detail on the multiple potential advantages of using drug nanocarriers for such purpose. However, one important issue being frequently neglected regards the development of vehicles for the administration of microbicide nanosystems. In this study, we propose and detail on the development of a nanoparticle-in-film system for the vaginal delivery of the microbicide drug candidate tenofovir. This is an innovative approach that, to our best knowledge, had never been tested for tenofovir. Results, including those from in vivo testing, sustain that the proposed system is safe and holds potential for further development as a vaginal microbicide product., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Aim: Evaluate the potential of selected SNPs as predictors of methotrexate (MTX) therapeutic outcome., Patients & Methods: In total, 35 SNPs in 14 genes involved in MTX intracellular pathways and Phase II reactions were genotyped in 233 rheumatoid arthritis (RA) patients treated with MTX. Binary logistic regressions were performed by genotype/haplotype-based approaches. Non-Response- and Toxicity-Genetic Risk Indexes (Non-RespGRI and ToxGRI) were created., Results: MTX nonresponse was associated to eight genotypes and three haplotypes: MTHFR rs1801131 AA and rs1801133 TT; MS rs1805087 AA; MTRR rs1801394 A carriers; ATIC rs2372536 C carriers, rs4673993 T carriers, rs7563206 T carriers and rs12995526 T carriers; CC for GGH rs3758149 and rs12681874; CGTTT for ATIC combination 1; and CTTTC for ATIC combination 2. From overall Non-RespGRI patients with indexes 6-8 had more than sixfold increased risk for MTX nonresponse than those patients with indexes 0-5. MTX-related toxicity was associated to five genotypes and two haplotypes: ATIC rs2372536 G carriers, rs3821353 T carriers, rs7563206 CC and rs12995526 CC; ADORA2A rs2267076 T; CTTCC for ATIC combination 1; and TC for ADORA2A rs2267076 and rs2298383. From overall ToxGRI, patients with indexes 3-4 had more than sevenfold increased risk for MTX-related toxicity than those patients with indexes 1-2., Conclusion: Genotyping may be helpful to identify which RA patients will not benefit from MTX treatment and, consequently, important to personalized medicine in RA. Nevertheless, further studies are required to validate these findings.

Recent estimates indicate that tuberculosis (TB) is the leading cause of death worldwide, alongside the human immunodeficiency virus (HIV) infection. The current treatment is effective, but is associated with severe adverse-effects and noncompliance to prescribed regimens. An alternative route of drug delivery may improve the performance of existing drugs, which may have a key importance in TB control and eradication. Recent advances and emerging technologies in nanoscale systems, particularly nanoparticles (NPs), have the potential to transform such approach to human health and disease. Until now, several nanodelivery systems for the pulmonary administration of anti-TB drugs have been intensively studied and their utility as an alternative to the classical TB treatment has been suggested. In this context, this review provides a comprehensive analysis of recent progress in nanodelivery systems for pulmonary administration of anti-TB drugs. Additionally, more convenient and cost-effective alternatives for the lung delivery, different types of NPs for oral and topical are also being considered, and summarized in this review. Lastly, the future of this growing field and its potential impact will be discussed., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The aim of this work was to use annealing as a tool to optimize the lyophilization cycle by decreasing its duration time, and simultaneously preserve the stability of poly(lactic-co-glycolic acid) nanoparticles and with upmost importance, maintain the structural stability of loaded insulin, used as model therapeutic protein. The contribution of a cryoprotectant in this preservation process was also evaluated. Insulin-loaded nanoparticles co-encapsulated with and without trehalose as cryoprotectant were produced, resulting in a particle size of about 250-300 nm, a PdI around 0.25 and a zeta potential in the range of -20 to -24 mV. The insulin association efficiency was higher than 90%, and the loading capacity was of 11-12%. The use of annealing allowed the decrease of duration time of primary drying in about 38%, representing a global decrease of lyophilization time of around 26%. The residual moisture content of all lyophilizates was around 1%, and the reconstitution of lyophilizates obtained using annealing was even faster than those without annealing. The co-encapsulated trehalose better preserved the nanoparticle size throughout the lyophilization process using annealing, compared to formulation containing no cryoprotectant. Fourier-transform infrared spectroscopy showed that the trehalose-containing nanoparticles presented higher insulin structural maintenance, compared to nanoparticles without cryoprotectant, presenting an insulin structural maintenance of 85.3 ± 0.7% and 86.0 ± 1.0% for annealing and no annealing, respectively. This formulation also presented the closest structural similarity with native insulin. Interestingly, the structural features of insulin loaded into nanoparticles upon lyophilization with and without annealing were practically identical, showing that annealing had no detrimental effect in insulin structure. Circular dichroism and fluorescence spectroscopy confirmed these results. Overall, this work gave rise to the importance of annealing in decreasing the duration time of lyophilization of protein-loaded poly(lactic-co-glycolic acid) nanoparticles, and simultaneously ensuring the stability of the carrier and loaded protein., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The purpose of this work was to evaluate the influence of the co-encapsulation of lyoprotectants with insulin into PLGA nanoparticles, on the stability of the protein and nanoparticles upon lyophilization. Different lyoprotectants were used, namely trehalose, glucose, sucrose, fructose and sorbitol at 10% (w/v). Insulin-loaded PLGA nanoparticles with co-encapsulated lyoprotectants achieved a mean particle size of 386-466nm, and a zeta potential ranging between -34 and -38mV, dependent on the lyoprotectant used. Formulations had association efficiencies and loading capacities of 85-91% and 10-12%, respectively. The lyophilization process increased the colloidal stability of nanoparticles, and maintained their spherical shape and smooth surface, particularly in presence of lyoprotectants. XRPD revealed that the lyophilizates of nanoparticles with co-encapsulated lyoprotectants were amorphous, whereas formulations with externally added lyoprotectants, except trehalose, showed crystallinity. FTIR assessment showed that co-encapsulating lyoprotectants better preserved insulin structure upon lyophilization with a spectral area overlap of 82-87%, compared to only 72% in lyoprotectant absence. These results were confirmed by circular dichroism spectroscopy. Surprisingly, the simultaneous co-encapsulation and addition of lyoprotectants was detrimental to protein stabilization. The insulin in vitro release studies demonstrated that formulations with co-encapsulated trehalose, glucose, sucrose, fructose and sorbitol achieved 83%, 69%, 70%, 77% and 74%, respectively after 48h. In contrast, formulations added with those lyoprotectants prior lyophilization showed a lower release rate not higher than 60% after 48h. This work gives rise to a different promising strategy of co-encapsulating lyoprotectants and therapeutic proteins, to better stabilize protein structure upon lyophilization., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Methotrexate (MTX) is widely used for rheumatoid arthritis (RA) treatment. Single nucleotide polymorphisms (SNPs) could be used as predictors of patients' therapeutic outcome variability. Therefore, this study aims to evaluate the influence of SNPs in genes encoding for MTX membrane transport proteins in order to predict clinical response to MTX., Methods: Clinicopathological data from 233 RA patients treated with MTX were collected, clinical response defined, and patients genotyped for 23 SNPs. Genotype and haplotype analyses were performed using multivariate methods and a genetic risk index (GRI) for non-response was created., Results: Increased risk for non-response was associated to SLC22A11 rs11231809 T carriers; ABCC1 rs246240 G carriers; ABCC1 rs3784864 G carriers; CGG haplotype for ABCC1 rs35592, rs2074087 and rs3784864; and CGG haplotype for ABCC1 rs35592, rs246240 and rs3784864. GRI demonstrated that patients with Index 3 were 16-fold more likely to be non-responders than those with Index 1., Conclusions: This study revealed that SLC22A11 and ABCC1 may be important to identify those patients who will not benefit from MTX treatment, highlighting the relevance in translating these results to clinical practice. However, further validation by independent studies is needed to develop the field of personalized medicine to predict clinical response to MTX treatment.

Age is a known susceptibility factor for the cardiotoxicity of several anticancer drugs, including mitoxantrone (MTX). The impact of anticancer drugs in young patients is underestimated, thus we aimed to evaluate the cardiotoxicity of MTX in juvenile and adult animals. Juvenile (3 week-old) and adult (8-10 week-old) male CD-1 mice were used. Each group was treated with a 9.0mg/kg cumulative dose of MTX or saline; they were maintained in a drug-free period for 3-weeks after the last administration to allow the development of late toxicity (protocol 1), or sacrificed 24h after the last MTX administration to evaluate early cardiotoxicity (protocol 2). In protocol 1, no adult mice survived, while 2 of the juveniles reached the end of the protocol. High plasma aspartate aminotransferase/alanine aminotransferase ratio and a high cardiac reduced/oxidized glutathione ratio were found in the surviving MTX-treated juvenile mice. In protocol 2, a significant decrease in plasma creatine-kinase MB in juveniles was found 24h after the last MTX-administration. Cardiac histology showed that both MTX-treated populations had significant damage, although higher in adults. However, MTX-treated juveniles had a significant increase in fibrotic tissue. The MTX-treated adults had higher values of cardiac GSSG and protein carbonylation, but lower cardiac noradrenaline levels. For the first time, mature adult animals were shown to be more susceptible to MTX as evidenced by several biomarkers, while young animals appear to better adjust to the MTX-induced cardiac injury. Even so, the higher level of fibrotic tissue and the histological damage showed that MTX also causes cardiac damage in the juvenile population., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Lung macrophages present an effective role in innate and immune response through specific and non-specific mechanisms, namely phagocytosis, antigen processing and presentation through major histocompatibility complex, activation of T cells and inflammatory cytokines release. Despite their protective role against injury in normal condition, they can cause several lung conditions, since they can mediate several processes, through recruitment of other inflammatory cells for alveolar space, release of proinflammatory cytokines or stimulation of collagen deposition. Lung macrophages constitute a good therapeutic target. With the development of nanotechnology, new carriers have been designed to target drugs towards these cells. One of the commonly used approaches is the attachment of ligands with affinity to receptors presented at lung macrophage surface, to the nanocarriers. This review will focus on the importance of lung macrophages in host-defense, their influence on different pulmonary diseases and different strategies for alveolar macrophage targeting.

Methotrexate (MTX) is used for rheumatoid arthritis (RA) treatment showing a wide toxicity profile. This study aimed to evaluate the influence of single nucleotide polymorphisms (SNPs) in genes encoding for MTX transporters with the occurrence of MTX-related toxicity (overall and gastrointestinal). A total of 233 Portuguese RA patients were genotyped for 23 SNPs. Haplotype analyses were performed and a toxicogenetic risk index (TRI) was created for SNPs that revealed to be statistically significant. Regarding MTX overall toxicity, an increased risk was associated to SLC19A1 rs7499 G carriers (p = 0.017), SLC46A1 rs2239907 GG (p = 0.030) and, SLCO1B1 rs4149056 T carriers (p = 0.040) and TT (p = 0.019). TRI revealed that patients with Index 3 were 18-fold more likely to present an adverse drug reaction when compared to those with Index 1 (p = 0.001). For MTX gastrointestinal toxicity, results demonstrated an increased risk associated with SLC19A1 rs7499 G carriers (p = 0.012) and GG (p = 0.045), SLC19A1 rs1051266 G carriers (p = 0.034), SLC19A1 rs2838956 A carriers (p = 0.049) and, SLCO1B1 rs4149056 T carriers (p = 0.042) and TT (p = 0.025). Haplotype analyses showed association between GGAG haplotype for SLC19A1 rs7499, rs1051266, rs2838956 and rs3788200 with MTX gastrointestinal toxicity (p = 0.029). TRI revealed that patients with Index 4 were 9-fold more likely to present a gastrointestinal disorder when compared to those with Index 1 (p = 0.020). This study demonstrated that SLC19A1, SLC46A1 and SLCO1B1 genotypes may help to identify patients with increased risk of MTX-related overall toxicity and that SLC19A1 and SLCO1B1 genotypes, and SLC19A1 haplotypes may help to identify patients with increased risk of MTX-related gastrointestinal toxicity., (© The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

This work aimed to evaluate the influence of a freeze-drying process using different cryoprotectants on the structure of insulin loaded into poly(lactic-co-glycolic acid) (PLGA) nanoparticles and to assess the stability of these nanoparticles upon 6 months of storage following ICH guidelines. Insulin-loaded PLGA nanoparticles with a size around 450 nm were dehydrated using a standard freeze-drying cycle, using trehalose, glucose, sucrose, fructose, and sorbitol at 10% (w/v) as cryoprotectants. All formulations, except those nonadded of cryoprotectant and added with trehalose, collapsed after freeze-drying. The addition of cryoprotectants increased the nanoparticles stability upon storage. FTIR results showed that insulin maintained its structure after encapsulation in about 88%, decreasing to 71% after freeze-drying. The addition of cryoprotectants prior to freeze-drying increased insulin structural stability an average of up to 79%. Formulations collapsed after freeze-drying showed better protein stabilization upon storage, in special sorbitol added formulation, preserving 76, 80, and 78% of insulin structure at 4 °C, 25 °C/60% RH, and 40 °C/75% RH, respectively. Principal component analysis also showed that the sorbitol-added formulation showed the most similar insulin structural modifications among the tested storage conditions. These findings suggested that regarding nanoparticles stability, cryoprotectants are versatile to be used in a standard freeze-drying, however they present different performances on the stabilization of the loaded protein. Thus, on the freeze-drying of the nanoparticles field, this work gives rise to the importance of the process of optimization, searching for a balance between a good obtainable cake with an optimal structural stabilization of the loaded protein.

Background: Therapeutic outcome of rheumatoid arthritis (RA) patients treated with methotrexate (MTX) can be modulated by thymidylate synthase (TS) levels, which may be altered by genetic polymorphisms in TS gene (TYMS). This study aims to elucidate the influence of TYMS polymorphisms in MTX therapeutic outcome (regarding both clinical response and toxicity) in Portuguese RA patients., Methods: Clinicopathological data from 233 Caucasian RA patients treated with MTX were collected, outcomes were defined and patients were genotyped for the following TYMS polymorphisms: 1) 28 base pairs (bp) variable number tandem repeat (rs34743033); 2) single nucleotide polymorphism C>G (rs2853542); and 3) 6 bp sequence deletion (1494del6, rs34489327). Chi-square and binary logistic regression analyses were performed, using genotype and haplotype-based approaches., Results: Considering TYMS genotypes, 3R3R (p = 0.005, OR = 2.34), 3RC3RG (p = 0.016, OR = 3.52) and 6bp- carriers (p = 0.011, OR = 1.96) were associated with non-response to MTX. Multivariate analysis confirmed the increased risk for non-response to MTX in 6bp- carriers (p = 0.016, OR = 2.74). Data demonstrated that TYMS polymorphisms were in linkage disequilibrium (p<0.00001). Haplotype multivariate analysis revealed that haplotypes harboring both 3R and 6bp- alleles were associated with non-response to MTX. Regarding MTX-related toxicity, no statistically significant differences were observed in relation to TYMS genotypes and haplotypes., Conclusion: Our study reveals that TYMS polymorphisms could be important to help predicting clinical response to MTX in RA patients. Despite the potential of these findings, translation into clinical practice needs larger studies to confirm these evidences.

Lung cancer, associated with the up-regulated epidermal growth factor receptor (EGFR) led to the development of EGFR targeted anticancer therapeutics. The biopolymeric nanoparticles form an outstanding system for the targeted delivery of therapeutic agents. The present work evaluated the in vitro effects of chitosan cross-linked γ-poly(glutamic acid) (γ-PGA) nanoparticles (Nps) loaded with docetaxel (DTXL) and decorated with Cetuximab (CET), targeted to EGFR over-expressing non-small-cell-lung-cancer (NSCLC) cells (A549). CET-DTXL-γ-PGA Nps was prepared by ionic gelation and CET conjugation via EDC/NHS chemistry. EGFR specificity of targeted Nps was confirmed by the higher uptake rates of EGFR +ve A549 cells compared to that of EGFR -ve cells (NIH3T3). The cytotoxicity of Nps quantified using cell based (MTT/LDH) and flowcytometry (Cell-cycle analysis, Annexin V/PI and JC-1) assays showed superior antiproliferative activity of CET-DTXL-γ-PGA Nps over DTXL-γ-PGA Nps. The A549 cells treated with CET-DTXL-γ-PGA NPs underwent a G2/M phase cell cycle arrest followed by reduction in mitochondrial membrane potential of A549 cells, inducing apoptosis and necrosis resulting in enhanced cancer cell death. CET-DTXL-γ-PGA Nps exhibited enhanced cellular internalization and therapeutic activity, by actively targeting EGFR on NSCLC cells and hence could be an effective alternative to non-specific, conventional chemotherapy by increasing its efficiency by many folds., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Thymidylate synthase (TYMS) has three polymorphisms that may modulate thymidylate synthase (TS) expression levels: (1) 28 base pairs (bp) variable number tandem repeat (VNTR) (rs34743033); (2) single nucleotide polymorphism (SNP) C>G at the twelfth nucleotide of the second repeat of 3R allele (rs2853542); and (3) 6 bp sequence deletion (1494del6, rs34489327). This study was conducted to evaluate the influence of TYMS polymorphisms on the survival of Portuguese patients with advanced non-small cell lung cancer (NSCLC) undergoing platinum-based chemotherapy. Our results showed no statistically significant differences between VNTR genotypes; although, considering the SNP C>G, homozygotes 3RG presented a better prognostic at 36 months (p=0.004) and overall survival (p=0.003) when compared to 2R3RG patients. Patients with "median/high expression genotypes" demonstrated a better survival at 12 months (p=0.041) when compared to "low expression genotypes". Furthermore, 6 bp- carriers (p=0.006) showed a better survival at 12 months when compared to 6 bp+ homozygotes patients. When analyzing TYMS haplotypes, better survival at 12 months was observed for patients carrying haplotypes with the 6 bp- allele (2R6 bp-; p=0.026 and 3RG6 bp-; p=0.045). This is the first report that evaluates the three major TYMS polymorphisms in the therapeutic outcome of NSCLC in Portugal. According to our results, the TYMS polymorphisms may be useful tools to predict which advanced NSCLC patients could benefit more from platinum-based chemotherapy regimens.

Aim: The aim of our study was to characterize the association of clinicopathological variables and the SLC19A1/RFC-1 G80A polymorphism in methotrexate (MTX)-related toxicity in Portuguese patients with rheumatoid arthritis., Patients & Methods: The study included 233 consecutively recruited patients with rheumatoid arthritis under MTX treatment. The SLC19A1 G80A polymorphism was evaluated by PCR-RFLP., Results: Statistical analysis revealed that SLC19A1 80G carriers had increased risk of gastrointestinal toxicity (odds ratio [OR]: 2.61, p = 0.019) and that regular folic acid supplementation was associated with both overall and gastrointestinal toxicity protection (OR: 0.15, p < 0.001 and OR: 0.19, p < 0.001, respectively). Multivariate analysis confirmed the association of SLC19A1 80G and regular folic acid supplementation to gastrointestinal toxicity (OR: 5.53 and 0.13, respectively). Moreover, a multivariate Cox regression model demonstrated a higher risk of earlier gastrointestinal toxicity in SLC19A1 80G carriers (hazard ratio: 3.63, p = 0.002)., Conclusion: SLC19A1 G80A genotyping may be a useful tool for clinicians to identify patients at higher risk for developing gastrointestinal toxicity related to MTX treatment.

Introduction: Drugs are indispensable for human welfare nevertheless there is a lack of safe drugs. Obtaining pharmacokinetics information, especially metabolism, is a requirement during preclinical and clinical drug development so that in vitro tools represent a simple path where it is possible to assess drug toxicity and predict drug safety in humans., Areas Covered: This review covers the latest in vitro tools developed for assessing drug toxicity in different organs. A special focus is given to hepatic models, as it is the main organ responsible for drug metabolism and consequently bioactivation and detoxification reactions. Three-dimensional culture models have been widely developed, resembling in vivo-like conditions, and are also discussed in this review., Expert Opinion: Several in vitro tools to assess hepatic drug metabolism have been developed, however, novel in vitro methods to investigate extra-hepatic drug metabolism still need to be improved. These methods are able to reduce the number of animal used in preclinical experiments, but in vivo tests are mandatory for drug approval and commercialization.

Methotrexate (MTX) is used in low doses to treat a variety of diseases. Although the mechanism responsible for its therapeutic action is unknown, MTX membrane transport proteins (influx and/or efflux) can be major determinants of pharmacokinetics, adverse drug reactions and clinical response profiles. With progess in pharmacogenomics, the improvement of the prediction of patients' therapeutic outcome treated with low doses of MTX will offer a powerful tool for the translation of transporter SNPs into clinical practice and will be essential to sustain a breakthrough in the field of personalized medicine. Therefore, this paper provides an update on the current data on SNPs in genes encoding low-dose MTX membrane transport proteins and their relevance as possible biomarkers of MTX therapeutic outcome.

Objective: Methotrexate (MTX), the most used drug in rheumatoid arthritis (RA) treatment, showing variability in clinical response, is often associated with genetic polymorphisms. This study aimed to elucidate the role of methylenetetrahydrofolate reductase (MTHFR) C677T and aminoimidazole carboxamide adenosine ribonucleotide transformylase (ATIC) T675C polymorphisms and clinicopathological variables in clinical response to MTX in Portuguese RA patients., Methods: Study included 233 RA patients treated with MTX for at least six months. MTHFR C677T and ATIC T675C polymorphisms were genotyped and clinicopathological variables were collected. Statistical analyses were performed and binary logistic regression method adjusted to possible confounding variables., Results: Multivariate analyses demonstrated that MTHFR 677TT (OR = 4.63; P = 0.013) and ATIC 675T carriers (OR = 5.16; P = 0.013) were associated with over 4-fold increased risk for nonresponse. For clinicopathological variables, noncurrent smokers (OR = 7.98; P = 0.001), patients positive to anti-cyclic citrullinated peptide (OR = 3.53; P = 0.004) and antinuclear antibodies (OR = 2.28; P = 0.045), with higher health assessment questionnaire score (OR = 2.42; P = 0.007), and nonsteroidal anti-inflammatory drug users (OR = 2.77; P = 0.018) were also associated with nonresponse. Contrarily, subcutaneous administration route (OR = 0.11; P < 0.001) was associated with response., Conclusion: Our study suggests that MTHFR C677T and ATIC T675C genotyping combined with clinicopathological data may help to identify patients whom will not benefit from MTX treatment and, therefore, assist clinicians in personalizing RA treatment.

This study aims to monitor the secondary structure behaviour of insulin when it is encapsulated into solid lipid nanoparticles (SLN), under the influence of several critical processing parameters. Insulin was used as a therapeutic protein model. Physicochemical properties of insulin-loaded SLN (Ins-SLN) were assessed, with special focus on the insulin secondary structure after its encapsulation into SLN and after freeze-drying using different cryoprotectants (glucose, fructose and sorbitol). Additionally, a 6-month stability study was performed to evaluate the maintenance of insulin secondary structure over time at different storage conditions (4 °C/60% RH, 25 °C/60% RH, 40 °C/75% RH). Ins-SLN were successfully produced with a mean and narrow particle size around 400 nm, zeta potential around -13 mV, an insulin association efficiency of 84%. Physical-chemical properties of SLN were maintained after freeze-drying. FTIR results showed that encapsulated insulin maintained a native-like structure in a degree of similarity around 92% after production, and 84% after freeze-drying. After 6 months, freeze-dried Ins-SLN without cryoprotectant stored at 40 °C/75% RH presented the same degree of structure preservation and morphology. Results revealed that insulin structure can be significantly protected by SLN matrix itself, without a cryoprotectant agent, even using a non-optimized freeze-drying process, and under the harsher storage conditions. Multivariable experimental settled the process parameters to fit with the desired product quality attributes regarding protein and nanoparticle stability., (Copyright © 2013 Elsevier B.V. All rights reserved.)

TS is critical for providing the requisite nucleotide precursors in order to maintain DNA synthesis and repair. Furthermore, it is an important target for several drugs such as 5-fluorouracil and methotrexate. However, several mechanisms of resistance to TS inhibitors have been explained as linked to TYMS overexpression. Some authors have described the relationship between genetic polymorphisms on TYMS, in particular rs34743033, rs2853542 and rs34489327, with the development of several diseases and with the clinical response to drug therapy and/or survival. Nevertheless, the obtained results described in the literature are controversial, which has lead to a search strategy to understand the impact of these polymorphisms on molecular epidemiology and pharmacogenetics. With the progress of these scientific areas, early identification of individuals at risk of disease along with improvement in the prediction of patients' outcome will offer a powerful tool for the translation of TYMS polymorphisms into clinical practice and individualization of treatments.

PLGA nanoparticles are useful to protect and deliver proteins in a localized or targeted manner, with a long-term systemic delivery pattern intended to last for a period of time, depending on polymer bioerosion and biodegradability. However, the principal concern regarding these carriers is the hydrolytic instability of polymer in aqueous suspension. Freeze-drying is a commonly used method to stabilize nanoparticles, and cryoprotectants may be also used, to even increase its physical stability. The aim of the present work was to analyze the influence of cryoprotectants on nanoparticle stability and porosity after freeze-drying, which may influence protein release and stability. It was verified that freeze-drying significantly increased the number of pores on PLGA-NP surface, being more evident when cryoprotectants are added. The presence of pores is important in a lyophilizate to facilitate its reconstitution in water, although this may have consequences to protein release and stability. The release profile of insulin encapsulated into PLGA-NP showed an initial burst in the first 2 h and a sustained release up to 48 h. After nanoparticles freeze-drying the insulin release increased about 18% in the first 2 h due to the formation of pores, maintaining a sustained release during time. After freeze-drying with cryoprotectants, the amount of insulin released was higher for trehalose and lower for sucrose, glucose, fructose and sorbitol comparatively to freeze-dried PLGA-NP with no cryoprotectant added. Besides the porosity, the ability of cryoprotectants to be adsorbed on the nanoparticles surface may also play an important role on insulin release and stability.

Therapeutic proteins represent a significant part of the new pharmaceuticals coming on the market every year and are now widely spread in therapy to treat or relief symptoms related to many metabolic and oncologic diseases. The parenteral route remains as a primary strategy for protein administration essentially due to its specific physicochemical properties. However, the research on alternative nonparenteral delivery routes continues. The high molecular weight (MW), hydrophilicity, and charged nature of therapeutically valued proteins render transport through membranes very difficult. In this regard, chitosan arises as a promising candidate for the development of protein-containing drug formulations, due to its exceptional biological properties. Chitosan-based delivery systems have been proposed as valid approaches to provide protective conditions to proteins from denaturation and loss of activity, during preparation and delivery, as well as during long-term storage of the prepared formulation. In this chapter, one production method of a chitosan-based nanoparticle formulation is presented, as well as several characterization techniques to assess both nanoparticles and proteins characteristics and stability.

Hemodialysis (HD) and peritoneal dialysis are associated with inflammatory events and immunological incompetence. The purpose of this study was to evaluate the effect of both uremia and dialysis modality on the production of cytokines and reactive oxygen species (ROS) by monocytes. four groups of subjects were studied: 28 chronic kidney disease (CKD) patients, 14 chronic HD patients, 14 patients on continuous ambulatory peritoneal dialysis (CAPD) patients, and 14 healthy volunteers, peripheral blood mononuclear cells (PBMC) were isolated from blood samples and incubated for 24 hr with or without lipopolysaccharide (LPS). TNF-alpha and IL-10 production by PBMC and serum levels of these cytokines were quantified by ELISA. Aliquots of whole blood were incubated in vitro and ROS production and phagocytosis were quantified by flow cytometry. Compared to the control group, Staphylococcus aureus-stimulated ROS production by monocytes was significantly lower in the HD group. The highest levels of unstimulated TNF-alpha production in vitro were observed in the HD group. In the CKD group, as well as in the whole population, there were a negative correlation between TNF-alpha production by unstimulated PBMC and ROS production by S. aureus-stimulated monocytes and a positive correlation between PMA-stimulated ROS production by monocytes and unstimulated and LPS-stimulated IL-10 production by PBMC suggesting that the pro-inflammatory state in CKD patients is associated with decreased response to infectious challenges.

Cardiovascular complications associated with 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) abuse have increasingly been reported. The indirect effect of MDMA mediated by a sustained high level of circulating biogenic amines may contribute to the cardiotoxic effects, but other factors, like the direct toxic effects of MDMA and its metabolites in cardiac cells, remain to be investigated. Thus, the objective of the present in vitro study was to evaluate the potential cardiotoxic effects of MDMA and its major metabolites 3,4-methylenedioxyamphetamine (MDA), N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA), and alpha-methyldopamine (alpha-MeDA) using freshly isolated adult rat cardiomyocytes. The cell suspensions were incubated with these compounds in the final concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 mM for 4 h. alpha-MeDA, N-Me-alpha-MeDA, and their respective aminochromes (oxidation products) were quantified in cell suspensions by HPLC-DAD. The toxic effects were evaluated at hourly intervals for 4 h by measuring the percentage of cells with normal morphology, glutathione (GSH), and glutathione disulfide (GSSG); intracellular Ca(2+), ATP, and ADP; and the cellular activities of glutathione peroxidase, glutathione reductase, and glutathione-S-transferase. No toxic effects were found after exposure of rat cardiomyocytes to MDMA or MDA at any of the tested concentrations for 4 h. In contrast, their catechol metabolites N-Me-alpha-MeDA and alpha-MeDA induced significant toxicity in rat cardiomyocytes. The toxic effects were characterized by a loss of normal cell morphology, which was preceded by a loss of GSH homeostasis due to conjugation of GSH with N-Me-alpha-MeDA and alpha-MeDA, sustained increase of intracellular Ca(2+) levels, ATP depletion, and decreases in the antioxidant enzyme activities. The oxidation of N-Me-alpha-MeDA and alpha-MeDA into the toxic compounds N-methyl-alpha-methyldopaminochrome and alpha-methyldopaminochrome, respectively, was also verified in cell suspensions incubated with these MDMA metabolites. The results obtained in this study provide evidence that the metabolism of MDMA into N-Me-alpha-MeDA and alpha-MeDA is required for the expression of MDMA-induced cardiotoxicity in vitro, being N-Me-alpha-MeDA the most toxic of the studied metabolites.

Mortality associated with endotoxin shock is likely mediated by Kupffer cells, alveolar macrophages, and circulating neutrophils. Acute dietary glycine prevents mortality and blunts increases in serum tumor necrosis factor-alpha (TNF-alpha) following endotoxin in rats. Furthermore, acute glycine blunts activation of Kupffer cells, alveolar macrophages, and neutrophils by activating a glycine-gated chloride channel. However, in neuronal tissue, glycine rapidly downregulates chloride channel function. Therefore, the long-term effects of a glycine-containing diet on survival following endotoxin shock were investigated. Dietary glycine for 4 wk improved survival after endotoxin but did not improve liver pathology, decrease serum alanine transaminase, or effect TNF-alpha levels compared with animals fed control diet. Interestingly, dietary glycine largely prevented inflammation and injury in the lung following endotoxin. Surprisingly, Kupffer cells from animals fed glycine for 4 wk were no longer inactivated by glycine in vitro; however, isolated alveolar macrophages and neutrophils from the same animals were sensitive to glycine. These data are consistent with the hypothesis that glycine downregulates chloride channels on Kupffer cells but not on alveolar macrophages or neutrophils. Importantly, glycine diet for 4 wk protected against lung inflammation due to endotoxin. Chronic glycine improves survival by unknown mechanisms, but reduction of lung inflammation is likely involved.

The purpose of this study was to determine whether early alcohol-induced liver injury (ALI) in females is associated with changes in CD14 on Kupffer cells, activation of hepatic nuclear factor (NF)-kappaB, and expression of tumor necrosis factor (TNF)-alpha mRNA. Male and female rats were given high-fat control or ethanol-containing diets for 4 wk using the intragastric enteral protocol. Physiological parameters were similar in both genders. Ethanol was increased as tolerance developed with higher blood levels than previously observed, resulting in a fourfold increase in aspartate aminotransferase (males 389 +/- 47 IU/l vs. females 727 +/- 66 IU/l). Hepatic pathology developed more rapidly and was nearly twofold greater and endotoxin levels were significantly higher in females after ethanol. Also, expression of CD14 on Kupffer cells was 1.5-fold greater and binding of transcription factor NF-kappaB in hepatic nuclear extracts and TNF-alpha mRNA expression were threefold greater in females. These data are consistent with the hypothesis that elevated endotoxin after ethanol triggers more activation of Kupffer cells via enhanced CD14 expression in females. NF-kappaB is activated in this process, leading to increases in TNF-alpha mRNA expression in the liver and more severe liver injury in females. It is concluded that gender differences in ALI are dependent on endotoxin and a signaling cascade leading to TNF-alpha.

The aim of this study was to investigate whether reduction in blood estrogen by removal of the ovaries would decrease the sensitivity of female rats to early alcohol-induced liver injury using an enteral ethanol feeding model, and if so, whether estrogen replacement would compensate. Livers from ovariectomized rats with or without estrogen replacement after 4 weeks of continuous ethanol exposure were compared with nonovariectomized rats in the presence or absence of ethanol. Ethanol increased serum alanine transaminase (ALT) levels from 30 +/- 6 to 64 +/- 7 U/L. This effect was blocked by ovariectomy (31 +/- 7) and totally reversed by estrogen replacement (110 +/- 23). Ethanol increased liver weight and fat accumulation, an effect that was minimized by ovariectomy and reversed partially by estrogen replacement. Infiltrating leukocytes were increased 6. 7-fold by ethanol, an effect that was blunted significantly by ovariectomy and reversed by estrogen replacement. Likewise, a similar pattern of changes was observed in the number of necrotic hepatocytes. Blood endotoxin and hepatic levels of CD14 messenger RNA (mRNA) and protein were increased by ethanol. This effect was blocked in ovariectomized rats and elevated by estrogen replacement. Moreover, Kupffer cells isolated from ethanol-treated rats with estrogen replacement produced more tumor necrosis factor alpha (TNF-alpha) than those from control and ovariectomized rats. It is concluded, therefore, that the sensitivity of rat liver to alcohol-induced injury is directly related to estrogen, which increases endotoxin in the blood and CD14 expression in the liver, leading to increased TNF-alpha production.

Extrahepatic factors, such as increased gut permeability and bacteria from the gut, have been shown to play a role in D-galactosamine toxicity in rats. Because bacterial endotoxin activates Kupffer cells, the purpose of this study was to clarify the role of Kupffer cells in the mechanism of D-galactosamine hepatotoxicity in rats and determine whether uridine, a compound that rescues animals from D-galactosamine toxicity, affects Kupffer cells. Rats were fed control or glycine (5%) containing diets to prevent Kupffer cell activation or treated with gadolinium chloride (GdCl3, 20 mg/kg) to destroy Kupffer cells selectively before injection of D-galactosamine (500 mg/kg, intraperitoneally). D-galactosamine caused panlobular focal hepatocellular necrosis, polymorphonuclear cell infiltration, and increased serum transaminases significantly at 24 hours. Dietary glycine or pretreatment with GdCl3 prevented these effects. D-galactosamine caused a transient increase in circulating endotoxin that was maximal at 1 hour and was blunted significantly by dietary glycine. Additionally, antisera to tumor necrosis factor-alpha (TNF-alpha) prevented hepatotoxicity caused by D-galactosamine. Moreover, apoptosis in hepatocytes caused by D-galactosamine occurred before necrosis (6 hours) and was prevented by glycine, GdCl3, TNF-alpha antiserum, and uridine. Thus, it was hypothesized that TNF-alpha from Kupffer cells causes apoptosis after D-galactosamine administration in the rat. Indeed, increases in TNF-alpha messenger RNA (mRNA) were detected as early as 2.5 hours after D-galactosamine treatment. Previous work proposed that uridine blocks D-galactosamine toxicity by preventing inhibition of mRNA synthesis. In view of these results, the possibility that uridine might affect Kupffer cells was investigated. Uridine significantly blunted the increase in [Ca2+]i and release of TNF-alpha caused by endotoxin in isolated Kupffer cells and prevented apoptosis caused by D-galactosamine treatment in vivo. These data support the hypothesis that uridine prevents D-galactosamine hepatotoxicity not only by rescuing the hepatocyte in the late phases of the injury but also preventing TNF-alpha release from Kupffer cells thereby blocking apoptosis that occurs early after D-galactosamine treatment. Taken together, these data strongly support the role of Kupffer cell activation by endotoxin early after D-galactosamine treatment as an important event in the mechanism of hepatotoxicity in the rat.

CD14 is a lipopolysaccharide (LPS) receptor distributed largely in macrophages, monocytes, and neutrophils; however, the role of CD14 in activation of Kupffer cells by LPS remains controversial. The purpose of this study was to determine if different methods used to isolate Kupffer cells affect CD14. Kupffer cells were isolated by collagenase (0.025%) or collagenase-Pronase (0.02%) perfusion and differential centrifugation using Percoll gradients and cultured for 24 h before experiments. CD14 mRNA was detected by RT-PCR from Kupffer cell total RNA as well as from peritoneal macrophages. Western blotting showed that Kupffer cells prepared with collagenase possess CD14; however, it was absent in cells obtained by collagenase-Pronase perfusion. Intracellular calcium in Kupffer cells prepared with collagenase was increased transiently to levels around 300 nM by addition of LPS with 5% rat serum, which contains LPS binding protein. This increase in intracellular calcium was totally serum dependent. Moreover, LPS-induced increases in intracellular calcium in Kupffer cells were blunted significantly (40% of controls) when cells were treated with phosphatidylinositol-specific phospholipase C, which cleaves CD14 from the plasma membrane. However, intracellular calcium did not increase when LPS was added to cells prepared by collagenase-Pronase perfusion even in the presence of serum. These cells were viable, however, because ATP increased intracellular calcium to the same levels as cells prepared with collagenase perfusion. Tumor necrosis factor-alpha (TNF-alpha) mRNA was increased in Kupffer cells prepared with collagenase perfusion 1 h after addition of LPS, an effect potentiated over twofold by serum; however, serum did not increase TNF-alpha mRNA in cells isolated via collagenase-Pronase perfusion. Moreover, treatment with Pronase rapidly decreased CD14 on mouse macrophages (RAW 264.7 cells) and Kupffer cells. These findings indicate that Pronase cleaves CD14 from Kupffer cells, whereas collagenase perfusion does not, providing an explanation for why Kupffer cells do not exhibit a CD14-mediated pathway when prepared with procedures using Pronase. It is concluded that Kupffer cells indeed contain a functional CD14 LPS receptor when prepared gently.

This study investigated the role of endotoxin in the hypermetabolic state or swift increase in alcohol metabolism (SIAM) due to acute ethanol exposure. Female Sprague-Dawley rats (100-120 g) were given ethanol (5 g/kg) by gavage. Endotoxin measured in plasma from portal blood was not detectable in saline-treated controls; however, 90 min after ethanol, endotoxin was increased to 85 +/- 14 pg/ml, and endotoxin clearance was diminished by approximately 50%. Oxygen uptake in perfused livers was increased 48% by ethanol, and production of PGE2 by isolated Kupffer cells was increased similarly. These effects were blunted by elimination of gram-negative bacteria and endotoxin with antibiotics before ethanol administration. To reproduce ethanol-induced endotoxemia, endotoxin was infused via the mesenteric vein at a rate of 2 ng. kg-1. h-1. Endotoxin mimicked the effect of ethanol on oxygen uptake. The specific Kupffer cell toxicant GdCl3 completely prevented increases in oxygen uptake due to endotoxin. These findings demonstrate that endotoxin plays a pivotal role in SIAM, most likely by stimulating eicosanoid release from Kupffer cells.

Activation of Kupffer cells by lipopolysaccharide (LPS) plays a pivotal role in the onset of pathophysiological events that occur during endotoxemia and intracellular calcium ([Ca2+]i) is involved in LPS-stimulated cytokine production. Recently, it was shown that Kupffer cells contain a glycine-gated chloride channel. Because taurine, a ubiquitous sulfur-containing beta-amino acid, acts similarly to glycine in neurons by causing hyperpolarization, it was hypothesized that taurine would act via a similar mechanism, blunting the LPS-induced increase in [Ca2+]i in Kupffer cells. To test this hypothesis, Kupffer cells were isolated from female Sprague-Dawley rats and cultured for 24 h. LPS-induced changes in [Ca2+]i were monitored fluorometrically in single cells, whereas levels of tumor necrosis factor alpha (TNF-alpha) released by Kupffer cells after exposure to LPS were measured by enzyme-linked immunosorbent assay. Taurine significantly blunted the LPS-induced increase in [Ca2+]i in a dose-dependent manner (IC50, 0.1 mM). This effect was reversed by strychnine (1 microM) and was prevented when chloride was removed from the extracellular media. Moreover, taurine increased 36Cl- uptake by Kupffer cells in a dose-dependent manner (EC50, 0.2 mM). Furthermore, strychnine (1 microM) reversed the effect of taurine on 36Cl- uptake. These results indicate that taurine activates a glycine-gated chloride channel in Kupffer cells causing chloride influx. In addition, LPS-induced TNF-alpha production was reduced by more than 40% by taurine, an effect that was also reversed by strychnine. In conclusion, taurine blocks the increase in [Ca2+]i due to LPS and significantly reduces TNF-alpha production by mechanisms involving chloride influx into the Kupffer cell.

Glycine prevents hepatic damage caused by hypoxia-reoxygenation, diminishes mortality due to endotoxin and minimizes alcoholic liver injury by decreasing blood ethanol. Our purpose was to investigate the effect of dietary glycine during recovery from early alcohol-induced injury, using a model that mimics the clinical presentation and histopathology with alcoholics. Male Wistar rats were exposed to ethanol continuously for 6 wk via intragastric feeding that resulted in typical histology of alcoholic liver injury, including steatosis, inflammation, necrosis and increased serum levels of aspartate aminotransferase and alanine aminotransferase. After cessation of ethanol, one group of rats received a control diet, the other a glycine-containing diet for 2 wk. During this period, all parameters studied tended to return to baseline values. However, serum aspartate aminotransferase and alanine aminotransferase recovered about 30% more rapidly in rats fed glycine. Further, the hepatic pathology score was also significantly lower in the glycine group than in controls (0.5 vs. 2.6). After 1 wk, steatosis was reduced significantly more in the glycine group (5. 6%) than in controls (8.9%). Glycine also diminished numbers of infiltrating leukocytes and necrotic cells significantly more than in controls. This beneficial effect of glycine may be partly explained by the fact that glycine increased influx of chloride into Kupffer cells leading to diminished tumor necrosis factor-alpha production. These results indicate that a glycine containing diet expedites the process of recovery from ethanol-induced liver injury and may lead to its clinical application in alcoholic hepatitis.

Methylene dianiline (DAPM) causes hepatic damage and bile duct necrosis in rats. This has been detected histologically and biochemically. The toxicity was dose related over the range 0-100 mg/kg but the dose response relationship showed a maximum at about 75-100 mg/kg. This was true for both histopathology and biochemical parameters of liver dysfunction. When animals were depleted of taurine using beta-alanine pretreatment, the toxicity of DAPM was increased. Conversely treatment of rats with taurine, significantly attenuated the rise in alanine transaminase (ALT). However depletion of taurine with guanidinoethanesulphonate (GES) attenuated rises in both transaminases. It is concluded that taurine may play a role in the toxicity of DAPM but that GES, although depleting taurine as does beta-alanine, causes additional effects such as increasing glutathione (GSH), perhaps leading to protection.

1. Taurine is a ubiquitous, free amino acid found in mammalian systems. 2. The biological functions of taurine are unclear. 3. Various in vivo data suggest that taurine has a variety of protective functions and deficiency leads to pathological changes. 4. Depletion in rats of taurine increases susceptibility to liver damage from carbon tetrachloride. 5. Susceptibility to a variety of hepatotoxicants correlates with the estimated hepatic taurine level. 6. In vitro data suggest that taurine can protect cells against toxic damage. 7. Taurine protects isolated hepatocytes against carbon tetrachloride, hydrazine and 1,4-naphthoquinone but not against allyl alcohol, alpha-naphthylisothiocyanate (ANIT) or diaminodiphenyl methane (DAPM) cytotoxicity. 8. The mechanisms of protection are unclear but may include modulation of calcium levels, osmoregulation and membrane stabilization.