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Tobacco smoke contains several compounds with oxidant and pro-oxidant properties with the capability of producing structural changes in biomolecules, as well as cell damage. This work aimed to describe and analyse the effect of tobacco smoke on human blood components, red blood cell (RBC) membrane, haemoglobin (Hb) and blood plasma by Atomic Force Microscopy (AFM) and Raman spectroscopy. Our results indicate that tobacco induced RBC membrane nano-alterations characterized by diminished RBC diameter and increased nano-vesicles formation, and RBC fragility. The Raman spectra profile suggests modifications in chemical composition specifically found in peaks 1135 cm−1, 1156 cm−1, 1452 cm−1 and intensity relation of peaks 1195 cm−1 and 1210 cm−1 of blood plasma and by change of peaks 1338 cm−1, 1357 cm−1, 1549 cm−1 and 1605 cm−1 associated with the pyrrole ring of Hb. The relevance of these results lies in the identification of a profile of structural and chemical alterations that serves as a biomarker of physiological and pathological conditions in the human blood components induced by tobacco exposure using AFM and the Raman spectroscopy as tools for monitoring them.

Ionizing radiation is nowadays effectively used in cancer treatments. However, the effect of irradiation in immune-system cells is poorly understood and remains controversial. The aim of this work was to determine the effect of γ-irradiation in the structural and functional properties of mice splenic cells.Structural traits of irradiated splenic cells were evaluated by Atomic Force Microscopy and Raman spectroscopy. Functional properties were measured by gene and protein expression by RT-qPCR and ELISA, respectively. The induced cytotoxic effect was evaluated by MTT assay and the phagocytic capability by flow cytometry.Membrane roughness and molecular composition of splenic adherent cells are not changed by irradiation doses exposure. An increase in transcription of pro-inflammatory cytokines was observed. While protein expression decreased in IL-2 dose-dependent, relevant differences were identified in the anti-inflammatory marker IL-10 at 27 Gy. An increase of cytotoxicity in irradiated cells at 7 Gy and 27 Gy doses was observed, while phagocytosis was slight increased at 7 Gy dose but not statistically significant.We have demonstrated that γ-irradiation affects the splenic cells and changes the cytokines profile toward a pro-inflammatory phenotype and a tendency to increase the cytotoxicity was found, which implies a stimulation of immune response induced by γ-irradiation.

In this work, we report the evaluation of lactosylated graphene oxide (GO-AL) as a potential drug carrier targeted at an asialoglycoprotein receptor (ASGPR) from hepatic cancer cells. Structural-modification, safety evaluation, and functional analysis of GO-AL were performed. The structure and morphology of the composite were analyzed by scanning electron microscopy (SEM) and atomic force microscopy (AFM), while Raman and FTIR spectroscopy were used to track the chemical modification. For the safe application of GO-AL, an evaluation of the cytotoxic effect, hemolytic properties, and specific interactions of the glycoconjugate were also studied. SEM and AFM analysis of the GO showed graphene sheets with a layer size of 2–3 nm, though a few of them reached 4 nm. The Raman spectra presented characteristic peaks of graphene oxide at 1608 cm−1 and 1350 cm−1, corresponding to G and D bands, respectively. Besides, Si–O peaks for the APTES conjugates of GO were identified by FTIR spectroscopy. No cytotoxic or hemolytic effects were observed for GO samples, thus proving their biocompatibility. The interaction of Ricinus communis lectin confirmed that GO-AL has a biorecognition capability and an exposed galactose structure. This biorecognition capability was accompanied by the determination of the specific absorption of lactosylated GO by HepG2 cells mediated through the asialoglycoprotein receptor. The successful conjugation, hemolytic safety, and specific recognition described here for lactosylated GO indicate its promise as an efficient drug-delivery vehicle to hepatic tissue.

The storage lesions and the irradiation of blood cellular components for medical procedures in blood banks are events that may induce nanochanges in the membrane of red blood cells (RBCs). Alterations, such as the formation of pores and vesicles, reduce flexibility and compromise the overall erythrocyte integrity. This review discusses the alterations on erythrocytic lipid membrane bilayer through their characterization by confocal scanning microscopy, Raman, scanning electron microscopy, and atomic force microscopy techniques. The interrelated experimental results may address and shed light on the correlation of biomechanical and biochemical transformations induced in the membrane and cytoskeleton of stored and gamma-irradiated RBC. To highlight the main advantages of combining these experimental techniques simultaneously or sequentially, we discuss how those outcomes observed at micro- and nanoscale cell levels are useful as biomarkers of cell aging and storage damage.

Nanodiamonds have been studied for several biomedical applications due to their inherent biocompatibility and low cytotoxicity. Recent investigations have shown perspectives in using fluorescent nanodiamonds as nanothermometers because of their optical properties’ dependence on temperature. Easy and accurate localized temperature sensing is essential in a wide variety of scientific fields. Our work demonstrated how the fluorescence spectrum of high-pressure high-temperature fluorescent nanodiamonds of three different sizes: 35 nm, 70 nm and 100 nm, changes with temperature within an important biological temperature range (25 °C to 60 °C). Taking advantage of this phenomenon, we obtained nanothermic scales (NS) from the zero phonon lines (ZPL) of the NV0 and NV− colour centres. In particular, the 100 nm-sized features the more intense fluorescence spectra whose linear dependence with temperature achieved 0.98 R2 data representation values for both NV0 and NV−. This model predicts temperature for all used nanodiamonds with sensitivities ranging from 5.73% °C−1 to 6.994% °C−1 (NV0) and from 4.14% °C−1 to 6.475% °C−1 (NV−). Furthermore, the non-cytotoxic interaction with HeLa cells tested in our study enables the potential use of fluorescence nanodiamonds to measure temperatures in similar nano and microcellular aqueous environments with a simple spectroscopic setup.

Background: Reports in a northwestern Mexico state linked arsenic (As) in drinking water to DNA damage in people from indigenous communities. However, this correlation remains under discussion due to unknown variables related to nutrition, customs, and the potential presence of other metal(oid)s. Methods: To determine this association, we sampled water from three Yaqui towns (C&oacute, corit, V&iacute, cam, and P&oacute, tam), and analyzed the metals by ICP-OES. We exposed four separate groups, with five male CD-1 mice each, to provide further insight into the potential effects of untreated drinking water. Results: The maximum concentrations of each metal(oid) in &micro, g&middot, L&minus, 1 were Sr(819) &gt, Zn(135) &gt, As(75) &gt, Ba(57) &gt, Mo(56) &gt, Cu(17) &gt, Al(14) &gt, Mn(12) &gt, Se(19). Histological studies revealed brain cells with angulation, satellitosis, and reactive gliosis with significant statistical correlation with Mn and As. Furthermore, the liver cells presented hepatocellular degeneration. Despite the early response, there is no occurrence of both statistical and significative changes in hematological parameters. Conclusions: The obtained results provide experimental insights to understand the potential effects of untreated water with low As and Mn contents in murine models. This fact is noteworthy because of the development of histological changes on both the brain and liver at subchronic exposure.

Molecular fingerprints revealed by Raman techniques show great potential for biomedical applications, like disease diagnostic through Raman detection of tumor markers and other molecules in the cell membrane. However, SERS substrates used in membrane molecule studies produce enhanced Raman spectra of high variability and challenging band assignments that limit their application. In this work, these drawbacks are addressed to detect membrane-associated hemoglobin (Hbm) in human erythrocytes through Raman spectroscopy. These cells are incubated with silver nanoparticles (AgNPs) in PBS before Raman measurements. Our results showed that AgNPs form large aggregates in PBS that adhered to the erythrocyte membrane, which enhances Raman scattering by molecules around the membrane, like Hbm. Also, deoxyHb markers may allow Hbm detection in Raman spectra of oxygenated erythrocytes (oxyRBCs). Raman spectra of oxyRBCs incubated with AgNPs showed enhanced deoxyHb signals with good spectral reproducibility, supporting the Hbm detection through deoxyHb markers. Instead, Raman spectra of oxyRBCs showed oxyHb bands associated with free cytoplasmic hemoglobin. Other factors influencing Raman detection of membrane proteins are discussed, like both z-position and dimension of the sample volume. The results encourage membrane protein studies in living cells using Raman spectroscopy, leading to the characterization and diagnostic of different pathologies through a non-invasive technique.

Storage and ionizing radiation of human red blood cells (RBC) produce alterations on RBC membranes and modify their normal shape and functionality. We investigated early morphological and biochemical changes in RBC due to those stressing agents at the nanoscale level and their impact on blood quality.Whole blood samples from healthy donors were γ-irradiated with 15, 25, 35, and 50 Gy. Non-irradiated and non-stored RBC were used as control samples. Irradiated blood samples were stored separately at 4 °C and analyzed immediately and after 5 and 13 d. Atomic force microscopy (AFM), osmotic fragility and Raman spectroscopy were used to detect morphological and biochemical changes.RBC function is challenged by both irradiation and storage. The storage procedure caused nanometric variations over the surface of RBC membrane for both irradiated and non-irradiated cells. The membrane of RBC became more fragile, while the biochemical fingerprint of hemoglobin (Hb) remained unaltered.Our work shows that the irradiation procedure leads to an increase in the number and size of nanovesicles along with the dose. The functionality of RBC can be affected from changes in the roughness, becoming more fragile and susceptible to breakage.

The targeted drug delivery has been studied as one of the main methods in medicine to ensure successful treatments of diseases. Pharmaceutical sciences are using micro or nano carriers to obtain a controlled delivery of drugs, able to selectively interact with pathogens, cells or tissues. In this work, we modified bovine serum albumin (BSA) with lactose, obtaining a neoglycan (BSA-Lac). Subsequently, we synthesized glyconanoparticles (NPBSA-Lac) with the premise that it would be recognized by microbial galactose specific lectins. NPBSA-Lac were tested for bio-recognition with adhesins of E. coli K88 and Ricinus communis agglutinin I (RCA). Glycation of BSA with lactose was analyzed by electrophoresis, infrared spectroscopy and fluorescence. Approximately 41 lactoses per BSA molecule were estimated. Nanoparticles were obtained using water in oil emulsion method and spheroid morphology with a range size of 300-500 nm was observed. Specific recognition of NPBSA-Lac by RCA and E. coli K88 was displayed by aggregation of nanoparticles analyzed by dynamic light scattering and atomic force microscopy. The results indicate that the lactosylated nanovectors could be targeted at the E. coli K88 adhesin and potentially could be used as a transporter for an antibacterial drug.

Norovirus (NoV) is the leading cause of epidemic and sporadic gastroenteritis worldwide; a high number of those cases are attributed to the consumption of contaminated food. Crop producers have used several strategies to inactivate the virus present in these products and thus stop the NoV transmission chain. Physical methods such as gamma radiation show excellent results in the inactivation of bacteria, but its effect on NoV has been little studied. This study aimed to evaluate the effect of gamma radiation for NoV inactivation, and over the surface topographic characteristics of strawberry cells, as a prototype of soft fruit. A 10% suspension of GII norovirus-positive stool samples were treated with either 200 mg/L of sodium hypochlorite (NaClO) or gamma-irradiated at doses of 5, 10, 15 and 20 kilograys (kGy). Viral inactivation was determined by measuring the integrity of viral capsid using RNase A alone or in combination with proteinase K followed by RT-qPCR. The effect over cellular surface topology characteristics of the fruit was measured by atomic force microscopy (AFM) and confocal microscopy. High doses of radiation (20 kGy) were necessary to detect a significant (p 0.05) decrease of up to 1.26 log

Hepatocellular carcinoma (HCC) ranks fifth in occurrence and second in mortality of all cancers. The development of effective therapies for HCC is urgently needed. Anticancer drugs targeted to the liver-specific asialoglycoprotein receptors (ASGPRs) are viewed as a promising potential treatment for HCC. ASGPRs facilitate the recognition and endocytosis of molecules, and possibly vehicles with galactose end groups, by the liver. In this study, bovine serum albumin (BSA) was conjugated with lactose using a thermal treatment. The formation of lactosylated BSA (BSA-Lac) was confirmed by a change of the chemical structure, increased molecular mass, and Ricinus communis lectin recognition. Subsequently, the low-crosslinking BSA-Lac nanoparticles (LC BSA-Lac NPs) and high-crosslinking BSA-Lac nanoparticles (HC BSA-Lac NPs) were synthesized. These nanoparticles presented spherical shapes with a size distribution of 560 &plusmn, 18.0 nm and 539 &plusmn, 9.0 nm, as well as an estimated surface charge of &minus, 26 &plusmn, 0.15 mV and &minus, 24 &plusmn, 0.45 mV, respectively. Both BSA-Lac NPs were selectively recognized by ASGPRs as shown by biorecognition, competition, and inhibition assays using an in vitro model of HCC. This justifies pursuing the strategy of using BSA-Lac NPs as potential drug nanovehicles with selective direction toward hepatocellular carcinoma.

Magnetite nanoparticles functionalized with alpha-tocopheryl succinate (Nps@α-TOS) increase anticancer activity of this vitamin E analogue. Previously, we reported Nps@α-TOS protect and enhance bioactivity of α-TOS in vitro . Nps@α-TOS selectively affected cervical cancer cells, without toxic effects for normal cells. The nanoparticles were internalized and accumulated around the nucleus. Severe morphological cell changes and lost viability were observed at 72 h post-treatment in contrast with non-effect in normal fibroblasts cells. These results suggested a cytotoxic effect in cancer cells and biocompatibility in normal cells. Further studies are needed in order to evaluate synergic anticancer bioactivity of Nps@α-TOS with other alternative treatments.

Nanodiamonds when carboxylated (cNDs) act as reducing agents and hence could limit oxidative damage in biological systems. Gamma (γ)-irradiation of whole blood or its components is required in immunocompetent patients to prevent transfusion-associated graft versus host disease (TA-GVHD). However, γ-irradiation of blood also deoxygenates red blood cells (RBCs) and induces oxidative damage, including abnormalities in cellular membranes and hemolysis. Using atomic force microscopy (AFM) and Raman spectroscopy, we examined the effect of cNDs on γ-irradiation mediated deoxygenation and morphological damage of RBCs. γ-Radiation induced several morphological phenotypes, including stomatocytes, codocytes and echinocytes. While stomatocytes and codocytes are reversibly damaged RBCs, echinocytes are irreversibly damaged. AFM images show significantly fewer echinocytes among cND-treated γ-irradiated RBCs. The Raman spectra of γ-irradiated RBCs had more oxygenated hemoglobin patterns when cND-treated, resembling those of normal, non-irradiated RBCs, compared to the non-cND-treated RBCs. cND inhibited hemoglobin deoxygenation and morphological damage, possibly by neutralizing the free radicals generated during γ-irradiation. Thus cNDs have the therapeutic potential to preserve the quality of stored blood following γ-irradiation.

Using detonation nanodiamonds and fluorescent nitrogen-vacancy center nanodiamonds, linked to gold nanoparticles, we synthesized two hybrid nanostructures (HGDs) that were subsequently conjugated with a fluorophore. An amplification effect induced by the gold nanoparticles increased the emission spectrum of the fluorophore, maximizing the possibilities for imaging applications of these HGDs. The incubation of the nanostructures with HeLa cells produced no alteration of cell viability after 3 h and showed the presence of nanostructures in the cell cytoplasm at 24 h. These observations also indicate the potential biomedical use of the proposed HGDs.

Nanodiamonds (NDs) possess exceptional physical, chemical, and biological properties, which make them suitable for potential biomedical applications. They are biocompatible and their usefulness as effective Raman/fluorescence probes for labeling as well as for drug delivery has been demonstrated. Related to their biocompatibility, the interaction between NDs and red blood cells (RBCs) is of great interest. In this work, the influence of carboxylated NDs (cNDs) in the re-oxygenation capability of both γ-irradiated and stored RBCs was studied. The standard 25 Gy γ dose recommended to prevent transfusion associated graft-versus-host disease was used. A 5-day maximum storage time was used to evaluate the “storage lesion.” The hemoglobin (Hb) oxygenation state was assessed by Raman microspectroscopy and the morphologic changes on cells were tracked by optical imaging. Our results show that irradiated RBCs have a better re-oxygenation capability and morphological recovery when they are in presence of cNDs.

Polyethylene glycol (PEG) molecules act as a reducing and stabilizing agent in the formation of silver nanoparticles. PEG undergoes thermal oxidative degradation at temperatures over 70 °C in the presence of oxygen. Here, we studied how the temperature and an oxidizing atmosphere could affect the synthesis of silver nanoparticles with PEG. We tested different AgNO3 concentrations for nanoparticles syntheses using PEG of low molecular weight, at 60 and 100 °C. At the higher temperature, the reducing action of PEG increased and the effect of PEG/Ag+ ratio on nanoparticles aggregation changed. These results suggest that different synthesis mechanisms operate at 60 and 100 °C. Thus, at 60 °C the reduction of silver ions can occur through the oxidation of the hydroxyl groups of PEG, as has been previously reported. We propose that the thermal oxidative degradation of PEG at 100 °C increases the number of both, functional groups and molecules that can reduce silver ions and stabilize silver nanoparticles. This degradation process could explain the enhancement of PEG reducing action observed by other authors when they increase the reaction temperature or use a PEG of higher molecular weight

The compounds 2,9,25,32-tetraoxo-4,7,27,30-tetrakis(carboxymethyl)-1,4,7,10,24,27,30,33-octaaza-17,40-dioxa[10.1.10.1]paracyclophane and 2,9,25,32-tetraoxo-4,7,27,30-tetrakis(carboxymethyl)-1,4,7,10,24,27,30,33-octaaza[10.1.10.1]paracyclophane binuclear copper complexes (Cu2PO and Cu2PC, respectively) were studied by determining their antioxidant capacity using the TROLOX equivalent antioxidant capacity (TEAC) assay, and their cytotoxicity on cultured cells, as well as the superoxide dismutase (SOD)-like activity. Cu2PO had an antioxidant capacity (0.1 g eq TROLOX mol−1) within the order of magnitude of ascorbic acid, and both, Cu2PO and Cu2PC were nontoxic to cultured peripheral mononuclear blood cells. The SOD-like activity was evaluated using the nitroblue tetrazolium assay, and both compounds presented an excellent activity: for Cu2PO, the IC50 was 52 nM and for Cu2PC an IC50 of 0.5 μM was obtained comparable to CuZn SOD IC50 17 nM (Fernandes et al., J Inorg Biochem 2007;101:849–858). These results suggest that synthetic binuclear macrocycles are good candidates to be used as synthetic bioactive molecules with applications in biomedicine.

The use of nanodiamonds in studies with living systems generally involves the modification of their surfaces with functional groups. Fluorescent molecules can be attached to these groups, so that one can know the exact position of the particles in each moment of the interaction with the cells. Here we modify the surface of detonation nanodiamonds and nitrogen-vacancy center nanodiamonds using carboxylation and hydroxylation procedures. Subsequent reactions with silicates and cysteine, before addition of fluorescein allow to obtain fluorescent nano-conjugates. We used confocal microscopy to observe the position of nanodiamonds interacting with HeLa cells. At 3 h post-incubation the green fluorescence is localized in extracellular rounded like-vesicles assemblies while at 24 h the conjugates can be observed inside the cells. The measurement of the fluorescence emitted by both conjugates allowed to find an enhanced emission of fluorescein isothiocyanate (FITC) when the nitrogen-vacancy center is present. We propose the existence of a fluorescence enhancement by electron transference process. The procedure described in this work allows the functionalization of nanodiamonds with FITC and other molecules using functional surface groups and small size mediators. Also, as was proved in our work, the nanodiamond-fluorescein conjugates can be used to track nanoparticles position within the cell. Localization studies are particularly important for drug delivery applications of nanodiamonds.

This work presents the fabrication and characterization of electro-sprayed core-shell particles that were composed of maize bran arabinoxylans (MBAX) with insulin in the core, and maize wastewater arabinoxylans (MWAX) with Bifidobacterium in the shell. Two concentrations of MBAX (3 and 6% w/v) and MWAX (6 and 10% w/v) were evaluated. The particles fabricated with MBAX at 6% (w/v) in the core and MWAX at 10% (w/v) in the shell were more stable, presented spherical shape and no aggregation being therefore selected to be loaded with insulin and probiotics. These particles presented a size of 2.9 mm. Scanning electron microscopy analysis of the particle cross section revealed the presence of both, a smooth (shell) and a porous (core) microstructure. Confocal laser scanning microscopy confirmed the core-shell structure of the particles and the viability of the probiotic entrapped. Gastrointestinal simulation strongly suggests that these particles are not degraded in the stomach and small intestine and that 76% of the carried insulin is released in colon. These results indicate that insulin and Bifidobacterium encapsulation by tetraaxial electro spraying can be a feasible and adequate technique to produce arabinoxylan capsules containing both insulin and probiotics. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018, 135, 46411.

The aim of this study was to characterize the immune responses of DCs after infection with four different EU strains of PRRSV and whether they show any ability to immunomodulate T cells activation. Our results show that all EU strains can efficiently infect and replicate in DCs. Nevertheless, SLA-II levels remained unaltered in DC infected by all EU PRRSV strains, whereas SLA-I expression was only reduced when strain 2992 was used. IL-10 production was induced by three EU PRRSV strains, being strain 2992 the highest inducer. However, no induction of Treg cells, measured by CD25 and Foxp3 expression on lymphocytes co-cultured with infected DCs, was found. TGF-β induction was not detected in DC infected with any EU strain tested. In conclusion, DCs infected with EU PRRSV strains exhibited an unbalanced ability to stimulate T cell response and was strain dependent. However, Treg cells were not induced, at least in vitro.

Antigen-presenting cells (APCs) are strategically placed in all anatomic sites with high antigen exposure such as the respiratory system. The aim of this study was to evaluate phenotypic and functional properties of APCs from the lung (L-Cs), mediastinal lymph node (LN-Cs) and bronchoalveolar lavage cells (BAL-Cs). The APCs were first analyzed based on forward scatter and side scatter profiles and the selection of MHC-II(high)CD172a(+) cells (referred to as APCs); then the expression of CD1a, CD163, CD206, CD16 and CD11R3 was evaluated in the APCs. The results showed that CD1a, CD163 and CD206 were differentially expressed among L-Cs, LN-Cs and BAL-Cs, suggesting the phenotype MHC-II(high)CD172a(+)CD1a(low/-)CD163(low)CD206(-) for L-Cs and MHC-II(high)CD172a(+)CD1a(+)CD163(low/-)CD206(+) for LN-Cs. BAL-Cs were MHC-II(high)CD172a(+)CD1a(-)CD163(high)CD206(+/-). The functional characteristics of L-Cs and LN-Cs were different from those of BAL-Cs, confirming that L-Cs and LN-Cs resemble specialized APCs. In conclusion, we present the characterization of APCs from L-Cs, LN-Cs and BAL-Cs of the porcine respiratory system.

Summary Background & aims Obesity was recognized as an independent risk factor for morbidity and mortality during last influenza A/H1N1 pandemic. Mechanisms involved in the high mortality risk from obesity during influenza A virus include reduced type I interferon production and delayed pro-inflammatory response, which lead to a higher rate of morbidity and mortality in murine models. In this study, we evaluated the production of type I interferons, pro-inflammatory and anti-inflammatory cytokines in peripheral blood mononuclear cells (PBMCs) from obese and lean subjects with and without confirmed infection of influenza A/H1N1. The expression levels of the suppressor of cytokine signaling-1 (SOCS1), SOCS3 and nuclear factor-kB were also evaluated. Methods Cytokines were measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and/or by ELISA in PBMCs stimulated with toll like receptor-3 (TLR-3) and TLR-7 ligands. The mRNA expression of SOCS1 and SOCS3 were evaluated by qRT-PCR. Results The obese volunteers infected with influenza A/H1N1 showed a diminished ability to produce type I interferon in response to TLR-3 ligand. Interestingly, the pro-inflammatory response was also affected in TLR-3 stimulated PBMCs. Obese influenza-free volunteers showed an increased basal expression of SOCS3, but not SOCS1. During influenza infection, SOCS1 and SOCS3 expression was higher in the lean infected volunteers in contrast to those who were obese infected. Conclusions These data suggest that obesity is related to TLR-3 impairment and explain, at least in part, the inadequate immune response of obese individuals during infection with influenza A/H1N1 virus.

Swine influenza virus (SwIV) is considered a zoonosis and the fact that swine may act as an intermediate reservoir for avian influenza virus, potentially infectious for humans, highlights its relevance and the need to understand the interaction of different influenza viruses with the porcine immune system. Thus, in vitro porcine bone marrow-derived dendritic cell (poBMDCs) were infected with a circulating SwIV A/Swine/Spain/SF32071/2007(H3N2), 2009 human pandemic influenza virus A/Catalonia/63/2009(H1N1), low pathogenic avian influenza virus (LPAIV) A/Anas plathyrhynchos/Spain/1877/2009(aH7N2) or high pathogenic avian influenza virus (HPAIV) A/Chicken/Italy/5093/1999(aH7N1). Swine influenza virus H3N2 infection induced an increase of SLA-I and CD80/86 at 16 and 24 h post infection (hpi), whereas the other viruses did not. All viruses induced gene expression of NF-κB, TGF-β, IFN-β and IL-10 at the mRNA level in swine poBMDCs to different extents and in a time-dependent manner. All viruses induced the secretion of IL-12 mostly at 24 hpi whereas IL-18 was detected at all tested times. Only swH3N2 induced IFN-α in a time-dependent manner. Swine H3N2, aH7N2 and aH7N1 induced secretion of TNF-α also in a time-dependent manner. Inhibition of NF-κB resulted in a decrease of IFN-α and IL-12 secretion by swH3N2-infected poBMDC at 24 hpi, suggesting a role of this transcription factor in the synthesis of these cytokines. Altogether, these data might help in understanding the relationship between influenza viruses and porcine dendritic cells in the innate immune response in swine controlled through soluble mediators and transcription factors., This work was partly funded by the Project No. CSD 2006-00007, AGL2006-13809-C03-01, AGL2009-12945-C02-01 and AGL2010-22200-C02-01 by the Spanish Government. PhD studies of Mrs. Tufária Mussá and Masssimiliano Baratelli are funded by doctoral grants from the AECID and MICIN respectively.

The aim of this study was to analyze the regulatory T cells (Tregs) induced by the porcine reproductive and respiratory syndrome virus (PRRSV) in pigs. Serum, blood, tonsil, and mediastinal lymph nodes' samples were obtained at different time post-infection (dpi). The frequencies of CD4(+)CD8(-)CD25(+)Foxp3(+), CD4(+)CD8(+)CD25(+)Foxp3(+), or CD4(-)CD8(+)CD25(+)Foxp3(+) phenotypes were determined in PBMC and lymph node cells, and cells producing IL-10 or TGF-β were analyzed. PRRSV increased the number of CD4(+)CD8(+)CD25(+)Foxp3(+) cells at 14 dpi, whereas CD4(+)CD8(-)CD25(+)Foxp3(+) remained constant until 28 dpi. Positive correlation exists between viremia and induced regulatory cells. CD4(+)CD8(+)CD25(+)Foxp3(+)-induced Treg cells were consistently observed in lymphoid tissues. Analysis of IL-10- and TGF-β-producing cell demonstrated that in response to PRRSV, CD4(+)CD8(-)Foxp3(low) and CD4(+)CD8(+)Foxp3(high) cells increase moderately the proportion of IL-10(+) cells. TGF-β was only observed in the CD4(+)CD8(+)Foxp3(high) population after PRRSV stimulation. In conclusion, PRRSV infection increases the frequency of Tregs with the phenotype CD4(+)CD8(+)CD25(+)Foxp3(high) and produces TGF-β.

"El virus del síndrome reproductivo y respiratorio porcino (PRRSV) establece un estado de persistencia en cerdo debido a su capacidad para modular la respuesta inmune. La respuesta celular de cerdos infectados con PRRSV se caracteriza por un bajo número de células productoras de interferon (IFN)-γ específicas a PRRSV, las cuales se detectan entre los 14 y 28 días post-infección (dpi). En muchos modelos de infección viral, el establecimiento de infecciones persistente o crónicas se asocia con la participación de los linfocitos T reguladores (Treg), los cuales tienen la función de suprimir la respuesta inmune. El objetivo de este trabajo fue determinar si el PRRSV induce la expansión de Treg y con ello identificar un posible mecanismo del virus para evadir la respuesta inmune. En el modelo in vitro se evaluó la capacidad de las células dendríticas infectadas con cepas americanas y europeas para inducir la expansión de células CD25+ Foxp3+ (Treg). Los resultados indicaron que las cepas europeas evaluadas no indujeron linfocitos Treg, al contrario de las cepas americanas que si indujeron Tregs y se caracterizaron por la producción de TGF-β. Lo anterior permitió hipotetizar que la inducción de Treg en cerdos infectados con PRRSV es dependiente de TGF-β. También se evaluó la presencia de Treg en animales adultos naturalmente expuestos al PRRSV. Se observó un incremento de Treg en animales con cargas virales bajas del virus en suero. Sin embargo no se pudo establecer si existe asociación con la secreción intermitente del virus en semen ya que fue un estudio exploratorio. Para evaluar la participación de las células Treg en el proceso de infección aguda del PRRSV, se infectaron lechones de entre 4 y 6 semanas de edad y se monitoreó la frecuencia de Treg en sangre periférica y tejidos asociados a la replicación de virus (ganglios mediastínicos y amígdalas). Los resultados demostraron que el PRRSV incrementó los Treg en circulación a partir del día 14 pi. Se observó que los Treg se incrementaron en las amígdalas hasta los 8-24 dpi respecto a los no infectados. Se caracterizó que el fenotipo inducido por el PRRSV es T reguladores tipo 1 (Tr1), los cuales producen principalmente interleucina (IL)-10. Los resultados obtenidos permiten suponer que el retraso en la aparición de la respuesta celular anti-PRRSV es resultado de la inducción de linfocitos Treg, los cuales se presentan en circulación y en tejidos asociados a la replicación del virus. Futuros estudios deberán

Porcine reproductive and respiratory syndrome virus (PRRSV) infects mature dendritic cells (mDCs) derived from porcine monocytes and matured with lipopolysaccharide. The infection of mDCs induced apoptosis, reduced the expression of CD80/86 and major histocompatibility complex class II molecules, and increased the expression of interleukin-10, thus suggesting that such mDC modulation results in the impairment of T-cell activation.

Las células dendríticas (DCs) tienen la capacidad de activar linfocitos T y modular su respuesta a Th1, Th2 o T reguladoras (Treg). La respuesta inmune celular ante el virus del PRRS se encuentra dlsrninuida y es ineficaz. Éste virus infecta y se replica en DCs, por ello la hipótesis fue que las DCs infectadas con virus PRRS suprimen la respuestas Th1 y Th2 en linfocitos T in vitro. El objetivo fue evaluar la capacidad de las DCs infectas con el virus PRRS para estimular una respuesta inmune. Las DCs se infectaron (m.o.i. 0.1) con 2 cepas americanas y 5 cepas europeas, se co-cultivaron con células no adherentes homólogas, La proliferación se analizó con CFSE y la inducción de células con fenotipo Treg con anticuerpos anti-Foxp3 y anti-CD25, por citometría de flujo. La cuantificación del mRNA de citocinas (IL-2, IL-4, IL-10 e IFN-γ) y factores de trascripción (T-bet, GATA3 y Foxp3) se analizaron por PCR en tiempo real a las 24 h de co-cultivo. La producción de cltocinas (IL-10 e IFN-γ) en el sobrenadante colectados al día 3 se determinó por ELISA. Los resultados muestran que las cepas americanas, pero no las europeas reducen la proliferación (p0.05), Las cepas americanas aumentaron (p0.05). No hubo diferencias en la expresión de IL-10, IFN-γ , IL-4, T-bet y Gata-3 (p>0.05). Para las cepas americanas se observó un incremento significativo en la expresión de TGF-β y el Foxp3 tiende a incrementar. Estos resultados muestran que las DCs infectadas con PRRS no son capaces de estimular la respuesta de células T y este efecto podría ser la consecuencia de la inducción de células Treg por las cepas americanas y debido a la acción del TGF-β.

Delayed development of virus-specific immune response has been observed in pigs infected with the porcine reproductive and respiratory syndrome virus (PRRSV). Several studies support the hypothesis that the PRRSV is capable of modulating porcine immune system, but the mechanisms involved are yet to be defined. In this study, we evaluated the induction of T regulatory cells by PRRSV-infected dendritic cells (DCs). Our results showed that PRRSV-infected DCs significantly increased Foxp3(+)CD25(+) T cells, an effect that was reversible by IFN-alpha treatment, and this outcome was reproducible using two distinct PRRSV strains. Analysis of the expressed cytokines suggested that the induction of Foxp3(+)CD25(+) T cells is dependent on TGF-beta but not IL-10. In addition, a significant up-regulation of Foxp3 mRNA, but not TBX21 or GATA3, was detected. Importantly, our results showed that the induced Foxp3(+)CD25(+) T cells were able to suppress the proliferation of PHA-stimulated PBMCs. The T cells induced by the PRRSV-infected DCs fit the Foxp3(+)CD25(+) T helper 3 (Th3) regulatory cell phenotype described in the literature. The induction of this cell phenotype depended, at least in part, on PRRSV viability because IFN-alpha treatment or virus inactivation reversed these effects. In conclusion, this data supports the hypothesis that the PRRSV succeeds to establish and replicate in porcine cells early post-infection, in part, by inducing Th3 regulatory cells as a mechanism of modulating the porcine immune system.

El ácido linoleico conjugado (CLA) es una familia de isómeros geométricos y posicionales del ácido linoleico. En diversos estudios en modelos animales y en humanos, los isómeros del CLA han mostrado cualidades de modulación del sistema inmunológico, como el incremento en la producción de anticuerpos, citocinas y proliferación de linfocitos. La inmunidad adaptativa es una de las defensas principales en la mucosa intestinal, donde el organismo está en contacto constante con patógenos, tales como Giardia lamblia. Este parásito es causante de una de las principales parasitosis en el mundo denominada giardiasis. La respuesta inmune contra G. lamblia se caracteriza por una producción elevada de anticuerpos tipo IgA. El objetivo de este trabajo fue evaluar el efecto del CLA en la respuesta inmune adaptativa en un modelo murino de giardiasis. Se realizaron dos bioensayos de infección de 40 días en 48 ratones C3H/HeN. Por medio de citometría de flujo se determinó el porcentaje de las poblaciones linfocíticas T y B en Placas de Peyer. Además se cuantificó la producción de citocinas ex vivo y la concentración de IgA total y específica en extracto fecal mediante ELISA. Se observó un pico a los 6 días post-infección en ambos grupos, con menor carga parasitaria en el grupo CLA en todos los tiempos evaluados (p < 0.05). Las poblaciones de linfocitos CD4 y CD8 se mantuvieron constantes entre grupos durante las 6 semanas postinfección (p > 0.05). Se observó un incremento de la población de linfocitos CD35/21+ en el grupo control el día 6 post-infección (p

En México las infecciones gastrointestinales son la segunda causa de morbilidad, principalmente por agentes bacterianos y parásitos. Sin embargo, no existe vigilancia epidemiológica para otros agentes, como por ejemplo, norovirus (NoV), que es la principal causa de gastroenteritis epidémica y esporádica a nivel mundial. Un alto número de infecciones intestinales se atribuyen al consumo de alimentos contaminados con NoV. Los productores de frutas y hortalizas han utilizado distintas estrategias para inactivar al virus, y así detener la cadena de transmisión. Una de ellas es la cloración, ya que es un método barato y de fácil aplicación. Por otro lado, los métodos físicos como la radiación gamma muestran excelentes resultados en la inactivación de bacterias, pero su efecto en NoV ha sido poco estudiado. El objetivo del presente trabajo fue evaluar el efecto de la cloración y la radiación gamma sobre la inactivación de NoV, y sobre las características topográficas celulares de fresa, como prototipo de frutos rojos blandos. Muestras de heces positivas a NoV fueron cloradas a una concentración final de 200 mg/L de hipoclorito de sodio (NaClO), y comparadas con muestras irradiadas a dosis de 5, 10, 15, y 20 kilograys (kGy). El efecto de ambos métodos en la inactivación de NoV, se midió mediante la cuantificación de copias del genoma viral por reacción en cadena de la polimerasa con transcriptasa inversa cuantitativa (RT-qPCR), posterior a un tratamiento enzimático. El efecto de los tratamientos sobre las características superficiales y fisiológicas de las células, se evaluó mediante microscopía de fuerza atómica (AFM) y microscopía confocal. Altas dosis de radiación gamma (20 kGy) fueron necesarias para conseguir una reducción significativa (p

La respuesta inmune efectora y reguladora en mucosa intestinal se encuentra en equilibrio debido a la relación entre poblaciones de células Th17 y T reguladoras (Treg), importantes en la respuesta a patógenos. Giardia lamblia es un parásito intestinal de importancia en salud pública, causa diarrea aguda, cambios en el epitelio duodenal y síndrome inflamatorio en colon posterior a la eliminación. Previamente en nuestro grupo de investigación se demostró que el ácido linoleico conjugado (CLA) tiene efectos sobre las células presentadoras de antígeno y que estimula la producción de IgA en intestino de ratones infectados con G. lamblia, pero se desconoce el efecto de polarización de células Th. El objetivo de este trabajo fue evaluar el efecto de la suplementación de CLA en el perfil transcripcional característico de Th17 y Treg en ratones infectados con G. lamblia, así como los cambios en la mucosa de colon después de la eliminación. Se infectaron dos grupos de ratones, uno suplementando con CLA (n=16) y otro grupo control (n=16). Se determinó el peso de los ratones en ambos grupos durante el bioensayo. A partir de células de intestino delgado, se realizó la extracción de ARN total con Trizol® y cuantificación de ARNm de citocinas y factores de transcripción a los 0, 6, 14 y 40 días post infección (dpi), por RT-qPCR. Se analizaron muestras histológicas de la región proximal de colon. El peso de los ratones no presentó diferencias significativas entre grupos. El CLA aumentó la expresión del factor de transcripción RORγT y la expresión de citocinas IL-10, IL-17A y TGF-β (P