Development of pH-Sensitive Magnetoliposomes Containing Shape Anisotropic Nanoparticles for Potential Application in Combined Cancer Therapy.

Late diagnosis and systemic toxicity associated with conventional treatments make oncological therapy significantly difficult. In this context, nanomedicine emerges as a new approach in the prevention, diagnosis and treatment of cancer. In this work, pH-sensitive solid magnetoliposomes (SMLs) were developed for controlled release of the chemotherapeutic drug doxorubicin (DOX). Shape anisotropic magnetic nanoparticles of magnesium ferrite with partial substitution by calcium (Mg0.75Ca0.25Fe2O4) were synthesized, with and without calcination, and their structural, morphological and magnetic properties were investigated. Their superparamagnetic properties were evaluated and heating capabilities proven, either by exposure to an alternating magnetic field (AMF) (magnetic hyperthermia) or by irradiation with near-infrared (NIR) light (photothermia). The Mg0.75Ca0.25Fe2O4 calcined nanoparticles were selected to integrate the SMLs, surrounded by a lipid bilayer of DOPE:Ch:CHEMS (45:45:10). DOX was encapsulated in the nanosystems with an efficiency above 98%. DOX release assays showed a much more efficient release of the drug at pH = 5 compared to the release kinetics at physiological pH. By subjecting tumor cells to DOX-loaded SMLs, cell viability was significantly reduced, confirming that they can release the encapsulated drug. These results point to the development of efficient pH-sensitive nanocarriers, suitable for a synergistic action in cancer therapy with magnetic targeting, stimulus-controlled drug delivery and dual hyperthermia (magnetic and plasmonic) therapy.

Magnetoliposomes Containing Multicore Nanoparticles and a New Antitumor Thienopyridine Compound with Potential Application in Chemo/Thermotherapy.

Multicore magnetic nanoparticles of manganese ferrite were prepared using carboxymethyl dextran as an agglutinating compound or by an innovative method using melamine as a cross-coupling agent. The nanoparticles prepared using melamine exhibited a flower-shape structure, a saturation magnetization of 6.16 emu/g and good capabilities for magnetic hyperthermia, with a specific absorption rate (SAR) of 0.14 W/g. Magnetoliposome-like structures containing the multicore nanoparticles were prepared, and their bilayer structure was confirmed by FRET (Förster Resonance Energy Transfer) assays. The nanosystems exhibited sizes in the range of 250-400 nm and a low polydispersity index. A new antitumor thienopyridine derivative, 7-[4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl]thieno[3,2-b]pyridine, active against HeLa (cervical carcinoma), MCF-7 (breast adenocarcinoma), NCI-H460 (non-small-cell lung carcinoma) and HepG2 (hepatocellular carcinoma) cell lines, was loaded in these nanocarriers, obtaining a high encapsulation efficiency of 98 ± 2.6%. The results indicate that the new magnetoliposomes can be suitable for dual cancer therapy (combined magnetic hyperthermia and chemotherapy).

Development of Thermo- and pH-Sensitive Liposomal Magnetic Carriers for New Potential Antitumor Thienopyridine Derivatives.

The development of stimuli-sensitive drug delivery systems is a very attractive area of current research in cancer therapy. The deep knowledge on the microenvironment of tumors has supported the progress of nanosystems' ability for controlled and local fusion as well as drug release. Temperature and pH are two of the most promising triggers in the development of sensitive formulations to improve the efficacy of anticancer agents. Herein, magnetic liposomes with fusogenic sensitivity to pH and temperature were developed aiming at dual cancer therapy (by chemotherapy and magnetic hyperthermia). Magnetic nanoparticles of mixed calcium/manganese ferrite were synthesized by co-precipitation with citrate and by sol-gel method, and characterized by X-ray diffraction (XRD), scanning electron microscopy in transmission mode (STEM), and superconducting quantum interference device (SQUID). The citrate-stabilized nanoparticles showed a small-sized population (around 8 nm, determined by XRD) and suitable magnetic properties, with a low coercivity and high saturation magnetization (~54 emu/g). The nanoparticles were incorporated into liposomes of dipalmitoylphosphatidylcholine/cholesteryl hemisuccinate (DPPC:CHEMS) and of the same components with a PEGylated lipid (DPPC:CHEMS:DSPE-PEG), resulting in magnetoliposomes with sizes around 100 nm. Dynamic light scattering (DLS) and electrophoretic light scattering (ELS) measurements were performed to investigate the pH-sensitivity of the magnetoliposomes' fusogenic ability. Two new antitumor thienopyridine derivatives were efficiently encapsulated in the magnetic liposomes and the drug delivery capability of the loaded nanosystems was evaluated, under different pH and temperature conditions.

Magnetoliposomes Based on Magnetic/Plasmonic Nanoparticles Loaded with Tricyclic Lactones for Combined Cancer Therapy.

Liposome-like nanoarchitectures containing manganese ferrite nanoparticles covered or decorated with gold were developed for application in dual cancer therapy, combining chemotherapy and photothermia. The magnetic/plasmonic nanoparticles were characterized using XRD, UV/Visible absorption, HR-TEM, and SQUID, exhibiting superparamagnetic behavior at room temperature. The average size of the gold-decorated nanoparticles was 26.7 nm for MnFe2O4 with 5-7 nm gold nanospheres. The average size of the core/shell nanoparticles was 28.8 nm for the magnetic core and around 4 nm for the gold shell. Two new potential antitumor fluorescent drugs, tricyclic lactones derivatives of thienopyridine, were loaded in these nanosystems with very high encapsulation efficiencies (higher than 98%). Assays in human tumor cell lines demonstrate that the nanocarriers do not release the antitumor compounds in the absence of irradiation. Moreover, the nanosystems do not cause any effect on the growth of primary (non-tumor) cells (with or without irradiation). The drug-loaded systems containing the core/shell magnetic/plasmonic nanoparticles efficiently inhibit the growth of tumor cells when irradiated with red light, making them suitable for a triggered release promoted by irradiation.

Three-Dimensional (3D) Microporous Iron Silicate with an Imandrite Type of Structure.

Small-pore iron silicate MS-1 (Minho-Sofia, solid number 1) with a 3D porous system, an analogue of the rare mineral imandrite, has been synthesized and characterized. This material is the lowest framework density iron silicate, one of the most siliceous (Si/Fe = 6) iron silicates, the first iron cyclosilicate achieved at hydrothermal conditions, and the only synthetic iron-based member of the lovozerite mineral group.

Double-walled iron oxide nanotubes via selective chemical etching and Kirkendall process.

Double-walled oxide nanotube structures are interesting for a wide range of applications, from photocatalysis to drug delivery. In this work, a progressive oxidation method to fabricate double-walled nanotube structures is reported in detail. The approach is based on the electrodeposition of metallic iron nanowires, in porous alumina templates, followed by a selective chemical etching, nanoscale Kirkendall effect, a fast oxidation and out-diffusion of the metallic core structure during thermal annealing. To validate the formation mechanism of such core-shell structure, chemical composition and atomic structure were assessed. The resulting hematite nanotubes have a high degree of uniformity, along several microns, and a nanoscopic double-walled structure.

Frequency of Equus caballus papillomavirus in equine aural plaques.

Despite the reported association between aural plaques and the presence of Equus caballus papillomavirus (EcPV), there are few data regarding the distribution of viral types in different geographic regions or possible correlations for different papillomaviruses and lesion characteristics. We detected the presence and frequency of EcPV (1-7) DNA in aural plaque biopsies of horses from different regions of Brazil and identified the patterns of these infections or coinfections and their possible association with lesion severity. A total of 108 aural plaque biopsies from horses in the 5 geopolitical regions of Brazil were examined. We performed PCR to detect EcPV DNA in the biopsies. At least 1 type of EcPV was detected in 97% of the samples. EcPV coinfection was observed in 59% of the samples. Compared to the other viruses, EcPV-4 was found at the highest frequency in coinfection (84%) or individually identified (32%). EcPV-2 and -7 were not detected. No significant association was found between lesion characteristics (type and distribution) and either the viral type detected or the presence of coinfection. EcPV is widely distributed in Brazil, both isolated and in coinfection; the viral type does not appear to influence the clinical characteristics of equine aural plaques.

Development of Multifunctional Liposomes Containing Magnetic/Plasmonic MnFe2O4/Au Core/Shell Nanoparticles.

Multifunctional liposomes containing manganese ferrite/gold core/shell nanoparticles were developed. These magnetic/plasmonic nanoparticles were covered by a lipid bilayer or entrapped in liposomes, which form solid or aqueous magnetoliposomes as nanocarriers for simultaneous chemotherapy and phototherapy. The core/shell nanoparticles were characterized by UV/Visible absorption, X-Ray Diffraction (XRD), Transmission Electron Microscopy (TEM), and Superconducting Quantum Interference Device (SQUID). The magnetoliposomes were characterized by Dynamic Light Scattering (DLS) and TEM. Fluorescence-based techniques (FRET, steady-state emission, and anisotropy) investigated the incorporation of a potential anti-tumor drug (a thienopyridine derivative) in these nanosystems. The core/shell nanoparticles exhibit sizes of 25 ± 2 nm (from TEM), a plasmonic absorption band (λmax = 550 nm), and keep magnetic character. XRD measurements allowed for the estimation of 13.3 nm diameter for manganese ferrite core and 11.7 nm due to the gold shell. Aqueous magnetoliposomes, with hydrodynamic diameters of 152 ± 18 nm, interact with model membranes by fusion and are able to transport the anti-tumor compound in the lipid membrane, with a high encapsulation efficiency (EE (%) = 98.4 ± 0.8). Solid magnetoliposomes exhibit hydrodynamic diameters around 140 nm and also carry successfully the anticancer drug (with EE (%) = 91.2 ± 5.2), while also being promising as agents for phototherapy. The developed multifunctional liposomes can be promising as therapeutic agents for combined chemo/phototherapy.

Increased expression of NLRP3 inflammasome in placentas from pregnant women with severe preeclampsia.

Preeclampsia is a pregnancy disorder characterized by imbalance between pro- and anti-inflammatory cytokines associated with high plasma levels of uric acid and Interleukin-1 beta (IL-1ß). The inflammasome is a protein complex that mediates innate immune responses via caspase-1 activation promoting secretion of IL-1ß and IL-18 in their active forms, and also release of the high-mobility group box 1 protein (HMGB1). As the placenta seems to play an important role in the pathogenesis of PE, the present study investigated the expression of genes and proteins related to the inflammasome in placentas from pregnant women with severe preeclampsia. Placental tissue was collected from 20 normotensive pregnant women and 20 preeclamptic women, and inflammasome components, NLRP3 (NOD-like receptor family, pyrin domain-containing protein 3), caspase-1, IL-1ß and IL-18, as well as tumor necrosis factor-alpha (TNF-α) and HMGB1 were evaluated by immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and also quantified by reverse transcription-qPCR (RT-qPCR). Compared with normotensive pregnant women, placenta from women with PE showed a significant increase in NLRP3, caspase-1, IL-1ß, TNF-α and HMGB1 mRNA. Immunohistochemical staining of NLRP3, caspase-1, IL-1ß and TNF-α in placental villi, as well as the levels of caspase-1, IL-1ß, TNF-α and HMGB1 in placental homogenate were significantly higher in the preeclamptic group than in the normotensive group. However, mRNA expression of IL-18 and its protein concentrations were lower in placentas from preeclamptic women. The results suggest that placentas from pregnant women with preeclampsia show higher expression of NLRP3 inflammasome, which may be involved in the exaggerated inflammatory state in preeclampsia.

Host-pathogen interactions in bovine mammary epithelial cells and HeLa cells by Staphylococcus aureus isolated from subclinical bovine mastitis.

Staphylococcus aureus is a common pathogen that causes subclinical bovine mastitis due to several virulence factors. In this study, we analyzed S. aureus isolates collected from the milk of cows with subclinical mastitis that had 8 possible combinations of bap, icaA, and icaD genes, to determine their capacity to produce biofilm on biotic (bovine primary mammary epithelial cells and HeLa cells) and abiotic (polystyrene microplates) surfaces, and their ability to adhere to and invade these cells. We also characterized isolates for microbial surface components recognizing adhesive matrix molecules (MSCRAMM) and agr genes, and for their susceptibility to cefquinome sulfate in the presence of biofilm. All isolates adhered to and invaded both cell types, but invasion indexes were higher in bovine primary mammary epithelial cells. Using tryptic soy broth + 1% glucose on abiotic surfaces, 5 out of 8 isolates were biofilm producers, but only the bap+icaA+icaD+ isolate was positive in Dulbecco's Modified Eagle's medium. The production of biofilm on biotic surfaces occurred only with this isolate and only on HeLa cells, because the invasion index for bovine primary mammary epithelial cells was too high, making it impossible to use these cells in this assay. Of the 5 biofilm producers in tryptic soy broth + 1% glucose, 4 presented with the bap/fnbA/clfA/clfB/eno/fib/ebpS combination, and all were protected from cefquinome sulfate. We found no predominance of any agr group. The high invasive potential of S. aureus made it impossible to observe biofilm in bovine primary mammary epithelial cells, and we concluded that cells with lower invasion rates, such as HeLa cells, were more appropriate for this assay.

Factors associated with equine aural plaque in Brazil.

BACKGROUND: Aural plaques can be found on the inner surfaces of one or both ears of horses. Despite their low malignancy, these lesions can sometimes cause discomfort and sensitivity in horses, and a loss in commercial value due to their aesthetic effect. There has been a study describing the epidemiological features and the clinical prevalence of equine aural plaques in Brazil. HYPOTHESIS/OBJECTIVES: To determine the clinical prevalence and selected associated factors of aural plaques. ANIMALS: In the study, 891 horses were assessed for aural plaques. The sample group had a median age of 5 years and comprised both sexes and various breeds from different regions of Brazil. METHODS: Horses were evaluated by a general observation of the body and a detailed observation of both ears. Data on the management system, characteristics of the lesions, the presence of ticks and ear grooming were collected for 109 clinically affected horses. An assessment of the frequency distribution of the disease and its characteristics was performed. Association tests were conducted to establish the relationships between the variables studied. RESULTS: In 85% (40 of 47) of farms assessed, at least one horse presented with aural plaques. In 14.8% (132 of 891) of the horses, lesions characteristic of aural plaque were detected. Significant associations between the prevalence of "coalescing" lesions and a "semi-intensive" management system and ear grooming were detected. CONCLUSIONS: The findings confirm the extensive distribution of this disease in Brazil and its association with several management factors.

Imiquimod treatment for Equus caballus papillomavirus infection in equine aural plaques.

BACKGROUND: Aural plaques are a dermatopathy associated with Equus caballus papillomavirus (EcPV). This disease affects horses of all ages, genders and breeds, and causes sensitivity of the ears. HYPOTHESIS/OBJECTIVES: The aim of this study was to evaluate the clinical efficacy of 5% imiquimod cream for the treatment of aural plaques and to compare the PCR detection of EcPV 3, 4, 5 and 6 before and after treatment. ANIMALS: Eight horses diagnosed with aural plaques (14 ears) were used. Three mares with unilateral aural plaques were used as untreated controls. METHODS: Imiquimod cream was applied every 48 h until complete resolution of the aural plaques was observed. Animals were evaluated clinically for 180 days after the end of treatment. PCR for detecting EcPV 3, 4, 5 and 6 was performed using aural plaque biopsies collected before and at 90 days after the end of treatment. RESULTS: Clinical resolution was observed in 93% of the treated ears. Imiquimod treatment promoted the clearance of EcPV in 71.4% of the treated ears. Clinical remission of the aural plaques and changes in EcPV DNA positivity between the first and second biopsies were not observed in the control group. In 75% of horses, sedation was required in order to carry out pretreatment cleaning. CONCLUSIONS: The results of this study support the hypothesis that 5% imiquimod cream may be used as an effective treatment for aural plaques in horses.

Phylogenetic-based nomenclatural proposals for Ophiocordycipitaceae (Hypocreales) with new combinations in Tolypocladium.

Ophiocordycipitaceae is a diverse family comprising ecologically, economically, medicinally, and culturally important fungi. The family was recognized due to the polyphyly of the genus Cordyceps and the broad diversity of the mostly arthropod-pathogenic lineages of Hypocreales. The other two cordyceps-like families, Cordycipitaceae and Clavicipitaceae, will be revised taxonomically elsewhere. Historically, many species were placed in Cordyceps, but other genera have been described in this family as well, including several based on anamorphic features. Currently there are 24 generic names in use across both asexual and sexual life stages for species of Ophiocordycipitaceae. To reflect changes in Art. 59 in the International Code of Nomenclature for algae, fungi, and plants (ICN), we propose to protect and to suppress names within Ophiocordycipitaceae, and to present taxonomic revisions in the genus Tolypocladium, based on rigorous and extensively sampled molecular phylogenetic analyses. When approaching this task, we considered the principles of priority, monophyly, minimizing taxonomic revisions, and the practical utility of these fungi within the wider biological research community.

Ferromagnetic sorbents based on nickel nanowires for efficient uptake of mercury from water.

This work reports the preparation of ferro-magnetic nickel nanowires (NiNW) coated with dithiocarbamate-functionalized siliceous shells and its application for the uptake of aqueous Hg(II) ions by magnetic separation. NiNW with an average diameter and length of 35 nm and 5 µm, respectively, were firstly prepared by Ni electrodeposition in an anodic aluminum oxide template. The NiNW surfaces were then coated with siliceous shells containing dithiocarbamate groups via a one-step procedure consisting in the alkaline hydrolytic co-condensation of tetraethoxysilane (TEOS) and a siloxydithiocarbamate precursor (SiDTC). A small amount of these new nanoadsorbents (2.5 mg·L(-1)) removed 99.8% of mercury ions from aqueous solutions with concentration 50 µg·L(-1) and in less than 24 h of contact time. This outstanding removal ability is attributed to the high affinity of the sulfur donor ligands to Hg(II) species combined with the high surface area-to-volume ratio of the NiNW.

Functionalization of nickel nanowires with a fluorophore aiming at new probes for multimodal bioanalysis.

This work reports research on the development of bimodal magnetic and fluorescent 1D nanoprobes. First, ferromagnetic nickel nanowires (NiNW) have been prepared by Ni electrodeposition in an anodic aluminum oxide (AAO) template. The highly ordered self-assembled AAO nanoporous templates were fabricated using a two-step anodization method of aluminum foil. The surface of the NiNW were then modified with polyethyleneimine (PEI) which was previously labeled with an organic dye (fluorescein isothiocyanate: FITC) via covalent bonding. The ensuing functionalized NiNW exhibited the characteristic green fluorescence of FITC and could be magnetically separated from aqueous solutions by using a NdFeB magnet. Finally, the interest of these bimodal NiNW as nanoprobes for in vitro cell separation and biolabeling was preliminary assessed in a proof of principle experiment that involved the attachment of biofunctionalized NiNW to blood cells.

Use of PCR to estimate the prevalence of Equus caballus papillomavirus in aural plaques in horses.

Aural plaques occur on the skin of the medial surface of the pinnae of horses. In this study the presence of Equus caballus papillomavirus (EcPV)-3 and -4 DNA was assessed in 45 such plaques using a 'touchdown' PCR. Papillomaviruses (PVs) were detected in 62.3% (28/45) of samples: EcPV-3 and -4 DNA in 8.89% (4/45) and 37.78% (17/45) of samples, respectively, with 15.56% (7/45) of samples exhibiting co-infection. Viral DNA was not detected in 37.78% (17/45) of samples, suggesting the possible existence of other equine PVs. Neither EcPV-3 nor -4 were detected in negative control skin. This study is the first to evaluate the prevalence of these two viruses in equine aural plaques.

Phylogenetic and structural studies of a novel equine papillomavirus identified from aural plaques.

Papillomaviruses (PVs) infect a wide range of animal species and show great genetic diversity. To date, excluding equine sarcoids, only three species of PVs were identified associated with lesions in horses: Equus caballus papillomavirus 1 (EcPV1-cutaneous), EcPV2 (genital) and EcPV3 (aural plaques). In this study, we identified a novel equine PV from aural plaques, which we designated EcPV4. Cutaneous samples from horses with lesions that were microscopically diagnosed as aural plaques were subjected to DNA extraction, amplification and sequencing. Rolling circle amplification and inverse PCR with specific primers confirmed the presence of an approximately 8 kb circular genome. The full-length EcPV4 L1 major capsid protein sequence has 1488 nucleotides (495 amino acids). EcPV4 had a sequence identity of only 53.3%, 60.2% and 51.7% when compared with the published sequences for EcPV1, EcPV2 and EcPV3, respectively. A Bayesian phylogenetic analysis indicated that EcPV4 clusters with EcPV2, but not with EcPV1 and EcPV3. Using the current PV classification system that is based on the nucleotide sequence of L1, we could not define the genus of the newly identified virus. Therefore, a structural analysis of the L1 protein was carried out to aid in this classification because EcPV4 cause lesion similar to the lesion caused by EcPV3. A comparison of the superficial loops demonstrated a distinct amino acid conservation pattern between EcPV4/EcPV2 and EcPV4/EcPV3. These results demonstrate the presence of a new equine PV species and that structural studies could be useful in the classification of PVs.

Lipopolysaccharide infusion up-regulates hepcidin mRNA expression in equine liver.

Hepcidin has been found to be the key regulator of iron metabolism that leads to hypoferremia during inflammation. Recent work has shown that equine hepcidin is predominantly expressed in the liver of horses. In this study, hepcidin gene expression was determined in the liver and bone marrow of six healthy horses after iv infusion of Escherichia coli O55:B5 LPS. The IL-6 gene expression was also determined in liver and bone marrow samples. Clinical and laboratory evaluations were measured at multiple time points between 0 and 240 h post-LPS infusion (PI). Liver and bone marrow biopsies were taken immediately before (baseline) and at 6 and 18 h PI. In response to endotoxin infusion, all horses showed characteristic clinical signs of endotoxemia. Plasma iron concentration was decreased significantly from the pre-infusion level at 8 h PI. Hypoferremia peak was observed at 12 h and returned to normal levels at 30 h PI. Relative real-time RT-PCR analysis showed that liver hepcidin and IL-6 mRNA expression was up-regulated at 6 h PI. Bone marrow hepcidin relative expression was not influenced by LPS infusion. In another experiment, equine monocyte cultures were stimulated with LPS (1 µg/ml). Monocyte hepcidin and IL-6 gene expression was significantly induced after 2 h of LPS stimulus and returned to baseline levels thereafter. The present study describes that, in horses, LPS infusion up-regulates hepatic hepcidin mRNA expression resulting in early observed hypoferremia and suggests that hepcidin may act as an acute-phase protein in horses.

Influence of experimental inflammatory response on hepatic hepcidin gene expression and plasma iron concentration in sheep.

Hepcidin is a highly conserved disulfide-bonded peptide that plays a central role in iron homeostasis. During systemic inflammation, hepcidin up-regulation is responsible for hypoferremia. This study aimed to analyze the influence of the inflammatory process induced by complete Freund's adjuvant (CFA) or lipopolysaccharide (LPS) on the liver expression of hepcidin mRNA transcripts and plasma iron concentration of sheep. The expression levels of hepcidin transcripts were up-regulated after CFA or LPS. Hypoferremic response was observed at 12 h (15.46 ± 6.05 µmol/L) or 6h (14.59 ± 4.38µmol/L) and iron reached its lowest level at 96 h (3.08 ± 1.18 µmol/L) or 16h (4.06 ± 1.58 µmol/L) after CFA administration or LPS infusion, respectively. This study demonstrated that the iron regulatory hormone hepcidin was up-regulated in sheep liver in response to systemic inflammation. These findings extend our knowledge on the relationship between the systemic inflammatory response, hepcidin and iron, and provide a starting point for additional studies on iron metabolism and the inflammatory process in sheep.

Pro- and anti-inflammatory cytokines produced by human monocytes challenged in vitro with Paracoccidioides brasiliensis.

Monocytes and macrophages play a central role in innate and adaptive immune response against systemic fungal infections. Imbalances in suppressor or stimulatory cytokine secretion caused by these cells may influence disease development, microorganism death, and the nature of the adaptive immune response. This study analyzed the monocyte cytokine profiles of healthy individuals challenged with high and low virulent strains of P. brasiliensis and mRNA cytokine expression kinetics by reverse transcription polymerase chain reaction (RT-PCR). Peripheral blood monocytes from healthy volunteers were cultured in vitro with and without virulent (Pb18) or low virulence (Pb265) strains from P. brasiliensis viable yeast cells. Interleukin-1 beta (IL-1beta), IL-6, IL-8, IL-10, tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta (TGF-beta1) were measured in culture supernatants by enzyme immunoassay (ELISA), and mRNA cytokine expression was determined by RT-PCR at 0, 4, 8, 12, 18 and 48 hr. Both P. brasiliensis strains induced monocyte production of IL-1beta, IL-6, IL-10 and TNF-alpha. Pb18 induced higher levels of IL-1beta, IL-6, and IL-10 than Pb265. IL-8 and TGF-beta1 levels were not significantly different from those cultured without stimulus. The mRNA cytokine expression was similar to supernatant cytokines measured by ELISA. In vitro monocyte challenge with virulent P. brasiliensis strain induces earlier and higher levels of pro- and anti-inflammatory cytokines than low virulence strain.