Microbial ecology is flourishing, and in the process, is making contributions to how the ecology and biology of large organisms is understood. Ongoing advances in sequencing technology and computational methods have enabled the collection and analysis of vast amounts of molecular data from diverse biological communities. While early studies focused on cataloguing microbial biodiversity in environments ranging from simple marine ecosystems to complex soil ecologies, more recent research is concerned with community functions and their dynamics over time. Models and concepts from traditional ecology have been used to generate new insight into microbial communities, and novel system-level models developed to explain and predict microbial interactions. The process of moving from molecular inventories to functional understanding is complex and challenging, and never more so than when many thousands of dynamic interactions are the phenomena of interest. We outline the process of how epistemic transitions are made from producing catalogues of molecules to achieving functional and predictive insight, and show how those insights not only revolutionize what is known about biological systems but also about how to do biology itself. Examples will be drawn primarily from analyses of different human microbiota, which are the microbial consortia found in and on areas of the human body, and their associated microbiomes (the genes of those communities). Molecular knowledge of these microbiomes is transforming microbiological knowledge, as well as broader aspects of human biology, health and disease. Microbial ecology used to be a small and specialized field that struggled to identify more than a tiny proportion of the Earth’s microbial biodiversity. Part of the problem was due to the prevalence of pure-culture methods, in which microorganisms had to be removed from their natural environments (which included communities of other organisms) and cultured in laboratories. Recent advances in molecular techniques, sequencing technologies and computational methods have enabled researchers to explore the microbial world at unprecedented levels, with a focus on the natural habitats of microorganisms. The combination of these advances has so far produced remarkable insight into the role of microorganisms in human health and their powerful effects on the natural world, while at the same time developing novel evidence about the evolution and diversification of life on Earth. In this article, we discuss how these advances have allowed researchers to create new lines of inquiry, we summarize important biological and philosophical results from recent publications, and we discuss how our improved understanding of microbial ecology may affect our lives in the coming years.The last decade has seen a transformation and democratization of DNA sequencing (Shendure and Ji These transformations in sequencing technology have correspondingly changed what it means to undertake a sequencing project. When sequences were very expensive (in the late 1970s and early 1980s), it was a substantial accomplishment to sequence even one gene from one species. Correspondingly, the focus was on identifying genes that acted as the best phylogenetic markers. These were short fragments of sequences from which inferences about the patterns of evolution were likely to match the inferred patterns of evolution of the corresponding species. These markers therefore provided efficient readouts of evolutionary history while minimizing sequencing costs. For example, ribosomal RNA genes, which play essential structural and catalytic roles in the ribosome and are thought to be almost exclusively vertically transmitted Lawrence , have beunknown species via bulk DNA extraction directly from the environment. This technique, pioneered by the Pace lab   from the genomes of well-studied organisms can often be assigned at least to the family level, and sometimes at the genus or species level, many sequences cannot confidently be assigned to known named taxonomic groups. The limitation here is primarily the amount of information present in short reads of marker genes for differentiating closely related taxa. Figure One shows that when working with the most informative region of the 16S rRNA gene for broad analyses of bacterial and archaeal communities, the fraction of reads that can be assigned to taxonomic groups increases as expected with the length of the sequence. In real-world experiments  typically find it at less than 1 % abundance in the gut of healthy adults , the abundance threshold that may be set deliberately or may be intrinsically limited by technology or study design , and the fraction of individuals that a taxon must appear into be considered “core” . Additionally, the cost of DNA sequencing posed a barrier until recently to collecting the detailed time-series and spatial datasets that are necessary for ecological modeling in microorganisms. However, some aspects of microbial ecology are substantially easier than in large-organism ecology. For example, the reliance on DNA sequence data means that with advances in technology, even a deep sampling of the population  can be performed rapidly, and observation biases are likely to be less profound than when attempting to glimpse rare and elusive insects or mammals. The ability to collect large-scale information about microbial populations is likely to allow classical ecological models to be applied to the microbial world far more effectively than has been possible in macroecology, because more types of microbes can be simultaneously observed with large population sizes and with replicated sampling.Ecological principles offer more than just ways to stratify the human population . At infancy, our microbial populations go through remarkable changes in structure prior to reaching a resemblance to most adult communities. Inoculation is not necessarily from our mothers, and is substantially influenced by delivery mode. Microbial communities of children delivered vaginally initially tend to resemble their mother’s vaginal communities, while the microbial communities of children delivered by C-section initially tend to resemble human skin communities. Skin inoculations may be obtained from the mother, the medical staff involved in the delivery, or hospital surroundings (many of which harbor communities resembling human skin) (Biasucci et al. A revelatory aspect to studies of the microbiome is that classical ecological models and datasets previously only obtainable for a few economically important systems, such as fisheries, are now testable on the microbial scale because of the ability to assess simultaneously the relative abundance of thousands of species in thousands of samples (Gonzalez et al. The analysis of time-series in microbial ecology has also been limited because the performance of standard signal processing methods is degraded with uneven sampling periods and small numbers of data points (Moller-Levet et al. Overall, the ability to collect far larger amounts of sequence data has led to much broader and deeper characterizations of the human microbiome and microbial communities in other habitats, especially when linked to rich contextual information about the provenance and status of each sample (Knight et al. Some of the solutions to these problems are being sought in large-scale projects such as the Earth Microbiome Project (Gilbert et al.


Saccharomyces cerevisiae (S. cerevisiae) are characterized by low structural stability, and that this is associated with reduced antigenicity and immunogenicity. However, little attention has been devoted to methods of improving the quality of the VLPs. Here, we investigated the effect of carbon source concentration in the medium on the antigenicity and immunogenicity of human papillomavirus (HPV) type 16 L1 VLPs expressed in S. cerevisiae from the galactose promoter. Media containing 2, 4, 6, and 8% carbon source, composed of both glucose and galactose in equal proportion, were used. VLP antigenicity was enhanced in cultures grown on media with 6 or 8% carbon source, compared to those from cultures with less than 6% carbon source. Moreover, the VLPs obtained from these cultures induced higher anti-HPV16 L1 IgG titers and neutralizing antibody titers in immunized mice than those purified from cultures with less than 6% carbon source. Our results indicate that the concentration of the carbon source in the medium plays a crucial role in determining the antigenicity and immunogenicity of HPV type16 L1 VLPs.There is accumulating evidence that virus-like particles (VLPs) recombinantly produced in  The ongoing technological advances in genetic engineering and in the production of recombinant proteins have enabled the development of subunit vaccines that utilize as antigens monomeric proteins derived from pathogens Virus-like particles (VLPs) are multimeric protein complexes similar in shape to naturally occurring virions Saccharomyces cerevisiae (S. cerevisiae) is one of the preferred expression systems used for producing VLPs S. cerevisiae possess the ability to self-assemble into VLPs. Another advantage of the yeast expression system is its low production cost. However, VLPs produced in this system are reported to have low structural stability The recently developed strategy for producing VLPs has provided new insights into mechanisms protecting against pathogens, and yielded an innovative platform for developing high-efficacy vaccines. However, obtaining high-quality VLPs presents a challenge because of their structural complexity. in vitro strategies for increasing VLP antigenicity and immunogenicity, such as redox refolding, VLP maturation, and salt treatment, have been tried with some success in vivo relies on intracellular assembly. In this study, we compared the antigenicity and immunogenicity of HPV type 16 L1 protein (HPV16 L1) VLPs produced in the S. cerevisiae expression system with different concentrations of carbon source. Previously, we found that the concentration and composition of the carbon source used for S. cerevisiae producing HPV16 L1 protein significantly affect the yield of the HPV16 L1 protein Many All animal experiments were treated in accordance with the guideline of Institutional Animal Care and Use Committee, Chung-Ang University IACUC, and the protocol was approved by the IACUC. The conditions of mice were monitored twice a day. Mice were anesthetized intraperitoneally with 10 μl of 4∶1 mixture of Zoletil 50  and Rompun  prior to blood collection.S. cerevisiae Y2805 was transformed with the resulting plasmid (YEGα-MCS-HP16 L1 gene-opt). Transformants were selected on SD-ura medium, a synthetic medium without uracil, and inoculated into 150 mL of YPDG medium. The YPDG medium contained 1% yeast extract, 2% peptone, and various concentrations of carbon source. For the purpose of this study, the selected concentrations were 2, 4, 6 and 8%, and the glucose to galactose ratio was set at 1∶1. Therefore, the percentages of glucose and galactose in the media were 1%, 2%, 3% and 4% each. All components used to prepare media were purchased from Duchefa (Netherlands). Cells were cultured for 6 days at 30°C with shaking at 230 rpm.The codon-optimized HPV16 L1 gene (HPV16 L1 gene-opt), designed to reduce the secondary structure of the mRNA g for 10 min. Thereafter L1 protein was recovered from the cell lysate as pellet by precipitation with 40% saturated ammonium sulfate. The pellet was resuspended in the buffer , and dialyzed against the same buffer for 4 h at room temperature (RT). After dialysis precipitation of contaminating proteins was induced in 10 mM sodium phosphate buffer  for 16 h at 4°C, and the precipitates were removed by centrifugation at 12000×g for 10 min. The supernatant (fraction containing L1 protein) was dialyzed against binding buffer for cation-exchange chromatography  for 3 h at RT and loaded onto a column packed with P-11 phosphocellulose cation-exchange resin . The column was washed with five column volumes of the binding buffer, and bound proteins were eluted by successive addition of buffer containing 0.6, 0.7, 0.8, 0.9 and 1 M NaCl. L1 protein fractions were collected and concentrated using an Amicon Ultra-4  and dialyzed against storage buffer .HPV16 L1 protein was purified as previously described Protein concentrations were measured by the Bradford protein assay  with bovine serum albumin  as a standard. The purity and quantity of L1 were determined by SDS-PAGE and Western blot analysis.http://rsbweb.nih.gov/ij/).SDS-PAGE and Western blotting were performed as described Electron microscopy was performed on a TEM200CX at a final magnification of 234000×. The purified HPV16 L1 VLPs were absorbed onto carbon-coated grids, stained with 2% phosphotungstic acid and analyzed under the microscope o-phenylenediamine , and optical density was measured at an absorbance of 492 nm.The ELISA was carried out as described DLS was performed using an ELSZ-2 system  as described Six-week-old female BALB/c mice  were immunized three times subcutaneously with purified HPV16 L1 VLPs at two-weekly intervals. The mice were divided into five groups of 8, 9, or 10 mice each. The control group received PBS, and the remaining groups received HPV16 L1 VLPs purified from cultures containing 2, 4, 6, or 8% carbon source. The mice received 10 or 1000 ng purified HPV16 L1 VLPs per dose in combination with aluminum hydroxide (200 μg per dose). The doses were as recommended in a previous report The anti-HPV16 L1 IgG titers of mouse sera were measured as described HPV16 PsV was prepared according to a published method The neutralizing antibody titer of a mouse serum was defined as the reciprocal of the highest dilution that caused a reduction of at least 50% in secreted embryonic alkaline phosphatase (SEAP) activity Production of HPV16 L1 protein was evaluated as a function of carbon source concentration , and theWe confirmed that the L1 protein finally recovered was able to form VLPs (data not shown). The concentration of L1 in each preparation was estimated by SDS-PAGE and Western blotting  prior toTo investigate the neutralizing epitopes in each VLP preparation, ELISAs were performed using neutralizing Mabs, H16.V5 and H16.E70 . It is sS. cerevisiae contain 2 – 4% carbon source due to the fact that higher concentrations can cause osmotic stress to the yeast and lead to higher alcohol production, which is potentially detrimental to cultures Typically, cultures designed to produce heterologous proteins in in vitroIn contrast with conventional fermentation strategies, in this study we obtained superior-quality HPV16 L1 VLPs from cultures containing 6 and 8% carbon source and after 6 days of culture. As shown in Previously we found that the levels of conformational neutralizing epitopes on the surface of HPV16 VLPs increased with increasing culture duration when the culture contained more than 6% carbon source in vitro treatment of redox reagent affects VLP conformation and maturation S. cerevisiae producing HPV L1  Representative plot of all VLPs: numbers in parentheses indicate the hydrodynamic diameters of the HPV16 L1 VLPs. (B) The mean ± SEM of four independent experiments.(TIF)Click here for additional data file.Figure S2GSSG and GSH level of S. cerevisiae producing HPV16 L1 protein. Cells were culture in YPDG medium containing 7% glucose and 1% galactose for 144 h at 30°C. This culture condition showed the highest production yield of the L1 protein (see reference (TIF)Click here for additional data file.


A 54-year-old woman with a past history of silicone augmentation mammoplasty was admitted with fever and dyspnea with diffuse interstitial shadows on computed tomography (CT). Although radiological findings were atypical, we diagnosed sarcoidosis by laboratory, microbiological, and bronchoalveolar lavage fluid analysis. Corticosteroids ameliorated the condition, but she had recurrent of fever and CT revealed miliary nodules while interstitial shadows disappeared. Liver biopsy showed that noncaseating granuloma and Ziehl-Neelsen stain was positive. We diagnosed miliary tuberculosis which developed during corticosteroid therapy. Antituberculotic therapy resulted in favorable outcome. Possibility exists that onset of sarcoidosis was induced by mammoplasty, namely, human adjuvant disease. Sarcoidosis is a systemic disease of unknown etiology that primarily affects the lungs and is characterized by noncaseous granuloma. On the other hand, tuberculosis is an infectious disease characterized by caseous granuloma. Differentiation of sarcoidosis from tuberculosis is important, as the treatment of sarcoidosis is opposite to that of tuberculosis. However, differentiation can be difficult because clinical and histological findings are similar. Given these similarities, etiological relationships between sarcoidosis and tuberculosis have been documented. The prevalence of sarcoidosis is high in patients who have previously suffered from tuberculosis –3, and tHuman adjuvant disease (HAD) is characterized by autoimmune-like symptoms following cosmetic mammoplasty . SarcoidHerein, we report the case of a patient with sarcoidosis following mammoplasty complicated by miliary tuberculosis during treatment with corticosteroids. Relationships between sarcoidosis, tuberculosis, and HAD are discussed.A 54-year-old woman was admitted to our hospital with fever, general malaise, and dyspnea. She had undergone bilateral breast augmentation surgery with silicone bags 5 years previously, with resurgery on the right side due to bag rupture 7 months before presentation. Seven days before admission, she became febrile and noticed dyspnea. She visited her family doctor and was diagnosed with pneumonia, but treatment with antibiotic therapy had no noticeable effect. She was then referred to our hospital for further examination and treatment.The patient was 158 cm tall, weighed 46 kg, and blood pressure was 100/50 mmHg, heart rate was 74 beats/min, body temperature was 37.0°C, and respiratory rate was 30 breaths/min on admission. Inspiratory rales were heard at both lung bases. The examination of the blood revealed severe thrombocytopenia with prolonged prothrombin time and high levels of fibrinogen degradation product (FDP) . ChemicaMycobacterium tuberculosis. Fever recurred on day 20 and liver biopsy was performed to identify the cause of fever. We considered the possibility of sarcoidosis based on elevated levels of angiotensin-converting enzyme (ACE) (63.0 IU/L) and lysozyme (11.8 mg/mL). On day 23, Liver biopsy confirmed Langhans' giant cell and noncaseating granuloma , likely caused by rifampicin. On day 59, the WBC count was fully recovered, and rifampicin was restarted at a low dose (150 mg). Fever was again seen on day 64, and CT was performed on day 68. Although miliary nodules in both lung fields had almost disappeared  and initiated methylprednisolone pulse therapy (1 g/day for 3 consecutive days) for interstitial pneumonia and gabexate mesilate and antithrombin III for DIC. Antibiotic  was administrated to cover the possibility of concomitant bacterial pneumonia. On day 4, respiratory status and interstitial opacity on CT were improved . Howeverranuloma . On day ranuloma . On day specimen , but negappeared , sickly appeared . These nappeared . On day appeared .Mycobacterium tuberculosis as proven by Ziehl-Neelsen staining and noncaseous granuloma in the liver. Since noncaseous granuloma is not specific for sarcoidosis and can also occur in tuberculosis, the series of events in the present case may be attributable to miliary tuberculosis from the beginning. However, this possibility is considered unlikely for the following reasons.The most characteristic feature of the present case was the coexistence of Mycobacterium tuberculosis on Ziehl-Neelsen-stained tissue or sputum culture. Although the yield of acid-fast smears from sputum is usually low (46.4–50%) [First, interstitial shadowing on CT and the respiratory status completely ameliorated after first steroid pulse without the use of antituberculotic therapy. The steroid therapy has brought down fever for 7 days. If she was affected by tuberculosis from the beginning, we could not control her fever only by steroid therapy. Second, bronchoscopy on day 18 failed to identify 6.4–50%) , tissue 6.4–50%) . Third, The classic findings of sarcoidosis on lung CT are widespread small nodules with bronchovascular and subpleural distributions, thickened interlobular septa, architectural distortion, and conglomerate masses . RadiogrThe etiology of sarcoidosis remains unclear. However, onset of sarcoidosis in some cases is causally related to mammoplasty –9. In thIn summary, we reported the case of a patient with sarcoidosis following mammoplasty complicated by miliary tuberculosis during treatment with corticosteroids. HAD may have induced or triggered the onset of sarcoidosis. Sarcoidosis needs to be distinguished from miliary tuberculosis because the therapeutic strategies for these two pathologies are in opposition.


Thus, anti-PD-L1 antibody might have a therapeutic potential in attenuating liver injury in sepsis.Liver plays a major role in hypermetabolism and produces acute phase proteins during systemic inflammatory response syndrome and it is of vital importance in host defense and bacteria clearance. Our previous studies indicated that programmed death-1 (PD-1) and its ligand programmed death ligand-1 (PD-L1) are crucial modulators of host immune responses during sepsis. Our current study was designed to investigate the role of PD-L1 in sepsis-induced liver injury by a mouse cecal ligation and puncture (CLP) model. Our results indicated that there was a significant increase of PD-L1 expression in liver after CLP challenge compared to sham-operated controls, in terms of levels of mRNA transcription and immunohistochemistry. Anti-PD-L1 antibody significantly alleviated the morphology of liver injury in CLP mice. Anti-PD-L1 antibody administration decreased ALT and AST release in CLP mice, decreased the levels of tumor necrosis factor (TNF)- Sepsis remains a major clinical challenge in intensive care units (ICU), characterized by the systemic inflammatory response and multiple organ failure . Liver iIt has been proved that the high rates of morbidity and mortality associated with sepsis may be contributed by immunosuppression caused by impaired pathogen clearance after infection –7. It isProgrammed death-1 (PD-1) is a newly identified coinhibitory receptor, which has been proved to be an important modulator of immune responses , 12. PD-+ T cells, CD8+ T cells, and CD19+ B cells. Expression of PD-1 on T cells and of PD-L1 on monocytes was dramatically upregulated in septic shock patients. PD-1/PD-L1 pathway blockade in vitro with anti-PD-L1 antibody decreased apoptosis of T cells induced by TNFα or T-cell receptor ligation. Meanwhile, this blockade potentiated the lipopolysaccharide-induced TNFα and IL-6 production and decreased IL-10 production by monocytes in vitro. In short, the PD-1/PD-L1 pathway might play an essential role in sepsis-induced immunosuppression. Our animal research found that PD-L1 blockade improved the survival of septic mice [α and interleukin (IL)-6 productions, decreased IL-10 production, and enhanced bacterial clearance. Anti-PD-L1 antibody administration may be a promising therapeutic strategy for sepsis-induced immunosuppression.Our previous work showed that the expression of PD-1 and PD-L1 was evaluated in septic patients . We examtic mice . The expLiver played a key role in the immune tolerance of a variety of diseases. However, the effect of PD-L1 blockade with antibodies on sepsis-induced liver injury and its molecular mechanism remains unclear. Thus, our current research was designed to investigate the role of PD-L1 in sepsis-induced liver injury by a mouse cecal ligation and puncture model. We want to determine the expression of PD-L1 in liver during sepsis and provide a preliminary result of the role of PD-L1 in sepsis-induced liver injury.Male 8- to 10-week-old C57BL/6 mice, weighing 22 g to 30 g each, were purchased from the Animals Experimentation Center of Second Military Medical University. All mice were housed in air-filtered, temperature controlled units with 12-hour light-dark cycles and had free access to food and water. All experiments were approved by the Institutional Animal Care and Use Committee of our university.μg anti-PD-L1 antibody (Anti-Mice CD274 (B7-H1) PE, Clone: MIH5, purchased from R&D Systems) in 200 μL of PBS was injected intraperitoneally 1 hour after the operation per mouse. Mouse from the isotype group was injected with a dose of 50 μg (200 μL) control antibody  accordingly.CLP-induced polymicrobial sepsis was performed as described previously . BrieflyMice were euthanized 24 hours after CLP or sham-operated surgery. Blood samples were taken from the heart. After separation from whole blood by centrifugation, plasma were used for the determination of alanine aminotransferase (ALT) and aspirate aminotransferase (AST) activities by commercially available ELISA kits  according to the manufacturer's instructions. μm tissue sections were cut from the paraffin blocks and stained with hematoxylin and eosin. The tissue sections were examined under light microscopy by a senior independent pathologist.The right lobe of liver was rapidly harvested after blood obtaining. The liver was then fixed in 10% formalin and then paraffin embedded. 5 μg/mL. Secondary antibodies were used in the morning to bind with primary antibodies after PBS washing and sections were incubated in room temperature for 20 min. The secondary antibodies were detected with DAB according to the manufacturer's instructions, and sections were counterstained with hematoxylin. Negative controls without primary antibody and positive control tissue were included in all experiments to ensure the staining quality.The left lobe of liver was also cut off to be frozen by microtome-cryostat. Consecutive 4-micron sections of the specimens were air-dried for 10 min and then fixed in acetone for 10 min. Endogenous peroxidase activity was blocked by treatment with 0.3% hydrogen peroxidase in PBS for 30 min at room temperature. Sections were then washed three times in PBS. After blocking nonspecific binding with normal solcoseryl for 20 min at room temperature, sections were incubated in a humidified chamber at 4°C overnight with primary antibodies, Anti-PD-L1 antibody, which were purchased from eBioscience  and used at the final concentrations of 20 α were measured by real-time polymerase chain reaction (RT-PCR). Small cubes of liver were obtained immediately after the death of mice. Total RNA in the cube was extracted using RNeasy Mini kit . 100 ng RNA was used for cDNA synthesis using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's protocol. Quantitative RT-PCR was performed using SYBR Green (TaKaRa) on an ABI PRISM 7900 Sequence Detector  with SDS 2.1 software. Each reaction was performed in quadruplicate, with final calculations resulting from means of quadruplicate wells. The ΔΔCq method was used to determine the difference of the mean expression levels of PD-L1, IL-6, IL-10, and TNF-α between study subjects with different genotypes of rs4755453. For each individual, the relative expression level ΔCq (Cq T − Cq E) of PD-L1, IL-6, IL-10, and TNF-α was normalized with GAPDH and then transformed into relative quantity using the RQ formula . The primers for PD-L1 were forward 5′-tgctgcataatcagctacgg-3′ and reverse 5′-gctggtcacattgagaagca-3′. The primers for IL-6 were forward 5′-atggatgctaccaaactggat-3′ and reverse 5′-tgaaggactctggctttgtct-3′. The primers for IL-10 were 5′-ccagttttacctggtagaagtgatg-3′ and reverse 3′-tgtctaggtcctggagtccagcagactc-5′. The primers for TNF-α were 5′-catcttctcaaaattcgagtgacaa-3′ and reverse 5′-tgggagtagacaaggtacaaccc-3′.To evaluate the gene expression of mediators in the inflammatory responses, the mRNA levels of PD-L1, IL-6, IL-10, and TNF-t tests were done when 2 groups were compared. Graphs are displayed as mean with error bars representing the standard error. A P value < 0.05 (two-tailed) was considered statistically significant.All data were analyzed using GraphPad Prism software 5.0.1 . Means and standard errors of the means were calculated in experiments. Paired To examine the localization of PD-L1 expressions in liver tissues, we performed immunohistochemical staining . Among aP < 0.01). In order to clarify the expression of PD-L1 in liver quantitatively, we performed real-time PCR. Our results indicated that there is a ~5.7-fold significant increase of PD-L1 mRNA expression in liver of mice 24 hours after CLP challenge than that in sham group  . In contrast, anti-PD-L1 antibody decreased levels of ALT and AST  (P < 0.01). Thus, anti-PD-L1 antibody could reduce the level of ALT and AST levels , indicating liver damage, compared with sham group  . There is no significant difference between CLP group and Isotype group in terms of each cytokine (P > 0.05)  . These rThe main finding of this study is that PD-L1 expression was regulated in the liver of mice with sepsis, based on levels of mRNA transcription and Immunohistochemistry of liver. PD-L1 blockade with antibodies could decrease the level of CLP-induced septic liver injury.CLP-induced sepsis caused a significant inflammatory response and damage in a liver characterized both biochemically (ALT and AST activity) and histologically (hematoxylin-eosin-stained section). Our findings are in accordance with the report by Koskinas et al. . They shα were involved in sepsis, which contributes to tissue and organ damage. Our study found that anti-PD-L1 antibody suppressed not only proinflammatory cytokines (IL-6 and TNF-α) but also anti-inflammatory cytokine (IL-10). These results are partially in accordance with our previous findings. Zhang et al. [α and Il-6 and decreased the level of IL-10 in CLP murine plasma. Collectively, these data indicated that the protective effects of anti-PD-L1 antibody on septic liver injury might be partially achieved by inhibition of proinflammatory cytokines.Sepsis was associated with overproductions of proinflammatory cytokines, which can lead to the recruitment of leukocytes and tissue damage , 21. A ng et al.  found thWith the use of CLP-induced sepsis, a well-established animal model for sepsis, our findings indicated that PD-L1 expression was regulated in the liver of mice with sepsis. Mice with sepsis had a significant elevation of PD-L1 expression in liver, based on the levels of mRNA and transcription immunohistochemistry staining of liver. It was indicated that PD-L1 was expressed on Kupffer cells (KCs) and liver sinusoidal epithelial cells in murine liver tissues (LSECs) . HepatocSeveral limitations in our study should be noted. We did not explore more specific mechanisms underlying the protective role of anti-PD-L1 antibody in sepsis-induced liver injury. It is reported that liver damage during sepsis is manifested by functional change rather than structure damage. A time course study of the role of PD-L1 in sepsis-induced liver injury is warranted. Our current research chose a single dose of anti-PD-L1 antibody to use on the mice, which was based on our previous findings. The effects of anti-PD-L1 antibody at different doses in the model also need to be clarified. Furthermore, whether the blockade of PD-L1 plays a vital role in other organ damages, that is, kidney, intestinal, and heart, during sepsis also needs to be clarified.To conclude, we found elevated PD-L1 expression in liver of mice with experimental sepsis. PD-L1 blockade with antibodies could exert protective roles against sepsis-induced liver injury, characterized by alleviating liver pathological injury, reducing ALT and AST release, and attenuating liver inflammatory responses. Thus, anti-PD-L1 antibody might have a therapeutic potential in reducing liver injury in sepsis.


We aimed to describe haemodynamic changes when haemodialysis is instituted in the critically ill. Three hypotheses are tested: (1) the initial session is associated with cardiovascular instability; (2) the initial session is associated with more cardiovascular instability compared to subsequent sessions; and (3) looking at unstable sessions alone, there will be a greater proportion of potentially harmful changes in the initial sessions compared to subsequent ones.Data were collected for 209 patients, identifying 1,605 dialysis sessions. Analysis was performed on hourly records, classifying sessions as stable/unstable by a cut-off >±20% change in baseline physiology (HR/MAP). Data from 3 hours prior to and 4 hours after dialysis were included, and average and minimum values derived. Three time comparisons were made . Initial sessions were analysed separately from subsequent sessions to derive two groups. If a session was identified as being unstable, then the nature of instability was examined by recording whether changes crossed defined physiological ranges. The changes seen in unstable sessions could be described as to their effects: being harmful/potentially harmful, or beneficial/potentially beneficial.P = 0.68, reinforcing the lack of significant variance.Discarding incomplete data, 181 initial and 1,382 subsequent sessions were analysed. A session was deemed to be stable if there was no significant change (>±20%) in the time-averaged or minimum MAP/HR across time comparisons. By this definition 85/181 initial sessions were unstable . Therefore Hypothesis 1 is accepted. This compares to 44% of subsequent sessions (95% CI 41.1 to 46.3). Comparing these proportions and their respective CI gives a 95% CI for the standard error of the difference of -4% to 10%. Therefore Hypothesis 2 is rejected. In initial sessions there were 92/1,020 harmful changes. This gives a proportion of 9.0% (95% CI SEM 7.4 to 10.9). In the subsequent sessions there were 712/7,248 harmful changes. This gives a proportion of 9.8% (95% CI SEM 9.1 to 10.5). Comparing the two unpaired proportions gives a difference of -0.08% with a 95% CI of the SE of the difference of -2.5 to +1.2. Hypothesis 3 is rejected. Fisher's exact test gives a result of Our results reject the claims that using haemodialysis is an inherently unstable choice of therapy. Although proportionally more of the initial sessions are classed as unstable, the majority of MAP and HR changes are beneficial in nature.


The excitation and absorption properties of grating coupled graphene surface plasmons were studied. It was found that whether a mode can be excited is mainly determined by the frequency of incident light and the duty ratio of gratings. In the structure consisting graphene bilayer, a blueshift of the excitation frequency existed when the distance between neighbor graphene layer were decreased gradually. In graphene-grating multilayer structures, a strong absorption (approximately 90% at maximum) was found in near-THz range. Recently, a lot of work has been done based on graphene due to its unique properties in electric, magnetic, thermal, etc.-3. Graphσ, which consists of both intraband and interband from Kubo formula[ω/μc was small[Up to date, the graphene is modeled usually to be an extremely thin film with a conductivity  formula-9. The ias small. Both tras small-12. In oIn the previous works, many numerical approaches were used to study the structured graphene, for example the finite element method (FEM), finite In this paper, we studied the binary grating bounded by graphene on both sides. The rigorous coupled wave analysis (RCWA),17 was uω ≾ μc (see[Previous research has shown that the conductivity of graphene came from the contribution of intraband and interband-22. The  μc (see). Then t μc  was positive, no solution of Equation 3 was found in real, which meant the TE mode GSP could not be excited.For TM mode, put Equation 2 into Equation 4, we foundneff = β/k0 = βc/ω as the effective index of GSP. After making a transformation of  → , the dispersion relations were obtained and plotted in Figure kΛ0 = 2π/λ0, λ0 = 1 μm. As a local mode, GSP modes were same as the surface plasmon polaritons (SPPs). They cannot be excited directly from the air. And in our work, gratings were used to provide an external wave vector to match the phase condition.Here, we defined h to be the depth of grating (thickness of gratings). The h was also the distance between two graphene layers. In multilayer structures of Figure h was the longitudinal period. The structures were designed to only contain two kinds of interfaces.In Figure J = σE, while graphene was included.In common, the conventional RCWA based on the Floquet's theorem was unabIn RCWA, the field was expanded into the form ofJ can also be expended to the sum of spatial harmonics with different wave vector components. To obtain the reflection, transmission, absorption, field distribution, and other optical properties of such structures as shown in Figure Hy field considering the induced current,So the current density Hy will also be continuous at the interface if each spatial harmonics subcomponent satisfied the boundary conditions independently,According to the principle of superposition, n was the order, ± in subscripts represented approaching to y0 from two different directions. After the modification on the RCWA program, we can utilize it to deal with the graphene-containing structures.in which For a structure with a binary grating bounded by graphene layers on two sides shown in Figure ε1-graphene-ε1 and ε1-graphene-ε2. When GSP is propagating along the interfaces, the phase shifts were φ1 and φ2 for the two kinds of interfaces, respectively. δ was the total phase loss considering two abrupt phase changes when GSP propagates across the joints between the two kinds of interfaces in a grating period. At the excitation frequency, the phase change in a grating period should satisfy the relationIn the structure shown in Figure N is the integer and can be rewritten aswhich was known to be the phase matching conditions,28. In Ef2 = L/Λ and f1 = 1 - f2, β1 and β2 were the wave vectors of GSP on two kinds of interfaces, respectively. When N was a nonnegative integer, the GSP mode could be excited, and N can be defined as the order of surface modes. The resonant frequencies can be obtained both from absorption spectrum in Figure δ = 0). They were given in Table where The field distributions of orders 1 and 2 of the structure in Figure f, where f = φ1/(φ1 + φ2), φ1, φ2 had the same meaning as Equation 9. From the discussion above, when the phase match conditions were satisfied, GSPs could be coupled and absorption peaks should appear. But some break points appeared as shown in Figure f of controlling the excitation of GSP.By using the modified RCWA, the absorption spectrum was obtained in Figure From the field distributions in Figure x0 is the distance of A and B in the form of light path (k0x0 = L1β1 = φ1 = (φ1 +φ2)f = 2πNf). Because in real space, different interfaces (ε1/ε1 and ε1/ε2) had different propagating constants, the expression might be complex. Here, the light path of x was used. It is found that scatting points A and B had a phase difference of π. This was caused by the different geometric symmetries. From Equation 11, when sin(k0x0/2) = 0, i.e., f = m/N , field amplitude F would always be 0, which meant that the field cannot be excited. It was a cancelation process of two sets of standing waves that are coherent. So, for GSP mode of N, N + 1 of none absorption points appeared.First two terms were GSP excited by one set of points  was the coupling coefficient and n, h, and ∆θ were order of GSP mode, thickness of grating, and phase difference of GSPs on two graphene layers, respectively. Essentially, the GSPs were surface waves so they interact with each other via evanescent interactions, and the coupling intensity decayed exponentially with h increasing. For fixed h, the lower order modes had larger skin depth (stronger coupling intensity) than the higher orders; then, the stronger coupling resulted in a large spectra shift. The phase difference of ∆θ also had affection to the absorption frequencies. However, in our case, the wavelength (15 meV ~ 82.8 μm) was much larger than the thickness of grating layer (h = 10 μm), it is reasonable to assume ∆θ is approximately 0. This can also be obtained clearly from the field distribution in Figure κ ∝ e-hq(n), whereIn Equation 12, κ(xup = xdown = x0eiωt- (no phase difference between GSP on neighbor layers), it is found that the resonant frequency becameSuppose the solution of having the form of h was small (h < 4 μm), the larger κ ∝ e-h was the larger shift of resonant frequency would be. And obviously, κ was approaching 0 rapidly when h was large enough, which meant that the resonant frequency became a stable value ofκ was also related to the order of GSP. The high order mode had a small skin deep with weak coupling intensity and less blueshift. When h tends to be 0, the grating became too thin to excite the surface mode. This was why the absorption disappeared when h = 0 in Figure When Moreover, the behavior of multilayer structures shown in Figure The field distributions of Figure On conclusion, the rigorous coupled wave analysis was modified to compute the excitation of graphene surface plasmon in graphene-containing binary gratings structures. Under the phase matching conditions, the excitation of the graphene surface plasmonics was determined by the distance between graphene layers and duty ratio of gratings, and the mode suppression can be realized by modifying the grating constant and duty ratio. A blueshift of the excitation frequency was obtained for enhanced coupling between GSP of neighbor graphene layers. Increasing the number of graphene layers had almost no effect on the excitation frequency of GSP but would lead to a high absorption with negligible reflection in near-THz range. Finally, the resonant frequency and absorptions can be easily modified by manipulating the structure parameter, including grating constant, duty ratio, and distance between the graphene layers and number of grating, and graphene-containing grating might become potential applications of THz region, such as optical absorption devices, optical nonlinear, optical enhancement, and so on.The authors declare that they have no competing interests.HYU, XJ, and XS conceived the idea. HYU, PL, HYA, and XS wrote the codes, calculated the results, and made the conclusions. HYU, XS, and PL contributed to the preparation and revision of the manuscript. All the authors read and approved the final manuscript.


The preclinical model of bleomycin-induced lung fibrosis, used to investigate mechanisms related to idiopathic pulmonary fibrosis (IPF), has incorrectly predicted efficacy for several candidate compounds suggesting that it may be of limited value. As an attempt to improve the predictive nature of this model, integrative bioinformatic approaches were used to compare molecular alterations in the lungs of bleomycin-treated mice and patients with IPF. Using gene set enrichment analysis we show for the first time that genes differentially expressed during the fibrotic phase of the single challenge bleomycin model were significantly enriched in the expression profiles of IPF patients. The genes that contributed most to the enrichment were largely involved in mitosis, growth factor, and matrix signaling. Interestingly, these same mitotic processes were increased in the expression profiles of fibroblasts isolated from rapidly progressing, but not slowly progressing, IPF patients relative to control subjects. The data also indicated that TGFβ was not the sole mediator responsible for the changes observed in this model since the ALK-5 inhibitor SB525334 effectively attenuated some but not all of the fibrosis associated with this model. Although some would suggest that repetitive bleomycin injuries may more effectively model IPF-like changes, our data do not support this conclusion. Together, these data highlight that a single bleomycin instillation effectively replicates several of the specific pathogenic molecular changes associated with IPF, and may be best used as a model for patients with active disease. Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by excessive matrix deposition that disrupts the normal architecture of the lung parenchyma. The key pathological features of IPF include fibroblastic foci that are highly synthetic, areas of epithelial cysts associated with the honeycombing appearance of the lung, and mild lymphoplasmacytic interstitial inflammation that is associated with areas of type II cell hyperplasia in vivo system for investigating candidate therapies. Its failure as a prognostic tool may be ascribed to the fact that the model has not been well characterized in terms of identifying the clinically relevant molecular changes and when they occur. Bleomycin is well understood to induce lung injury that results in an acute inflammatory response that is unlikely to reflect the processes driving the disease in patients. The inflammatory phase is, however, followed by fibrotic changes that replicate certain pathological features consistent with those associated with IPF. Therefore, some have argued that it may be more appropriate to evaluate compounds after or during the onset of the fibrosis phase of the response (i.e. using therapeutic dosing regimens), which may be a more disease-relevant paradigm The lack of effective therapies is arguably due to an incomplete understanding of the molecular mechanisms driving the disease and the failure of preclinical experimental models to correctly predict the clinical efficacy of several molecules Using classic histopathology and physiology methods, we report that the repetitive model offered no significant improvement over the single challenge model. Integrative bioinformatic and pharmacological approaches revealed corresponding molecular changes in the lungs of bleomycin-treated mice and IPF patients, especially in genes associated with mitosis and extracellular matrix signaling. Interestingly, these same pathways appeared to be altered in fibroblasts isolated from IPF patients with rapidly progressing, but not slowly progressing disease. It did not appear that these changes in expression were directly associated with TGFβ signaling and furthermore, an inhibitor to the TGFβR1  could not completely attenuate bleomycin-induced fibrosis in mice. These data support the premise that the bleomycin model can recapitulate many of the complex profibrotic responses that are also elevated in the lungs of IPF patients, particularly in patients with active disease.To establish a dose of bleomycin that induced fibrosis but did not result in mortality, a preliminary bleomycin dose-response study was performed. Bleomycin induced a dose-dependent increase in lung fibrosis . SignifiIn response to repetitive bleomycin administration, we observed increased inflammatory cell infiltrate in the BALF of mice . InteresH (lung tissue elastance) and work of inflation (WoI) correlated significantly to collagen levels in the lung , although the changes were relatively modest and significant at 35 days post-administration , an active fibrosis phase (days 7–14) and a late fibrosis phase (days 21–35), with saline-treated samples clustering separately . Peak geGenes altered during the inflammation phase generally consisted of immune response and wound healing genes including various cytokines and chemokines. In addition to genes associated with immune and wound healing categories, genes altered during the active fibrosis phase were also associated with cell cycle-related processes. These changes in gene expression were validated by the increased expression of inflammatory cytokines early in the response to bleomycin , followeClustering analysis of genes involved in fibrosis-related pathways revealed preferential up-regulation of genes during the active fibrosis phase. Matrix metalloprotease (MMP) genes including MMP12, MMP19, as well as lysyl oxidase-like (LOXL) genes implicated in the pathogenesis of fibrotic diseases were induced from 7 days onwards following bleomycin treatment . PositivThe translational relevance of the bleomycin response signatures were assessed in clinical IPF datasets using gene set enrichment analysis (GSEA). Gene expression data from two publicly available clinical IPF datasets were compiled: GSE2052 in vivo datasets, GSEA also revealed that gene sets defined by changes in the clinical IPF datasets were enriched within the expression profiles of lung samples generated from the bleomycin model (data not shown). These results further highlight commonalities in genes altered in response to bleomycin treatment with those altered in clinical IPF samples.Closer examination of the leading edge genes  shows up-regulation of a common subset of genes across different clinical datasets . Genes iNext, we prospectively compared the gene expression profiles of fibroblasts obtained from patients with rapidly progressing and slowly progressing IPF to fibroblasts obtained from non-IPF control subjects, as these cells are centrally involved in the disease pathogenesis Given that several pathways associated with fibrotic processes were elevated in response to bleomycin-induced lung injury, we wanted to assess whether TGFβ was the dominant driver of fibrosis in this model. To this end, we assessed an ALK-5 inhibitor, SB525334, under both prophylactic and therapeutic conditions. SB525334 significantly inhibited bleomycin-induced fibrosis changes under both conditions . The maxThe data presented in this manuscript provide clear evidence that the bleomycin model of lung fibrosis induces molecular changes directly relevant to IPF and in particular may be more relevant for modeling “active” rather than slowly progressing disease. Additionally, these data indicate that there are multiple pathways involved in eliciting the pathophysiological changes in the model that are consistent with those observed in the lungs of patients with IPF. As such, it would appear that therapies aimed at affecting several of these key signaling networks either by targeting “nodes”, using combination therapy approaches, or broad spectrum inhibitors of key enzymes may be the only means of making a significant impact on disease progression in all patients with IPF.The response to bleomycin occurred in three distinct phases, which we have termed the (1) inflammation phase, (2) active fibrosis phase, and (3) late fibrosis phase. The inflammation phase was largely characterized by the presence of neutrophils in the airways and the increased expression of immune processes in the lung tissue. While some have proposed that neutrophils may be playing a role in IPF H) and our new measure of WoI Another commonly stated limitation of the bleomycin model is that the fibrosis is not progressive and in fact, resolves with time. Interestingly, we observed that there was still measureable fibrosis and changes in pulmonary mechanics in the late fibrosis phase (out to 5 weeks post-bleomycin). Furthermore, we took samples 56 days after bleomycin instillation in a separate study (data not shown) and measureable fibrosis was still present contrary to previous reports and dogma As an attempt to improve the single challenge system we performed repetitive bleomycin instillations, but found that repetitive exposures provided no additional value in terms of producing a more substantial or progressive fibrotic response in the lung. The one difference to the single challenge model was that epithelial remodeling was more evident at most of the time points assessed in the repetitive challenge system. Epithelial remodeling in IPF is associated with the deterioration of the lung parenchyma and the honeycombing pattern of the lung characteristic of IPF The most interesting observations from these studies occurred during the active fibrosis phase of the response to bleomycin. This phase was characterized by increased levels of (protein and lipid) pro-fibrotic mediators associated with increased matrix deposition in the lung that correlated with alterations in pulmonary mechanics. These changes were also temporally correlated with the increased expression of a variety of fibrosis-related gene sets including TGFβ signaling, matrix proteases, and structural proteins – observations that confirm previous findings reported by others Here, we have shown for the first time a direct correlation between the molecular changes observed in the lungs of bleomycin-treated mice and patients with IPF using novel translational and quantitative bioinformatics approaches. GSEA allows one to define gene signatures from one experimental system and subsequently quantify whether that signature is enriched in another biological system. The gene sets defined by changes during the active fibrosis phase (days 7–14) of the mouse bleomycin model generated the highest enrichment scores amongst the samples from IPF patients obtained from two separate publicly available data-sets. In fact, the changes occurring in the late fibrosis phase were also enriched in the clinical samples, an observation that is consistent with the lack of fibrosis resolution at these later times. To understand the biology behind the correlative gene changes, we overlapped a union of the leading edge gene lists with canonical signaling pathways. Interestingly, the leading edge genes were largely involved in mitotic processes likely due to the proliferation of mesenchymal cells. Additionally, there were changes in signaling pathways  associated with fibroblast proliferation and signaling in response to changes to the extracellular matrix. These data highlight the multifactorial molecular mechanisms involved in the pathogenesis of IPF Budd et al, under review). Additionally, the cells from rapidly progressing patients are able to induce substantial lung fibrosis when injected into immunocompromised mice (versus a mild response when using slowly progressing IPF patient fibroblasts) Although these are interesting observations, additional clinical information such as lung function data, high resolution computed tomography scores, and rate of disease progression associated with publicly available clinical datasets would improve the interpretation of these datasets. Such information would provide greater insights about whether these molecular changes that correlate with the model are generic amongst all IPF patients or whether the correlation is strongest within certain IPF patient sub-populations. Interestingly, many of the overlapping changes between the model and clinical samples were likely to be due to changes in fibroblast activation and proliferation. These same processes were enriched in fibroblasts isolated from the lungs of IPF patients with rapidly progressing disease. Surprisingly, these processes did not appear to be altered when comparing fibroblasts from patients with slowly progressing IPF to non-IPF controls. Together, these observations would suggest that the bleomycin model may be more effective at mimicking active disease processes that may be more associated with patients with a rapidly progressing disease course. That said, there are caveats to these data that need to be taken into account. First, the patients’ cells are not freshly isolated or laser capture microdissected, but rather they have been in culture for several passages, which may confound interpretation. Second, the controls are not healthy individuals, but cancer patients undergoing lung resection. Finally, these data were generated from 4–6 patients within each group and additional biological replicates would improve confidence in these observations. That said, it is still very interesting to note that the cells from the rapidly progressing IPF patients do have enhanced responses to PDGF  and TGFβ , whereas these responses are not as profoundly altered in fibroblasts from slowly progressing IPF patients relative to the non-IPF controls  was only enriched in the IPF patients who had experienced an acute exacerbation compared to non-IPF control subjects, possibly indicating its importance in active disease. Similarly, the down-regulated TGFβ gene set (comprised mainly of negative regulators of the TGFβ pathway) was only down-regulated in one clinical data-set. Although no one would discount TGFβ’s contribution to the lung remodeling associated with IPF, it does highlight the fact that it is unlikely to be the only pathway driving disease pathogenesis. These observations are also consistent with the data demonstrating that the ALK-5 inhibitor, SB525334, was not capable of fully blocking the fibrotic response elicited by bleomycin. This molecule consistently attenuates the response in our laboratory as well as in others The data described in this paper contradict current dogma and suggest that bleomycin is able to induce clinically meaningful molecular responses in the lungs of mice. Using innovative bioinformatic approaches we directly linked the molecular changes occurring in the bleomycin model to those occurring in the lungs of IPF patients in a quantitative manner. Genes involved in mitotic processes were expressed at higher levels in lung tissues from bleomycin-treated mice and IPF patients as well as fibroblasts isolated from IPF patients with rapidly progressing disease. These data along with pharmacological data indicate that bleomycin triggers complex, multifactorial (TGFβ dependent and independent) responses that result in pathophysiological changes in mice that are consistent with IPF. Furthermore, our analysis suggests that the changes in this model best represent changes occurring in patients with active (i.e. rapidly progressing) disease.ad libitum. All animal care and experimental procedures were approved by the Roche Animal Care and Use Committee, which is a facility accredited by the American Association for the Accreditation of Laboratory Animal Care (AAALAC).Male C57BL6/J mice (7–9 weeks old) were purchased from the Jackson Laboratory , housed under pathogen-free conditions with food and water Bleomycin sulphate  was dissolved in 0.9% saline and loaded into a IA-1C liquid MicroSprayer  as previously described 2O positive end-expiratory pressure).Invasive lung function measurements were taken at specified time points following saline or bleomycin administration as previously described Exercise capacity was measured based on a method previously published by Koch and colleagues Immediately following lung function measurements, mice were exsanguinated, bronchoalveolar lavage fluid (BALF) was collected, and total and differential cell counts were performed, as previously described For the analysis of 4-hydroxyproline (HP), BALF samples were extracted with acetonitrile at volume of 1:6, respectively. After centrifugation, acetic acid (0.1% in water) was then added to the supernatants at volume of 1.5:1, respectively. Samples were mixed and centrifuged before injection for LC/MS/MS analysis. LC/MS/MS analysis was conducted by gradient HPLC with selective reaction monitoring (SRM). The calibration range was 40- 2,000 ng/ml. Since the control BALF matrix contained a basal level of HP (approximately 200 ng/mL), the calibration curve is corrected with the basal concentration determined by the standard addition. The LC/MS/MS system used for the analysis consisted of an AB-Sciex API4000 with an electrospray source connected to an Agilent 1200 pump, an Waters 2777 autosampler, and an Agilent 1100 series column oven. Chromatographic analysis was conducted by HILIC HPLC with an Ascentis Express HILIC  column. The SRM transition is m/z 132 to m/z 41.2O pressure with 10% neutral buffered formalin through the tracheal cannula and immersed in formalin for at least 24 h. After being processed into paraffin blocks, the lungs were sectioned (5 µm) and stained with either hematoxylin and eosin (H&E) or immunolabeled with an anti-collagen I antibody , to assess fibrotic changes in the lungs as previously described After performing lung function measurements, whole lungs  were inflated under 25 cm HAdditionally, staining for alpha-smooth muscle actin , and heat-shock protein-47 (HSP-47)  was done using a 3-step biotin-streptavidin-HRP detection method, and DAB  as the detection chromogen. Tissue sections were counterstained with hematoxylin, dehydrated, and mounted with Permount mounting media.Total RNA was isolated from the lung tissue of bleomycin and saline treated mice across 7 time points . The mice used for genomic profiling were not used for measuring lung function, performing bronchoalveolar lavage, and they were not used for histopathology analysis. The entire left and right lung tissues were homogenized in QIAzol reagent. Purified total RNA was amplified and labeled using NuGen Ovation kits  and samples were hybridized to Affymetrix Mouse 430 2.0 arrays. Array washing, staining and scanning was performed according to standard Affymetrix protocols . Probe level data was curated by first mapping individual probe sequences to their most current mouse genome sequences. Probes which were non-uniquely mapped to specific genes or contained outdated mappings were discarded, and the remaining probes were summarized into probesets and normalized using RMA. Potential outlier samples were assessed using principal component analysis (PCA) on all normalized probesets across all samples, resulting in 1 sample  being removed from subsequent analysis. Probes level data was subsequently summarized to unique genes based on a variance filter, yielding one expression value per unique gene across 111 samples. Mouse genes were then mapped to their human orthologs for subsequent pathway analysis. This resulted in 14643 unique mouse genes which mapped to human orthologs and were subsequently used for analysis. All data is MIAME compliant and all raw.CEL files from the arrays are deposited in NCBI’s GEO database (GSE40151).Differentially expressed genes were determined using an ANOVA, with pairwise comparisons between bleomycin and saline treatment at each time point. P-values for differentially expressed genes in pairwise comparisons were adjusted using a Benjamini-Hochberg correction to account for multiple hypothesis testing http://pid.nci.nih.gov/index.shtml). This repository includes pathway data imported from BioCarta and Reactome. Enrichment of functional ontologies and pathways for all DEGs was determined by a hypergeometric test, with p-values and FDR calculated to determine significance of enrichment.Gene ontology (GO) functional analysis and pathway enrichment analysis were performed on DEGs between saline and bleomycin treated mice at each time point using internally curated GO biological process annotations. Similarly, pathway enrichment for all DEGs was determined using internally curated data from the NCI Pathway Interaction Database , collagens (COLs), TGFβ signaling , alternative macrophage activation, Wnt signaling, and an Gene set enrichment analysis (GSEA) Clustering analysis was subsequently performed on leading edge genes from GSEA, in order to more closely examine genes within bleomycin signatures most contributing to enrichment of the signatures in clinical datasets. A union of leading edge genes from GSEA of IPF versus control comparisons from two datasets  was compiled and expression of these genes was assessed in the two clinical datasets with IPF-only subjects versus controls.In addition, GSEA was used to determine enrichment of genes differentially expressed in clinical IPF datasets in bleomycin versus saline treated mice. DEGs were determined in each IPF dataset between IPF and non-IPF groups . DEGs were determined based on being 1.5-fold altered between groups with an FDR <0.05. A union of DEGs from comparisons in each dataset was compiled resulting in two gene sets consisting of 30 genes . Enrichment of these gene sets was determined against genes from each time point in the mouse bleomycin data, ranked based on a composite of fold-change and FDR differences between bleomycin and saline treated samples per time point, with FDRs of gene set enrichment determined as described above.GSEA was also used to determine enrichment of internally curated canonical pathways from the NCI Pathway Interaction Database in both the mouse bleomycin and cultured human fibroblast datasets. Canonical pathways enriched in the bleomycin dataset were defined by ranking genes based on a composite score of fold-difference and FDR between bleomycin and saline-treatments for the 14 day time point (active fibrosis phase). Canonical pathways were then used as gene sets for GSEA, with FDR values being determined for gene set enrichment by permuting genes. Canonical pathway enrichment was similarly determined in the fibroblast dataset by generating ranked gene as described above in rapid progressing IPF vs. non-IPF samples. Significantly enriched canonical pathways  were then compared between the bleomycin-treated 14 day time point and fibroblasts from rapid progressing IPF patients.Primary cultures of LFs were isolated from the distal parenchyma of patients from unaffected donor controls (lung cancer patients) and patients with IPF as previously described Data are expressed as mean ± SEM unless specified otherwise. Unless stated otherwise elsewhere in the methods, statistical analyses of time course studies were determined using a Student’s t-test using GraphPad Prism (version 5.03).Figure S1Bleomycin induces dose-dependent increases in lung fibrosis. Fibrosis scores evaluated in H&E stained lung sections 21 days after a single instillation of bleomycin. Data expressed as mean ± SEM of n = 8 except for the 5 U/kg group where n = 5. Significance relative to the saline treated was determined using a one-way ANOVA and Dunnett’s post-hoc test and is denoted as, *p<0.05; or ***, p<0.001.(TIF)Click here for additional data file.Figure S2Bleomycin induces inflammatory cell infiltrate in the airways of mice. The numbers inflammatory cells increase in the BALF fluid after a single (A – C) or repetitive (D – F) bleomycin (black bars) or saline (white bars) administration. Differential cell counts revealed that neutrophils (A and D), lymphocytes (B and E), and macrophages (C and F) were all elevated in the BALF. Data are expressed as mean ± SEM of n = 7–8 mice. Significance (relative to the time-matched control at each time point) was determined using a Student’s t-test and is denoted as follows: *p<0.05; **p<0.01; and ***p<0.001.(TIF)Click here for additional data file.Figure S3Bleomycin induces collagen deposition in the lung. Measureable changes in matrix remodeling were observable after a single (A and B) or repetitive (C and D) bleomycin (black bars) or saline (white bars) administration. Lung collagen (A and C), and BALF hydroxyproline levels (B and D) were all elevated in response to bleomycin. Data are expressed as mean ± SEM of n = 7–8 mice. Significance (relative to the time-matched control at each time point) was determined using a Student’s t-test and is denoted as follows: *p<0.05; **p<0.01; and ***p<0.001.(TIF)Click here for additional data file.Figure S4Changes in lung mechanics correlate to changes in lung fibrosis. Changes in H (A) and volume normalized work of inflation (WoI) (B) after a single bleomycin instillation significantly correlated to the amount of lung tissue stained positive for collagen I as determined by Pearson correlation coefficients (R). The correlations for H (C) and WoI (D) with collagen I were similar after repetitive bleomycin instillations.(TIF)Click here for additional data file.Figure S5Bleomycin increased collagen deposition and myofibroblast activation. Serial lung tissue sections from one representative animal illustrate that areas positive for collagen I (D) were also positive for α-smooth muscle actin (E), as well as HSP47 (F). As a reference, sections from saline treated animals stained for collagen I (A), α-smooth muscle actin (B), and HSP47 (C) are also shown. Images were captured at 200X magnification.(TIF)Click here for additional data file.Figure S6Exercise capacity is reduced in bleomycin treated mice. The exercise capacity of mice treated with saline (filled circles) or 2 U/kg bleomycin (filled squares) was measured on a motorized treadmill. Data expressed as mean ± SEM of n = 19–20. Significance relative to the saline treated was determined using a one-way ANOVA and Dunnett’s post-hoc test and is denoted as, *p<0.05; or ***, p<0.001.(TIF)Click here for additional data file.Figure S7Bleomycin induces cytokine, chemokine, and pro-fibrotic mediator secretion in the BALF. BALF levels of KC (A), IL-16 (B), CCL17 (TARC) (C), total IL-12 (D), TGFβ (E), and LPA (18:1 isoform) (F)were measured. Saline treated controls represented by white bars and bleomycin treated animals by black bars. The following cytokines had no measurable change: IFN-g, TNF-α, IL-1β, IL-2, IL-4, IL-5, IL-10, GM-CSF, VEGF, IL-13, IL-17 (data not shown). Data are expressed as mean ± SEM of n = 8 mice. Significance (relative to the time-matched control at each time point) was determined using a Student’s t-test and is denoted as, *p<0.05.(TIF)Click here for additional data file.Figure S8Supervised hierarchical clustering of custom panels of genes across all mouse samples. Clustering of genes was performed for a panel of (A) genes involved in Wnt signaling, (B) genes altered downstream of PI3 kinase, and (C) genes involved in alternative macrophage activation. Samples are ordered based on treatment and time point.(TIF)Click here for additional data file.Figure S9Heatmap of gene set enrichment of custom signatures in clinical IPF datasets. GSEA was performed for gene sets of custom gene panels from fibrosis-related mechanisms including MMP, LOXL, collagen, and TGFβ signaling. Enrichment of each gene set (denoted in rows) was determined against ranked lists of genes from clinical datasets comparing IPF vs. non-IPF conditions from two datasets . Enrichment scores were plotted in a heatmap where gene sets enriched in IPF samples  were denoted in red while gene sets enriched in non-IPF samples were denoted in blue. Intensity of each cell was based on enrichment score .(TIF)Click here for additional data file.Figure S10Plasma exposure levels for SB525334 in bleomycin treated mice after initiating a diet using Purina Rodent Chow #5001 mixed with SB525334.(TIF)Click here for additional data file.Data File S1A union of leading edge genes from GSEA of bleomycin-induced gene sets in IPF vs. non-IPF subject comparisons from two clinical cohorts . This alphabetized list of genes corresponds to a union of the genes highlighted in (PDF)Click here for additional data file.Data File S2Canonical pathways enriched among the leading edge genes from GSEA of bleomycin-induced gene sets in IPF vs. non-IPF subject comparisons from two clinical cohorts . The canonical pathways, p value of the enrichment of leading edge genes in these pathways, and the leading edge genes contained within the pathways are highlighted in this table.(PDF)Click here for additional data file.


The role of extracellular signal-regulated protein kinase (ERK) in intestinal ischemia/reperfusion (I/R) injury has not been well investigated. The aim of the current study was to examine the effect of inhibition of the ERK pathway in an in vitro and in vivo model of intestinal I/R injury.ERK1/2 activity was inhibited using the specific inhibitor, U0126, in intestinal epithelial cells under hypoxia/reoxygenation conditions and in mice subjected to 1 hour of intestinal ischemia followed by 6 hours reperfusion. In vitro, cell proliferation was assessed by MTT -2,5-diphenyl tetrazolium bromide) assay, apoptosis by DNA fragmentation, and migration using an in vitro model of intestinal wound healing. Cells were also transfected with a p70S6K plasmid and the effects of overexpression similarly analyzed. In vivo, the effects of U0126 on intestinal cell proliferation and apoptosis, intestinal permeability, lung and intestinal neutrophil infiltration and injury, and plasma cytokine levels were measured. Survival was also assessed after U0126. Activity of p70S6 kinase (p70S6K) was measured by Western blot.In vitro, inhibition of ERK1/2 by U0126 significantly decreased cell proliferation and migration but enhanced cell apoptosis. Overexpression of p70S6K promoted cell proliferation and decreased cell apoptosis. In vivo, U0126 significantly increased cell apoptosis and decreased cell proliferation in the intestine, increased intestinal permeability, intestinal and lung neutrophil infiltration, and injury, as well as systemic pro-inflammatory cytokines, TNF-α, IL-6 and IL-1β. Mortality was also significantly increased by U0126. Inhibition of ERK1/2 by U0126 also abolished activity of p70S6K both in vitro and in vivo models.Pharmacologic inhibition of ERK1/2 by U0126 worsens intestinal IR injury. The detrimental effects are mediated, at least in part, by inhibition of p70S6K, the major effector of mammalian target of rapamycin pathway. Intestinal ischemia/reperfusion (I/R) occurs in a wide range of clinical scenarios, including small bowel transplantation, midgut volvulus, major trauma, thrombosis of the superior mesenteric artery and vein, hemorrhage and cardiopulmonary bypass. Intestinal I/R leads to the release of cytotoxic substances from the ischemic tissues and the accumulation of inflammatory mediators in the intestine. This results in mucosal injury, hyperpermeability, and inflammation. The release of proinflammatory factors and bacteria-derived endotoxins from the injured intestine may also instigate a systemic inflammatory response and lead to multiple organ dysfunction syndrome -3. ThereThe extracellular signal-regulated protein kinases (ERK) 1 and 2 (ERK1/2) signaling pathway is a cascade consisting of at least three families of protein kinases, including Raf (MAPKKK or MEKK), MAPKKs (MEK 1 and MEK 2), and MAPK (ERK 1 and ERK 2 or p42/p44 MAPKs). Each molecule acquires protein kinase activity after phosphorylation, and is subsequently able to phosphorylate and then activate the next member of the signaling cascade. The ERK pathway not only regulates a wide range of cell behaviors, such as, growth, proliferation, migration, differentiation, apoptosis and autophagy, but also mediates inflammatory responses ,6. The E2-adrenoceptor agonist, in transient cerebral I/R [ERK1/2 has been implicated in tissue injury induced by I/R, but results have been inconsistent. Zhu et al. demonstrated that activation of ERK1/2 mediated neuroprotection of dexmedetomidine, a potent and highly selective αbral I/R . Similarbral I/R . Howeverbral I/R . Similarbral I/R . There ibral I/R . Therefol-glutamine, 1% penicillin and 1% streptomycin in the incubator with a humidified atmosphere of 5% CO2/95% air at 37°C. Cells were used between passages 17 and 25.The intestinal epithelial IEC-6 cell line which was derived from adult rat intestinal crypts was purchased from the American Type Culture Collection. Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 2 mM Cells were transfected with empty vector pRK7 plasmid, p70S6K plasmid (wild-type p70S6K expressing vector pRK7-HA-S6K1-WT)  using th2/5% CO2/94.5% N2 for indicated periods of time, cells were reoxygenated by placing cells into in an incubator under 37°C/ 5% CO2 conditions with 10% serum (normoxic conditions) for indicated periods of time. To inhibit ERK1/2 phosphorylation/activity, cells were treated with 10 µM of U0126 (Sigma) [A well-established hypoxia/reoxygenation (H/R) model was employed using IEC-6 cells to mimic I/R in vivo . After i (Sigma) ,16, a sp3 cells were added into each well in a 96-well plate. After 24 hours, cells were cultured in medium without FBS under hypoxic conditions for 12 hours in a hypoxic chamber, and then 10 µM of U0126 or vehicle was added into cells. After 1 hour, 10% FBS was supplemented to medium. Cells were transferred to an incubator under normoxic conditions for 3 days. Cells were then incubated with MTT (Sigma)  for 3 hours. After removal of medium, dimethyl sulfoxide (200 µl/well) was added to wells. A microplate reader was used to measure the absorbance of dissolution sample at 570 nm and subtracted background at 670 nm.MTT -2,5-diphenyl tetrazolium bromide) assay was employed for cell growth assessment . Briefly4 cells/well). After 24 hours, fresh FBS-free medium containing U0126 (10 µM) or vehicle was added. Cells were cultured under hypoxic conditions for 24 hours followed by incubation in normoxic conditions for 6 hours. To measure the DNA fragmentation, the medium was discarded, and the attached cells were washed twice with PBS and lysed with lysis buffer (200 µl /well). Cell lysates were collected into 1.5ml-tubes and centrifuge at 200 Χ g for 10 minutes. A 100-µl supernatant of the cell lysate per well was taken and added into the ELISA plate and measured the immunocomplex at 405/490 nm in a microplate reader. The value was normalized by cell numbers in corresponding wells in parallel experiments. Cells grown in basal conditions served as untreated control.A Cell Death Detection ELISA (enzyme-linked immunosorbent assay) kit (Roche) was used to measure the DNA fragmentation in cells . Cells w5 cells in a 12-well plate for 24 hours. Cells were then cultured with FBS-free medium under hypoxic conditions for additional 12 hours. The confluent cell monolayer was scraped with a pipette tip across the diameter of the well. After removal of cell debris by washing with PBS two times, cells were then treated with U0126 (10 µM) or vehicle for 1 hour followed by adding 10% FBS. Cells were cultured for another 24 hours under normoxic conditions. The wound area at the beginning of wounding and at the end of treatment was photographed respectively under an inverted phase-contrast microscope with an attached camera. The wound area was measured using OPTIMAS 6.1 image analysis software (Optimas Corp.). The recovered surface area was calculated by subtracting the wound area at the end of treatment from the corresponding original wound area.A H/R model of in vitro intestinal wound healing was employed to mimic I/R in vivo . Each weAll animal protocols were approved by the University of Texas, Houston Medical School Animal Welfare Committee.2 asphyxiation immediately and recorded as dead.Male, 8-10 weeks old C57BL/6J mice were randomly assigned into 4 groups (n=5/group): sham, sham-U0126, IR, IR-U0126. Mice were fasted with free access to water overnight before laparotomy. U0126   was giveIntestinal and lung tissues were fixed in buffered formaldehyde solution (10% in PBS) for 24 hours and embedded in paraffin. The tissue was then sectioned, deparaffinized, stained with hematoxylin and eosin (HE). To evaluate histopathologic changes in the intestine, Park/Chiu scoring system was used in a blinded manner using a light microscope on a 0 to 5 scale, where 5 is the most severely injured ,18. HistA commercial Myeloperoxidase (MPO) Colorimetric Activity Assay Kit (BioVision Research Products) was employed to quantitatively measure the MPO activity in the intestinal and lung tissue. The MPO activity reflects the extent of neutrophil infiltration into the tissue. The MPO value was normalized to milligrams of protein determined by Bio-Rad Protein Assay (Bio-Rad Laboratories).The relative level of cytokines TNF-α, IL-6 and IL-1β in plasma was determined by enzyme-linked immunosorbent assay with a Mouse Cytokine ELISA Strip I for Profiling 8 Cytokines kit (Signosis) according to manufacturers’ instruction.The ex-vivo isolated everted ileum sac method  was usedIntestinal tissues and IEC-6 cells were quickly lysed with radioimmunoprecipitation assay buffer in the presence of protease inhibitors. Western blot analysis was performed as previously described . AntibodCell apoptosis in the intestinal tissue was evaluated using an In Situ Cell Death Detection Kit (Roche) as described previously .Ki-67 is a nuclear protein that is expressed in proliferating cells. It has been used as a marker for cell proliferation. Immunohistochemistry for Ki-67  (Merck Millipore) was performed using the Dako-Cytomation EnVision + System-HRP (AEC) kit (Dako) in paraffin-embedded intestinal sections as described previously . Ki-67 pData is presented as mean ± SEM and were compared by one way analysis of variance with the nonparametric Mann-Whitney test used to determine differences between groups. The Mantel–Haenzel log rank test was used to compare survival rates. A p value less than 0.05 was considered statistically significant.ERK1/2 is activated by phosphorylation. To test the efficacy of U0126 inhibition of ERK1/2 in intestinal cells under H/R conditions and after I/R in the intestines, we detected protein levels of phosphorylated ERK1/2 by Western blot. U0126 virtually abolished the phosphorylation of ERK1/2 in IEC-6 cells under H/R conditions . In vivoUnder H/R conditions, there was no difference between control and vehicle treated groups in cell proliferation (control vs. vehicle: 2.40±0.10 vs. 2.33±0.15. P>0.05), migration (control vs. vehicle: 101.67±13.31 vs. 95.67±10.69. P>0.05) or apoptosis (control vs. vehicle: 6.07±0.73 vs. 6.53±1.27. P>0.05) . U0126 sIn sham groups, treatment of U0126 did not affect intestinal cell apoptosis  or proliferation (Ki-67 proliferation index) . However, after intestinal I/R, U0126 significantly enhanced intestinal cell apoptosis  and reduced cell proliferation  . These dMPO activity, an index of neutrophil infiltration, was similar in the sham and sham-U0126 groups (sham vs. sham-U0126: 0.25±0.06 vs. 0.24±0.16. P>0.05). However, MPO activity was markedly increased in the intestine after I/R (sham vs. IR: 0.25±0.06 vs. 0.96±0.07. p<0.01) and was further increased by U0126 treatment (IR vs. IR-U0126: 0.96±0.07 vs. 2.24±0.26. p<0.01) .Permeability was similar in the intestine between sham and sham-U0126 groups (sham vs. sham-U0126: 17.64±1.99 vs. 22.86±5.90P>0.05), but was increased after I/R  and U0126 treatment exacerbated the intestinal permeability (IR vs. IR-U0126: 81.30±18.16 vs. 157.08±23.30. p<0.01) .Similarly, there was no difference between sham and sham-U0126 in mucosal injury (sham vs. sham-U0126: 0.10±0.22 vs. 0.20±0.27. P>0.05), but I/R induced severe mucosal injury in the intestine (sham vs. IR: 0.10±0.22 vs. 2.50±0.50. p<0.01), and treatment with U0126 further worsened intestinal injury (IR vs. IR-U0126: 2.50±0.50 vs. 4.50±0.50. p<0.01) .Consistent with previous reports ,21, systIntestinal I/R caused significant lung inflammation (sham vs. IR: 0.56±0.07 vs. 5.57±1.59. p<0.01) and injury (sham vs. IR: 0.20±0.45 vs. 3.30±0.48. p<0.01). U0126 further exacerbated both inflammation (IR vs. IR-U0126: 5.57±1.59 vs. 11.11±2.53. p<0.01) and injury (IR vs. IR-U0126: 3.30±0.48 vs. 4.38±0.41. p<0.05). There were no differences in inflammation (sham vs. sham-U0126: 0.56±0.07 vs. 0.59±0.20. p>0.05) or injury (sham vs. sham-U0126: 0.20±0.45 vs. 0.20±0.45. p>0.05) between sham groups .Since inhibition of ERK1/2 activity by U0126 led to more severe local and remote organ (lung) damage, we assessed the impact of U0126 on mortality and demonstrated a marked increase in mortality in U0126-treated animals (p<0.01) . MortaliSimilar to ERK pathway, the mTOR/p70SK pathway is also a major regulator of cell growth, proliferation and motility -24 as weU0126 is a signal transduction inhibitor that specifically targets the ERK pathway in mammalian cells . U0126 hERK1/2 activation controls cell proliferation and apoptosis . StudiesInhibition of ERK1/2 may also increase inflammation. Our results showed that inhibition of ERK1/2 facilitated neutrophil infiltration into the intestinal tissue and promoted the production of proinflammatory factors systemically during intestinal I/R. Furthermore, ERK1/2 inhibition exacerbated inflammation-related remote organ damage. Consistent with our results, Noh et al. reported that upregulation of ERK1/2 kinase activity promoted the anti-inflammatory cytokine IL-10 expression in lipopolysaccharide-stimulated conditions .The lung is frequently injured after intestinal I/R. Once activated by proinflammatory mediators, neutrophils are able to penetrate the endothelial layer and migrate into the alveolar space ,36. RecrThe precise molecular mechanism of ERK1/2 in regulation of intestinal injury and inflammation after I/R remains to be elucidated. PPARγ is an anti-inflammatory mediator in the intestine and contains a mitogen-activated protein kinase site. Phosphorylation of this site by ERK1/2 resulted in inhibition of PPARγ . HoweverIt is well-known that ERK1/2 regulates mTOR /p70S6K pathway ,27. We hIn conclusion, we have demonstrated that ERK1/2 activation is crucial to protection against intestinal I/R injury. Inhibition of ERK1/2 decreased cell proliferation, suppressed cell migration, and increased cell apoptosis, as well as exacerbating the breakdown of the intestinal barrier. These effects were due, at least in part, to increased local and systemic inflammation. Additionally, p70S6K mediated the effects of ERK1/2 inhibition on intestinal I/R injury. This finding enhances the understanding of the mechanisms responsible for intestinal I/R injury and may potentially be translated into new therapeutic approaches for prevention and recovery of intestinal I/R damage. The results of this study may also be applicable to other organs damaged by I/R since ERK1/2 is universally expressed by most tissues/organs, such as lung, kidney, liver and heart.


TLR2 might be important in a small group of Caucasian sarcoidosis patients. The present study aimed to establish whether these findings are relevant to the Japanese population.Sarcoidosis is a systemic inflammatory disease characterized by the formation of non-caseating granulomas, with varied clinical manifestations. The common etiology of sarcoidosis is uncertain, but it is thought to be triggered by an exogenous antigenic stimulus, such as some bacterial proteins. Toll-like receptors (TLRs) recognize microbial components and elicit innate as well as adaptive immune responses. It has been reported that polymorphisms in TLR2 and assessed the allelic diversity between 257 Japanese sarcoidosis patients and 193 Japanese healthy controls.We genotyped 5 single-nucleotide polymorphisms (SNPs) in TLR2 alleles and haplotypes in the sarcoidosis cases were found in comparison with the controls. However, marginal associations were observed for TLR2 at rs3804099 and rs3804100 in sarcoidosis patients with cutaneous manifestations.No significant differences in the frequency of TLR2 polymorphisms are not significantly related to the pathogenesis of sarcoidosis in the Japanese population.Our results suggest that  Sarcoidosis is a systemic inflammatory disorder resulting in non-caseating granulomas in multiple organs, such as: lung, skin, eye, lymph nodes, central and peripheral nervous system, and heart -3. JapanThe exact cause of sarcoidosis is unknown, but the fact that the frequency and course of the disease varies widely among racial groups suggests that genetic factors may be the basis of disease susceptibility. African Americans are more commonly and severely affected by sarcoidosis than Caucasian Americans. The annual sarcoidosis incidence for African Americans is threefold higher, compared with Caucasian Americans; at 35.5 versus 10.9 cases per 100,000, respectively . In the Mycobacterium tuberculosis and Propionibacterium acnes has been detected in some sarcoid lesions by using polymerase-chain-reaction (PCR) methods [Environmental factors are also thought to contribute to the disease progression. The DNA of  methods -13. Rece methods . These sTLR polymorphisms are closely associated with many autoimmune diseases [TLR2 are associated with impaired responses to bacterial infection in human [TLR2 promoter lesion (rs4696480) was associated with sarcoidosis in a Dutch Caucasian population, but could not confirm this in their validation cohort. They inferred from these findings that a TLR2 variant could play a role in a small percentage of patients. The association between TLR2 polymorphisms and sarcoidosis needs to be confirmed by further replication studies, particularly in other ethnic groups. In the present study, we therefore evaluated the association of multiple SNPs in TLR2 in Japanese patients.Toll-like receptors (TLRs) recognize microbial components and elicit innate as well as adaptive immune responses. Stimulation with TLR ligands induces the production of proinflammatory cytokines and type I interferons in cells of the innate immune system through intracellular signaling cascades -17. Accudiseases -20. Amondiseases ,22. Polyin human -27. Recein human  reportedTwo hundred fifty-seven unrelated patients with a diagnosis of sarcoidosis and 193 healthy controls were recruited from Yokohama City University, Hokkaido University, Fujita Health University, Tokyo University, Keio University, and Kumamoto City hospital. All patients and control participants were of Japanese ethnicity. Sarcoidosis patients were diagnosed according to the diagnostic criteria developed by the Japanese Society of Sarcoidosis and Other Granulomatous Disorders (JSSOG) previously described . Uveitisrs1898830, rs11938228, rs3804099, re3804100, and rs7656411 . Probe fluorescence signals were detected by TaqMan Assay for real-time PCR  following the manufacturer’s instructions.Peripheral blood lymphocytes were collected, and genomic DNA was extracted from peripheral blood cells using the QIAamp DNA Blood Maxi Kit . We evaluated five single-nucleotide polymorphisms (SNPs): s7656411 . These S2 test. The Haploview 3.32  program was used to compute pair-wise linkage disequilibrium (LD) statistics [Hardy–Weinberg equilibrium was tested for each SNP among the controls. Differences in allele and genotype frequencies between cases and controls were assessed by the χatistics . Standaratistics . HaplotyTLR2 gene: rs1898830, rs11938228, rs3804099, re3804100, and rs7656411 . The allele frequencies of the five SNPs in both the cases and controls are listed in rs3804099 and rs3804100 were higher when compared with the frequency in 193 controls . However, these statistical differences disappeared after the Bonferroni correction was applied (p>0.05).Linkage disequilibrium (LD) blocks of five SNPs in  defined . The TLRTLR2 polymorphisms affect the development of sarcoidosis in the Japanese population. Our results showed that all the TLR2 polymorphisms so far examined were not significantly associated with any clinical subtype of sarcoidosis including ocular involvement in the Japanese population. However, a marginally significant p-value was observed for the SNPs rs3804099 and rs3804100 in patients with cutaneous manifestations, in comparison with the healthy control group. Healthy normal human skin contains two distinct major subsets of resident dendritic cells: Langerhans cells (LCs) and dermal DCs (DDCs). These DCs are located in the outer skin layers of the epidermis and play a critical role as the first line of defense against pathogens invading the skin. These DCs in the skin, especially DDCs, express TLR2 as well as TLR1 and TLR6. DDCs recognize bacteria and trigger the innate immune response [TLR2 polymorphisms and skin manifestations of sarcoidosis. The variants of the TLR2 gene in DDCs may play a causative role in the development of cutaneous sarcoidosis in a site-specific manner.The current study was designed to determine whether response . Our resSeveral reports have suggested that genetic variants of innate immune receptors might be associated with the risk of developing sarcoidosis . Innate TLR4 polymorphisms in the Caucasian population [TLR4 polymorphisms and sarcoidosis in the Japanese population [TLR4 is a major receptor for lipopolysaccharide (LPS), a component of gram-negative bacterial cell walls. TLR4 and TLR2 signal transduction results in the activation of inflammatory pathways involving nuclear factor-kappa B (NF-kB). Conflicting reports about the association between TLR4 and sarcoidosis have recently been published. It was shown that there is a significant association between patients with chronic sarcoidosis and pulation . Whereaspulation ,37. Our pulation .NOD2 with early-onset sarcoidosis and Blau syndrome. However, no significant associations between the genetic polymorphisms in the NOD2 gene and the risk of adult sarcoidosis were detected [Nucleotide-binding oligomerization domain 2 (NOD2), a member of the NLR (Nod-like receptor) family, is an intracellular microbial sensor. This protein detects muramyl dipeptide (MDP); a component of bacterial peptidoglycans, and induces innate immune responses. Several reports have suggested there may be an association of polymorphisms in detected ,38-41.In the present study, genetic variations in TLR2 did not affect ocular sarcoidosis risk. However, some types of genetic predispositions underlying the pathogenesis of sarcoidosis can lead to ocular inflammation. Microbial pathogens have long been suspected as the cause of sarcoidosis. Therefore, further studies are needed to analyze other genes involved in the innate immune response against bacterial antigens.TLR2 do not appear to be significantly relevant to sarcoidosis in the Japanese population. However, in cutaneous sarcoidosis, rs3804099 and rs3804100 SNPs in TLR2 are slightly associated with clinical disease. Further studies, especially in other ethnic populations, are required to elucidate what association there may be between sarcoidosis and TLR2.In summary, the minor allele frequencies of


Scar sarcoidosis is a rare and uncommon but specific cutaneous manifestation of sarcoidosis. In general it arises in pre-existing scars deriving from mechanical traumas. As most surgeons dealing with scars might not be aware of cutaneous sarcoidosis and its different types of appearance the appropriate staging and treatment might be missed or at least delayed. To our knowledge this is the first case in literature of scar sarcoidosis on a finger.We present a case of a 33-year-old carpenter who developed scar sarcoidosis on his right index finger 4 years after the tendon of the long digital flexor got accidentally cut by an angle grinder. He was referred due to a swelling of the finger suspected to be a malignant soft tissue tumour. The circumference of the affected finger had almost doubled, adding up to 94 mm. Incision biopsy revealed typical noncaseating granulomas. Further investigation showed a systemic extent of the disease with involvement of the lung. A systemic treatment with oral steroids led to an almost full regression of the swelling with restoration of function and resolution of lung infiltrates.In case of a suspicious and/or progressive swelling a definite diagnosis should be achieved by biopsy within a short time to enable a proper treatment. If scar sarcoidosis is proven further investigation is necessary to exclude a systemical involvement. A surgical treatment of the swelling is not indicated. Sarcoidosis is a systemic inflammatory disorder with formation of noncaseating granulomas. The incidence of this multisystem disease is 10–15 cases per 100.000 per year. Broad American studies show that sarcoidosis is 10–17 times more common in African American than in Caucasians-6. Scar We report on a 33-year-old carpenter with a solid swelling in the area of the palmar base and middle phalanx of his right index finger Figure A and B. The patient told us that this scar derived from an injury 4 years ago when the tendon of the deep digital flexor was cut by an angle grinder. After suturing the tendon appearance and function of the finger had been fully restored.Apart from enlarged soft tissue X-rays of the finger were unsuspicious. MRI-scans showed a considerable palmar soft tissue swelling with an enhancement of contrast medium with contact to the flexor tendon Figure A and B. For diagnosis and exclusion of the existence of a malignant and/or local aggressive soft tissue tumour we performed a small incision biopsy. The result of the histopathological examination was scar sarcoidosis with detection of typical noncaseating granulomas Figure . The patOral corticosteroid therapy with an initial daily dose of 40 mg prednisone was started and led to an efficient diminution of the swelling of the right index finger. Hilar lymphadenopathy and lung infiltrates resolved. After 12 months therapy with a stepwise decrease of the corticosteroid dosage the swelling of the finger had almost vanished and function and range of motion was totally restored Figure A and B. Besides lymphadenopathy an involvement of lung or other organs is a feature of sarcoidosis. Even destruction and deformation of bones have so far been described. In a reScar sarcoidosis is a form of cutaneous sarcoidosis. It occurs after mechanical harms of the skin like cuts, but even a venous puncture or a tattoo seem to be sufficient to cause it,3,7,10. Every suspicious or progressive swelling needs a complete work-up with consideration of differential diagnoses and radiological examinations . In case of cutaneous or subcutaneous swelling and a discoloration in the area of an old scar beside keloid a scar sarcoidosis is a possible differential diagnosis. For histological proof and exclusion of a malignant process a biopsy is to be performed. The biopsy offers the right diagnosis and thus leads to an appropriate, reasonable and successful treatment of sarcoidosis. In most cases satisfactory clinical results can be achieved by systemic or topic application of corticosteroids. Sometimes a long term therapy is needed, even if the local manifestation seems to be gone. Further local surgical procedures are not indicated.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.MH and JH are responsible for conception and writing of the manuscript, AS and GG have been involved in drafting and revising the manuscript. CS and CD were responsible for acquisition and analysis of data and findings. All authors read and approved the final manuscript.