Elevated oxidative resistance of differentiated podocytes was associated with increased activities of antioxidant enzymes and H-ferritin expression. Immunohistochemical analysis of normal human kidney specimens revealed that podocytes highly express H-ferritin in vivo as well.Podocytes are highly specialized, arborized epithelial cells covering the outer surface of the glomerular tuft in the kidney. Terminally differentiated podocytes are unable to go through cell division and hereby they are lacking a key property for regeneration after a toxic injury. Podocytes are long-lived cells but, to date, little is known about the mechanisms that support their stress resistance. Our aim was to investigate whether the well-known morphological changes during podocyte differentiation are accompanied by changes in oxidative resistance in a manner that could support their long-term survival. We used a conditionally immortalized human podocyte cell line to study the morphological and functional changes during differentiation. We followed the differentiation process for 14 days by time-lapse microscopy. During this period nondifferentiated podocytes gradually transformed into large, nonproliferating, frequently multinucleated cells, with enlarged nuclei and opened chromatin structure. We observed that differentiated podocytes were highly resistant to oxidants such as H During the glomerular morphogenesis, four resident cell types are responsible for the formation of a functional glomerular filtration barrier in the kidney. A complex system of interference between parietal epithelial cells of Bowman's capsule, podocytes , fenestrated endothelial cells, and mesangial cells maintains the integrity of the glomerular structure. The parietal epithelial cells and the podocytes originate from the metanephric mesenchyme and are identifiable at week 5 of gestation in humans .During embryonic development, the ability of podocytes to proliferate ceases, and the cells undergo cytoskeletal rearrangement, form foot processes, and express molecules that contribute to the formation of the slit diaphragm. Mature podocytes are highly specialized cells with an interdigitating network of foot processes which control the filtration of proteins from the capillary lumen into Bowman's space . Narrow The glomerular filtration barrier is the primary site of sieving and is exposed to an excessive amount of circulating reactive oxygen species (ROS) . AccumulAntioxidant enzymes including glutathione-peroxidase (GPX), catalase, and superoxide dismutase (SOD) are crucial elements of the cellular defence system against ROS . Damagin in vitro [ in vivo in conditional FtH knockout animals [The antioxidant nature of ferritin was provedin vitro  and more animals . This mo animals , 16. in vitro growth conditions using a conditionally immortalized human podocyte cell line [ in vitro and in vivo. This paper provides evidence that the differentiation of immortalized human podocytes is a complex process showing characteristic phenotypic plasticity. Phenotypic changes during podocyte differentiation are not just a certain filtering function of podocytes but are of crucial importance in helping survival of these highly differentiated cells in certain oxidative conditions.Our aim was to explore novel functional changes during podocyte differentiation that could support oxidative resistance and long-term survival of these cells. We hypothesized that antioxidant enzymes and FtH are an integral part of podocyte defence mechanism. In this work we applied time-lapse video microscopy to follow morphological changes of individual podocytes in the course of differentiation underell line . We showμg/mL amphotericin B), insulin (10 μg/mL), transferrin (5.5 μg/mL), and selenium (5 ng/mL Na selenite). At ~50–60% confluence, the cells were transferred to 37°C (nonpermissive condition) for 14 days to silence the temperature sensitive tsA58 T antigen. Passage numbers were used between 14 and 20. Cell culture materials were obtained from PAA Laboratories Ltd. .Human podocytes were kindly provided by Moin Saleem (University of Bristol) and cultured as described previously . Brieflyμg/mL for 40 minutes was applied to stain F-actin. To optimize detection, streptavidin/biotinylated horse-radish peroxidase system was applied for amplification of the signal. Finally, the samples were covered with a DAPI  containing antifade mounting medium  to stain the nuclei and minimize photobleaching of the samples. Pictures were taken using a Leica DM4000 fluorescent microscope equipped with a Leica DFC310 FX digital color camera .Human immortalized podocytes were grown on poly-D-lysine-four-well chamber slides to 50–60% confluence. After removing the medium, cells were washed twice thoroughly with PBS followed by fixation with 4% freshly prepared paraformaldehyde and blocking with 5% donkey serum for 20 minutes. The cells were stained with primary anti-synaptopodin  and secondary Cy3-conjugated antibodies. For the visualization of the cytoskeleton, fluorescein isothiocyanate- (FITC-) labelled phalloidin  at a dose of 50 2 incubator, were equipped with high-sensitivity digital cameras and connected to a dual image-acquisition computer system. Illumination was developed to minimize heat- and phototoxicity. Operation of the spectrally cold-white light emitting diodes was synchronized with image-acquisition periods. Frames were recorded every minute and the whole video sequence was converted to database form. The time of exposure was indicated in the right lower corner of each frame. Exposures were converted to video films by speeding up the projection to 30 exposures/seconds.Long-term time-lapse microscopy analysis was performed on the culture under nonpermissive condition from day 0 to day 14 to follow the process of differentiation. Time-lapse microscopy was performed as previously described . Brieflyμm sections from the human kidney cortex was stained after antigen retrieval. The sections were subsequently incubated in 3% H2O2 for 15 minutes to quench endogenous peroxide activity and blocked for 20 minutes at room temperature to suppress nonspecific binding of subsequent reagents. Subsequent FtH visualization in podocytes in the glomeruli monoclonal antibody from Paolo Arosio  was used in combination with Wilms tumor 1 (WT-1) antibody . The sections were incubated for 15 minutes with avidin-biotin-HRP complex before applying biotin labelled secondary antibodies. Vectastain Elite ABC dual detection system  was used following the manufacturer's guidelines. To visualize antigens of interest Vector VIP  and DAB  were applied as substrates. Appropriate positive and negative control slides were analysed in parallel.Formalin-fixed paraffin-embedded tissue in 4 Modified Giemsa staining was performed on parallel cell cultures at different time points. The monolayer of cells was fixed with methanol for 5 minutes. After air-drying, the cells were stained with freshly prepared Giemsa solution (Sigma-Aldrich) for 10 minutes. The redundant staining solution was washed away with distilled water and the samples were let to air-dry again. Pink colour represents cytoplasmic staining and blue nuclear staining. Magnification was 10x.To compare the cyclic character of chromatin unfolding and chromosome condensation of both nondifferentiated and differentiated podocytes, isolation of nuclei was performed as described previously . Briefly2PO4 buffer . Cells were lysed by sonication on ice (3 × 5 s), and cell lysate was obtained by centrifugation . Enzyme activities were measured from cell lysates as described previously [Nondifferentiated and differentiated podocytes were harvested in KHeviously .μg protein/lane) was subjected to 6% nondenaturing PAGE. FtH was detected using human FtH monoclonal antibodies at a dilution of 1 : 1000 and horseradish peroxidase- (HRP-) conjugated goat anti-mouse IgG as secondary antibody at a dilution of 1 : 15000. Antigen-antibody complex was detected by a HRP chemiluminescence system according to the manufacturer's instructions . Quantification was performed using video densitometry .Confluent podocytes grown on 6-well plates originating from both permissive and nonpermissive conditions were treated for 1 hour with heme in HBSS . After that, heme was removed and cells were incubated in RPMI supplemented with 10% FBS for further 8 hours. Cells were harvested as described previously  and equaμmol/L) or H2O2 (125–500 μmol/L) or the combination of heme and H2O2 in HBSS. When used in combination, cells were pretreated with heme for 1 hour prior to H2O2 treatment. Appropriate vehicles were analysed in parallel. Cell viability was determined by MTT-reduction  measured at 590 nm.Nondifferentiated and differentiated cell cultures of human immortalized podocytes were grown on 96-well plates and exposed to heme (1.25–5 μmol/L) or H2O2 (125–500 μmol/L) in HBSS for 4 hours. Cells were scraped in 200 μL of KH2PO4 buffer  containing SDS (6%). To each sample 400 μL of TBARS reagent  was added. After incubating at 90°C for 30 minutes the samples were cooled down and extracted with 100 μL n-butanol with vigorous vortexing. Phases were separated by centrifugation , and optical density of the organic phase was measured at 532 nm.Nondifferentiated and differentiated human podocytes were grown on 6-well tissue culture plates. Cells were exposed to heme . Differences between two groups were analysed by unpaired We placed nondifferentiated human podocytes expressing the thermosensitive A58 T antigen at 50–60% confluency from 33°C to 37°C to induce differentiation. We followed changes in cell number and morphology by video microscopy for 14 days. By analysing the growth curve we observed that the initial exponential growth phase lasted for 9 days . After cTo prove that differentiation of podocytes was complete during the 14 days, we evaluated synaptopodin expression in undifferentiated and differentiated cells by immunostaining   . In cont2O2 or heme nondifferentiated podocytes underwent cell death, whereas mature podocytes showed high resistance. To assess whether this phenomenon was accompanied by differences in oxidative stress, we measured the oxidative stress marker TBARS levels in both nondifferentiated and differentiated podocytes under normal and stress conditions. We found that nondifferentiated podocytes responded by a dose-dependent elevation of TBARS levels to both H2O2 and heme stress, whereas the same triggers did not cause significant increases in TBARS levels in differentiated podocytes . In contrast, in differentiated podocytes, which show high FtH expression at basal level, low doses of heme (1.2 and 2.5 μmol/L) failed to induce FtH expression further more. Higher doses of heme triggered the induction of FtH suggesting that these differentiated podocytes remained responsive to this challenge but their resistance is shifted. Next, we examined whether FtH is expressed in significant amount in human kidney podocytes. Adult human kidney sections were stained with WT-1 antibody to visualize podocytes (purple) structurally in the glomerulus. For functional assessment, the sections were counterstained with FtH antibody. Glomeruli in the cortex contained several podocytes with strong FtH staining (brown), which supports our in vitro findings of high FtH content and iron sequestering capacity of resting podocytes  .To date, few studies have investigated and compared nuclear properties of glomerular podocytes. The primary role of the nucleus is to store information and ensure a site for cell retrieval and replication . We stud2O2-induced oxidative stress. Because of the Fenton reaction, ROS-mediated oxidative stress is highly amplified in the presence of redox-active iron. Therefore, we tested oxidative resistance of differentiated and nondifferentiated podocytes in the presence of heme that act as a Fenton catalyst [2O2 on cell viability, we found that there was a significant difference in the oxidative resistance capacity of undifferentiated and differentiated podocytes. Heme alone caused a strongly significant decrease in cell viability on nondifferentiated podocytes in a dose-dependent manner, while the same doses did not exert any cytotoxic effect on differentiated cells. Treatment of nondifferentiated podocytes with heme prior to H2O2 challenge resulted in an even lower rate of cell viability. In contrast, mature podocytes survived this lethal combination of heme and H2O2. H2O2 and heme-provoked cell death of nondifferentiated podocytes were accompanied by elevated TBARS levels, whereas the same triggers did not cause significant increases in TBARS levels in differentiated podocytes. Moreover, increased oxidative resistance of mature podocytes is associated with elevated activities of GPX, catalase, and SOD compared to nondifferentiated cells.Podocytes are subjected to several injurious and deleterious factors, for example, oxidative stress factors, while producing the glomerular filtrate. Oxidative stress resistance and the exact mechanisms by which these cells survive are yet uncovered. Here we report that the oxidative resistance of differentiated podocytes is much higher than that of undifferentiated cells against Hcatalyst , 37. Whe via sequestering iron from Fenton-like reactions. The deletion of FtH is embryonically lethal in mice; therefore, a conditional FtH deficient mice model was generated [ in vivo evidence supports the idea that FtH provides protection to tissue damage. Recently, Zarjou et al. showed that conditional deletion of FtH in renal proximal tubules increased nephrotoxicity in different models of acute kidney injury [ FtH in the liver increases tissue damage and mortality in mice models of severe malaria [FtH has been shown to be effective in the defence against oxidative cell injury , 14 via enerated  to allowy injury . Gozzeli malaria . Moreove malaria . in vivo relevance of our observation, we examined the FtH content of human kidney specimens. Sections of kidney cortex rich in glomeruli were stained immunohistochemically. In support of our theory, we detected a massive FtH store in the cytoplasm of podocytes surrounding the glomerular capillaries that might be implicated in long-term survival of podocytes.This finding is in agreement with our hypothesis that FtH may play a role in protecting podocytes from deleterious injuries. Differentiated podocytes that are more resistant to oxidative stress express more FtH than nondifferentiated cells. To assess theThis study provides the first evidence that, during embryonic differentiation, podocytes go through fundamental morphological and functional changes assisting them to intensify defence mechanisms against oxidative stress damage and increase their resistance essential for long life duration . Antioxi2 incubator, was equipped with high-sensitivity digital cameras and connected to an image-acquisition computer system. Frames were recorded every minute and the whole video sequence was converted to database form. The time of exposure was indicated in the right lower corner of each frame. Exposures were converted to video films by speeding up the projection to 30 exposures/seconds.Method: Time-Lapse Videomicroscopy. Long-term time-lapse microscopy analysis was performed on the culture under nonpermissive condition from day 0 to day 14 to follow the process of differentiation. An inverse microscope, placed in a COResults: Podocyte Growth and Differentiation under Nonpermissive (37°C) Condition. We placed nondifferentiated human podocytes expressing the thermosensitive A58 T antigen at 50–60% confluency from 33°C to 37°C to induce differentiation. We followed changes in cell number and morphology by video microscopy for 14 days. we observed that the initial exponential growth phase lasted for 9 days. After cell number reached its peak between days 9 and 10,we saw a slight decrease in cell number followed by stabilization of the culture (days 10–14) when we could hardly detect any mitotic event.When the temperature was shifted to 37°C the morphology of nondifferentiated podocytes gradually transformed into that of differentiated cells. At permissive temperature (33°C) nondifferentiated podocytes showed typical epithelial cobblestone morphology. In contrast, differentiated podocytes are characterized by enlarged cell bodies with an irregular shape and the formation of processes involving shorter and rounded as well as longer and spindle-like projections. Formations of spindle-like projections were often preceded by partial retraction of cytoplasmatic protrusions. Many differentiated cells developed cellular hypertrophy, some of the cells died, and others started to arborize. We detected numerous binucleated but nonproliferating cells. After day 9 we observed a reduction of cell number, but themotility of cells remained high even after differentiation, and the culture proved capability of migration until all bare fields of the culturing vessel were completely covered.


Sc) of the cellular prion protein (PrPc). Prions replicate autocatalytically in neurons and other cell types by inducing conformational conversion of PrPc into PrPSc. Within neurons, PrPSc accumulates at the plasma membrane and in vesicles of the endocytic pathway. To better understand the mechanisms underlying neuronal dysfunction and death it is critical to know the impact of PrPSc accumulation on cellular pathways. We have investigated the effects of prion infection on endo-lysosomal transport. Our study demonstrates that prion infection interferes with rab7 membrane association. Consequently, lysosomal maturation and degradation are impaired. Our findings indicate a mechanism induced by prion infection that supports stable prion replication. We suggest modulation of endo-lysosomal vesicle trafficking and enhancement of lysosomal maturation as novel targets for the treatment of prion diseases.Prions are proteinaceous infectious particles which cause fatal neurodegenerative disorders in humans and animals. They consist of a mostly β-sheeted aggregated isoform (PrP Upon direct interaction between PrPc and PrPSc, new PrPSc molecules are generated. In contrast to PrPc, PrPSc has a high β-sheet content, and forms aggregates and amyloid fibres3Sc aggregates accumulate in the brain of individuals and cause fatal neurodegenerative disorders, e.g. Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, or chronic wasting disease in cervids56Prions are a class of infectious agents that are composed exclusively of protein, namely a misfolded isoform of the cellular prion protein PrPc is a plasma membrane protein attached to the cell surface by a glycosylphosphatidyl-inositol (GPI) anchorc is constantly converted into PrPSc, for which PrPc expression101112Sc can be found at the plasma membrane and along the endocytic pathway1416171819Sc in endocytic vesicles affects their subcellular trafficking.On a cellular level, PrPRab proteins comprise a large family of small GTPases which are localized to distinct intracellular membranes and regulate vesicle trafficking. They switch between an inactive, GDP-bound cytosolic state and an active, GTP-bound membrane associated state. Rab proteins are activated by the exchange of GDP with GTP which is catalysed by guanine nucleotide exchange factor (GEF), and membrane association is mediated by C-terminal prenylation. The active form interacts with effector proteins and is inactivated by GTP hydrolysis. Inactive, GDP-bound rabs are recognized and bound to rab GDP dissociation inhibitor (rabGDI) in the cytosol. Membrane properties such as the lipid composition can influence the recruitment of rab proteins to certain compartments22Sc depositionscStudies on axonal transport in motor neurons of prion infected mice revealed an impairment of retrograde transport, which involves a rab7-mediated pathwaySc aggregates in vesicles along the endocytic pathway led us to investigate a possible influence of prion infection on endocytic vesicle trafficking in neuronal cell lines (CAD5 and N2a) persistently infected with prion strain RML and/or 22L.These findings and the described distribution of PrPSc accumulation that can support the persistent replication of prions by preventing PrPSc degradation in lysosomes. Notably, perturbations of the lysosomal degradation pathway may be linked to neurodegeneration and neuronal death, as observed in prion infected individuals. Based on our results, we suggest induction of lysosomal maturation as a target for treatment of prion diseases.We found that the amount of membrane-bound rab7 is reduced upon prion infection. Since functional rab7 is involved in lysosomal maturation, we compared the efficiency of lysosomal degradation in N2a and 22LN2a cells. We found a significantly lower degradation rate in prion infected cells. Our data suggest a mechanism induced by PrPSc aggregates are mainly located at the cell surface and in vesicles along the endocytic pathway14151719In prion infected neuronal cells PrPSc propagationSc Sc were detectable, in both non-infected and PPS treated CAD5 cells no PrPSc was found  and untreated (−Gli) N2a and 22LN2a cells upon PK digestion of cell lysates were analysed to confirm the absence of PrPSc  and membrane preparations of these cells did not harbor detectable levels of PK-resistant PrP  which induces autophagyof PrPSc . Whereasof PrPSc  as well of PrPSc  containetant PrP . Next, wtant PrP .In summary, with this set of experiments we were able to demonstrate that crude membrane preparations of prion-infected cells contain less rab7 than those of non-infected cells or cells cured from prion infection. This effect was not specific for the cell line or prion strain used for infection, as all prion-infected cell lines, i.e. 22LCAD5, RMLCAD5 and 22LN2a showed a reduction of membrane-bound rab7.in vitro and in vivo3233343738We and others have shown previously that prion infection results in up-regulation of cholesterogenic genes, concomitant with increased levels of unesterified cholesterol both Therefore, we incubated crude membrane preparations from N2a and 22LN2a cells with 0 or 3 μM of recombinantly expressed and purified rabGDI which solubilises membrane-bound rab proteinsAltogether, these data indicate that the strength of rab7, rab9 and rab11 membrane association is not altered upon prion infection.Our experiments demonstrate that prion infection results in reduced levels of membrane bound rab7 in persistently prion infected neuronal cells. Association of rab7 with late endosomal membranes is critical for lysosomal maturation, and diminished rab7 expression levels can interfere with lysosomal degradation41424Cl throughout the experiment. None of these treatments was significantly toxic to the cells as demonstrated by XTT assay  which is a commonly used marker protein for lysosomal degradationTT assay . Cells wTT assay . Signalstectable . We havenificant . PK treaanalysis .If lysosomal maturation is affected by the lack of membrane-bound rab7 we expected that the numbers of lysosomes will be reduced in 22LN2a cells. To verify this, we co-stained N2a cells, 22LN2a cells and 22LN2a + Gli cells that were cured from prion infection with anti-lamp1 for detection of late endosomes and lysosomes, and with lysotracker red-dnd99 which accumulates in a pH-dependent manner in lysosomes . WhereasSc can, albeit with a long half-life, be degraded in lysosomes, the impairment of lysosomal degradation may contribute to sustained prion propagation in persistently infected cells.Our findings are summarized in We demonstrate here that in two neuronal cell lines, CAD5 and N2a, infected with one or both prion strains 22L and RML the levels of membrane-bound rab7 are reduced, whereas the level of total rab7 in 22LN2a cells is comparable to that in non-infected N2a cells. Curing the cells from prion infection with two different anti-prion drugs can restore the amounts of membrane-bound rab7 to the levels found in non-infected CAD5 or N2a cells. None of the drugs exerted effects on the levels of any of the tested membrane-bound rab proteins in non-infected cells. This indicates that the decreased levels of membrane-bound rab7 are induced by prion infection. The reduction of membrane-bound rab7 is not due to an increased sensitivity to rabGDI extraction.Sc formationSc accumulationA number of valuable studies has been performed that aimed to identify the subcellular compartment of prion conversion171844In vivo studies demonstrated defects in rab7-mediated retrograde axonal transport in motor neurons of prion infected micein vivo studySc signals which was ascribed to an inhibition of early-to-late endosome maturationIn contrast, the aim of our current study was to investigate prion-host cell interactions, specifically how prion accumulation affects subcellular vesicle trafficking. Proteomics studies have revealed increased expression levels of RabGDI alpha upon expression of mutant PrP resulting in reduced membrane association of rab11Sc can be degraded to a certain extent by lysosomes at a steady state3048Sc degradation, the retention time of PrPSc in late endosomes/multivesicular bodies which are major sites of prion conversionLysosomes are critical for protein degradation in the endocytic pathway, and as a consequence of the described alterations, we observed that the overall lysosomal degradation capacity is impaired upon prion infection. PrPEscherichia coli K1E. coli K1 on lysosomal fusion depends on the presence of long chains of polysialic acidc and PrPSc are sialoglycoproteinsSc aggregation has been linked to neurodegenerationc sialylation also controls PrPSc amplification rate and biochemical propertiesSc has similar effects on lysosomal maturation.Evasion of lysosomal degradation is a survival mechanism that is used by bacteria or viruses, e.g. in vitro and in vivo systems indicates an impact of prion infection on cholesterol metabolism and membrane properties. Cholesterol synthesis is increased both in cultured neurons32333738What might interfere with rab7 recruitment to membranes in prion infected cells? A huge body of evidence provided by our group and others both in Sc clearance. This can contribute to sustaining persistent prion replication. In addition, continuous blocking of endo-lysosomal vesicle trafficking can result in damage of neurons. Our finding that prions can modulate pathways that are non-beneficial for their propagation expands the range of information that can be encoded by protein conformation. Based on our data, we suggest modulation of vesicle trafficking as a target for treatment of prion diseases.In summary, our study sheds new light into prion-host cell interactions, and points at a prion-induced mechanism that prevents PrPProteinase K and Pefabloc proteinase inhibitor were obtained from VWR. Immunoblotting was done using the enhanced chemiluminescence blotting technique (ECL plus) from Pierce. The monoclonal anti-PrP antibody (mAb) 4H11 has been describedThe cell lines N2a  and 22LN2a have been described3260Cell lysis and immunoblot was performed as described previouslyThe procedure has been described previouslyE. coli strain BL21 and purified as described2 1 mM, GDP 1 mM, BSA 0.5 mg/ml, Pefabloc) containing 0 or 3 μM of purified rabGDI for 1 h at 37 °C. Insoluble (membrane bound) proteins were separated by centrifugation  from solubilised rab proteinsRabGDI cloned into pRSET  was expressed in VSV-glycoprotein pseudotyped retroviruses were produced using the pVPack system (Stratagene) and pBABE-EGFR (Addgene) co-transfected into HEK 293FT cells. Forty-eight hours post transfection cell culture supernatants were collected and cell debris was removed by centrifugation . Transduction of N2a and 22LN2a cells was performed as described4Cl (10 mM) was added one hour before and during cycloheximide treatment and EGF stimulation. Equal amounts of protein were analysed by immunoblot for EGFR expression. Signals were quantified using Image Quant TL  or Quantity one software (Biorad) and normalized using β-actin signals. Statistical evaluation of results from 3 independent experiments was done using one-way ANOVA and post-hoc analysis with Tukey’s test (GraphPad Prism software).N2a-EGFR and 22LN2a-EGFR were pre-treated with cycloheximide (25 μg/ml) for 90 min before stimulation with EGF (50 ng/ml). Cells were lysed after cycloheximide treatment (0 min) and 15, 60, 120 and 180 min after EGF addition. To inhibit lysosomal degradation, NH4Cl/glycine (50 mM/20 mM), triton-X 100 , and gelatine  for 10 min each. Primary antibody recognizing lamp-1 (BD Bioscience) and cy-2 conjugated secondary antibody were incubated for one hour each at room temperature. For lysosomal staining, lysotracker red dnd-99 (50 nM) was added for 30 min to the cell culture medium prior to fixation. Images were acquired using a Zeiss LSM710 confocal microscope.Cells were fixed with paraformaldehyde (4%) prior to treatment with NHHow to cite this article: Shim, S. Y. et al. Prion infection impairs lysosomal degradation capacity by interfering with rab7 membrane attachment in neuronal cells. Sci. Rep.6, 21658; doi: 10.1038/srep21658 (2016).


The PDI family form disulfide bridges in substrates via thiol-disulfide exchange reactions. We show in the present study that disulfide exchange can occur directly between individual PDI proteins. Implication is that only certain members need to be oxidized or reduced to maintain function. The formation of disulfides in proteins entering the secretory pathway is catalysed by the protein disulfide isomerase (PDI) family of enzymes. These enzymes catalyse the introduction, reduction and isomerization of disulfides. To function continuously they require an oxidase to reform the disulfide at their active site. To determine how each family member can be recycled to catalyse disulfide exchange, we have studied whether disulfides are transferred between individual PDI family members. We studied disulfide exchange either between purified proteins or by identifying mixed disulfide formation within cells grown in culture. We show that disulfide exchange occurs efficiently and reversibly between specific PDIs. These results have allowed us to define a hierarchy for members of the PDI family, in terms of ability to act as electron acceptors or donors during thiol-disulfide exchange reactions and indicate that there is no kinetic barrier to the exchange of disulfides between several PDI proteins. Such promiscuous disulfide exchange negates the necessity for each enzyme to be oxidized by Ero1 (ER oxidoreductin 1) or reduced by a reductive system. The lack of kinetic separation of the oxidative and reductive pathways in mammalian cells contrasts sharply with the equivalent systems for native disulfide formation within the bacterial periplasm. A family of proteins, collectively termed the protein disulfide isomerases (PDIs), is responsible for formation of correct disulfides in proteins entering the secretory pathway. This protein family is characterized by the presence of one or more domains structurally related to thioredoxin . Most meAs oxidoreductases, PDI proteins typically catalyse thiol-disulfide exchange reactions. Consequently, whether these enzymes function in the formation or breaking of a disulfide depends on the reduction potential of their CXXC active site disulfide, their ability to be recycled efficiently and their functional interaction with their substrate proteins ,6. For e7/8) and VKOR (vitamin K epoxide reductase) [A number of processes that oxidize PDI proteins have been characterized which require enzymes such as Ero1 (ER oxidoreductin 1), PrxIV (peroxiredoxin IV), glutathione peroxidase Gpx/8 and VKductase) . The Eroductase) –13. The ductase) ,15. The ductase) . Althougductase) . Mixed dWhereas substantial progress has been made to understand how PDI proteins are oxidized, no enzyme-catalysed pathway for their reduction has been characterized to date. The necessity to reduce disulfides, both non-native and native, by PDI proteins is crucial during folding of complex disulfide-containing polypeptides, during ER-associated degradation (ERAD) and in the regulation of the unfolded protein response (UPR) –20. The In an attempt to extend our understanding of how individual PDI family members are oxidized or reduced, we have examined the ability of various PDI proteins to exchange disulfides with one another. Using enzyme assays with purified, recombinant PDI proteins, we demonstrate that several PDI proteins can interact and exchange disulfides. In addition, the formation of mixed disulfide complexes in stable cell lines reveals that PDI proteins can react with each other within a cellular context. These results suggest the existence of a disulfide exchange cascade that transfers oxidizing equivalents between individual PDI proteins. Such ready transfer of disulfides between individual PDI proteins contrasts with the situation within the bacterial periplasm where the oxidative and reductive pathways are kinetically separated with little or no cross-talk between the disulfide exchange proteins involved in disulfide formation or reduction .N-ethylmaleimide (NEM) and 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) were purchased from Sigma and Life technologies respectively.The following mouse monoclonal antibodies were used: anti-V5 (Invitrogen), anti-V5-conjugated agarose beads (Sigma–Aldrich), anti-His  and anti-GAPDH . The rabbit polyclonal antisera raised against P5, PDI and ERp57 have been described previously . N-ethylL-glutamine. Stable cell lines expressing the substrate-trap mutants (CXXA) of the PDI oxidoreductases have been described previously [HT1080 cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g/l glucose, 10% FBS and 0.56 mM eviously ,26.The expression and purification of thioredoxin, wild-type (wt) PDI, P5 and ERp57 have been described previously ,27. PDI PDI proteins were reduced with 10 mM DTT for 10 min at room temperature. DTT was removed using a 5 ml of Hitrap desalt column  pre-equilibrated with 50 mM Tris/HCl, pH 7.5, buffer containing 150 mM NaCl and 1 mM EDTA (buffer A), according to the manufacturer's protocol. To oxidize the proteins, samples were incubated with 10 mM GSSG for 10 min at room temperature. GSSG was removed by gel filtration using a Superdex 10/300 GL column  pre-equilibrated with buffer A. The redox status of all PDI proteins was verified by carrying out AMS alkylation and separation by SDS/PAGE.To determine if the PDI protein can exchange disulfides, equimolar amounts  of reduced and oxidized recombinant PDI oxidoreductases were incubated in buffer A with samples taken at specific time points. The reactions were stopped by boiling in SDS sample buffer  supplemented with 10 mM AMS to freeze the redox states. Finally samples were analysed under non-reducing SDS/PAGE followed by Coomassie staining or Western blotting to visualize the redox states of the individual PDI proteins. Quantification of band intensities was carried out using ImageJ software.Confluent HT1080 cells and HT1080 cells stably overexpressing substrate-trapping PDI oxidoreductases were rinsed twice with PBS supplemented with 20 mM NEM. Cells were lysed in buffer containing 50 mM Tris/HCl, pH 7.4, 1% (v/v) Triton X-100, 150 mM NaCl, 2 mM EDTA and 0.5 mM PMSF (lysis buffer) supplemented with protease inhibitor cocktail (Roche). Immunoisolation and SDS/PAGE were performed as described above. For Western blotting, proteins were transferred to nitrocellulose membranes (LI-COR Biosciences), which were blocked in either 5% (w/v) dried milk or 3% BSA (for ERp57 blots) in 10 mM Tris/HCl buffer, pH 7.5, containing 150 mM NaCl and 0.1% (v/v) Tween 20 for 1 h. Blots were incubated with primary antibody for 1 h at room temperature. LI-COR IRDye fluorescent secondary antibodies were used for detection at a 1:10000 dilution. Blots were scanned using an Odyssey Sa Imaging System (LI-COR Biosciences).in cellulo redox state of thioredoxin and ERdj5 with AMS were determined as described previously [Hela cells stably expressing doxycycline inducible human thioredoxin, engineered with a V5-tag and KDEL ER-retrieval sequence at the C-terminus, were generated using the Flp-In T-Rex Core Kit (Invitrogen). The HT1080 cell line stably overexpressing wt ERdj5 has been described previously . The in eviously .N2) initiated by the attack on a disulfide bond by a nucleophilic thiolate anion. The reaction proceeds via a transient mixed-disulfide intermediate which becomes resolved, resulting in the reduced reactant becoming oxidized and the oxidized reactant becoming reduced .Disulfide bond formation catalysed by the PDI proteins typically involves a bimolecular nucleophilic substitution reaction  were incubated with reduced ERp57 and ERp72 . We inclThe ability of other PDI proteins to exchange disulfides with ERp57 and ERp72 was determined and the time courses of oxidation quantified C and 3D.Having established that various oxidized PDIs can exchange disulfides with reduced forms of ERp57 and ERp72, we next examined the possibility of electron transfer between reduced PDI proteins and oxidized ERp72. An equivalent experiment with ERp57 could not be performed due to the insolubility of the oxidized protein. Stoichiometric amounts of reduced PDIs including ERp46, ERp18, P5, ERp57, PDI and PDI ΔS1S2 were incubated with oxidized ERp72 . A repreWe have shown that various ER-localized PDIs, with similar active site chemistries and redox potentials can engage in disulfide exchange with varying efficiencies resulting in mixtures of partially reduced and oxidized proteins. In particular, our results show that PDI and ERp72 can engage in reversible disulfide exchange; it did not matter which protein was reduced or oxidized, disulfide exchange was still relatively efficient. In order to examine to what extent the direction of disulfide exchange can be driven by the reduction potential of the active-site disulfide, we assayed the ability of thioredoxin, a cytosolic protein with a comparatively low redox potential (−270 mV)  to exchaThe ability of thioredoxin to efficiently reduce the PDI proteins provided us with the opportunity to evaluate how efficient the reductive pathway is within the ER at recycling oxidized thioredoxin domains with low reduction potentials. Within the bacterial periplasm, DsbC is maintained primarily in the reduced state  by a robThiol-disulfide exchange reactions typically involve the formation of a transient mixed-disulfide intermediate between enzyme and substrate, which is resolved to yield the final reaction products A. Previoin vitro, we used the substrate-trap approach to investigate if members of the PDI family can form mixed-disulfides complexes with each other in cells grown in culture. HT1080 stable cell lines overexpressing V5-tagged CXXA mutants of PDI proteins were lysed in buffer containing NEM to trap any mixed disulfides. The expression of each PDI protein was verified by carrying out a V5-Western blot (To extend these studies, and in light of our observations that various PDIs can exchange disulfides ern blot A. When tTo determine the presence of other PDI proteins in the mixed-disulfide complexes, we immunoisolated the exogenously expressed PDI protein and separated the immunoisolated material on reducing and non-reducing SDS/PAGE. Western blot analyses were then carried out using antisera raised against specific PDI proteins, including P5, PDI and ERp57 (in vitro assays (Western blots were also carried out to determine interactions between the various PDI proteins and endogenous PDI E. The reo assays A and 3B.Western blotting of immunoisolated material using anti-ERp57 also revealed most prominently an interaction with ERdj5 F. The noIn the present study, we demonstrate the ability of various purified PDI proteins to exchange disulfides with each other and show that similar exchange reactions can occur within mammalian cells. It would appear that PDI and ERp46 are most likely to be the conduits between Ero1 and the ER disulfide exchange proteins and that ERdj5 is most likely to provide the conduit between the ER disulfide exchange proteins and any putative reduction pathway. Hence, the oxidative and reductive pathways in the ER lumen need only interface with a restricted number of PDI proteins to allow passage of electrons between most of the other members of the family .The fact that exchange reactions can occur relatively efficiently raises the question of how the opposing pathways of disulfide formation and reduction are regulated within the mammalian secretory pathway. Within the bacterial periplasm, the two pathways are kinetically segregated so that although the active site reduction potentials of the disulfide exchange proteins DsbA and DsbC are similar ,35, DsbAThe fact that ERdj5 and ER-localized thioredoxin are mostly oxidized would also suggest that the ER reductive pathway is inefficient or tightly regulated to prevent excessive reduction in the PDI proteins and to favour disulfide formation. Such a reductive pathway may only be activated upon hyperoxidizing conditions within the ER lumen such as occurs during the UPR  or durinWhereas most of the PDI proteins tested were able to exchange disulfides, there was some selectivity both with purified proteins and with the substrate-trapping mutants expressed in cells. In particular, reduced ERp72 was unable to transfer electrons to oxidized ERp18, whereas oxidized ERp72 can be reduced by ERp18. If the reaction was solely determined by the active site chemistries then the reaction should proceed in both directions until equilibrium is reached. As their calculated redox potentials are quite similar ,39, therin cellulo contrasts with their ability to exchange disulfides in vitro, for example between PDI and ERp57 or ERp72. Demonstration of disulfide exchange with purified proteins shows that this reaction can occur efficiently but it does not mean that this will occur in cells where the individual components may be in complex with other proteins precluding their interaction. For example, ERp57 is in complex with calnexin, calreticulin and also ERp27 [The apparent absence of mixed disulfides formation between some PDI proteins tested In summary our results raise some intriguing questions about how the balance of oxidation and reduction in disulfide bonds is regulated in the ER lumen and suggest the absence of a clear kinetic segregation of the pathways. Given the promiscuity of disulfide exchange, further work needs to be carried out to identify the mechanism for reduction in PDI proteins such as ERdj5 and to determine how this pathway is regulated to prevent excessive reduction in disulfides to favour disulfide formation.


Individual implants from both homozygous and heterozygous TER null outgrowths showed growth senescence beginning at transplant generation two, earlier than implants from TER+/+ mammary glands which continued to show growth. This result suggests that either mammary epithelial stem cells maintain their telomere length in order to self renew, or that the absence or reduction of telomerase template results in more frequent death/extinction of stem cells during symmetric divisions. A third possibility is the inability of signaling cells in the niche to replicate resulting in reduction of the maintenance signals necessary for stem cell renewal. Consistent with this, examination of senescent outgrowths revealed the absence of estrogen receptor alpha (ERα+) epithelium although progesterone receptor (PR+) cells were abundant. Despite their inability to establish mammary growth in vivo, TER+/− cells were able to direct neural stem cells to mammary cell fates.It has been proposed that the erosion of telomere length is a limiting factor in replicative capacity and important in cell senescence. To determine if this activity was essential in the mouse mammary gland  The action of mammary stem cells and their mitotic progeny is fundamental to normal mammary growth, differentiation, maintenance and regeneration in successive cycles of pregnancy, lactation and involution. An important feature of the mammary gland is the regenerative capacity of its epithelium, which is demonstrated upon successive reproductive cycles. Any portion of the mammary epithelial tree can reproduce the entire functional mammary epithelium upon transplantation into the mammary fat pad -5. Norma−/− females with those from TER−/− generation 5.The appearance of growth senescence can be linked to the disruption of the mammary stem cell niche, as not all random fragments chosen reached senescence during the regeneration of the mammary epithelium in any given transplant generation. In cases where fragments continued to grow, we propose that competent stem cell niches must be present. Additionally, we concluded that growth senescence was a direct result of the aging of local mammary stem cells or their niche as they lose their ability to self-renew . The queDuring cellular aging, the repetitive sequences at chromosome ends called telomeres protect intact chromosomes from degradation during successive rounds of cell division. In humans telomeres comprise 10-15 kb and in mice more, 25-50 kb of the repetitive TTAGGG sequences , 10. The+/− and TER−/− transgenic mice show reduction of telomere length upon successive generations [+/−) and both first (TER−/−g1) and fifth generations (TER−/−g5) that have shortened telomeres due to a loss of telomerase activity were transplanted serially in the cleared fat pads of wild type female hosts. As controls, serially transplanted wild type tissues that became growth senescent were analyzed identically to the TER+/− and TER−/− growth senescent implants. To evaluate the capacity of in vivo, growth senescent mammary epithelial populations to redirect non-vmammary cells to mammary epithelial cell fates, TER+/− and WAP-TGF-beta1 mammary cell populations were mixed with reporter-marked neural stem cells (NSC). The WAP-TGF-beta1 [in vivo and their growth senescent ducts were shown to be deficient (lacking) in ER-α+ epithelial cells in the present work.We hypothesized that regenerative capacity of the mammary gland would be influenced by the presence or absence of telomerase activity since expansion of the stem cell population must occur during branching ductal development in outgrowths. To answer this we utilized a telomerase deficient mouse model in which one or both alleles for the RNA template for the telomerase enzyme have been removed to model a prematurely aged population, to compare growth rate and senescence to that of normal mammary epithelium. Both TERerations . TelomerGF-beta1 , 18 mamm+/+), heterozygous (TER+/−), and telomerase knockout (TER−/−) mice were implanted into the cleared inguinal fat pad of three-week old nu/nu female mice and allowed to grow for at least 12 weeks. This initial transplantation yielded a majority of positive takes in all three groups, 20/20, 9/10, and 8/10 respectively. The outgrowths in the wild type and heterozygous groups filled 90-100% of the fat pads and the outgrowths from the knockout group filled similarly albeit two of the transplants grew between 60-80% filling. Fragments were taken from the center of each outgrowth and serially transplanted into new cleared fat pads of three week old mice and then repeated consecutively, thus termed transplant generations two (T gen 2), three (T gen 3), and four (T gen 4). Serially transplanted fragments of wild type epithelium consistently led to positive outgrowths that filled 90-100% of the recipient fat pads (Table +/− or TER−/−) led to a decrease in positive takes and fat pad filling. Effects could be seen by transplant generation three and maintained through T gen 4 with only 15% of fat pad filling occurring . TERT expression is low in most cell types, leading to a progressive shortening of telomere length and subsequent senescence of these cells. To determine whether transplantation of other cells with a senescent phenotype had a similar growth curve as TER null cells, we injected either one hundred thousand TERhs Table .+/− or one hundred thousand WAP-TGF-β1 mouse mammary cells with fifty thousand NSCs and injected them into the cleared fat pads of recipient mice. Interestingly initial outgrowths of these chimeras occurred in six of ten glands by both cell types . Briefly, we mixed fifty thousand TERes Table . Presences Table .+/− mammary cells with wild type NSCs resulted in NSC/mammary epithelial hybrid outgrowths, neither the same number of mammary cells from these two populations nor fragment implants from the chimeric outgrowths were able to generate secondary outgrowths when placed into the cleared mammary fat pads of immune-compromised hosts  as compared to wild type controls  specifically, only form rudimentary mammary ducts prior to the onset of puberty . The samal cells . To detes Figure .in vivo growth capacity of mammary fragment implants compared to wild type. Indeed, mammary fragments from tissue lacking telomerase activity and shortened telomeres showed early growth senescence compared to wild type. These results support the conclusions drawn by Young and Daniel [+/+ implants rarely if ever showed growth senescence in four transplantation generations while TER+/− and TER−/− transplant fragments showed early growth senescence despite implant fragments being taken from the outgrowth center of the previous generation.Our original premise was to test whether the absence of telomerase activity hastened the loss of the d Daniel  which de−/− senescent outgrowths repeat these findings. Our attempts demonstrated that no growth could subsequently occur in epithelium-free mammary fat pads implanted with TER−/− or TER+/− fragments from senescent outgrowths and thus no alveolar development was observed. It was reported that no phenotype was observed in the renewing tissues, such as the intestinal, germinal or hematopoietic tissues, in mice null for the telomerase RNA template in early generations, and defects only emerged after four breeding generations [th generation TER−/− females did not show more rapid attainment of growth senescence  required for in vivo growth, yet fail to maintain this property in subsequent transplant generations. Examination of the positive outgrowths obtained when mixing the neural stem cells with TER+/− and WAP-TGFbeta1 mammary cells indicate that no ER-alpha positive cells are present. This could be an explanation for the failure of secondary outgrowths from transplants of these tissues. Nevertheless, the non-mammary cells were redirected to mammary epithelial cell fates and in combination with the growth-deficient mammary cells produced robust primary outgrowths in post-partum females.It is somewhat more difficult to explain why or how mixing non-mammary cells with mammary cells incapable of −/− females. This suggests that cell division as demonstrated by Daniel and Young in 1971 [We conclude that the increase in reaching growth senescence in the mammary epithelial transplants is the result of telomere shortening, this must occur during the development of the outgrowth from the implanted fragment. No decrease in the rate of positive takes or the amount of implanted fat pad filled in the early passages is observed even in implants from generation #5 TER in 1971  is param+) are required for continued in vivo proliferation in post-pubertal female hosts. Growth senescent outgrowths  contain PR+ cell and can respond to pregnancy by producing lobulo-alveolar structures that express milk proteins  and have been previously described [+ mice were engineered as previously described [Female Athymic NCr Nu/Nu mice were used for transplantation studies. TER wild type, TERescribed . WAP/TGFescribed . All micThe surgical procedures for clearing the mammary epithelium from the #4 inguinal fat pads of 3 week-old female mice and the method of implanting either tissue fragments or cell suspensions have been described in detail in earlier publications -26. GeneRandom fragments (∼1.0 mm3) of mammary epithelium were taken from virgin female glands. The fragments were implanted as described above and the hosts were bred after 3 weeks. The implanted glands as well as host glands were taken at 8 weeks post-op. Fragments were obtained from the outgrowths to be used in subsequent transplant generations. Eight to ten fragments were selected at random from the glands. This procedure was repeated for 5 transplant generations or until growth senescence was achieved. Alternatively cell suspensions were injected in a ratio of 100,000 cells/10μl volume of non-supplemented Dulbecco's Modified Eagle's Medium (DMEM) or sterile phosphate-buffered saline, pH 7.2 with a Hamilton syringe equipped with a 30 gauge needle.Mammary glands were harvested and dissociated with 0.1% collagenase (Sigma) overnight at 37°C in complete tissue culture medium with 10% fetal calf serum. The following day samples were triturated with a 10 mL sterile pipette and passed through a 19-gauge needle. Resulting organoids were cultured in plastic flasks in DMEM supplemented with 10% fetal bovine serum, insulin (1.0 μg/ml), and epidermal growth factor (10 ng/ml). Fibroblasts were removed by differential trypsinization after 72 hours and mammary epithelial cells collected 24 hours later.NSC isolation was performed as previously described . IsolatiFetal cultures are normally passaged approximately every 5 days. After the first passage (which removes most of the remaining contaminants), the cultures are composed of 95% NSCs. Clonal and real-time lineage analyses confirm their self-renewal properties and multipotential. Adult cultures are composed of both multipotent , 29 stemFetal cultures are derived from E13.5 mouse embryo cortex, whereas adult cultures are derived from the sub ventricular zone of 2- to 3-month-old mice. After dissection, the culture conditions are identical for these two stem cell sources. Cells were then maintained in culture as previously described .+ cells were fixed and stained as previously described [2 in phosphate-buffered saline (1X PBS) overnight at 4°C and processed for X-gal as previously described [Briefly, the number #4 inguinal fat pads were excised from the transplant-bearing mice and spread onto glass slides. The glands were spread to expose as much area to the glass and to flatten the sample to improve viewing. The glands were then fixed in Carnoy's fixative  for 4 h at room temperature. They were then stained with carmine alum, dehydrated through a series of alcohols, cleared in xylene, and sealed with Permount and a glass coverslip. Whole mounts of inguinal glands containing LacZescribed , 30. Briescribed . For X-gFor histological examination, X-gal + glands were embedded in paraffin and cut in 5 μm sections and mounted on positively charged slides. Sections were subsequently cleared in xylenes and rehydrated through ethanol gradients. Antigen retrieval was performed by heating slides in a boiling water bath for 20 min. in either 10mM citrate buffer pH 6.0  or Tris-EDTA pH 9.0 (Dako). Endogenous peroxidase activity was blocked by using 3% hydrogen peroxide for 15 minutes at room temperature. Slides were blocked with normal horse serum  for 1 hour at room temperature and then incubated overnight with primary antibodies at 4°C. Primary antibodies used were rabbit anti-PR , rabbit anti-ERα sc#-542 . Secondary antibody staining was performed using the R.T.U. Vectastain (goat anti-rabbit/mouse) kit (Vector Laboratories). Staining was visualized using the DAB peroxidase substrate kit (Vector Laboratories) per manufacturer's recommendations. Slides were counterstained with Mayer's hematoxylin (Sigma-Aldrich) and negative tissue controls were included in all immunohistochemical analyses.


Particularly, the chitosan/copper/zinc hydrogel exhibited significantly higher antimicrobial effect against L. fermentum, one of the primary targets of antibiotic growth promoters. Additionally, the chitosan matrix ameliorated the cytotoxicity levels of metal supplements when screened against a murine macrophage cell line RAW 264.7 and in TE-71, a murine thymic epithelial cell line. In this proof-of-concept study, we show that by using chitosan as a delivery platform, micronutrient-based metal feed additives could be used to minimize the undesirable levels of microbial population without causing significant cytotoxic effect under in vitro conditions. These findings provide the platform for further studies in target animal models to quantify the required physiological concentrations of copper and zinc when delivered via a chitosan hydrogel platform to elicit a growth promoting effect without causing any toxicity.Large-scale use of antibiotics in food animal farms as growth promoters is considered as one of the driving factors behind increasing incidence of microbial resistance. Several alternatives are under investigation to reduce the amount of total antibiotics used in order to avoid any potential transmission of drug resistant microbes to humans through food chain. Copper sulfate and zinc oxide salts are used as feed supplement as they exhibit antimicrobial properties in addition to being micronutrients. However, higher dosage of copper and zinc (often needed for growth promoting effect) to animals is not advisable because of potential environmental toxicity arising from excreta. Innovative strategies are needed to utilize the complete potential of trace minerals as growth promoting feed supplements. To this end, we describe here the development and preliminary characterization of hydrothermally treated chitosan as a delivery vehicle for copper and zinc nanoparticles that could act as a micronutrient-based antimicrobial feed supplement. Material characterization studies showed that hydrothermal treatment makes a chitosan hydrogel that rearranged to capture the copper and zinc metal particles. Systemic antimicrobial assays showed that this chitosan biopolymer matrix embedded with copper (57.6  The World Health Organization recently cautioned that antimicrobial resistance amongst infectious agents has reached alarming levels and that it could turn the twenty-first century into a time where common wound injuries can become lethal infections . Over us4) and zinc in the form of zinc oxide (ZnO) have shown to produce a growth promoting effect via reduction of fermentation loss of energy by altering the gut microbiome  recommends them as minor feed supplements in cattle and swine production . Howevercrobiome . Higher crobiome . Despitecrobiome  would prcrobiome . Furtherd-glucosamine molecules and has been investigated extensively for its potential as an animal feed additive in poultry, swine  treatment of chitosan results in depolymerization of chitosan into shorter chain polymers with increased functional groups availability for loading high amounts of antimicrobial copper and similar metals . FurtherOne pot HT method of making water-dispersible chitosan–metal composite hydrogel was performed as described earlier . BrieflyA Zeiss ULTRA-55 FEG SEM was used to study the gross morphological characteristics of the chitosan–metal complex. The liquid samples were spin coated (2000 rpm) on boron-doped silicon wafers  and the images were acquired at 5 kV.Carbon-filmed gold grids (400 square mesh)  were used for preparing samples where the materials were dip coated and air dried at room temperature before they were analyzed in a FEI Tecnai F30 microscope at the Material Characterization Facility (MCF) at the University of Central Florida. An electron beam intensity of 100 kV was used for studying all the materials.E. coli (ATCC 8739) and Staphylococcus aureus (ATCC 25923) were grown in tryptic soy media (Sigma-Aldrich). Enterococcus faecalis (ATCC 29212) was grown in brain heart infusion (BHI) media (BD Biosciences). Lactobacillus fermentum (ATCC 9338) was grown in MRS medium (BD Biosciences). All incubations were done at 37°C.The growth inhibition effect of the chitosan–metal composites was tested against a range of microorganisms that acted as model organisms for gut microflora of livestock gastrointestinal tract. μl in the final volume of 200 μl in each well. As per clinical and laboratory standards institute (CLSI) requirements, for all the bacterial species, a culture concentration of 0.5 McFarland standards was used as a starter culture and from which 1 × 105 CFU/well  was added. A classical antibiotic kanamycin (50 μg/ml) was used as positive control for killing. After 24 h of incubation at 37°C, 10 μl of alamar blue dye  was added to each well. The plate was then kept back in the incubator for one more hour before the absorbance was measured at both 570 and 600 nm for each well. The reduction of the dye in percentage  value was calculated by using the formula as suggested by the manufacturer. The motivation behind choosing these specific concentrations of metals (copper and zinc) in these assays was to ascertain the difference in antimicrobial activity against gut microbes between the traditional method (no chitosan) and using chitosan hydrogel matrix as a delivery system.Initial screening for determining the most effective metallic concentration was performed using a microplate alamar blue assay (MABA) as described elsewhere . The MABThe absolute values of the reduction in bacterial numbers after treatment with various concentrations of chitosan–metal composite were determined using bacterial killing assay (CFU assay). The protocol used in the MABA assay was followed for this assay for treatment of bacteria with samples but at the end of 24 h incubation, serial dilutions of the bacteria from each well were made in phosphate-buffered saline and plated on corresponding agar plates. The colonies were counted after overnight incubation and expressed in logarithmic scale.μg/ml of metallic zinc concentration) of the hydrogel. These numbers were selected to identify the specific concentration where there is a difference in cytotoxicity levels between the samples that contained chitosan and those that did not.  After 24 h of incubation at 37°C in the presence of 5% CO2, the media containing hydrogel were removed and fresh media [DMEM  complete media containing 10% fetal calf serum and 1% antibiotic and antimycotic] were added to avoid any interference from the material during measurement of fluorescence. Then, to each well in the 96-well flat bottom plates, 20 μl of alamar blue reagent was added. After 3 h of incubation with the alamar blue reagent, the contents of the plates were transferred to black 96-well plate  and were read for fluorescence (590 nm) in a TECAN infinite M200PRO plate reader. Triplicate wells were used for each sample and the wells containing only the macrophages and media (no samples added) were used as growth control. The percentage viability of cells in the chitosan–metal samples treated wells were deduced by normalizing the viable cells percentage in the growth control wells.To determine any potential cytotoxicity effects of the newly synthesized chitosan–metal hydrogel, RAW 264.7 murine macrophage and TE-71 mouse thymic epithelial cell lines were treated with various concentrations  image of the chitosan–metal hydrogel showed that the copper and zinc nanoparticles (of size <5 nm) were uniformly distributed throughout the HT chitosan matrix background Figures D,E. Addid” spacing in the SAED pattern , copper chloride, copper zinc alloy, metallic zinc, zinc oxide, zinc nitrate, and zinc chloride  on the high-resolution transmission electron microscopy (HRTEM) images, the lattice spacing of the metal nanocrystallites Figure  were anaL. fermentum .In both MABA and CFU assay, the chitosan hydrogel loaded with copper and zinc salts showed an enhanced killing effect (>99.99%) on  Figures  and 4 atE. coli, S. aureus, and E. faecalis as representative organisms of zoonotic and opportunistic pathogens. At 800 μg/ml of metallic zinc , while there was a 1.24 log reduction of E. coli growth (94.95% reduction of bacteria) by HTCh-RTCuZn. At 800 μg/ml, the metallic zinc containing chitosan–metal hydrogel reduced the load of E. faecalis only by 0.61 logs (75.55% reduction), whereas RTCuZn (no chitosan) reduced the load by 90.91%. Also, the salt mixture completely inhibited (>99.99%) growth of S. aureus even at 300 μg/ml concentrations. Although not significantly, the growth inhibitory effect of salt mixture was lower in E. coli (91.58% reduction) at 800 μg/ml of metallic zinc when compared to HTCh-RTCuZn (94.95%).For this study, we used  Figures  and 4, tμg/ml , the cytotoxicity levels were not significantly different between the samples. In TE-71 epithelial cells, the metals were more toxic and started killing cells at metallic zinc concentrations as low as 25 μg/ml, whereas at the same concentration (25 μg/ml), the chitosan-containing samples were not toxic to the cells  and the murine thymic epithelial cell line TE-71  were used to screen for any cytotoxic effect caused by chitosan–metal hydrogel. In case of RAW264.7 cells, only about 5% cells treated with HT chitosan–metal hydrogel were killed, whereas cells treated with just the mixture of copper and zinc salts (with no chitosan) exhibited significant reduction in viable cell numbers (92% of cells killed) at a metallic zinc concentration of 50 Commercially feasible potential alternatives are in greater need to reduce the total amount of classical antibiotics that are used as AGPs in food animal production facilities , 20. MetIn our previously published studies, we showed that HT treatment generates water-dispersible chitosan, a property that would allow for uniform mixing of chitosan with the animal feed in industrial scale livestock farms . The othLactobacillus species of bacteria have been indicated as one of the primary targets of AGPs because they produce bile salt hydrolase, which has shown to have a negative impact on host animal digestion and absorption of nutrients  did not significantly inhibit the bacterial growth even at the same metallic zinc concentrations. The antimicrobial part of this study used both a metabolic activity based assay  and viability count (CFU) assay to cross-verify the effect on the microbes, since metabolic activity may not always correlate with the total number of viable bacteria. For example, in case of E. coli treated with HTCh-RTCuZn 800 sample, the dye reduction was statistically significant  than that of our previous findings  were killed at higher concentrations  while Gram-positive organisms were killed at lower concentrations  suggesting a cell wall targeting activity of our chitosan–metal hydrogel  considered in antimicrobial studies were used, all the samples (with and without chitosan) exhibited significant cytotoxicity in a 96-well plate (data not shown). The cytotoxicity exhibited at those concentrations was understandable considering the low number of macrophages/epithelial cells that were exposed per microgram of metal in a smaller surface area/volume. HT-chitosan alone did not exhibit significant cytotoxicity (at least until 28.8 μg/ml). The toxicity to cell lines was primarily caused by the zinc concentration as the material containing HTCh-RTCu  exhibited reduced toxicity (concentrations adjusted to reflect the copper concentration in the corresponding HTCh-RTCuZn samples) when compared to cells treated with HTCh-RTZn (at 50 μg/ml) (Figure The HTCh-RTCuZn significantly reduced the cytotoxic effects of the metallic salts on two different types of eukaryotic cells Figure . In both-RTZn at 0 μg/ml (PR conceived and designed the study, collected data, performed critical analysis and interpretation of data, and wrote the manuscript. Also accepts to be accountable for all aspects of this work. SS helped in conception and design of the study, critical analysis, interpretation of data, and editing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Culex spp. mosquitoes. In North America (NA), lineage 1 WNV caused the largest outbreak of neuroinvasive disease to date, while a novel pathogenic lineage 2 strain circulates in southern Europe. To estimate WNV lineage 2 epidemic potential it is paramount to know if mosquitoes from currently WNV-free areas can support further spread of this epidemic.West Nile virus (WNV) is a highly pathogenic flavivirus transmitted by Culex pipiens mosquitoes originating from north-western Europe (NWE) in direct comparison with those from NA. We exposed mosquitoes to infectious blood meals of lineage 1 or 2 WNV and determined the infection and transmission rates. We explored reasons for vector competence differences by comparing intrathoracic injection versus blood meal infection, and we investigated the influence of temperature. We found that NWE mosquitoes are highly competent for both WNV lineages, with transmission rates up to 25%. Compared to NA mosquitoes, transmission rates for lineage 2 WNV were significantly elevated in NWE mosquitoes due to better virus dissemination from the midgut and a shorter extrinsic incubation time. WNV infection rates further increased with temperature increase.We assessed WNV vector competence of Our study provides experimental evidence to indicate markedly different risk levels between both continents for lineage 2 WNV transmission and suggests a degree of genotype-genotype specificity in the interaction between virus and vector. Our experiments with varying temperatures explain the current localized WNV activity in southern Europe, yet imply further epidemic spread throughout NWE during periods with favourable climatic conditions. This emphasizes the need for intensified surveillance of virus activity in current WNV disease-free regions and warrants increased awareness in clinics throughout Europe. Culex pipiens for lineage 2 WNV in a systematic, direct comparison with North American Culex pipiens and with the lineage 1 WNV strain. We demonstrate that European mosquitoes are highly competent for both WNV lineages, which underscores the epidemic potential of WNV in Europe. However, the transmission rate for lineage 2 WNV was significantly lower in North American mosquitoes, which indicates different risk levels between both continents for lineage 2 but not lineage 1 WNV. Based on our result, we propose that WNV surveillance in mosquitoes and birds must be intensified in Europe to allow early detection, timely intervention strategies and prevent outbreaks of WNV neurological disease.West Nile virus (WNV) is on the rise in Europe, with increasing numbers of human cases of neurological disease and death since 2010. However, it is currently unknown whether or not WNV will continue to spread to north-western Europe (NWE), in a fashion similar to the WNV epidemic sweep in the United States (1999–2004). The presence of competent mosquitoes is a strict requirement for WNV transmission, but no laboratory studies have been conducted with the new European lineage 2 WNV outbreak strain. Our study is the first to investigate transmissibility in NWE  Flaviviridae, genus Flavivirus) is an important mosquito-borne human pathogen associated with febrile illness, which may develop into severe neuroinvasive disease and death [West Nile virus  [Culex species found positive during WNV surveys is the common house mosquito Culex pipiens [Culex pipiens, Culex tarsalis and Culex quinquefasciatus [Culex pipiens mosquitoes are competent vectors for NA isolates of lineage 1 WNV [During enzootic transmission, WNV circulates primarily between mosquitoes of the ica (NA)  and Euroica (NA) ,15 are sica (NA) . In Euro pipiens . In NA tasciatus ,19. Laboge 1 WNV . The vecge 1 WNV . The vecAs the global activity of these pathogenic WNV lineages has significantly increased over the past two decades, we set out to assess the potential for virus transmission in areas that are currently free of lineage 1 and/or lineage 2 WNV strains. The results show that European mosquitoes from an area free of WNV disease have the intrinsic capability to transmit both lineage 1 and lineage 2 WNV. However, comparing transmission rates at varying temperatures provides evidence that the differences in climatic conditions currently restrict the spread of WNV throughout Europe.Culex tarsalis cells  were grown on Leibovitz L15 and Schneiders (Gibco) medium supplemented with 10% fetal bovine serum . Hela (ATCC CCL-2), DF-1 (ATCC CRL-12203) and Vero E6 (ATCC CRL-1586) cells were cultured with DMEM Hepes  buffered medium supplemented with 10% FBS containing penicillin (100IU/ml) and streptomycin (100μg/ml). When Vero E6 cells were incubated with mosquito lysates or saliva the growth medium was supplemented with fungizone  and gentamycin (50μg/ml). P2 virus stocks of the NY’99 and Gr’10 isolates were grown on C6/36 cells and titrated on Vero E6 cells.C6/36 (ATCC CRL-1660) and Culex pipiens colony originated from Brummen, The Netherlands (°05'23.2"N 6°09'20.1"E) and was established in 2010 and maintained at 23°C. The NA Culex pipiens colony [The NWE 8 WNV infectious particles per ml. Mosquitoes were allowed to membrane feed, using the Hemotek system and a parafilm membrane, in a dark climate controlled room . After 1 hour, mosquitoes were sedated with 100% CO2 and the fully engorged females were selected. Injections: Mosquitoes were sedated with CO2 and placed on a semi-permeable pad, attached to 100% CO2. Mosquitoes were infected by intrathoracic injection using the Drummond nanoject 2 . Infected mosquitoes were incubated at their respective temperatures with a 16:8 L:D cycle and fed with 6% sugar water during the course of the experiment.2–5 day old mosquitoes were infected either via ingestion of an infectious blood meal or via intrathoracic injections. Infectious blood meals: Whole chicken blood was mixed with the respective P2 virus stock to a final concentration of 1.4*10Legs and wings of sedated mosquitoes were removed and their proboscis was inserted into a 200ul filter tip containing 5 ul of salivation medium ). Mosquitoes were allowed to salivate for 45 minutes. Mosquito bodies were frozen in individual Eppendorf tubes containing 0.5 mm zirconium beads  at -80°C. The mixture containing the saliva was added to 55 ul of fully supplemented growth medium.Frozen mosquito bodies were homogenized in the bullet blender storm  in 100 μl of fully supplemented medium and centrifuged for 90 s at 14000 rpm in a table top centrifuge. 30ul of the supernatant from the mosquito homogenate or the saliva containing mixture was incubated on a monolayer of Vero cells in a 96-wells plate. After 2–4 hours the medium was replaced by 100 μl of fresh fully supplemented medium. Wells were scored for WNV-specific cytopathic effects (CPE), confirmed with immunofluorescence assay (IFA) against WNV E  at threeMaps displaying the mean diurnal temperature during July and August of the indicated year . Human cWNV infections in mosquito bodies and saliva were scored positive or negative and significant differences were calculated using the Fisher’s exact test (P<0.05). Differences in WNV titres (TCID50/ml) in infected mosquito bodies and heads were calculated using the Mann Whitney test (P<0.05).Culex pipiens mosquitoes from NWE (The Netherlands), and a NA Culex pipiens colony [8 TCID50/ml of either WNV-lin2 or WNV-lin1 isolates were fed to the NWE and NA Culex pipiens mosquitoes. Fully engorged females were selected and kept at an ambient temperature of 23°C. Immediately after completion of the blood meal, a subset of fully engorged females was tested for the presence of infectious WNV to confirm that both mosquito populations had ingested equal amounts of infectious virus particles  have a markedly different vector competence for WNV-lin2, suggests a degree of genotype-genotype specificity in the interaction between virus and vector. Alternatively, the presence of certain endosymbionts or insect-specific flaviviruses can have an effect on the vector competence as well [Here we show that both pathogenic lineages of WNV can effectively infect mosquitoes from NWE. Our finding that two geographically separated  as well ,31. As tThe presence of highly competent vector species in WNV disease-free areas suggests that extrinsic factors such as temperature play an essential role in the current distribution of WNV. In NWE, the lower average summer temperature (<20°C) may provide a possible explanation for the current WNV epidemics, which remain restricted to southern Europe.Culex pipiens forms may increase the incidence of human WNV disease, as these ‘bridge-vectors’ are considered less ornithophilic and more likely to feed on other vertebrates, including humans. These hybrids are relatively common in North America, but not in north-western Europe [Culex pipiens populations are in transmitting WNV is currently unknown, but this can be investigated by using wild caught Culex pipiens mosquitoes from a variety of sources and regions.However, other extrinsic factors can shape the vectorial capacity and may compensate for a reduced vector competence at low temperature by facilitating larger mosquito populations. The recent resurgence of WNV disease in the United States was most likely fuelled by climatic conditions that were favourable for local vector populations ,33. In an Europe . How effAdditionally, viral adaptations that increase the replication efficiency at lower temperatures could further facilitate and enhance transmission of WNV throughout Europe. Indeed, the WNV-lin1 isolate has already proven to be able to adapt to the different climatic conditions in the Americas , while oCulex pipiens. Based on our results and experimental evidence by others that European birds are suitable amplifying hosts [Finally, travel and trade continuously (re-)introduce WNV to areas free of overt WNV-disease. In addition, WNV transmission in the absence of noticeable disease has been suggested based upon serological surveys in (sentinel) birds , 37, whing hosts , we prop


Idiopathic pulmonary fibrosis (IPF) is a relentlessly progressive and usually fatal lung disease of unknown etiology for which no effective treatments currently exist. Hence, there is a profound need for the identification of novel drugable targets to develop more specific and efficacious therapeutic intervention in IPF. In this study, we performed immunohistochemical analyses to assess the cell type-specific expression and activation of protein kinase D (PKD) family kinases in normal and IPF lung tissue sections. We also analyzed PKD activation and function in human lung epithelial cells. We found that PKD family kinases  were increased and activated in the hyperplastic and regenerative alveolar epithelial cells lining remodeled fibrotic alveolar septa and/or fibroblast foci in IPF lungs compared with normal controls. We also found that PKD family kinases were increased and activated in alveolar macrophages, bronchiolar epithelium, and honeycomb cysts in IPF lungs. Interestingly, PKD1 was highly expressed and activated in the cilia of IPF bronchiolar epithelial cells, while PKD2 and PKD3 were expressed in the cell cytoplasm and nuclei. In contrast, PKD family kinases were not apparently increased and activated in IPF fibroblasts or myofibroblasts. We lastly found that PKD was predominantly activated by poly-L-arginine, lysophosphatidic acid and thrombin in human lung epithelial cells and that PKD promoted epithelial barrier dysfunction. These findings suggest that PKD may participate in the pathogenesis of IPF and may be a novel target for therapeutic intervention in this disease. Idiopathic pulmonary fibrosis (IPF), the most common form of the idiopathic interstitial pneumonias, is a chronic, relentlessly progressive and usually fatal lung disease of unknown etiology for which no effective pharmacologic treatments currently exist The serine/threonine protein kinase D (PKD) family kinases include PKD1 , PKD2 and PKD3 (PKCν) To determine whether PKD is involved in the pathogenesis of IPF, we compared the cell type-specific expression and activation of PKD isoforms in IPF lung tissues with normal controls and found that PKD family kinases were increased and activated in bronchiolar and alveolar epithelial cells as well as macrophages in IPF. We further found that PKD was predominantly activated by poly-L-arginine, lysophosphatidic acid (LPA), and thrombin in human lung epithelial cells and that PKD promoted epithelial barrier dysfunction.This study involved the use of de-identified lung tissue section slides from control normal subjects and IPF patients. Paraffin-embedded IPF lung tissue slides were kindly provided by the Lung Tissue Research Consortium and the paraffin-embedded normal lung tissue slides were obtained commercially. The present study was reviewed and approved by the Institutional Review Board of the University of Texas Health Science Center at Tyler.Escherichia coli 0111:B4) were from Sigma . LPA was from Avanti . TLR ligands Type B CpG oligonucleotide (ODN 2006), polyinosinic-polycytidylic acid (poly (I:C)), peptidoglycan (PGN), and Pam3CSK4 were from InvivoGen .Antibodies specifically against PKCμ/PKD1 (A-20) and vinculin were from Santa Cruz Biotechnology . PKD2 antibody was from Millipore . PKD3 antibody was from Bethyl Laboratories . Phospho-PKD (Ser744/748) antibodies and reagents for chemiluminescence detection were from Cell Signaling . Vectastain Elite ABC kit, Vector NovaRED substrate, and Vector hematoxylin QS nuclear counterstain were from Vector Laboratories . Recombinant human transforming growth factorβ (TGFβ), epidermal growth factor (EGF), tumor necrosis factorα (TNFα), platelet-derived growth factorβ (PDGFβ), and fibroblast growth factor (FGF) were from R & D Systems . Thrombin, poly-L-arginine and lipopolysaccharides . Formalin-fixed paraffin-embedded normal lung tissue slides from 7 subjects ranged in age from 24 to 80 years were obtained from BioChain , US Biomax , IHCWorld , ProSci , and Alpha Diagnostic , respectively.2O2 for 5 min and washed in phosphate-buffered saline with 0.1% tween-20. Immunohistochemical staining was performed using a Vectastain Elite ABC kits according to the manufacturer's instructions. Primary antibodies were diluted and applied to sections and incubated for 40 min. The dilutions of rabbit polyclonal primary antibodies were: PKD1 (A-20), 1∶500; PKD2, 1∶300; PKD3, 1∶500; and phospho-PKD (Ser744/748), 1∶50. Non-immune normal rabbit immunoglobulin served as a negative control. After incubation with biotinylated secondary antibody and ABC reagents, all the sections were incubated with peroxidase substrate Vector NovaRed for an equal amount of time to allow for suitable staining. The slides were then lightly conterstained with Hematoxylin QS, mounted with VectaMount mounting medium, examined and photographed using an Olympus BX41 microscope equipped with an Olympus DP25 digital camera. Adobe Photoshop 7.0 software was used for image processing. The staining was evaluated according to the number and intensity of immunopositive cells and scored semiquantitatively using a scale from − to + + +: negative (−), weak (+), moderate (++), or strong (+++) in different types of pulmonary cells as described previously Lung section slides were deparaffinized by incubation at 56°C for 30 min and subsequent xylene washes, then rehydrated by using a graded ethanol series. For antigen retrieval, the sections were incubated at 95°C for 8 min in 10 mM sodium citrate buffer (pH 6.0), then soaked in the buffer for another 30 min at room temperature. The slides were then treated with 3% H16HBE14o- human bronchial epithelial cells 3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml each of leupeptin and aprotinin). Whole cell lysates at equal protein amounts were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membrane was probed with various primary antibodies as indicated and detected using the ECL system with horseradish peroxidase-conjugated secondary antibodies according to the manufacturer's protocol .Western blot analysis was performed essentially as we described previously 2) or changes relative to the control group as we described recently TEER was measured with an EVOMX voltohmmeter (World Precision Instruments). The data, which subtract the basal resistance of cell-free collagen-coated Transwell inserts from each experimental point, were presented either as absolute values  at a dilution of 1∶500 and found that PKD1 was undetectable in bronchiolar epithelium of all the normal lungs examined n = 7; . InteresThe main histopathological feature of IPF/UIP is a temporally heterogeneous appearance of the fibrotic areas with alternating areas of less affected or normal parenchyma, scattered fibroblastic foci, and honeycombing We next assessed the expression level of PKD2 in normal and IPF lung tissue sections by using a PKD2 specific antibody We lastly examined PKD3 expression level in normal and IPF lung tissue sections by using a PKD3 specific antibody. Generally PKD3 was barely detectable in normal lung bronchiolar and alveolar epithelia as well as macrophages. In two cases (29%), PKD3 immunoreactivity could be slightly detected in the nuclei of normal lung BECs and AECs . Like PKPKD is normally activated by a novel PKC-mediated phosphorylation of the activation loop two key serine residues To identify agonists that activate PKD in lung epithelial cells, we next treated primary human small airway epithelial cells and A549 alveolar cell line with various receptor ligands or stimuli and performed Western blotting analysis to assess PKD activation by using the phospho-specific PKD-pSer744/748 antibody. Interestingly, we found that PKD was predominantly activated by poly-L-arginine, LPA, and thrombin through a strong phosphorylation of PKD on Ser-744/748 in both primary airway epithelial and A549 alveolar cells . LPA andSince PKD family kinases are increased and activated in IPF bronchiolar and alveolar epithelia, we next sought to assess the effect of PKD overexpression on lung epithelial cell biology. We have recently shown that overexpression of PKD family kinases disrupts the formation of apical intercellular junctions and their reassembly, impairs the development of TEER, and increases paracellular permeability to sodium fluorescein in 16HBE14o- human airway epithelial monolayers IPF, the most common form of the idiopathic interstitial pneumonias, is a chronic, relentlessly progressive and usually fatal lung disease of unknown etiology, and for which no effective treatments exist so far We and others have reported that PKD family kinases are critical for cell proliferation, the production and secretion of cytokines and growth factors in numerous types of cells in response to various stimuli We found that PKD family kinases were increased and activated in alveolar macrophages in almost all IPF subjects examined. It has been recently shown that PKD1 is essential for TLR ligand-induced macrophage activation and cytokine production Increasing evidence indicates that the disorders of small airways are involved in the pathogenesis of IPF We also found that LPA, thrombin, and poly-L-arginine strongly activated PKD in both primary small airway epithelial and A549 alveolar cells. LPA and thrombin are profibrotic factors and are implicated in the pathogenesis of pulmonary fibrosis In summary, we have obtained substantive evidence indicating that PKD family kinases are increased and activated in IPF bronchiolar and alveolar epithelia as well as lung macrophages. PKD is predominantly activated by LPA, thrombin and poly-L-arginine in lung epithelial cells and promotes lung epithelial barrier dysfunction and permeability. PKD family kinases may represent a potential target for the development of novel and efficacious therapeutic intervention in IPF.