Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid expression system in silkworm larvae. Two constructs, full-length L1 (500 a.a) and C-terminal-deleted short L1 (479 a.a), and three PCR-manipulated antigenic loops at amino acids 55–56, 174–175, and 348–349 regions were incorporated with whole enhanced green fluorescent protein (EGFP). Expressed in full, short L1 proteins and variants were purified in heparin affinity column chromatography and confirmed by SDS-PAGE and western blot. The presence of self-assembled virus-like particles (VLPs) and EGFP incorporation on the surface of VLPs were confirmed by the observation of transmission electron and immunoelectron microscopies, respectively. HPV 6b L1 major capsid protein was successfully expressed in silkworm, and effective manipulation on the antigenic regions showed the path to versatile vaccine development based on HPV L1-VLPs.Human papillomavirus (HPV) 6b L1 capsid protein was expressed using the  Virus-like particles (VLPs) are empty virus particles, which lack virus-derived genome DNA or RNA, and composed of virus-capsid or matrix proteins. A non-enveloped virus, VLPs are mainly composed of virus-capsid proteins, which can be self-assembled both in vitro and in vivo. VLPs have been utilized as a vaccine because of its high immunogenicity and capability of inducing cellular and humoral immune responses . AlternBorrelia burgdorferi were inserted into the immunodominant c/e1 epitope region of hepatitis B virus capsid protein without the disruption of VLP’s shape . An enhz et al. ; Nassal z et al. ). In genIn this study, human papillomavirus (HPV) 6b L1 protein was expressed in silkworm larvae, and HPV 6b L1-VLPs were purified from the fat body of silkworms. HPV is a non-enveloped virus that has approximately 50–60 nm in diameter and ~8 kbp of double-stranded circular DNA genome. HPV L1 protein can be self-assembled as a VLP when this protein is expressed in various expression systems . To disHPV 6b L1 protein has a basic domain at its C-terminus, which can bind to genomic DNA and pack into virus particles  . ChimerThree L1-EGFP fusion proteins did not have green fluorescence, but EGFP was displayed on the surface of its capsomeres. To date, a genetic whole protein display system on the VLP has not been established, except for hepatitis B virus nucleocapsid protein VLP . HPV 6b L1 full-length gene . Table EcoRI-KpnI site in pFastBac1. The recombinant BmNPV CP- bacmid containing each L1-EGFP fusion gene was constructed according to the protocol described above.Each L1-EGFP fusion gene was obtained by two-step PCR. To obtain L1-EGFP55, two truncated L1 genes coding a.a.1–55 and 56–500 were amplified by PCR using primers Eco-HPV 6b L1-F and HPV 6b L1-55R-LINKER and HPV 6b L1 LINKER-56F and Kpn-HPV 6b L1-R . Five micrograms of extracted BmNPV CP- bacmid DNA, together with a helper plasmid, was mixed with 1/10 volume of DMRIE-C reagent (Invitrogen) and incubated at room temperature for an hour. This mixture was injected into silkworm larvae. The DNA-injected silkworm larvae were reared using Silkmate 2S  as a diet for 6–7 days and followed by the collection of fat body from silkworm larvae. Collected fat body was stored at −80°C before use.A recombinant BmNPV CPThe fat body was suspended in a homogenate buffer (50 mM Tris–HCl buffer [pH 7.5] containing 150 mM NaCl) and sonicated to extract expressed proteins. The homogenate was centrifuged at 30000 × g for 15 min. A supernatant was applied to the HiTrap heparin affinity column chromatography . After loading the sample, the column was washed with 20-column volumes of homogenate buffer, followed by an elution by NaCl concentration gradient to 2 M. Fractions containing L1 protein were collected and dialyzed against homogenate buffer overnight. The dialyzed sample was applied to Mono S 5/50GL column chromatography . The column was washed with 10-column volumes of homogenate buffer, and proteins were eluted by NaCl concentration gradient to 1 M.The samples were subjected to SDS-PAGE on a 10 or 12% polyacrylamide gel with the Mini-protean II system . The total number of proteins on the SDS-PAGE gel was detected with Coomassie Brilliant Blue (CBB) R-250. In the case of western blot, the proteins found in the gels were blotted onto a polyvinylidene fluoride (PVDF) membrane using the Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). After being blocked in 5% skim milk in a Tris-buffered saline containing 0.1% Tween 20 (TBST), the membrane was incubated in a 1:10000 diluted mouse anti-HPV 16 L1 antibody  for an hour. This antibody can also recognize HPV 6b L1 protein. The membrane was washed with TBST and then incubated in a 1:20000 diluted either anti-mouse or anti-rabbit labeled with horseradish peroxidase (HRP)  for an hour. Detection was performed using ECL Plus Western Blotting Reagent . Specific bands were detected using a Fluor-S/MAX Multi-Imager (Bio-Rad). Protein band intensity was analyzed by Quantity One software (Bio-Rad).The homogenate of the fat body was laid on a 25–60% sucrose density gradient. This suspension was centrifuged at 96000 × g for 3 h at 4°C. Each 0.5 ml of the fraction was collected at the top of the tube. Each fraction was analyzed by SDS-PAGE and western blot.Purified VLPs were immobilized on the grid and blocked with 4% BSA in PBS (pH 7.4). This grid was soaked in either mouse anti-HPV 16 L1 antibody diluted by 30-fold with 1% BSA in PBS for 2 h. After washing with PBS, the grid was soaked in either 10 nm gold-conjugated goat polyclonal anti-mouse IgG+IgM (H+L)  diluted by 25-fold with 1% BSA in PBS for 1 h. After washing with PBS, the grid was stained with 2% phosphotungstic acid. VLPs were observed by JEM-2100F  at 200 kV.


We demonstrate that near-edge X-ray-absorption fine-structure spectra combined with full-field transmission X-ray microscopy can be used to study the electronic structure of graphite flakes consisting of a few graphene layers. The flake was produced by exfoliation using sodium cholate and then isolated by means of density-gradient ultracentrifugation. An image sequence around the carbon K-edge, analyzed by using reference spectra for the in-plane and out-of-plane regions of the sample, is used to map and spectrally characterize the flat and folded regions of the flake. Additional spectral features in both π and σ regions are observed, which may be related to the presence of topological defects. Doping by metal impurities that were present in the original exfoliated graphite is indicated by the presence of a pre-edge signal at 284.2 eV. These occupied bands have counterparts on the unoccupied side of the Fermi edge (σ* and π* bands). In graphene sheets, the pz orbital lies perpendicular to the basal plane of the planar lattice, while the sp2 orbitals are set in the basal plane. The high directionality of these electronic orbitals is strongly reflected in the electronic properties of graphene-based materials [The demonstration of the remarkable transport properties of graphene in 2004 by Geim and Novoselov triggered intense interest in its electronic structure –11. A keaterials .−1, π*) final states being selectively excited when the E-vector lies perpendicular to the basal plane, and  final states being selectively excited when the photon E-vector lies in the basal plane. The presence of defects, nonplanarity and edges relax these symmetry selection rules. The peak energy positions and line shapes of the measured NEXAFS resonances reflect the unoccupied density of states, although the energies and intensities are modified by core–hole interactions [E-vector. Deviation from the expected signal intensity can be associated with nonplanarity, structural defects, etc. [The near-edge X-ray-absorption fine-structure technique (NEXAFS) is ideally suited for the study of graphene-based materials, because the carbon K-edge is very sensitive to the bonding environment, providing diagnostic information about the structure, defects and doping . Photoabractions . When liractions . When coractions –18. Linets, etc. .24H39O5Na) is used to promote graphite exfoliation, resulting in graphene–surfactant complexes having buoyant densities, which vary with the graphene thickness. Raman studies on samples produced by the same technique found no impurities, such as amorphous carbon, in the sample [Here we use NEXAFS spectromicroscopy, performed with the Helmholtz Zentrum Berlin (HZB) full-field transmission X-ray microscope (TXM) installed at the electron storage ring BESSY II , to image sample –22.The setup of the HZB full-field X-ray microscope  is analoWe examined several different flakes to ensure that we had selected a typical specimen; electron microscopy images are given in E parallel to the basal plane). However, we see a residual intensity due to the folding of a few graphene layers, implying that there is a nonzero angle between the polarization vector and the basal plane in places. This is most notable in the central spectrum, confirming that this region corresponds to a fold in the flake.The peak near 292 eV corresponds to the σ* threshold. This peak is composed of two distinct features at 291.5 eV and 292.6 eV. The sharp feature at 291.5 eV is an exciton , its shaE-vector of the light is always in the horizontal plane, which is thus in the plane of the graphene sheets for the unfolded area, but approximately perpendicular to the folds. Thus the blue spectrum , which could appear, for example, due to residual intercalation in the flake.We also scanned over the O 1s spectral range 528–556 eV, and the absence of signal confirms that the sample is essentially oxygen free .In summary, the distinctly different X-ray spectra of the flat and folded regions of the sample, which are related to the strong dichroism of the C 1s spectrum of graphene, have been used in a fitting procedure to generate maps of the flat and folded regions of the flake. A low-lying peak at 284.2 eV is associated with doping states in the electronic structure of the flake induced by metallic impurities present in the graphite that was exfoliated to make the multilayer graphene sample. We tentatively associate a peak at 287.4eV with defects created during the exfoliation using sodium cholate  and acquisition time.The data analysis was accomplished by using aXis2000 . The NEXThe sample was obtained from NanoIntegris  in the form of “PureSheets” (MONO). The aqueous solution (0.5 μg per 10 μL) with an ionic surfactant (2% w/v) contains pristine multistacked graphene flakes with different numbers of sheets, which have not been oxidised, reduced, or chemically modified in any way.Schematic of the morphology of the studied sample, electron microscopy images of typical samples and oxygen K-edge spectrum.File 1Additional figures.


Integration of electronics and photonics for future applications requires an efficient conversion of electrical to optical signals. The excellent electronic and photonic properties of graphene make it a suitable material for integrated systems with extremely wide operational bandwidth. In this paper, we analyze the novel geometry of modulator based on the rib photonic waveguide configuration with a double-layer graphene placed between a slab and ridge. The theoretical analysis of graphene-based electro-absorption modulator was performed showing that a 3 dB modulation with ~ 600 nm-long waveguide is possible resulting in energy per bit below 1 fJ/bit. The optical bandwidth of such modulators exceeds 12 THz with an operation speed ranging from 160 GHz to 850 GHz and limited only by graphene resistance. The performances of modulators were evaluated based on the figure of merit defined as the ratio between extinction ratio and insertion losses where it was found to exceed 220. In recent years it has become evident that bandwidth-limited electrical interconnects can no longer meet the growing demand on data processing and telecommunication traffic. Besides the limited capacity, electrical wires suffer from large energy consumption, signal attenuation and significant operational costs as interconnects densities rise. As opposed to it, photonics enables huge amounts of data to be moved at very high speeds with extremely low power over very small optical waveguides. One of the key components of the future telecommunication networks are optical modulators which serve as the gateway from the electrical to the optical domain. These are particularly attractive for low energy transmitters because they do not have a threshold that could limit the minimum operating energy and they may be easier to integrate with the available silicon platform1211Recently, the broadband electro-absorption modulator based on interband absorption of graphene was demonstrated with an overall length of 40 μm and with a modulation depth of 0.16 dB/μm. This was achieved by placing a double-layer graphene on top of a silicon waveguide0), and accordingly shifts a Fermi level: 0 is the offset voltage caused by natural doping, α is estimated from a simple capacitor model (α = ε0εd/de), ℏ is the Planck constant, vF is the Fermi velocity, and n is the electron/hole doping concentration.The proposed double-layer graphene optical modulator is based on the rib waveguide configuration with the Si rib waveguide deposited on the buried-oxide layer. To maximize the influence of the graphene on the modulator performances, the double-layer graphene was placed between Si layer (slab) and Si ridge . The dou1(ω) + iσ2(ω) of graphene, consisting of interband and intraband contributions , was obtained from the complex optical conductivity σ(ω) = σibutions .The dielectric constant of graphene varies very fast from ε(0.4 eV) = 13.66 + i6.71 to ε(0.7 eV) = −4.21 + i0.69 with a dip in the magnitude curve corresponding to μ = 0.51 where “dielectric graphene” is transforming to “metallic graphene” with ε(0.51 eV) = −0.50 + i0.50.i.e., between a buried-oxide and the Si ridge  and high-κ dielectric respectively .eff = 2.353 for the hBN spacer to neff = 2.450 for the high-κ dielectric spacer. Secondly, for the modulator with the low-κ dielectric spacer (hBN), the minimum mode effective index occurs at μ = 0.495 eV whereas for the high-κ dielectric spacer it shifts to μ = 0.500 eV. At the same time, the maximum mode effective index is not affected by the spacer and remains at μ = 0.530 eV. Apart from it, the difference between maximum and minimum mode effective index increases from Δn = 0.118 for the low-κ dielectric spacer (hBN) to Δn = 0.144 for the high-κ dielectric spacer. In terms of a chemical potential, the change between minimum and maximum mode effective index is Δμ = 0.035 eV for the hBN spacer and Δμ = 0.030 eV for the high-κ dielectric spacer. However, to achieve the same charge carrier mobility in graphene, a lower voltage change is required for the high-κ dielectric spacers. The required voltage change was calculated to be ΔV = 0.505 V for the hBN spacer while for the high-κ dielectric spacer it drops more than four times to ΔV = 0.141 V. Additionally, to introduce a π-phase shift between both arms of the graphene-based Mach-Zehnder interferometer, an active arm length of 6.57 μm is needed for structure with the hBN spacer and only 5.4 μm with the high-κ dielectric spacer.As shown in bit = 0.23 fJ/bit with calculated capacitance of 46 fF and ΔV = 0.141 V.For a 6.57 μm-long and 0.4 μm-wide modulator with the hBN spacer, the capacitance of the device was calculated to be 18 fF, which in tandem with the voltage change of ΔV = 0.505 V needed to ensure a full modulation results in an energy per bit on the order of 1.16 fJ/bit. Conversely, for a 5.4 μm-long and 0.4 μm-wide modulator with a high-κ dielectric it was calculated to be only Ebit = 0.26 fJ/bit for modulator with the hBN spacer and Ebit = 0.96 fJ/bit with the high-κ dielectric spacer respectively.Since the optical absorption in graphene can be controlled through electrical gating, the graphene can be used as the active medium in an optical electro-absorption modulator. Therefore, conductivity change induced by an applied gate voltage has a strong effect on the propagating mode . RegardlOne of the ways to increase the modulator attenuation is to push mode down into a slab such that graphene resides closer to the mode field maximum. Thus, the interaction between mode field and a graphene is maximized. It can be achieved either by increasing the slab thickness or by decreasing the ridge dimensions. In our calculations the slab thickness and ridge width were kept constant while the ridge height was decreased from 260 nm to 200 nm . The anaApart from high modulation speed, small footprint and high modulation strength (efficiency), the novel integrated modulators require large optical bandwidth for applications in on-chip optical interconnects. However, due to the poor electro-optic properties of regular materials the conventional electro-optic modulators suffer either from very large footprint or narrow bandwidth. In comparison to compound semiconductors, the ultrahigh carrier mobility and optical absorption of graphene which is independent of wavelength, enables a new optical modulators with ultra-broad optical bandwidth.To evaluate the bandwidth of the presented rib waveguide photonic modulator, the dependence of chemical potential on graphene's dielectric constant and, in consequence, on the mode effective index and MPA was studied for different wavelengths covering the entire telecommunications bandwidth. As shown in Thus, for a chemical potential of 0.512 eV, the dip in the magnitude of dielectric constant is observed at a wavelength of 1550 nm, what fulfill the requirements of maximum losses of the modulator. Away from the central wavelength, the MPA decreases. Consequently, the wavelength spanning from 1520 nm to 1580 nm only results in a decrease in modulation depth of ~1.5 dB/μm.Consequently, for a lower potential, μ = 0.46 eV, the dip in the real part of the dielectric constant moves towards longer wavelengths with a maximum MPA corresponding to λ ≈ 1710 nm. Thus, for a 1 μm-long modulator, a 3 dB optical bandwidth was calculated to be 14.1 THz near 1550 nm, 12.3 THz near 1480 nm and 13.6 THz near 1710 nm for a chemical potential of 0.512 eV, 0.54 eV and 0.46 eV respectively. For a lower chemical potential, μ = 0.42 eV, a 3 dB modulation requires at least 1.5 μm-long waveguide with the optical bandwidth calculated for a 2 μm-long modulator exceeding 15.1 THz.Apart from the chemical potential, the wavelength dependence of the MPA and mode effective index was studied for different spacer materials and for both supported modes – TE and TM . For a hFor the TE supported mode  with a mTherefore, this modulator shows broadband operation so hundreds of channels from different systems can be processed in the same device because of weak wavelength dependence.Due to the exceptionally high carrier mobility and high saturation velocity, the operation bandwidth is not likely limited by the carrier transit time. The relaxation time is inversely proportional to the degree of crystalline disorder in the graphene so with a high quality of graphene it can operates on the timescale of picoseconds, which implies that graphene-based electronics may operate at 500 GHz. In practice, the maximum operating bandwidth is limited by the RC constant of the device.i.e., small losses for OFF voltage state, to obtain a 3 dB modulation it requires to introduce a modulator to a high loss regime, which is achieved by increasing the chemical potential. For a 0.72 μm-long modulator with a low-κ dielectric spacer (ε = 3.9), a 3 dB modulation is achieved with an operation speed of 0.51 THz while keeping an energy per bit at a low level of 0.96 fJ/bit. Conversely, for modulator with a high-κ dielectric spacer, the active length of around 0.595 μm is sufficient to achieve a 3 dB modulation with an operation speed of 0.16 THz and Ebit = 0.26 fJ/bit. Thus, it can be confirmed there is fundamental energy-speed tradeoff. Additionally, it should be noted that adaptation of longer modulators will not affect the modulation speed but will improve the modulation depth which will be however, at the cost of Ebit. Furthermore, it can be summarized that low-κ dielectric spacers offer a higher operation speed but at the cost of energy and footprint  simulations at the telecom wavelengths using commercial software COMSOL. The FEM is a well know technique for numerical solution of partial differential equations or integral equations, where the region of interest is subdivided into small segments and the partial differential equation is replaced with a corresponding functional. In the calculations, the refractive indexes of the Si rib, the SiO0 = e2/(4ℏ) = 60.8 μS is the universal optical conductance, by using: g = 0.7 nm is a thickness of the graphene layer.To evaluate the mode effective index and mode power attenuation of the considered structure the gate-dependent complex dielectric constant of graphene has to be calculated. The complex dielectric function ε(ω) can be obtained from the complex optical conductivity of graphene, consisting of interband and intraband contributions, 1 = 1.2 ps for interband conductivity and τ2 = 10 fs for intraband conductivity.As the complex conductivity σ depends on the angular frequency ω, the charge particle scattering rate Γ = 1/τ with τ being the relaxation time, the chemical potential μ and temperature TJ.G. conceived the idea, designed the structures, performed theoretical calculations and FEM simulations and wrote the manuscript. D.T. supervised the project.


Interleukin-8 (IL-8) is a potent chemo-attractant cytokine responsible for neutrophil infiltration in lungs with idiopathic pulmonary fibrosis (IPF). The IL-8 protein and mRNA expression are increased in the lung with IPF. We evaluated the effect of single nucleotide polymorphisms (SNPs) of the IL-8 gene on the risk of IPF.One promoter (rs4073T>A) and two intronic SNPs (rs2227307T>G and rs2227306C>T) of the IL-8 genes were genotyped in 237 subjects with IPF and 456 normal controls. Logistic regression analysis was applied to evaluate the association of these SNPs with IPF. IL-8 in BAL fluids was measured using a quantitative sandwich enzyme immunoassay, and promoter activity was assessed using the luciferase reporter assay.p = 0.006 and 0.007, respectively). The IL-8 protein concentration in BAL fluids significantly increased in 24 subjects with IPF compared with 14 controls (p = 0.009). Nine IPF subjects homozygous for the rs4073 T>A common allele exhibited higher levels of the IL-8 protein compared with six subjects homozygous for the minor allele (p = 0.024). The luciferase activity of the rs4073T>A common allele was significantly higher than that of the rs4073T>A minor allele (p = 0.002).The minor allele frequencies of rs4073T>A and rs2227307T>G were significantly lower in the 162 subjects with surgical biopsy-proven IPF and 75 subjects with clinical IPF compared with normal controls in the recessive model (OR = 0.46 and 0.48, The common allele of a promoter SNP, rs4073T>A, may increase susceptibility to the development of IPF via up-regulation of IL-8. Idiopathic pulmonary fibrosis (IPF) is a devastating disease of the idiopathic interstitial pneumonia family. It predominantly affects the lung parenchyma and is characterized by progressive dyspnea and worsening lung function . AlthougCharacteristic of IPF is neutrophilia of the bronchoalveolar lavage fluid. The recruitment and activation of neutrophils plays a fundamental role in the development of lung injury, which precedes aberrant wound repair in the pathogenesis of IPF. InterleIn humans, the gene encoding IL-8 is located on chromosome 4q12-q21 and consists of four exons and three introns . Polymorn = 162) or using radio-clinical criteria (n = 75), i.e., the presence of clinical, functional, and high-resolution computed tomography patterns strongly consistent with IPF. None of the patients with IPF had any evidence of the underlying collagen vascular diseases clinically or by laboratory diagnosis. The institutional review board by Soonchunhyang University hospital for human studies approved the protocol, and informed written consent was obtained from all subjects.Subjects with IPF were recruited from the Korean Cohort of Interstitial Lung Disease. The study population comprised 237 patients with IPF recruited from January 1984 to November 2004 from eight university hospitals. Normal (control) subjects (n = 456) were the spouses of the patients or volunteers from the general population. Control subjects were at least 50 years old, had no respiratory symptoms, exhibited normal FVC and FEV1 , and normal findings on a simple chest posterior-anterior view x-ray. The diagnosis of IPF was based on an international consensus statement by ATS/ERS with compatible findings via surgical lung biopsy  was used to design both the PCR primers and the MGB TaqMan probes. One allelic probe was labeled with the FAM dye and the other was labeled with fluorescent VIC dye. The PCRs were run on the TaqMan Universal Master mix without UNG (Applied Biosystems), with a PCR primer concentration of 900 nM and a TaqMan MGB-probe concentration of 200 nM. The reactions were carried out in a 384-well format in a total reaction volume of 50 ul using 20 ng of the genomic DNA. The plates then were placed in a thermal cycler  and heated to 50°C for 2 min and 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The TaqMan assay plates were then transferred to a Prism 7900HT instrument (Applied Biosystems), which measured the fluorescence intensity in each well of the plate. The fluorescence data files from each plate were analyzed using automated software (SDS 2.1). Detailed information concerning the primers is presented in additional file To genotype polymorphic sites, primers and probes were designed for TaqMang for 5 minutes. IL-8 in BAL fluids was measured using a quantitative sandwich enzyme immunoassay kit . The lower limit of detection for IL-8 was 15.6 pg/mL. Values below this limit were assumed to be 0 pg/mL for the statistical analysis. The inter- and intra-assay coefficients of variance were below 10%. Protein concentration of BAL samples was measured for standardization using a micro BCA protein assay kit .BAL had been performed in the most affected lobe by computed tomography in the 24 subjects without any immunosuppressive therapy and in the right middle lobe of 14 normal controls, as described previously. The supGGTACC(KpnI)ACATTACTCAGAAA-3', reverse; 5'-CCTACGCGT(MluI)GTCTCTGAAAGTTTG-3') for construction of the IL8 reporter plasmid. The amplified fragment of the promoter region of the IL8 gene (-79 to -743 bp from the transcription start site) was cloned using the pGEM-T easy vector system , was ligated with pGL-3 basic Luc+ reporter vector (Promega). Cloned DNA sequences were determined by a DNA direct-sequencing service . One day before transfection, 293 T cells were seeded at 5 × 105 cells per well (6-well plate) in 2 ml with 10% FBS. A 2-μg aliquot of the IL8-pGL3 basic constructor plasmid and 50 ng of PSV-galactosidase reporter vector  were diluted in 250 μl OptMEM  without serum. The 4 μl of lipofectamine 2000  was diluted in 250 ul OptMEM (GIBCO BRL) per well. The diluted DNA was combined with the diluted lipid . Then, 500 μl of transfection complex was added, and the cells were incubated at 37°C with 5% CO2 in humidified air for 48 h. β-galactosidase activity was measured by ortho-nitrophenyl-D-galactopyranoside (ONPG) hydrolysis using β-Gal Assay kit (Promega). The cells were solubilized by scraping with 400 μl of cell lysis buffer of Luciferase Assay System kit (Promega). Luciferase activity was measured using the Luciferase Assay System and luminometer . And the relative luciferase activity was normalized to the protein concentration and β-galactosidase activity.The promoter region of IL-8 was amplified using PCR. The genomic DNA fragment was isolated from B cell lines of the IPF subjects using a genomic DNA preparation kit . The first PCR product was amplified using the following primers: forward; 5'-TGCCTTTGGAAGATTCTGCT-3', reverse; 5'-GCCAGCTTGGAAGTCATGTT-3'. The primary PCR reaction mixture was diluted and used as a template for a nested PCR reaction using the nested primers containing restriction enzyme sequences  developed by Stephens et al. [We applied widely used measures of linkage disequilibrium to all pairs of biallelic loci: Lewontin's D' (|D'|)  and r2. s et al. . The gens et al. . Mantel-s et al. .p < 0.01). The subjects with biopsy-proven IPF and those with clinical IPF had the comparable impairment of FVC and DLCO.Clinical profiles of the study subjects are summarized in Table IL8 gene were genotyped in IPF patients and normal subjects . The LDs were calculated, and haplotypes of IL8 polymorphisms were constructed  and two intronic SNPs (rs2227307T>G and rs2227306C>T) within the T>A and tp = 0.009, Figure p = 0.024). The IL-8 concentration in BAL fluids did not differ among individuals with the rs2227307T>G genotype .The amount of IL-8 protein was measured in BAL fluids from 24 subjects with IPF and 14 NC. IL-8 concentrations were significantly increased in IPF patients compared with NC  vector and ONPG activity. The luciferase activity of the rs4073T>A TT allele was significantly higher than that of the rs4073T>A AA allele  were evaluated in patients with systemic sclerosis with (n = 78) or without fibrosing alveolitis (n = 50), those with cryptogenic fibrosing alveolitis (n = 71), and normal healthy subjects in the UK [IL8 with the risk of pulmonary fibrosis. The discrepancy between ours and the previously reported results may be due to the small study population in the previous study[Our logistic regression analysis of a case-control study determined that the diseases . Two SNPn the UK . These sous study or to etous study and bronous study. In addious study. This daous study. Further>A compared to those with the minor allele. This result indicates that the rs4073T>A allele within the promoter may result in increased IL-8 production when compared with the minor allele.We did not validate the association between the SNPs of the IL-8 gene in an independent replication population. We evaluate the effect of the SNP on IL-8 gene or protein expression instead. We measured IL-8 protein concentrations in the lung. IL-8 protein was increased in the BAL fluids of patients with IPF compared with normal controls. The IL-8 protein level in BAL fluid was significantly increased in the subjects with IPF having the common allele of rs4073TT>A TT allele was significantly stronger than that of the rs4073T>A AA allele. This is in accordance with a previous study, which reported that the rs4073T>A TT allele exhibited 2- to 5-fold stronger transcriptional activity than did the rs4073T>A AA counterpart [T allele on the promoter may enhance the IL-8 transcription compared with the rs4073 A allele. Putative transcription factor binding sites in the promoter of the IL8 gene were searched using the TFSEARCH and TESS websites. The candidate binding protein for the transcription of IL8 at rs4073 was eEF1A1 : Idiopathic pulmonary fibrosis; (IL-8): Interleukin-8; (ONPG): ortho-nitrophenyl-D-galactopyranoside; (|D'|): Lewontin's D'; (MHC): Mantel-Haenszel chi-square;The authors declare that they have no competing interests.MHA performed all experimental steps; BLP, SHL, and HDS analyzed statistics and wrote the manuscript; SWP, JSP, DJK and ASJ provided experimental assistance; JSP, HKS, SU, YK, YWK, SKH, KSJ, KYL, SHJ, JWP, BWC, IWP, MPC, JWS, DSK and YSS supervised this project; CSP conceptualized of the study and wrote the first draft of the manuscript. All authors read and approved the final manuscript.http://www.textcheck.com/certificate/ox3Vhg.The authors thank the editors from textcheck.com, both native speakers of English, for their proofreading for grammar and typographic errors. For a certificate, see The fluorescence labeled allelic probe for amplification of IL8, IL8RA and IL8RB genes. The data provided represent the probe for amplification of IL8, IL8RA and IL8RB genes.Click here for fileSNPs on the map of the IL8 gene, linkage disequilibrium, and haplotypes of IL8 genes. The figure provided represent the map of the IL8 gene, linkage disequilibrium, and haplotypes of IL8 genes.Click here for fileThe Minor allele frequency (MAF), Heterozygosity, Hardy-Weinberg equilibrium (HWE) of IL8 gene polymorphisms. The data provided represent the MAF, HWE of IL8 gene polymorphisms.Click here for fileThe candidate binding protein for the transcription of IL8 at rs4073. The figure provided represent the putative transcription factor binding sites in the promoter of the IL8 gene.Click here for file


Trans-activation Response DNA-binding Protein-43 (TDP-43) lesions are observed in Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Lobar Degeneration with ubiquitin inclusions (FTLD-TDP) and 25–50% of Alzheimer's Disease (AD) cases. These abnormal protein inclusions are composed of either amorphous TDP-43 aggregates or highly ordered filaments. The filamentous TDP-43 accumulations typically contain clean 10–12 nm filaments though wider 18–20 nm coated filaments may be observed. The TDP-43 present within these lesions is phosphorylated, truncated and ubiquitinated, and these modifications appear to be abnormal as they are linked to both a cellular heat shock response and microglial activation. The mechanisms associated with this abnormal TDP-43 accumulation are believed to result in a loss of TDP-43 function, perhaps due to the post-translational modifications or resulting from physical sequestration of the TDP-43. The formation of TDP-43 inclusions involves cellular translocation and conversion of TDP-43 into fibrillogenic forms, but the ability of these accumulations to sequester normal TDP-43 and propagate this behavior between neurons pathologically is mostly inferred. The lack of methodology to produce soluble full length TDP-43 and recapitulate this polymerization into filaments as observed in disease has limited our understanding of these pathogenic cascades.The protocols described here generate soluble, full-length and untagged TDP-43 allowing for a direct assessment of the impact of various posttranslational modifications on TDP-43 function. We demonstrate that Casein Kinase II (CKII) promotes the polymerization of this soluble TDP-43 into 10 nm diameter filaments that resemble the most common TDP-43 structures observed in disease. Furthermore, these filaments are recognized as abnormal by Heat Shock Proteins (HSPs) which can inhibit TDP-43 polymerization or directly promote TDP-43 filament depolymerization.These findings demonstrate CKII induces polymerization of soluble TDP-43 into filaments and Hsp90 promotes TDP-43 filament depolymerization. These findings provide rational for potential therapeutic intervention at these points in TDP-43 proteinopathies. Trans-activation response DNA-binding protein (TDP-43) is highly conserved among species and ubiquitously expressed in humans Since the identification of TDP-43 in neuronal cytoplasmic inclusions (NCI) and dystrophic neurites (DN) in FTLD and neuronal intranuclear inclusions (NII), skein-like inclusions and glial inclusions in ALS, it has become apparent that abnormal TDP-43 localization and accumulation into amorphous aggregates and filamentous structures must be related to numerous pathological stressors in vitro and ubiquitin-positive, sarksoyl-insoluble TDP-43 aggregation in vivoThe observation abnormal nuclear and cytoplasmic TDP-43 aggregation can correlate with disease progression in ALS and FTLD indicates that if this accumulation can be inhibited the resulting phenotypic changes may be alleviated magnifying the importance of understanding the mechanisms leading to these TDP-43 lesions TDP-43 forms amorphous aggregates when isolated but can assemble into filamentous structures when prompted by an activating or nucleating event indicating that endogenous TDP-43 solubility is likely regulated by its binding partners and that when not sequestered it is vulnerable to either aggregation or polymerization in vitro assembly of tagged, full-length, wild-type TDP-43 demonstrated that the protein is extremely aggregation-prone when purified to homogeneity in2 as reported, but this led to negligible Thioflavin S fluorescence characteristic of cross-β amyloid structures as shown in Previous reports on the in vivo, reactions were set up using CKI or CKII, for which TDP-43 is a known substrate. These results demonstrated this preparation of rTDP-43 is recognized by these kinases and is in a conformation that permits phosphorylation. As shown in Non-specific aggregation provides little detail as to the possible cellular activity of these preparations so to characterize the potential functionality of this soluble TDP-43 its ability to interact with known binding partners was examined. First, the ability to bind a physiologically relevant substrate was confirmed. TDP-43 is reported to specifically bind TG-repeated single-stranded DNA sequences, but not AC-repeated DNA sequences, and this assay has been used to demonstrate functionality using other tagged rTDP-43 preparations Previous reports demonstrated that phosphorylation of enriched rTDP-43 fractions with CKI would promote the polymerization of 10 nm wide filaments that could be immuno-gold labeled using anti-TDP-43 antibodies Aggregation of rTDP-43 preparations is well documented, but the presence of pore-like structures has not been reported. As shown in It is difficult to assess the true filament assembly properties of TDP-43 using traditional high-throughput techniques as the filaments are reported to be predominantly ThT- negative and TDP-43 aggregates into amorphous precipitates so readily it precludes the use of light scattering or pelleting assays that rely on ultracentrifugation in vitro while also promoting Tau function in vivoGiven that the same kinases are implicated in both the polymerization of TDP-43 and Tau filaments, and HSPs can inhibit the polymerization of Tau oligomers and filaments TDP-43 expressed using recombinant methods has been shown to be highly prone to aggregation, and this has limited research efforts directed at understanding how full-length wild-type TDP-43 can assemble into ordered filaments. Here, the data demonstrate the TDP-43 Figure 1The development of biochemical techniques to produce soluble wild-type recombinant TDP-43 without protein tags or the use of chemical stabilizers and denaturants provides the cleanest system to study the effects of post-translational protein modification on TDP-43 function. This should permit the effects of modifications such as phosphorylation, acetylation and proteolysis on TDP-43 function, protein folding, stability, oligomerization and polymerization to be studied effectively without the concerns that arise from the use of artificial constructs or the chemical modifiers used in other studies. Furthermore the assembly of this soluble rTDP-43 into 9.9 +/− 0.9 nm diameter filaments that characterize the predominant 10–12 nm forms of filamentous TDP-43 inclusions observed in ALS, FTLD and AD provides the best resource to date for studying the effects of TDP-43 filament assembly on TDP-43 function and the ability of these filaments to sequester wild-type TDP-43 or other cellular components. These filaments should more accurately represent the structures found in disease because they are formed from purified TDP-43 which does not contain potential bacterial proteins that may have contaminated enriched TDP-43 fractions used in the only other study reporting 10 nm wide filaments with TDP-43 phosphorylation The results reported here using CKII most closely recapitulate what might be expected when soluble and mislocalized, but not yet fibrillogenic, TDP-43 finds its way to the cytoplasm where it might be further modified and converted to polymerization competent forms. CKII levels are found to be up regulated in AD cases The recognition and disassembly of these CKII induced rTDP-43 filaments by Hsp90 suggest the phosphorylated TDP-43 is abnormal and contains exposed Hsp90 binding sites further strengthening the argument that these filaments are pathologically relevant. The generation of filamentous TDP-43 should facilitate the determination of the role particular TDP-43 filaments play in sequestering normal TDP-43 intraneuronally or through cell-to-cell propagation interneuronally, and the functional consequences and cellular responses of this conversion of normal TDP-43 into filamentous aggregates can be examined. Polymerized rTDP-43 filaments could be introduced into cells using protein transfection reagents, and the ability of filamentous TDP-43 to recruit normal endogenous TDP-43 from the nucleus could be determined using immunofluorescence, while the functional effects of seeding could be assessed through the examination of TDP-43 RNA client transcripts Filamentous TDP-43 inclusions form pathological lesions in some of the most prominent and debilitating neurodegenerative diseases including the motor neuron disease ALS where TDP-43 mutations are causative The polymerization of soluble wild-type TDP-43 into 10 nm filaments characteristic of ALS, FTLD and AD upon CKII phosphorylation suggests this signaling pathway is involved in disease pathogenesis. The novel techniques described here for untagged TDP-43 purification and the unprecedented demonstration that CKII can drive TDP-43 filament assembly that outpaces its generic aggregation indicates that free TDP-43 deposition may be controlled in a disease specific manner resulting from the confluence of multiple independent mechanisms. The identification of CKII as a kinase that can promote TDP-43 assembly without any further modification means this pathway may be susceptible to therapeutic modification in the treatments of these diseases, and the binding of Hsp90 to TDP-43 following phosphorylation by CKII demonstrates this protein is recognized as abnormal. The potential of targeting this CKII phosphorylated TDP-43 though pharmacological manipulation of the Hsp90 chaperones system is enhanced dramatically by the demonstration Hsp70 and Hsp90 can both inhibit CKII mediated TDP-43 filament assembly and promote the depolymerization of TDP-43 filaments once formed. Indeed CKII phosphorylation of aggregates composed of only carboxy-terminal fragments of TDP-43 has been shown to target these for disassembly in cell culture, but it was not determined whether the HSPs then promote any beneficial TDP-43 degradation also http://www.mayo.edu/research/departments-divisions/department-neuroscience-florida/brain-bank). Written informed consent from the donor or the next of kin was obtained for brain autopsy and the use of tissues for research purposes. All studies involving autopsy materials were approved by the Mayo Foundation Institutional Review Board. All animal procedures were approved by the Mayo Institutional Animal Care and Use Committee (Protocol # A30911) and are in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication 80–23).Human postmortem brain tissue was provided by the Mayo Clinic Brain Bank  was purchased from Fisher Scientific Incorporated in Pittsburg PA, and Phosphatase Inhibitor Cocktail II (#5726) and III (#P0004) were from Sigma Corporation in Cream Ridge NJ. DNAase (#5025), ADP-agarose (#A2810) and dithiothreitol (#D0632) were from Sigma-Aldrich Corporation in St. Louis MO. Bicinchoninic acid reagents  and bovine serum albumin  were from Pierce Thermo Fisher Incorporated in Rockford IL. Electron microscopy carbon/formvar coated 400 mesh copper grids (#FCF400-Cu) were purchased from Electron Microscopy Sciences in Hatfield PA. The kinases used were obtained as follows: Casein Kinase I (#P6030S) and Casein Kinase II (#P6010L) were from New England Biolabs in Ipswich MA. Streptavidin labeled horse radish peroxidase (#016-030-084) was from Jackson ImmunoResearch Laboratories in West Grove PA.2, 1 mM dithiotreitol, 1 mM PMSF, 1x PIC and 21 units/ml DNAase, pH 7.4) and lysed by probe sonication for 30–90 seconds on ice at 50% power in a Bronson sonicator. Cell debris and inclusion bodies were removed by centrifugation at 12,000×g for 20 minutes, and saturated ammonium sulfate solution was added to 15% saturation while on ice for 10 minutes. The precipitated proteins were collected by centrifugation at 12,000×g for 10 minutes, and the pellets were resuspended in 8 ml of assembly buffer  by rocking at 4°C for 30 minutes. The samples were clarified at 12,000×g for 10 minutes and dialyzed 3 times against 200 volumes of assembly buffer. The rTDP-43 was then clarified again at 12,000×g for 10 minutes, and then aliquotted and snap frozen in liquid nitrogen before storage at −80°C. Before use, the soluble rTDP-43 was collected after clarification at 100,000×g for 37 minutes, and the protein concentration was determined by BCA analysis with BSA standards. Purity was analyzed by SDS-PAGE and Coomassie brilliant blue staining.Human TDP-43 cDNA was cloned into pET30b for expression under the T7 promoter. Overnight 3 ml culture were prepared in Luria broth with 20 µg/ml kanamycin using frozen glycerol stocks and grown overnight with shaking at 37°C. These were used to inoculate 300 ml of LB media again with 20 µg/ml kanamycin, and these were grown with shaking at 37°C until the OD600 reached 0.5 absorbance. These cultures were then chilled at 4°C for 30 minutes without shaking before induction and then transferred to a 16°C shaker for overnight induction with 0.3 µM Isopropyl β-D-1-thiogalactopyranoside. Cells were then collected by centrifugation, washed 1x in Tris-buffered saline , collected and frozen at −80°C. These cells were then resuspended in lysis buffer  12 or (AC)12 DNA probes were incubated with soluble 0.1–1.0 µM TDP-43 proteins in TDP assembly buffer for an hour at 37°C. The incubation reaction was then filtered through a pretreated nitrocellulose membrane overlaid on an assembly buffer equilibrated nylon membrane in a 96-well slot blot apparatus. After washing with 3 ml of assembly buffer per well, the bound biotin-labeled (TG)12 or (AC)12 DNA probes were cross-linked to the membranes by shortwave ultraviolet radiation for 2 minutes on each side. The membranes were then blocked with 5% milk in TBS with 0.1% Triton X-100 for 60 minutes and then incubated with streptavidin-HRP diluted at 1∶5000 for 60 minutes at room temperature. The biotin-labeled DNAs on the membranes were developed with standard ECL reagents. To reduce nonspecific single-stranded DNA binding to the nitrocellulose membranes that was not TDP-43 dependent, the membranes were presoaked for 60 min in 0.4 M KOH, rinsed in water and then equilibrated in assembly buffer for 30 minutes before slot blotting. The nylon membranes were not treated, but they were also incubated in assembly buffer for 30 minutes prior to the filtration 2, pH 7.5) as recommended by the kinase provider. These reactions contained 2.5 µM rTDP-43 supplemented with 500 µM ATP and the kinase indicated. Control reactions used heat denatured kinases or 1 x KB in substitution of the kinase volume. Typically 1 µl of kinase was used in a 30 µl reaction. All reactions were incubated at 37°C without shaking for the time indicated, and then these were immediately prepared for western blotting or electron microscopy as described. Experiments to examine the effects of Hsp90 on rTDP-43 assembly were carried out as described except Hsp90 was added at 0.0–2.5 µM for the duration of the phosphorylation assembly reactions. Experiments to examine the effects of Hsp90 on pre-assembled rTDP43 filaments were carried out as described except the concentration of rTDP-43 and kinases and ATP were proportionally scaled up by a third to 3.25 µM rTDP-43, 1.3 µl CKII and 667 µM ATP and incubated as described for 2 hours to reach maximal filament assembly before adding 4× Hsp90 so the final rTDP-43 concentration was again 2.5 µM and the Hsp90 was 0.0–5.0 µM. These disassembly reactions were then incubated as described for 0–4 hours, and grids for EM were taken during this time as indicated.Recombinant TDP-43 filament phosphorylation assembly reactions were set up in 1 x AB buffer supplemented with 1x kinase buffer  for 60 minutes. Blots were then incubated overnight with either total TDP-43 antibody or phosphorylation specific S409/410 TDP-43 antibodies. Blots were developed using ECL Plus reagents and traditional film.Tissue was collected from the parahippocampal gyrus of formalin-fixed brains from pathologically confirmed cases of FTLD-TDP. All tissue was dehydrated in ethanol and embedded in LR White Reaction samples were diluted 4x in 1XTBS, and 10 µl was adsorbed onto carbon/formvar-coated 400 mesh copper grids (EM Sciences) for 45 seconds. These were then stained with 2% uranyl acetate for 45 seconds, and the grids were examined with a Philips 208S electron microscope . For quantification, twelve images were collected randomly at three predetermined coordinate locations that remained fixed throughout these studies.These 12 images were collected at 5,000× magnification, and the average filament number, average filament length and total filament length per field was measured manually using Image J freeware TDP-43 filament assembly was monitored by fluorescence of thioflavin S binding using a Cary Varian Eclipse Spectrofluorimeter  with an excitation wavelength of 440 nm and a slit width 10 nm, and emission spectrum collected from 460–600 nm and a 10 nm slit width. Measurements were performed at room temperature after incubating 30 µl of TDP-43 phosphorylation reaction with 90 µl of 0.006 mg/ml thioflavin S for 30 minutes. Negative controls included buffer alone, kinase and buffer, and reactions containing TDP-43 alone, but each with thioflavin S as above. Thioflavin S binding intensity was measured by integrating the curve between the ranges of 460–600 nm using the Cary Eclipse Scan software.


The right temporo-parietal junction (TPJ) is widely considered as part of a network that reorients attention to task-relevant, but currently unattended stimuli . Despite This viUnderstanding TPJ is important not only because attention is a core cognitive function that contributes to many other domains of information processing, but also because TPJ has been identified as a key structure in seemingly unrelated domains . In all of these domains, the attentional account of TPJ has played a critical role to either explain function or fuel debate over functional specialization . In bothThe purpose of this review is to critically evaluate two core assumptions about the role of TPJ in attentional control in order to highlight the need to reconsider theories of TPJ function. As an alternative account, we then propose the “contextual updating” hypothesis in which the function of TPJ is to update internal models of the current behavioral context for the purpose of generating appropriate actions; contextual updating is therefore particularly important when unexpected stimuli occur. A similar idea has long existed within the event-related potentials (ERP) literature with respect to the P300 component, which is thought to have a number of neural sources including TPJ .While the contextual updating hypothesis implies an unitary account for TPJ function, we readily acknowledge that it is likely that there are multiple specialized sub-regions in “TPJ” . HoweverThe review is structured in five sections: the first reviews the evidence for the role of TPJ in attention based on human functional magnetic resonance imaging (fMRI) and neuropsychology; the second reconsiders the evidence for two commonly accepted characteristics of TPJ in attention; the third discusses the contextual updating hypothesis for TPJ with respect to studies of attention; the fourth section evaluates (necessarily in brief) the generalizability of the contextual updating hypothesis to other psychological domains; finally, in the fifth section, we discuss recent anatomical work that contributes importantly to the anatomical definitions for the region of cortex broadly referred to as TPJ.22.1Perhaps the most prevalent theory of TPJ function, particularly in the right hemisphere, comes from Maurizio Corbetta and colleagues . In thisIn addition to spatially “invalid” targets, TPJ is also sensitive to relevance defined by other stimulus dimensions such as: target-similarity , cues th2.2Support for the notion that right TPJ plays a crucial role in reorienting attention to unexpected sensory stimuli comes from spatial neglect. Spatial neglect is a neuropsychological phenomenon that is defined by behavioral symptoms of a deficit in spontaneously reorienting attention to stimulus information in the contralesional visual field. Spatial neglect cannot be attributed to either sensory or motor defects alone  and is a3While the neuroimaging and neuropsychological data converge in support of the 3.1If TPJ is to send a fast-latency “interrupt” signal to the dorsal network that reorients attention, it logically follows that TPJ must generate an earlier output signal in response to sensory inputs than the dorsal system regions. However, the evidence from electrophysiology and TMS suggests that FEF actually responds earlier, not later, to sensory information than TPJ. For example, the latency of stimulus-evoked responses in FEF range from 50 to 138 ms  and whennot stimulus onset) and lasted 200 ms with an approximate peak at 200 ms  following selective damage to each hemisphere . Those fA more recent model for neglect by There appear to be clear hemispheric differences in attentional control, but there is also increasing evidence that the left TPJ plays an important role in control processes related to attention. For example, many studies in which right TPJ is found in response to task-relevant stimuli also report left lateralized activation in similar conditions, although the left hemisphere activation is rarely investigated in detail . A notabThe left TPJ is also frequently activated in studies of memory . This ha4In the previous section we reviewed evidence against the idea that TPJ provides an early stimulus-driven signal that triggers attentional reorienting. However, the existing data also clearly indicate that TPJ is engaged in processing task-relevant stimuli, particularly when they are unexpected. Here we suggest that the observed effects in TPJ can be understood in terms of “contextual updating” (see above) . AlthougThe contextual updating hypothesis is consistent with extant findings of TPJ activation in standard attentional tasks. For example, invalid targets in the Posner paradigm are infrequent and violate expectations and therefore produce an update to stimulus-response mappings of what action to take and what to expect in the future. Similarly, a valid target would also be predicted to activate TPJ, albeit to a lesser degree, because it signals the need to make a context-appropriate response based on the new occurrence of a target stimulus, but there is no violation of expectations .The contextual updating hypothesis provides similar explanations for why target-colored distracters produce TPJ activation . The appThe contextual updating hypothesis also fits in well with the overall goal of organisms to establish predictive expectancies that facilitate behavioral actions . We do nThe flexible integration between sensory, motor, and decisional regions can also be illustrated between domains. Although there are still many details that need to be specified with regard to the contextual updating hypothesis, the framework is a significant deviation from the current theories and therefore stands as a reasonable alternative hypothesis: instead of initiating “bottom-up” attentional reorienting, we hypothesize that TPJ has a more general post-perceptual function in evaluating and integrating stimulus information with internal models of task performance and expectations. We note that the contextual updating hypothesis does not leave attentional reorienting without a mechanism. It is reasonable that unattended, but relevant stimuli be assigned attentional priority through “dorsal attentional” network regions in a manner similar to other salient or relevant stimuli . More st5Any theory of TPJ function must go beyond any one sub-discipline in order to account for its involvement in different cognitive processes. The contextual updating hypothesis is appealing because similar ideas have been suggested in many domains. For example, it has been proposed that right TPJ encodes an internal model of the body and engages in a “test-for-fit” process to determine whether sensory events belong to one's own body . This hySimilarly, the contextual updating hypothesis helps to reconcile the debate between TPJ as a cognitive module for theory-of-mind (ToM) and attention tasks by suggesting a common computation occurs in both situations. The contextual updating hypothesis specifically refers to the TPJ activation and does not suggest that attentional reorienting itself does not occur during ToM . While there may be anatomically separate functional modules in the posterior parietal cortex that map onto existing cognitive domains , we arguAlthough a comprehensive review of other cognitive domains is beyond the scope of this current review, we note that the idea of contextual updating would be compatible with a number of other studies showing TPJ involvement in altruism  and empaThe broader characterization of TPJ in contextual updating is consistent with its role in attentional and social-cognitive domains. Although these situations are superficially very different, they all involve a stimulus-triggered event update of internal mental models of the external environment (or person), which are then used to predict future outcomes . A challenge for future studies of TPJ will be to provide insight into how the general function of contextual updating is made specific for a highly variable range of tasks and cognitive domains .6A critical aspect of the discussion about TPJ relates to its anatomical location. Thus far, we have used the term TPJ loosely to refer to a cluster of regions in the inferior parietal lobule extending into the superior temporal gyrus see . One posNevertheless, there are general conventions and TPJ is typically described as being near “the posterior sector of the superior temporal sulcus (STS) and gyrus (STG) and the ventral part of the supramarginal gyrus (SMG)” p. 307  cf. Fig. More prOne exciting recent development has been creation of a new cytoarchitectonic maps based on postmortem human brains . WhereasInterestingly, studies that have used the cytoarchtectonic parcelation scheme to identify fMRI activations in TPJ, have been more mixed. For example, in a cued attention task, invalid targets activated both PGa and PFm, regions in the anterior portion of the angular gyrus and posterior supramarginal gyrus . This is7Regions of the inferior parietal lobe within the supramarginal and angular gyri have commonly been referred to collectively as the temporoparietal junction. In the extant literature on attention, the exact location of TPJ has varied between studies considerably since the definition is frequently based on functional contrasts across a group rather than individual functional or anatomical locations. It is possible that different studies are actually referring to different subregions that are near anatomical neighbors. Although it may well be the case that the results we reviewed will someday be discovered to be anatomically distinct, the review was aimed at addressing the question of what common computation the results attributed to TPJ might perform; even if the specific nature of representations between subregions are separable, neighbors are likely to be computationally similar.Here we have proposed that a likely framework for TPJ function can be found in the idea of contextual updating: that the purpose of TPJ is to update internal models of the environment (including other people) for the purpose of constructing appropriate expectations and responses. These may occur for attention to simple stimuli as well as for understanding social cognition and our bodies. The anatomical connectivity between TPJ and the medial temporal lobe and frontal regions situates it well to integrate internal (or memory) representations of the current context with context-appropriate sensory-to-motor transformations. Such a framework describes well the myriad deficits that are involved with brain damage to this region and the many different tasks that produce activation in this region, but there is still much to be done to characterize the functional profile of TPJ as well its anatomical demarcations. Nevertheless, we hypothesize that the context of current events and behavioral goals will be critical to the function of this region. Much is still to be discovered about the function of TPJ, but the evidence indicates that we need to reevaluate the idea that it is involved in sending a fast-latency interrupt signal to dorsal attentional control regions and that its function is right-lateralized.


Propionibacterium acnes is the only microorganism that has been isolated from sarcoid lesions. Many P. acnes have been detected in sarcoid lymph nodes using quantitative PCR and in sarcoid granulomas by in situ hybridization. P. acnes trigger factor protein causes a cellular immune response only in sarcoid patients and induces pulmonary granulomas in mice sensitized with the protein and adjuvant, but only those with latent P. acnes infection in their lungs. Eradication of P. acnes by antibiotics prevents the development of granulomas in this experimental model. Although P. acnes is the most common commensal bacterium in the lungs and lymph nodes, P. acnes-specific antibody detected the bacterium within sarcoid granulomas of these organs. P. acnes can cause latent infection in the lung and lymph node and persist in a cell-wall-deficient form. The dormant form is activated endogenously under certain conditions and proliferates at the site of latent infection. In patients with P. acnes hypersensitivity, granulomatous inflammation is triggered by intracellular proliferation of the bacterium. Proliferating bacteria may escape granulomatous isolation, spreading to other organs. Latent P. acnes infection in systemic organs can be reactivated by another triggering event, leading to systemic sarcoidosis.Sarcoidosis is a systemic granulomatous disease of unknown etiology.  Sarcoidosis is one of the best-known systemic granulomatous diseases. Despite intensive investigation, however, the etiology of sarcoidosis has remained unresolved for more than 100 years . SeveralMycobacterial and propionibacterial organisms are the most commonly implicated etiologic agents based on studies using polymerase chain reaction (PCR) of microbial DNA from these organisms in tissues from sarcoid patients around the world –7. DiffeMycobacterium tuberculosis and a recombinant trigger factor protein from Propionibacterium acnes, have been examined in sarcoid patients and control subjects [Immune responses against microbial antigens from these organisms, such as ESAT-6 and KatG peptides from subjects –12. ImmuGranuloma formation results from the persistence of a nondegradable product or a hypersensitivity response . The twoP. acnes is so far the only microorganism isolated from sarcoid lesions by bacterial culture [P. acnes is an anaerobic, nonspore-forming, gram-positive rod bacterium indigenous to the skin and mucosal surfaces. A series of Japanese studies has provided accumulating evidence for a role of P. acnes in sarcoidosis. In this paper, we propose mechanisms of granuloma formation in response to this indigenous bacterium in subjects with sarcoidosis based on our results obtained using histopathological and experimental approaches and introduce a new concept of endogenous infection caused by hypersensitivity to indigenous bacteria.  culture , 15. P. The lung and its draining lymph nodes are the organs most commonly affected by sarcoidosis. As the lung constantly encounters airborne substances, including pathogens, many researchers have considered infection to trigger sarcoidosis and have thus tried to identify possible causative transmissible agents and their contribution to the mechanism of sarcoid granuloma formation , 17. P. acnes, and no other microorganism, was isolated from the large number of samples [P. acnes was isolated in culture from biopsy samples of 31 (78%) of 40 lymph nodes from 40 patients with sarcoidosis [P. acnes during biopsy, and the results of both studies were the same.In the late 1970s, a large Japanese research project conducted by many clinicians and microbiologists with support by a grant from the Japanese Ministry of Health was organized to seek the pathogens responsible for sarcoidosis. Extensive trials were performed to isolate microorganisms, including bacteria, viruses, and fungi, from tissue samples  affected by sarcoidosis. Only  samples . P. acnecoidosis , whereasP. acnes was isolated from 24 of 43 lungs and 8 of 11 mediastinal lymph nodes, mostly in pure culture. P. acnes was isolated from 10 of 20 gastric and 3 of 12 intestinal lymph nodes; intestinal bacteria were also numerous. The number of P. acnes cells isolated was usually no more than 500 colony forming units (CFUs)/g in the lungs and lymph nodes. Of 43 lungs from patients without sarcoidosis, only 4 (9%) had exceptionally high numbers of P. acnes cells. Random amplified polymorphic DNA analysis was used to compare the DNA of 45 isolates of P. acnes from these patients, 39 isolates from sarcoid lymph nodes, and 67 isolates from normal skin, conjunctiva, and intestine. The P. acnes strains in the lung and mediastinal lymph nodes differed genetically from those in the skin. Therefore, contamination from the skin during operative or culture procedures seems unlikely. These findings suggest that P. acnes normally resides in peripheral lung tissue and mediastinal lymph nodes.Ishige et al. cultured peripheral lung tissue and various lymph nodes obtained from patients with diseases other than sarcoidosis . P. acneP. acnes are essential for linking this indigenous bacterium to the cause of sarcoidosis because infectious granulomas are commonly caused by intracellular pathogens. Furukawa et al. examined the cell invasiveness and serotype of P. acnes isolates from lymph nodes affected by sarcoidosis, together with isolates from nonsarcoid tissue obtained from the lymph nodes, lungs, prostate, skin, conjunctiva, and intestine [P. acnes isolates into HEK293T cells was examined by cell invasion assay according to the method described by Cue and Cleary [Studies of cell invading ntestine . The invd Cleary  and intrSome investigators in Europe using PCR assays detected mycobacterial DNA in samples of affected tissue from patients with sarcoidosis –23, but P. acnes, P. granulosum, and M. tuberculosis in histologic sections of lymph nodes from patients with sarcoidosis, tuberculosis, or gastric cancer [M. tuberculosis were found in samples from all 15 patients with tuberculosis, 3 patients with sarcoidosis, and 1 control sample. Genomes of P. acnes were found in 12 of 15 patients with sarcoidosis, 2 tuberculosis patients, and 3 controls. The difference in the estimated number of P. acnes genomes between individuals with and without sarcoidosis was similar to that in the number of M. tuberculosis between people with and without tuberculosis. Biopsy samples from the three patients with sarcoidosis but without P. acnes all contained many P. granulosum DNA. These findings suggest that propionibacteria resided in or proliferated ectopically in the sarcoid lesions, whether or not there was a connection with the disease. Propionibacteria are more likely than mycobacteria to cause sarcoidosis.Ishige et al. used quantitative PCR to search for bacterial genomes of c cancer . They exP. acnes, P. granulosum, M. tuberculosis, M. avium subsp. paratuberculosis, and Escherichia coli (as the control) were estimated by quantitative real-time PCR. Either P. acnes or P. granulosum was found in all but two of the sarcoid samples. M. avium subsp. paratuberculosis was not found in any sarcoid sample. M. tuberculosis was found in only 0% to 9% of the sarcoid samples, but in 65% to 100% of the tuberculosis samples. In sarcoid lymph nodes, the total numbers of genomes of P. acnes or P. granulosum far exceeded those of M. tuberculosis. P. acnes or P. granulosum was found in 0% to 60% of the tuberculosis and control samples, but the total numbers of genomes of P. acnes or P. granulosum in these samples were lower than those found in sarcoid samples. Propionibacteriium spp. are more likely than Mycobacteriium spp. to be involved in the etiology of sarcoidosis, not only in Japanese but also in the European patients with sarcoidosis.The international collaborative study evaluated the possible etiologic link between sarcoidosis and the suspected bacterial species . FormaliIn situ localization of P. acnes genomes in sarcoid lymph nodes may help to elucidate an etiologic link between sarcoidosis and this indigenous bacterium. Formalin-fixed and paraffin-embedded biopsy samples of lymph nodes from nine patients with sarcoidosis, nine patients with tuberculosis, and nine patients with nonspecific lymphadenitis as controls were examined by quantitative real-time PCR (QPCR) for P. acnes and by in situ hybridization (ISH) using catalyzed reporter deposition (CARD) for signal amplification with digoxigenin-labeled oligonucleotide probes that complemented 16S rRNA of P. acnes [μm2 of tissue sections from inside and outside sarcoidosis and tuberculosis granulomas and from control lymph nodes were counted. The number of genomes determined by QPCR was examined for correlation with the mean signal count by ISH with CARD. In sarcoid samples, one or several signals were detected in the cytoplasm of some epithelioid cells in granulomas . The accumulation of P. acnes genomes in and around sarcoid granulomas suggests that this indigenous bacterium is related to the cause of granulomatous inflammation in sarcoidosis.P. acnes . The siganulomas . The meaP. acnes, Negi et al. screened for this indigenous bacterium in sarcoid and nonsarcoid tissues using immunohistochemical methods with novel P. acnes-specific monoclonal antibodies that react with cell-membrane-bound lipoteichoic acid (PAB antibody) and ribosome-bound trigger factor protein (TIG antibody). They examined formalin-fixed and paraffin-embedded samples of lungs and lymph nodes from 196 patients with sarcoidosis and corresponding control samples from 275 patients with nonsarcoidosis diseases. The samples were mostly from Japanese patients, with 64 lymph node samples from German patients [Granulomatous reactions are basically a defense mechanism that the body uses to fight off poorly degradable antigens. granulomas begin as a small collection of lymphocytes and macrophages surrounding poorly degradable antigens. The aggregating macrophages, called an early focus of granuloma, then change to epithelioid cells and become organized into a cluster of cells, called an immature granuloma. Further progression results in ball-like clusters of cells and fusion of macrophages into giant cells, called a mature granuloma. The questions that must be asked in searching for the cause of sarcoidosis, therefore, are: “What is the antigen that the granulomas are fighting?” and “How is the antigen localized within the sarcoid lesion?” To evaluate the pathogenic role of patients .Immunohistochemistry with the PAB antibody revealed small round bodies within sarcoid granulomas in 20/27 (74%) video-assisted thoracic surgery lung samples, 24/50 48%) transbronchial lung biopsy samples, 71/81 (88%) Japanese lymph node samples, and 34/38 (89%) German lymph node samples. The PAB antibody did not react with nonsarcoid granulomas in any of the 45 tuberculosis samples or the 34 samples with sarcoid reaction. The appearance of the small round bodies detected by the PAB antibody within sarcoid granulomas did not differ between lungs and lymph nodes. In sarcoid granulomas with many small round bodies, the cytoplasm of some granuloma cells was filled with small round bodies, consistent with the intracellular proliferation of the bacterium . In many% transbrIn nongranulomatous areas small round bodies detected by the PAB antibody were found in alveolar macrophages of lungs and paracortical macrophages of lymph nodes from many sarcoid and some nonsarcoid patients. In the lymph nodes, paracortical macrophages with many small round bodies detected by the PAB antibody  were obsP. acnes, but rather intact forms of intracellular bacteria because the original distribution pattern  of these bacterial components was preserved in terms of the morphologic structure of the bacterium. Furthermore, conventional electron microscopy revealed that these bodies lack a cell-wall structure and occasionally exhibit protrusions from the body that appear to be yeast-like proliferating features , characteristic of cell-wall-deficient (L-form) bacteria (P. acnes.Hamazaki-Wesenberg (HW) bodies frequently appear in sarcoid lymph nodes although these bodies are not specific to sarcoidosis –32. The bacteria . HW bodiP. acnes causes latent infection and persists in macrophages. HW bodies are dormant and cell-wall-deficient P. acnes. This dormant form of P. acnes can be activated endogenously under certain environmental conditions and proliferate in cells at the sites of latent infection. Small round bodies proliferating in macrophages are infective forms of P. acnes. When these bodies spread out of macrophages, they infect other cells or organs via the lymph and blood streams. Sarcoid granulomas are formed as a host defense mechanism at the sites of activated bacteria proliferating intracellularly in patients with hypersensitive immune responses to P. acnes to prevent the spread of the infectious agent.Histopathological analysis with the PAB antibody led us to formulate a hypothesis for the mechanism of granuloma formation in sarcoidosis . P. acneP. acnes because an epitope of lipoteichoic acid detected by this antibody is more exposed in cell-wall-deficient forms than in conventional forms of the bacterium. The high frequency and specificity of P. acnes detected by the PAB antibody within sarcoid granulomas suggest an etiologic link between sarcoidosis and this indigenous bacterium. The PAB antibody may be useful for diagnosing sarcoidosis caused by P. acnes, when the reactivity is detected in idiopathic granulomas  of the P. acnes trigger factor, which has 529 amino acid residues and a calculated molecular mass of 57,614 Da. The C-terminal sequence (Asp-463 to Lys-529) seems to be unique to P. acnes, with no similarity to sequences of other bacterial proteins deposited in the Swiss-Prot database. Conformational analysis of the Ser-491 to Lys-529 regions at the C terminus revealed it to be highly antigenic. RP35 caused sarcoidosis-specific proliferation of peripheral blood mononuclear cells (PBMCs) from 9 (18%) of 50 patients with sarcoidosis  or ESAT-6 recombinant protein were not significantly different. Sarcoidosis may arise from an imbalance of Th1/Th17 immune responses to viable P. acnes.Recently, Furusawa et al.  reportedP. acnes was obtained in studies of BAL cells. When stimulated with a crude extract of P. acnes with pyridine, BAL cells from patients with sarcoidosis proliferated more than BAL cells from healthy subjects or from patients with lung cancer [P. acnes antigen was greater in sarcoidosis patients than in healthy subjects or patients with other lung diseases [P. acnes DNA was detected in BAL cells from 21 (70%) of 30 sarcoid patients and 7 (23%) of 30 control patients with other lung diseases [In situ signals of P. acnes DNA were detected in the cytoplasm of a few alveolar macrophages among the BAL cells from sarcoid patients, but from no other kinds of BAL cells, including alveolar lymphocytes and neutrophils. Gallium-67 uptake by lung parenchyma was found in about half of the 30 sarcoid patients with P. acnes DNA, but in none of the other sarcoid patients [Additional evidence of the hypersensitivity of sarcoid patients to g cancer . Interlediseases . P. acnediseases . In situpatients . P. acnes activates NF-κB in both an NOD1- and NOD2-dependent manner [P = 0.006 for trend). Functional studies indicated that the NOD1 796A-allele is associated with reduced expression leading to diminished NF-κB activation in response to intracellular P. acnes , TLR4, and TLR9 mediate the effects of P. acnes infection [P. acnes. TLRs are likely to serve as first-line receptors for P. acnes, but NOD proteins might play a major role in a subsequent phase of intracellular infection. NOD1 was recently reported to be a critical regulator of beta-defensin 2 during Helicobacter pylori infection [κB enables P. acnes to survive and persist intracellularly, leading to the pathogenesis of sarcoidosis.nfection , 46. Thenfection . It is pP. acnes. A single intravenous injection of P. acnes into mice leads to the development of many granulomas in the liver [P. acnes into sensitized rats [P. acnes was used as a sensitizer, and a challenge by a single intravenous injection of the bacterium was essential for granulomas to form in the lungs.In experimental animals, granulomatous lesions can be induced by he liver –50, but zed rats  and rabbzed rats . In thesP. acnes trigger factor protein (RP35) or heat-killed P. acnes causes pulmonary granulomas in some (25%–57%) mice sensitized with the protein and complete Freund's adjuvant (CFA) [P. acnes as a challenge was not essential for granulomas to form in the lungs. Granulomas were scattered throughout the lungs, especially in subpleural areas (P. acnes. nt (CFA) . An intral areas . The graP. acnes trigger factor, such as has been experimentally induced, has been reported in some patients with sarcoidosis. Second, situations resembling intravenous challenge with P. acnes are rare in humans, and sarcoidosis can start in asymptomatic persons without evidence of septicemia.This experimental protocol may provide a satisfactory model of sarcoidosis. First, hypersensitivity to P. acnes trigger factor protein or heat-killed P. acnes in CFA induces granulomatous inflammation confined to the lungs. This finding suggests that such antigens from P. acnes exist in the lungs of mice even before the experiment. Experimental models of allergic diseases, such as encephalomyelitis , thyroidP. acnes was cultured from the lungs, liver, and lymph nodes from some of the untreated normal mice, and culture was most often successful with the lungs. There was an unexpected concordance in the rate (33%) of culture from normal lungs and the frequency of detection of pulmonary granulomas in mice sensitized with the trigger factor protein. The concordance suggests that mice without granulomas may have been free from P. acnes in the normal indigenous flora of their lungs before and during the experiment. Similar to the results obtained by bacterial culture of human samples from lung and lymph nodes, P. acnes trigger factor antigen developed more severe and diffuse pulmonary granulomatosis than did sensitized mice. In fact, the severe granulomatous inflammation that developed could be even identified macroscopically  before and during the experiments prevented granuloma formation in these experimental models . P. acnes was taken up by lung cells, and the P. acnes-bearing cells expressed F4/80 rather than CD11c or DEC205, consistent with the ability of macrophages to phagocytose and deliver antigens to dendritic cells in the lung. As far away as the end of the airway, airborne organisms are impacted and eliminated by mechanical defenses, including mucociliary clearance and coughing. Nevertheless, a small number of P. acnes might escape this system and reside on the alveolar surface. P. acnes genomes were detected in normal pulmonary lymph nodes as well as the lungs, and lymphocytes from lymph nodes showed P. acnes-specific proliferation, suggesting that these cells had already been exposed to P. acnes by lung-derived antigen-presenting-cells and had established a memory response. Additionally, these results indicate that P. acnes were continuously transported to pulmonary regional lymph nodes in the steady state. Because of this constant delivery of antigens to the pulmonary lymph nodes for a long period, the small number of indigenous P. acnes in the normal lung would be enough to produce a specific immune response, but not for the formation of a steady-state granuloma. Although mycobacterial, atypical mycobacterial, and other propionibacterial antigens are potential candidate endogenous microorganisms that trigger pulmonary granuloma formation, genomic analyses revealed an absence of these organisms in the lungs of specific-pathogen-free C57BL/6 mice. Nishiwaki et al. further examined the mechanism of pulmonary granulomatosis caused by the sensitization of heat-killed P. acnes , using aP. acnes . In the P. acnes-sensitized lymph node CD4+ T cells into naïve mice resulted in granulomatous changes in the lung, indicating that extrapulmonary lymph node CD4+ T cells primed with P. acnes can interact with pulmonary resident cells via the circulation and induce granuloma formation in the normal lung. It was therefore hypothesized that a continuous supply of P. acnes-sensitized T cells should lead to chronic pulmonary granuloma formation, and consequently, we performed continuous remote sensitization of normal mice with P. acnes. These mice exhibited distinct pulmonary granulomas, distributed in lymph-rich spaces such as the subpleural, peribronchial, and perivascular areas and had typical cellular components of granuloma and preferential Th1 cytokine expression. These features are similar to those of pulmonary sarcoidosis. In addition, the ratio of CD4 to CD8 BAL lymphocytes was elevated in the group immunized twice, and serum calcium levels were also increased. Thus, the characteristics of this P. acnes-immunization model, without any direct exposure of antigen to the lung, showed several similarities to those of sarcoid patients.The adoptive transfer of P. acnes cells in the lung affected pulmonary granuloma formation. As expected, preloading of P. acnes exacerbated pulmonary disorders, whereas reduction of the P. acnes population by antimicrobial treatment reduced the pulmonary lesions. These findings suggest a pivotal role of normally localized P. acnes in the formation of pulmonary granuloma by extrapulmonary P. acnes sensitization, as well as the potential clinical usefulness of antimicrobial eradication targeting lung-indigenous P. acnes for the treatment of pulmonary granulomatosis induced by similar pathogenesis.That study also examined whether changes in the number of preexisting Pneumocystis carinii pneumonia, are well known to be associated with immunodeficiency in AIDS patients. Combination type infections, such as Candida and Aspergillus, not only cause opportunistic infections, but may also cause hypersensitivity pneumonitis. The hypersensitivity type of endogenous infection does not cause any tissue damage until the hypersensitive immune response is triggered. P. acnes as a cause of sarcoidosis can therefore be classified within the group of endogenous diseases that results from hypersensitivity.In the past, once the germ theory of disease was accepted, microbes were considered to be pathogens if they met the stipulations of Koch's postulates. Although there are many microbes, however, most human infections are caused by only a few. Some microbes have been classified as pathogens although they do not cause disease in every host. In addition, some microbes have been classified as nonpathogenic although they cause disease in certain hosts. For these reasons, in a redefinition of the concepts of virulence and pathogenicity of microbes, Casadevall and Pirofski suggested a classification system for pathogens based on their ability to cause damage as a function of the host's immune response . Koch's P. acnes is the most [P. acnes is found in 20% of nonsarcoid lymph nodes by bacterial culture [P. acnes in nongranulomatous areas of the lungs and lymph nodes from nonsarcoid patients suggests that latent infection and endogenous reactivation of this indigenous bacterium occurs in these organs, even in patients without sarcoidosis. Sarcoidosis involves many organs, and the lungs and mediastinal lymph nodes are involved at the highest frequency [P. acnes in these organs may explain why they are frequently involved in sarcoidosis.the most  common cthe most . Some P. culture , 15% of  culture , 18% of  culture . Occasiorequency . CommensP. acnes, indigenous low-virulence bacterium, can cause latent infection in the lungs and lymph nodes and persist in a cell-wall-deficient form. This dormant form of P. acnes can be activated endogenously under certain environmental conditions and then proliferate in cells at the site of the latent infection. In patients hypersensitive to this endogenous bacterium, granulomatous inflammation is triggered by intracellular proliferation of the bacterium. Some proliferating bacteria may escape from isolation by the granuloma and spread to other organs via the lymph and blood streams. The spread of infective P. acnes might cause a new latent infection in systemic organs, such as eyes, skin, and heart. Latent infection established in certain systemic organs will be reactivated simultaneously by the next triggering event, resulting in the onset of systemic sarcoidosis  latent infection with cell-wall-deficient P. acnes, (2) endogenous activation of dormant P. acnes triggered by certain environmental factors, and (3) a hypersensitive Th1 immune response towards the intracellular proliferation of P. acnes. The formation of sarcoid granulomas might be induced by a Th1 immune response to one or more antigens of P. acnes proliferating in the affected organ or tissue in an individual with a hereditary or acquired abnormality of the immune system. Sarcoidosis is most likely the result of a complex interaction between infection, immunity, and allergic reaction. There are three conditions essential to the development of sarcoidosis caused by M. tuberculosis infection usually occurs in childhood and produces lesions termed the “primary complex,” which is a combination of lesions in the lung and lung hilar lymph nodes. Around 90% of subjects with primary infection by M. tuberculosis exhibit a so-called “unapparent infection;” that is, they are asymptomatic. Latent infection by this pathogen is characterized by healed lesions comprising consolidated scar tissue or necrotic lesions that often becomes calcified. The persistent mycobacteria in this dormant phase are thought to be cell-wall-deficient. Active tuberculosis occurs when the latent mycobacterial infection is endogenously activated under certain environmental conditions, especially in older people. The risk for activation is also significantly increased by immunosuppressive triggers, such as HIV infection and diabetes. Recent immunologic data provide evidence of latent tuberculosis in about one-third of the global population, which corresponds to more than 2 billion individuals [Tuberculosis shares many common features with sarcoidosis, not only their histopathological features, but also aspects of their pathogenesis. Many tuberculosis cases arise from the endogenous activation of latent tuberculosis infection. Primary ividuals .α drugs administered to patients with rheumatoid arthritis [α treatment is thought to reactivate latent tuberculosis infection [P. acnes might be reactivated by antitumor necrosis factor-α treatment, resulting in sarcoidosis in certain susceptible subjects among these patients.Endogenous reactivation of latent bacteria is well known to occur in tuberculosis, which shares many common features with sarcoidosis, not only the histopathological features, but also the pathogenic features. Many cases of adult tuberculosis are caused by endogenous activation of latent tuberculosis infection , 61. Tubrthritis , 63. Antnfection . In the P. acnes infection.Immunosuppressive, mainly corticosteroid, therapy has been used for sarcoidosis for more than 50 years, but the long-term effects of steroidal treatment in chronic sarcoidosis are still disputed . FurtherAntibiotics not only kill the bacteria proliferating in cells, but they also prevent the endogenous activation of latent bacteria. Long-term administration of antibiotics may therefore be effective for patients with progressive sarcoidosis by preventing inflammatory relapses caused by reactivation of the latent bacteria . If lateP. acnes antigens responsible for granuloma formation, such as the trigger factor protein. Recent advances in the modulation of epitope-specific immune responses by synthetic peptides might help to improve the treatment of sarcoidosis. Another approach for treating sarcoidosis is specific suppression of the hypersensitivity to P. acnes. In an observational study, Bachelez and colleagues [P. acnes is sensitive, for the treatment of chronic forms of cutaneous sarcoidosis. The authors treated 12 patients with biopsy-proven cutaneous sarcoidosis with 200 mg daily minocycline over a median 12-month period. With a median followup of 26 months, the authors noted complete and partial responses to treatment in 8 and 2 patients, respectively. The mean time to reach a maximal response was 3.2 months. Of the 8 patients who had a complete response, minocycline could be withdrawn in 7 patients, 3 of whom experienced recurrent lesions and received further treatment with 200 mg daily doxycycline. Complete remission was maintained for a mean of 15.3 months. Regression of pulmonary infiltrates and mediastinal lymphadenopathy was noted in the 2 patients with concurrent pulmonary involvement. Minocycline is the first-choice antibiotic for patients with acne vulgaris caused by lleagues  reportedP. acnes in the pathophysiology of sarcoidosis and the uncertainty of the long-term effects of corticosteroids. The lesions were worsening before the antibiotic therapy was initiated, so the improvements appeared to be due to the therapy. Park and colleagues [The results of a nationwide questionnaire survey, performed by a Japanese research group in 2005 (reported in Japanese), indicated that antibiotic therapy was effective in 43% of 87 patients with sarcoidosis treated with many kinds of antibiotics, including minocycline, doxycycline, and clarithromycin. Baba and colleagues  used minlleagues  reportedlleagues  reportedin vitro studies and corroborated by clinical trials. Tetracycline suppresses neutrophil migration and chemotaxis [in vitro [P. acnes proposed in this chapter suggest that these antibiotics have many roles in the process of granuloma formation, including the events triggering granuloma formation caused by intracellular proliferation of the bacterium.Based on the studies described in this section, the antimicrobial properties of tetracyclines are effective for treating sarcoidosis. Many researchers have questioned the antimicrobial role of tetracyclines because tetracyclines also have anti-inflammatory properties which were demonstrated by emotaxis , and minemotaxis . Both miin vitro . Althoughttp://marshallprotocol.com/ and also at http://autoimmunityresearch.org/.The Marshall protocol  is an erP. acnes is currently the only microorganism that has been isolated from sarcoid lesions by bacterial culture. In the late 1970s, Homma et al. reported frequent isolation of P. acnes from biopsy specimens of Japanese patients with sarcoidosis. In the 1980s, Abe et al. isolated the same microbe from 31 (78%) of 40 sarcoid lymph nodes. These Japanese researchers also cultured this organism from 20% of 141 nonsarcoid lymph nodes. In 1999, Ishige et al. reported many genomes of P. acnes in biopsy samples of lymph nodes from 12 of 15 Japanese patients with sarcoidosis. Biopsy samples of the three patients without P. acnes all showed many genomes of P. granulosum. Similar results were obtained in an international collaborative study of samples from European patients with sarcoidosis. P. acnes genomes in and around sarcoid granulomas, suggesting that this indigenous bacterium is related to granulomatous inflammation in sarcoidosis. In 2012, Negi et al. reported that a novel monoclonal antibody specific to P. acnes that recognizes lipoteichoic acid of the plasmalemma reacted with small round bodies within sarcoid granulomas in 88% of lymph node biopsy samples, 77% of video-assisted thoracic surgery lung samples, 57% of transbronchial lung biopsy samples from Japanese patients with sarcoidosis, and in 89% of lymph node biopsy samples from German patients with sarcoidosis. Reactivity to the antibody was not observed in nonsarcoid granulomas, including those from patients with tuberculosis and so-called sarcoid reaction. The high frequency and specificity of P. acnes detected within sarcoid granulomas indicate that this indigenous bacterium might be the cause of granuloma formation in many patients with sarcoidosis. In 2002, Yamada et al. found P. acnes is the most common commensal bacterium in the lungs and lymph nodes of subjects without sarcoidosis. P. acnes is found in 20% of nonsarcoid lymph nodes by bacterial culture and in 15% of nonsarcoid lymph nodes by polymerase chain reaction. In nonsarcoid samples examined by immunohistochemistry, P. acnes was found in 18% of lung samples and in 22% of lymph node samples. Occasional detection of P. acnes in nongranulomatous areas of the lungs and lymph nodes from nonsarcoid patients indicates that host factors may be more critical than agent factors in the sarcoidosis etiology. P. acnes causes a cellular immune response in some sarcoid patients, but not in subjects without sarcoidosis. The P. acnes trigger factor protein induces pulmonary granulomas in mice sensitized with the protein and adjuvant, but only in mice with latent P. acnes infection in their lungs. This pathogenesis was confirmed by further experiments using mice sensitized with P. acnes and adjuvant, in which the eradication of P. acnes by antibiotics prevented granulomas in this experimental model. The findings from this experimental model of sarcoidosis might explain the recently reported effectiveness of tetracyclines for treating sarcoidosis. A particular protein, referred to as a trigger factor, from P. acnes can be activated endogenously under certain environmental conditions and then proliferate in cells at the site of latent infection. In patients who are hypersensitive to this endogenous bacterium, granulomatous inflammation is triggered by intracellular proliferation of the bacterium. Some of the proliferating bacteria may escape from isolation by the granuloma and spread to other organs via the lymphatic and blood streams. The spread of infective P. acnes might cause a new latent infection in systemic organs such as the eyes, skin, and heart. Latent infection established in certain systemic organs will be reactivated simultaneously by the next triggering event, resulting in the onset of systemic sarcoidosis.This indigenous low-virulence bacterium can cause latent infection in the lungs and lymph nodes and persist in a cell-wall-deficient form. This dormant form of