The histopathology of cardiac xenograft rejection has evolved over the last 20 yr with the development of new modalities for limiting antibody-mediated injury, advancing regimens for immune suppression, and an ever-widening variety of new donor genetics. These new technologies have helped us progress from what was once an overwhelming anti-Gal-mediated hyperacute rejection to a more protracted anti-Gal-mediated vascular rejection to what is now a more complex manifestation of non-Gal humoral rejection and coagulation dysregulation. This review summarizes the changing histopathology of Gal- and non-Gal-mediated cardiac xenograft rejection and discusses the contributions of immune-mediated injury, species-specific immune-independent factors, transplant and therapeutic procedures, and donor genetics to the overall mechanism(s) of cardiac xenograft rejection. Deceased human organ donation rates do not meet the demand for clinical transplantation. Changes in donor definition and legislation have not substantially closed this gap in supply. Potential alternatives to cardiac allotransplantation include mechanical devices, regenerative medicine applications, and xenotransplantation. Genetic modifications of organ-source pigs in concert with evolving immunosuppressive strategies have resulted in significant progress in cardiac xenotransplantation. Cardiac xenograft survival in the heterotopic pig-to-primate model has increased from a few hours to a median survival of 3 months with individual survival beyond 8 months –3.Major contributions to this progress have been recognition of (i) the importance of antibodies directed to pig galactose-α1,3-galactose  epitope in xenograft rejection –7; (ii) The authors, members of the NIAID-supported Consortium on Immunobiology of Xenotransplantation, have extensively reported on cardiac xenotransplantation. In this review, the group assesses the histopathology of anti-non-Gal antibody-mediated cardiac xenograft rejection and discusses the implications this may have for future research strategies.The initial barrier to xenotransplantation was hyperacute rejection (HAR) caused by complement-mediated endothelial cell (EC) destruction directed by preformed anti-Gal antibody. The histopathology of HAR is predominantly characterized by rapid graft failure and widespread intravascular hemorrhage Fig. . This isEnduring reduction in anti-Gal antibody in vivo was successfully achieved using continuous or intermittent infusion of non-antigenic Gal polymers ,27,31–34This change in histopathology was attributed to sustained depletion of anti-Gal antibody. A recent histopathology comparison of cardiac xenografts under conditions where pre-transplant anti-Gal antibody was uniformly depleted and post-transplant induction of anti-Gal antibody was either partially muted by immunoapheresis, blocked by in vivo Gal polymers, or made irrelevant using GTKO donor hearts supports this conclusion . Under tThe initial study of heterotopic GTKO cardiac xenotransplantation reported no HAR and a median survival of 78 days . This stA detailed histology and immunohistology analysis was consistent with a progressive humoral rejection, resulting in widespread platelet-rich/fibrin-rich microvascular thrombi, myocardial ischemia, and necrosis, with focal interstitial hemorrhage . ImportaThese transplants showed that using GTKO organs effectively eliminated a role of anti-Gal antibody in graft rejection, but also clearly demonstrated the significance of anti-non-Gal antibody in the development of graft failure. Under these conditions, non-Gal-mediated GTKO heart rejection involved three major processes: (i) direct antibody-mediated EC injury, supported by the vascular deposition of antibody and terminal complement complexes in 7 of 8 grafts; (ii) EC activation, as evidenced by increased expression of pTF and vascular loss of CD39; and (iii) EC apoptosis that occurred relatively late in the rejection process. The development of these pathophysiologic processes progressed in parallel with histologic changes (microvascular thrombosis and coagulative necrosis), suggesting that TM within the graft resulted from the effects of immunoglobulin and complement, that is to say immune-mediated rejection.However, other processes may also contribute significantly to graft thrombosis. These include systemic activation of recipient innate immune cells, leading to consumptive coagulopathy (CC) –42, as wCytotoxic anti-non-Gal antibody, with a titer typically 2- to 3-fold lower than anti-Gal antibody, is broadly present in human and non-human primate serum . Despiteα1,3-galactosyltransferase gene knockout pigs heart xenografts have also been reported to undergo early immune injury from preformed anti-non-Gal antibody, which did not result in HAR ,41. In tThese results demonstrate that preformed anti-non-Gal antibody contributes to early graft injury, but also show clearly that under some conditions, systemic activation of the recipient innate immune cells can occur. This activation, resulting in increased recipient bTF expression on peripheral blood mononuclear cells, may be stimulated through antibody-mediated rejection or through cell–cell interactions that are independent of antibody-induced EC activation ,50,51. IThe differential expression of TF either from circulating recipient monocytes or from donor graft endothelium (described above) is a key observation, which suggests that a combination of distinct mechanisms—local non-Gal antibody-mediated immune injury and systemic recipient innate cell activation—may contribute to overall intragraft thrombosis. What is clear is that similar heterotopic and orthotopic xenograft survival can be obtained with ,35,41 ande facto inherent property of porcine organs. We suggest that a combination of four major factors—(i) antibody-mediated injury; (ii) pig-specific immune-independent factors; (iii) transplantation and certain transplant-related therapies; and (iv) donor porcine genetics—need to be considered as potential factors that contribute to recipient innate cell activation and its role in CC  and thrombomodulin (TBM), which have been recently reviewed . PorcineCross-species cellular interactions between porcine ECs and resting human platelets and monocytes may also lead to systemic platelet activation and EC apoptosis ,69. Co iAnti-CD154 mAb has been used as a chronic immunosuppressive agent in several pig-to-primate organ and cellular xenotransplantation models. Combined with other therapies, anti-CD154 mAb efficiently blocks induction of anti-pig antibody ,41,42,75The transplantation model—heterotopic or orthotopic—may affect the development of CC, which has been primarily associated with heterotopic cardiac xenotransplantation ,35,41,55In contrast, CC has generally not been reported after orthotopic cardiac xenotransplantation in the context of conventional immunosuppression ,80–87 deAn increasing variety of genetically modified pigs are becoming available Table , which mCardiac xenotransplantation has also been reported using GTKO pigs expressing human CD46 . In thisPigs expressing human TBM have been reported   and an iThe relative contribution of anti-glycan compared with anti-protein antibody to xenograft rejection as well as the pathogenicity of any individual non-Gal specificity is not clear. This is an important point, as additional genetic modification to eliminate or substitute common porcine protein target antigens is probably not feasible, but elimination of porcine expression of other glycans  is. The pathogenicity however of antibodies to other glycans, other than Gal, has not yet been shown –122.Little is known about the origin of non-Gal antibodies bound to the vasculature of cardiac xenografts. Are these germ-line-encoded antibodies, cross-reactive affinity-matured antibody derived from an unrelated immune challenge, or the product of an induced xenograft-specific immune response? Depletion of mature B cells perioperatively has been associated with better graft outcome and reduced humoral sensitization ,2, but tThird, it is essential to evaluate the contribution that new pigs expressing anti-coagulant transgene function will have on xenograft rejection, thrombosis, and systemic coagulopathy. Transgenic pigs expressing human TBM, EPCR, or CD39 have been produced ,126–129.Expression of CD39 in conjunction with CD73 leads to local production of adenosine, which is a potent inhibitor of thrombosis. Transgenic mice expressing human CD39, although fully viable, show an increased capacity to produce adenosine, have impaired platelet aggregation, and prolonged bleeding times. In a mouse cardiac transplant model, these mouse hearts are protected from thrombosis . In a waInitial experience with heterotopic heart transplantation in the hCD39, hCRP, and hTBM multitransgene GTKO pig-to-baboon model indicates that in the presence of CVF and anti-CD154 mAb therapy, high level expression of hCD39 by the porcine vasculature is associated with less thrombocytopenia and better maintenance of plasma fibrinogen levels in recipient baboons .These data suggest that high level expression of hCD39 on this genetic background with concurrent validated expression of hCRP may have two benefits: first to prevent ischemia–reperfusion injury that might help prevent PCXD and second as an anti-thrombotic to prevent intragraft thrombosis. Further testing these and other new genetically modified donor swine will be required in both heterotopic and orthotopic cardiac transplant models to gauge the impact of these genes on PCXD, intragraft thrombosis, and systemic coagulation parameters in recipient baboons. In any case, CVF should be avoided as GTKO pigs expressing hCRPs are now available, and CVF may exacerbate recipient innate cell activation.Cardiac xenograft survival has improved significantly since the introduction in the early 1990s of the first transgenic pigs in which a hCRP was expressed ,9. GTKO


Fabrication of self-assembled nanostructures is one of the important aspects in nanoscience and nanotechnology. The study of self-assembled soft materials remains an area of interest due to their potential applications in biomedicine. The versatile properties of soft materials can be tuned using a bottom up approach of small molecules. Peptide based self-assembly has significant impact in biology because of its unique features such as biocompatibility, straight peptide chain and the presence of different side chain functionality. These unique features explore peptides in various self-assembly process. In this review, we briefly introduce chemical reaction-mediated peptide self-assembly. Herein, we have emphasised enzymes, native chemical ligation and photochemical reactions in the exploration of peptide self-assembly. The spontaneous formation of ordered structures at the nanoscale is usually referred to as self-assembly . When thMolecular self-assembly  is the sThe development of nanoscale structures and devices can be accomplished through “bottom-up approach” or “top-down” methods. In the bottom-up approach, small building blocks assemble into larger structures ,32 Figu. ExampleIn this review, we aim to describe various mild chemical reactions in the development of peptide self-assembly . A chemiet al. flanked a short self-assembling peptide sequence with cysteine (Cys) residues that enabled the macrocyclization of these peptides [The self-assembly of short peptides can be controlled by imposing a conformational constraint that leads to the prevention of the β-sheet structure  43]. Ni. Ni43]. et al. reported a dissipative self-assembly system in which a synthetic DSA fibrous network uses chemical fuel as an energy source [l)-cystine (DBC) is converted into self-supporting gel by reaction with a chemical fuel methyl iodide at pH = 7 leading to the formation of diester. Hydrolysis of the methyl esters of the gelator, which is labile under ambient conditions, leads to energy dissipation and dis-assembly of the formed structures. Yang et al. reported redox controllable self-assembly properties of selenium containing peptides [Van Esch y source   as chemical fuel [An N-capped 4-phenyl-selenyl butanoic acid peptide was converted to selenoxide upon oxidation with Hr saline . Howeverr saline . The dynthiazole . Ajayan thiazole . Das et cal fuel . The libcal fuel .et al. reported an azobenzene-linked symmetrical gemini α-helical peptide which reversibly transforms between the trans- (Z-) and cis-structure (U-structure) under UV (λ = 365 nm) and subsequently visible light irradiation [et al. developed light-harvesting peptide nanotubes that integrate photosynthetic units for mimicking natural photosynthesis [et al. demonstrated that a bis maleic acid shows irreversible photoinduced gelation in water that works on photochemical isomerization of nongelling maleic acid amide to gelling fumaric acid amide [The self-assembly of bio-organic molecules into nanostructures is an attractive route to fabricate functional materials. For example, diphenylalanine , an aromatic dipeptide consisting of two covalently linked phenylalanine units, can form various nanostructures such as nanotubes ,68, nanoadiation   hydrogel  Figure ; the seq. Thus, the enzyme thermolysin is preferably used to exploit peptide self-assembly. Self-assembly of macroscopic materials from small molecular building blocks provides a route to design molecular biomaterials. The controlled development of biomaterials is in high demand in the context of biomedical applications. The stimuli that trigger self-assembly include various physical chemical perturbations. Enzyme triggered self-assembly is particularly interesting due to mild and physiological reaction conditions. Enzymes allow biocatalytic reactions in a biological environment which could lead to hydrogelation of peptides. Herein, some examples demonstrate the hydrogelation of peptides via reverse hydrolysis of amide bonds in peptides.Amide bond formation of peptides is exactly opposite to the hydrolysis of amide bonds in peptides. Usually some proteases show the ability to reversibly synthesize the amide bond as well as hydrolyze the amide bond in dilute aqueous conditions. It has been reported that self-assembly drives the reverse hydrolysis of peptides and prefers the most stable product formation in gel phase mediumet al. have used a protease enzyme that normally hydrolyzes peptide bonds in aqueous medium. They described a conceptually novel approach by using thermolysin to perform the reverse reaction , which can produce amphiphilic peptide hydrogelators and self-assembles to form nanofibrous structures [et al. reported bio-catalytic evolution of dynamic combinatorial libraries via amide bond formation. Self-assembly evolves thermodynamically downhill library members via enzyme-catalysed amide bond formation [Ulijn ructures   materials can be easily controlled and tuned by using biological catalysts taking advantage of chemo-, regio- and enantioselective synthesis and of mild reaction conditions. In general, there are two routes to drive enzymatic molecular self-assembly through either breaking or making of covalent bonds. Interestingly, both routes lead to self-assembly by maintaining the hydrophobic and hydrophilic balance between the self-assembling molecules. Important efforts have been made to gain more insight into molecular self-assembly.Lipase is an important class of enzyme that catalyses the hydrolysis of fats (lipid). Lipases are the most broadly deployed biocatalysts because of their ability to produce chiral products  with higLys-OtBu . It has These studies have proved that non-covalent interactions such as π-π stacking, hydrogen bonding and hydrophobic interactions play a key role in the development of such systems. Recently, thermolysin was used to generate dynamic combinatorial libraries for the discovery of stable self-assembling nanostructures. Although lipases are primarily used in esterification and transesterification of carboxylic acids, there are few examples where lipases have been used to develop peptide self-assembly through amide bond synthesis. Palocci and colleagues reported peptide self-assembly via coupling of Fmoc-phenylalanine and diphenylalanine using lipase as the catalyst at physiological conditions  Figure . This iset al. demonstrated lipase catalyzed sugar-containing self-assembled organogels with nanostructured morphologies [et al. reported lipase incorporated peptide nanotubes [et al. described lipase-catalysed incorporation of gastrodigenin  to Nmoc-protected peptides [p-hydroxybenzyl alcohol can efficiently be incorporated into peptide bolaamphiphiles [Dordick hologies   is an important extension of the chemical ligation discovered by Kent and co-workers in 1994 . The reaC-terminal peptides or protein thioesters are essential in NCL. The synthetic thioester preparation is generally carried out via conventional carboxyl activation chemistry rather than the N→S route so elegantly directed by intein. Thioesters can also be prepared via tert-butyloxycarbonyl (Boc)-based solid-phase peptide synthesis (SPPS) on a thioester resin or 9-fluorenylmethoxycarbonyl (Fmoc)-based SPPS, which involves mild bases during synthesis [As posited in a “Thioester World”, thioesters are possible precursors to life . It is rynthesis . Melnyk ynthesis . Once, tynthesis  and is cet al. described the combination of chemical ligation with peptide self-assembly to deliver extremely long polypeptide chains with stipulated, repeated sequences. The self-assembling fibers were used to align peptide from their N- to C-terminals, which facilitates the ligation reactions without the usual requirement of an N-terminal catalytic cysteine residue [et al. illustrated the use of NCL as a strategy to form covalently cross-linked polymer hydrogels under mild conditions and in the absence of catalysts [N-terminal cysteine polymer bioconjugate were synthesized to bring up hydrolgelation by native chemical ligation reaction and the viscoelastic nature of hydrogel was studied by oscillatory rheology.Woolfson  residue . Collier residue . Messersatalysts   activated C-terminal ester  [p-nitrophenol oxo-esters are more labile acyl donors than thioester in NCL reactions.Amino acid and peptide oxo-esters have played an important role in peptide chemistry for many years. Various amino acid esters are commonly used as protecting groups for peptide synthesis in conventional solution phase methodology. Moreover, activated phenyl ester derivatives have been used for chemical ligation of peptides. Several modifications of thioester mediated NCL reactions have been made in an effort to expand the utility of the method. Danishefsky ic ester , which wal ester . The oxo (>48 h) . Long NC (>48 h)  under th (>48 h) ,109. Howp-nitrophenol ester is easy and less problematic. Weissenborn et al. described oxo-ester mediated NCL on oxo-ester activated surfaces and found that 2,3,4,5,6-pentafluorophenyl (PFP) is a more efficient acyl donor than p-nitrophenol and N-hydroxysuccinimide (NHS) activating agents [et al. reported a simple and less activated phenyl oxo-ester of peptide for chemoselective NCL reactions [et al. investigated peptide-guided assembly of poly(ethyleneoxide)-peptide conjugate via intramolecular O→N acyl transfer to restore native amide bonds [p-nitrophenyl esters in peptide self-assembly via native chemical ligation [C-terminal p-NP esters. The modified ligation precursors undergo self-assembly via NCL with N-terminal cysteine residues. Self-assembly via NCL was studied with Nmoc-protected amino acid and peptide p-NP esters. Five compounds 1–5  group. The C-terminals of 1–4 were protected with phenyl selenoester, which could readily undergo NCL reaction at room temperature with N-terminal cysteine and N-terminal cysteine based peptide Cys-Gly. Considering the active role of Cys-Gly in increased risk of women breast cancer, we have used Cys-Gly to ligate with Nmoc-protected selenoesters.Over the past decades, selenium and organoselenium compounds are gaining increasing attention due to their properties as antioxidant and antitumor agents, as apoptosis inducers, and in the effective chemoprevention of cancer in a variety of organs ,114,115.The NCL reaction proceeds through the thioester-linked intermediate where acyl transfers from Se→S  are follThere are many applications where self-assembled peptide nanostructures could play an important role as part of biosensing platforms, as efficient drug-delivery systems, cell cultures or as a hydrogels for tissue reparation. Like some of the traditional antibiotics, the short cationic antimicrobial peptides can kill the microbes by interacting and disrupting bacterial cell membranes. Effectiveness of antimicrobial activity depends on the cationic charges and the hydrophobicity of peptides . The selIn situ formation of self-assembled soft materials has significant importance in biology, and peptide based self-assembly is biocompatible and rapid, which allows gelation at physiological conditions to make it an ideal biomaterial.This review has aimed to provide readers with comprehensive details about peptide self-assembly via mild chemical reactions and various chemical reactions utilized by different research groups for the construction of self-assembled nanostructures in aqueous medium were discussed. Self-assembly is an important process in bottom-up nanotechnology and various covalent and non-covalent interactions govern the self-assembly process. Chemoselective native chemical ligation offers a novel approach for fabrication of self-assembled architectures.


The stress response enzyme heme oxygenase 1 (HO-1) is expressed ubiquitous in the body tissue and mediates, besides its major biological function of heme degradation, a variety of anti-inflammatory effects. Although these effects have been described in various models, underlying mechanisms remain elusive. Few studies revealed an influence of HO-1 on the bone marrow [We investigated the particular role of the bone marrow in terms of HO-1-dependent pulmonary inflammation.Wildtype mice inhaled LPS for 30 minutes. After 24h, PMNs were detected in lung compartments  using a flow cytometry-based technique. Bone marrow and blood were analyzed by flow cytometry after four hours. HO-1 was pharmacologically induced by Cobalt(III)Protoporphyrin-IX-Chloride (CoPP) and inhibited by TinProtophorphyrin-IX (SnPP). Chemokines were measured in the BAL and bone marrow by ELISA.in vivo migration assay showed reduced PMN migration into the BAL. Differentiation of PMNs in the BAL revealed a decrease of segmented PMNs after HO-1 activation. In this group, segmented PMNs were also decreased intravascularly and concordantly, mature and immature PMN populations were higher in the bone marrow. Inhibition of HO-1 by SnPP aggravated parameters of tissue inflammation, led to destruction and increased PMN migration into the BAL. Differential BAL counts in this group revealed an increase of segmented and banded PMNs with enhanced PMN release from the bone marrow. The chemokine SDF-1, which mediates homing of leukocytes into the bone marrow, was increased by CoPP and reduced by inhibition of HO-1. When SDF-1 was blocked by the specific antagonist AMD3100, HO-1 activation was no longer effective in curbing PMN trafficking to the inflamed lungs. Additionally, we compared the inhibition of HO-1 by SnPP alone and in combination with CoPP. Enzyme activity was more reduced after CoPP/SnPP treatment, resulting in deteriorated lung injury with higher PMN counts in the lung interstitium, and pronounced tissue destruction.Immunohistochemistry revealed less lung destruction after HO-1 stimulation by CoPP, and We show evidence that the anti-inflammatory effects of HO-1 are largely mediated by inhibiting the release of segmented PMNs from the bone marrow rather than direct effects within the lung.


Immune checkpoints blockade inhibitors, such as anti-CTLA-4 and anti-PD-1, are now proven effective cancer treatments. In melanoma, the patients who experience clinical benefits usually have T cell-inflamed tumor microenvironment. Understanding the molecular mechanism explaining lack of T cell infiltration in a major subset of patients should lead to development of new therapeutic strategies that improve spontaneous T cell infiltration into tumors and render these patients responsive to immunotherapies. It has been proposed that the T cell-inflamed (hot) and non-T cell inflamed (cold) phenotypes might be due to the differential expression of immunogenic antigens. To test this hypothesis, we compared the levels of three types of antigens: cancer-testis (CT) antigens, differentiation antigens, and somatic mutational antigens, in hot and cold tumors.Using the TCGA data set for malignant melanoma, the patients were segregated according to the presence or absence of the T cell-inflamed gene expression signature in the tumor microenvironment. Transcriptional profiling revealed no differences in the levels of cancer-testis (CT) antigens or differentiation antigens between the hot and the cold tumors. Using exome sequencing of tumor versus germline, a range of 18 to 3001 of non-synonymous mutations was observed in both cohorts. Using the syfpeithi algorithm for HLA-A*0201 patients, a median of 123 mutations having a high immunogenicity score were found in the T cell-inflamed cohort versus 176 in the non-T cell-inflamed. To confirm actual immunogenicity, 321 peptides from hot tumors and 409 peptides from cold tumors have been synthesized. Using a high-throughput T2 binding assay, peptides from both cohorts were found to bind to HLA-A*0201. In vitro priming of T cells using autologous dendritic cells also revealed that peptides from both cohorts could prime human antigen-specific CD8+ T cells. Our results indicate that there is not a lack ofimmunogenic antigens in non-T cell-inflamed melanomas. Other mechanisms must explain T cell exclusion in those cases, such as the recently described Wnt/b-catenin pathway activation. New strategies to promote T cell entry into non-inflamed tumors have a chance for success if immunogenic antigens are indeed present and can be accessed by host immune cells.


Chemical inducers of dimerization (CIDs) have been developed to orchestrate protein dimerization and translocation. Here we present a novel photocleavable HaloTag- and SNAP-tag-reactive CID (MeNV-HaXS) with excellent selectivity and intracellular reactivity. Excitation at 360 nm cleaves the methyl-6-nitroveratryl core of MeNV-HaXS. MeNV-HaXS covalently links HaloTag- and SNAP-tag fusion proteins, and enables targeting of selected membranes and intracellular organelles. MeNV-HaXS-mediated translocation has been validated for plasma membrane, late endosomes, lysosomes, Golgi, mitochondria, and the actin cytoskeleton. Photocleavage of MeNV-HaXS liberates target proteins and provides access to optical manipulation of protein relocation with high spatiotemporal and subcellular precision. MeNV-HaXS supports kinetic studies of protein dynamics and the manipulation of subcellular enzyme activities, which is exemplified for Golgi-targeted cargo and the assessment of nuclear import kinetics. Localization of signaling enzymes is key to controlling protein and lipid kinase cascades in physiology and disease. Control Naturally occurring light-sensitive protein domains have been used to design genetically encoded light-controlled protein–protein interaction modules. These so-called optogenetic systems contain a photoisomerizable chromophore, which undergoes a conformational change upon illumination at a defined wavelength. Optogenetic systems have been used to control the activation of single signaling proteins by protein caging (light-inducible GTPase Rac), or in a Presently, cell-permeable CIDs that can be efficiently manipulated intracellularly have not been reported. Some spaHere we present a novel photocleavable CID, which forms a covalent link between HaloTag- and SNAPAs depicted schematically in Figure t<10 min, Figure MeNV-HaXS was optimized to match the cell permeability of the noncleavable dimerizer of HaloTag and SNAP-tag fusion proteins called HaXS8. Time-depDue to the matched dimerization properties of MeNV-HaXS and HaXS8, the noncleavable HaXS8 is a valuable control compound to monitor the efficiency of photocleavage, and to detect potential side effects exerted by UV irradiation. The efficiency of the intracellular photocleavage of MeNV-HaXS was tested in HeLa cells expressing HaloTag-GFP and SNAP-tag-GFP fusion proteins. A HaloTag-SNAP-tag complex preformed with MeNV-HaXS could be cleaved in bulk by illumination with a 360 nm lamp . Quantification of protein dimers before and after exposure to UV light revealed that MeNV-HaXS-induced dimers were quantitatively cleaved after 10 min of illumination, whereas HaXS8-containing dimers remained intact Figure .MeNV-HaXS thus offers the possibility to trigger covalent dimerization, and to subsequently release associated proteins in a controlled way. This allows the manipulation of protein localization, which can be exploited to mimic cellular signaling events in a timed and localized fashion. Many signaling events take place at defined intracellular locations. The combination of a tagged anchor protein and MeNV-HaXS exposure permits the depletion of tagged signaling enzymes from their productive sites. Presently, MeNV-HaXS has been validated for the targeting of tagged proteins to intracellular organelles such as Golgi Figure , plasma Targeted irradiation of MeNV-HaXS-anchored protein complexes using a microscope equipped with an XY scanning excitation laser for FRAP  indeed released tagged proteins from the illuminated spots. HeLa cells were cotransfected with cytosolic teal fluorescent (cyan) SNAP-tag fusion protein (SNAP-mTFP1) and the Golgi anchor Halo-RFP-Giantin, which was constructed by the fusion of a red fluorescent protein  to a HaloTag and a C-terminal Golgi-targeting motif derived from giantin.m of MeNV-HaXS or 5 μm of HaXS8 efficiently translocated cytosolic SNAP-mTFP1 to the cytosolic surface of the Golgi membrane . This demonstrates that MeNV-HaXS can be utilized to manipulate protein localization with excellent subcellular precision on a timescale of seconds.Incubation with 5 μe Figure . After ae Figure . No signe Figure , confirmAs a scanning FRAP laser is not part of standard fluorescence microscope equipment, we explored the possibility of using global field of view illumination with standard DAPI excitation filters  to induce the photocleavage of MeNV-HaXS-induced protein complexes. We found that an illumination time of <20 s was sufficient to completely liberate SNAP-mTFP1 from Golgi membranes Figure . A correAlthough slower due to the limited excitation energy, photocleavage using DAPI excitation filters on a conventional fluorescence microscope greatly expands the range of applications offered by the MeNV-HaXS system.In contrast to previously reported CIDs, MeNV-HaXS can instantaneously release its cargo when illuminated. To further evaluate this concept, a nuclear probe was forced to dock on a Golgi anchor. Expressed alone in HeLa cells, the nuclear localization sequence (NLS)-containing cyan fluorescent probe (NLS-CFP-SNAP) accumulated in the nucleus. In the presence of co-expressed Golgi anchor  and added MeNV-HaXS, NLS-CFP-SNAP was trapped at perinuclear sites on the Golgi Figure . SubsequIn summary, MeNV-HaXS is the first cell-permeant CID giving rise to the formation of covalently linked and photocleavable protein complexes. Like other CID approaches, the design of fusion proteins bearing interacting functional protein domains must be carefully considered. The covalent link formed by MeNV-HaXS simplifies monitoring and validation of protein complexes greatly. MeNV-HaXS is therefore a novel valuable tool that combines intracellular protein dimerization with photocleavage triggered by a wavelength compatible with widely used fluorescent reporter proteins. The possibility to control protein localization by two independent events can be exploited to sequester any protein of interest away from its functional compartment. CID-dependent trapping of enzymes to nonfunctional sites has been established as an elegant approach to interrupt signaling pathways.,20 Optic


Despite advances in combinatorial chemotherapy regimens and the advent of intraperitoneal chemotherapy administration, current therapeutic options for ovarian cancer patients are inadequate. Immunotherapy offers a novel and promising therapeutic strategy for treating ovarian tumors. Following the demonstration of the immunogenicity of ovarian tumors, multiple immunotherapeutic modalities have been developed. Antibody-based therapies, immune checkpoint blockade, cancer vaccines, and chimeric antigen receptor-modified T cells have demonstrated preclinical success and entered clinical testing. In this review, we discuss these promising immunotherapeutic approaches and emphasize the importance of combinatorial treatment strategies and biomarker discovery. In 2012, there were an estimated 239,000 new cases of ovarian cancer worldwide leading to over 140,000 deaths . Novel t+ tumor-infiltrating T cells correlated with increased overall survival [+, CD8+ T cells as important antitumor effectors [In the last two decades, advances in the understanding of ovarian cancer immunogenicity have opened the door to immunotherapeutic approaches to ovarian cancer treatment. A crucial early step in establishing the validity of ovarian cancer immunotherapy was the observation that CD3survival . Later wffectors . The ideffectors . These mIn the last fifteen years, antibody-based therapies for hematologic cancers and solid tumors have become well-established therapeutic strategies. Following rituximab’s 1997 FDA-approval for treating chemotherapy-resistant non-Hodgkin lymphomas, 18 other molecular antibodies have gained FDA approval for use in oncologic care. Antibody therapy is a promising area of research and increasingly antibody therapies are being used in ovarian cancers.Bevacizumab  is a humanized monoclonal antibody (mAb) that binds to all isoforms of the vascular endothelial growth factor (VEGF) receptor ligand. VEGF is a key mediator of developmental angiogenesis and has been shown to regulate the vascularization of tumors . Anti-VECatumaxomab  is a bispecific, trifunctional antibody directed against the epithelial cell adhesion molecule (EpCAM) and the T cell antigen CD3. Primary, metastatic, and recurrent epithelial ovarian cancers express EpCAM at a significantly elevated level compared to normal human surface epithelium . CatumaxIn vitro, treating ovarian cancer cell lines with cetuximab inhibits cell growth, potentiates apoptosis, and impairs tumor metastasis [Cetuximab  is a chimeric, IgG1, mAb that binds to the extracellular domain of the epidermal growth factor receptor (EGFR) preventing EGFR-signaling and accelerating receptor internalization . Cetuximtastasis .A phase II trial of single-agent cetuximab in persistent/recurrent ovarian or primary peritoneal carcinoma found minimal clinical benefit: nine patients had stable disease, while only one of 25 patients achieved a partial response . HoweverThe limitations of single-agent cetuximab, but success of anti-EGFR therapy in combination with chemotherapeutics highlights an important preclinical reality in immunotherapy: chemotherapy and immunotherapeutics can act synergistically to enhance antitumor immunity and improve therapeutic outcomes. Chemotherapy has historically been thought to be immunosuppressive, but recent work suggests the opposite. In a variety of tumor types, patients with greater numbers of TILs have a better clinical response to cytotoxic chemotherapy ,28. OxalTumor-associated macrophages (TAMs) are a major stromal component in solid tumors. In the ovarian tumor microenvironment, TAMs are the most abundant subset of infiltrating immune cells . Howeverin vitro B7-H4+ macrophages significantly decrease T cell proliferation and T cell activation upon TAA recognition [+ TAMs with single chain fragments of antibody variable regions (scFvs) that target and block B7-H4, can reverse tumor immunosuppression and induce T cell activation [In ovarian tumors, M2 macrophages contribute to multiple mechanisms of TAM-mediated immunosuppression. Ovarian tumor macrophages upregulate and secrete the chemokine CCL22 which promotes T regulatory cell (Treg) trafficking to the tumor. In mice bearing primary human ovarian tumors, treatment with an anti-CCL22 mAb decreased Treg migration into tumors . In addiognition . Treatmetivation .+ T cells to CD4+ T cells while decreasing the number of FoxP3+ Tregs [While anti-CCL22 mAbs and anti-B7-H4 scFvs exert antitumor effects by modulating macrophage-T cell interactions, targeting the macrophage colony stimulating factor-1 receptor (CSF-1R) directly depletes immunosuppressive TAMs. Colony stimulating factor, also known as macrophage colony stimulating factor (CSF-1 or M-CSF), regulates the migration, proliferation, survival, and function of macrophages . Macroph3+ Tregs . In 20113+ Tregs . TargetiImmune checkpoints are inhibitory pathways that downregulate activated T cells following antigen presentation and costimulatory signaling by APCs. By controlling the intensity and duration of the immune response, immune checkpoint signaling prevents collateral self-tissue damage. During tumorigenesis, however, cancer cells express proteins that activate immune checkpoint pathways and induce immune suppression thereby evading targeting and removal by the immune system. The clinical successes of antibodies modulating immune checkpoints continue to fuel the enthusiasm surrounding immunotherapeutic approaches to cancer treatment.The cytotoxic T-lymphocyte-associated protein 4 (CTLA-4 or CD152) plays a vital role in regulating T-cell activation . Activat+ and CD8+ effector T cells, leading to tumor clearance. In a variety of preclinical tumor models, the administration of an antagonistic anti-CTLA-4 antibody induced tumor rejection [In the immunosuppressive tumor microenvironment, blocking CTLA-4 has the potential to directly activate CD4ejection . The sucejection . The majThe programmed cell death protein-1 (PD1) and its ligand (PD-L1) represent a promising immune checkpoint pathway that can be targeted to reverse tumor-mediated immunosuppression. Ligation of PD1 suppresses the lytic activity of immune effector subsets . In ovarIn addition to transmembrane receptor targets, metabolic enzymes are being investigated as therapeutic strategies for reversing immunosuppression within the tumor microenvironment. Indoleamine 2,3-dioxygenase (IDO) is the leading metabolic immune regulator in clinical development.+ T cells into Treg cells [+CD25− T cells into Tregs. With mounting evidence for the immunosuppressive effects of IDO, its role in tumor immune evasion is increasingly under investigation.IDO is an intracellular enzyme that catalyzes the initial and rate-limiting step of the oxidative catabolism of the amino acid tryptophan . Tryptopeg cells . Inhibitet al. who reported constitutive IDO expression in most human tumors and linked elevated IDO levels to a low frequency of TILs in murine cancer models [+ TILs [The role of IDO in human cancer was first documented in 2003 by Uyttenhove r models . In ovar [+ TILs . IDO exp [+ TILs . In pati [+ TILs . Gene ex [+ TILs .The first IDO-targeted therapy to enter preclinical testing was 1-methyl-tryptophan (1-MT), a small molecule inhibitor of IDO. In an IDO-overexpressing ovarian cancer model, combination treatment of paclitaxel and 1-MT synergistically prolonged mouse survival relative to paclitaxel monotherapy . This reCurrently, the IDO inhibitor Indoximod® (NewLink Genetics) is in five clinical trials with encouraging early-phase clinical data . In a phIn addition to the IDO inhibitors developed by NewLink Genetics, INCB024360 (Incyte Corp.), an IDO1 inhibitor, has entered clinical testing. In preclinical data, INCB024360 was shown to significantly inhibit tumor growth and to induce T and NK cell proliferation and IFN-γ production . In the Therapeutic cancer vaccines have been an area of research interest since the 1920s, when injections of lymph node extracts were used to treat Hodgkin’s lymphoma. The promise of vaccine strategies is the potential to “teach” individual patients’ immune systems to recognize, target, and eradicate tumor cells in an approach that employs both adaptive and innate immunity. Vaccines aim to provoke a tumor-specific immune response by increasing TAA presentation by APCs thus generating tumor-antigen specific cytotoxic T lymphocytes. Unlike chemotherapy, surgery, radiotherapy, or antibody therapy, an effective vaccine-induced immune response could establish a state of immunological memory that persists after tumor clearance and indefinitely suppresses tumor regrowth. In recent decades, multiple approaches to therapeutic cancer vaccines have been developed and dendritic cell vaccines have emerged as efficacious in ovarian cancer.ex vivo with MUC-1. In the CAN-003 Phase II study, 63 epithelial ovarian cancer patients in complete remission received CVac. In the patients who had achieved a remission after second-line therapy, PFS and OS were increased [Dendritic cells (DCs), the most potent class of APCs, are responsible for processing cancer antigens from tumor cells and presenting peptide fragments to naive T cells, B cells, and NK cells. DC vaccines attempt to enhance DC uptake and presentation of TAAs, galvanizing an antitumor response. In ovarian cancer, a promising TAA for DC vaccines is mucin 1 (MUC-1). MUC-1 is a heavily glycosylated, type 1 transmembrane protein that is overexpressed in a large number of cancers including colorectal, pancreatic, and ovarian . While mncreased .Ex vivo DC vaccines require isolating and stimulating DCs on a patient-specific basis and are thus costly, labor-intensive, and mostly limited to large, academic medical centers. An attractive alternative is to use an “off-the-shelf” therapeutic that is not patient-specific and stimulates DCs to uptake TAAs in vivo. One example of an early, preclinical success with this approach in ovarian cancer is the administration of a vaccine based on the MSLN-Hsp70 fusion protein [Mycobacterium tuberculosis (Hsp70). In this system, the dendritic cells are activated by the tuberculosis Hsp70 and, because the fusion protein localizes DC activation to MSLN-expressing cells, immediately recognize the tumor antigen. Administration of the bifunctional fusion protein in mouse models of ovarian cancer significantly enhanced survival and slowed tumor growth while augmenting tumor-specific CD8+ T cell responses. protein . The MSLA potential avenue for improving the efficacy of DC vaccines is the inclusion of multiple tumor antigens during DC priming. When immunotherapeutic intervention is limited to a single target antigen, there is the possibility of tumor escape. Tumor escape occurs when the tumor mutates to downregulate the immunogenic antigen and thus evades immune system surveillance and continues to proliferate. By preparing vaccines from whole tumor cells, the immune system can be trained to recognize a broad range of TAAs. Whole-tumor preparations also eliminate the need for researchers to identify an optimal antigen target; unknown tumor antigens may drive or contribute to the cellular immune response. Recent work utilized a lysate of tumor cells to demonstrate the effectiveness of whole tumor DC-based vaccination against ovarian cancer in both a preclinical and clinical setting . In the + T cells and NY-ESO-1-specific antibody responses were undetectable, but after vaccination with OLP and administration of both Montanide and Poly-ICLC, 91% of patients demonstrated both a NY-ESO-1-specific CD8+ T cells and a NY-ESO-1-specific antibody response. Additional targets for peptide vaccine strategies in ovarian cancer include p53, Her-2/neu, and CA125 [Peptide vaccines rely primarily on the immunogenicity of the injected peptides to stimulate an immune response. In the cancer setting, the peptides chosen for the vaccine are TAAs. Recently, overlapping long peptides (OLP) from NY-ESO-1 were used as a peptide vaccine in combination with two different adjuvant preparations: Montanide and Poly-ICLC . After Ond CA125 -76. WhilRecombinant viral vaccines utilize genetically modified viruses as vectors for introducing TAA-encoding DNA into cells within the body . Viruses+ T cell responses were induced in 32% of patients (14% had a preexisting response), while CD4+ T cell responses were induced in 22% of patients (68% had a preexisting response). In the ovarian cancer patients, the median PFS was 21 months and the median overall survival (OS) was 48 months. Poxviral vectors are also the basis for the PANVAC vaccine platform, which is actively being tested in ovarian cancer patients (NCT00088413). PANVAC is a cancer vaccine therapy that contains transgenes for the TAAs MUC-1 and carcinoembryonic antigen (CEA) as well as transgenes for three human T cell costimulatory molecules, collectively known as TRICOM  [The cancer testis antigen NY-ESO-1 is a well-documented target for immunotherapy of ovarian cancer and has been the focus of multiple cancer vaccine studies -80. Recetigen-3) . In a pitigen-3) .The convenience, low toxicity, and potential therapeutic activity of vaccine strategies make them an irresistible target of future immunotherapeutic research. As optimal vaccine platforms, antigen targets, and adjuvant conditions are identified, vaccines will become an increasingly valuable therapeutic option for treating ovarian cancer.in vivo survival and expansion of tumor-specific lymphocytes. First described in 1988, ACT initially demonstrated strong responses in melanoma, but has since been tested in other tumor types including ovarian cancers [Adoptive Cell Therapy (ACT) is an immunotherapeutic technique that uses autologous or allogeneic antitumor lymphocytes to induce cancer regression. In autologous ACT, lymphocytes are isolated via apheresis, cultured, and assayed for tumor recognition. Highly reactive cultures are expanded and reinfused into the cancer patient. Lymphodepleting chemotherapy preceding infusion eliminates immunosuppressive cells and supports the  cancers ,85. A 19 cancers . However cancers . The ant+ and CD8+ T cells at the tumor site and increased the number of antigen-specific host CD4+ T cells in the tumor and draining lymph nodes [In the ID8 mouse model of ovarian cancer, T cells transduced to express an NKG2D-based CAR demonstrated an endogenous antitumor immunity and long-term, tumor-free survival . Surviviph nodes . With enph nodes .While single-agent immunotherapies have produced promising clinical responses, unleashing the maximal antitumor immune response is likely to require combinatorial therapeutic strategies . As illuThe initial combination immunotherapies for ovarian cancer patients were based on immune checkpoint blockade strategies. In the ID8-VEGF model of ovarian carcinoma, researchers observed that up to half of TILs were double positive for both CTLA-4 and PD-1 and displayed a decreased proliferation capacity and inability to produce effector cytokines . Co-admiCombination immunotherapy trials have also expanded to include vaccine strategies, mAb therapy, and ACT. In a trial for patients with recurrent ovarian cancer, a DC-based autologous whole-tumor lysate vaccine was tested in combination with the anti-angiogenic mAb bevacizumab (NCT01312376). Of the six patients who participated, four patients demonstrated a clinical benefit and an increase in tumor-reactive T cells following vaccination; tumor reactivity was quantified by measuring IFN-γ secretion . From thIn the near future, the number of combination immunotherapy studies targeting ovarian cancer patients will dramatically increase. However, the application of multiple immunotherapies simultaneously requires careful considerations. Primarily, with combination treatments there is a potential for overlapping toxicities and elevated risk of sequela due to immune system disinhibition. Effective adverse-event management will be a crucial element of successful combination immunotherapy. Secondly, there is a need to optimize the timing of agent administration; in some situations, sequential administration may prove more advantageous than concurrent administration. If immune-related toxicities can be controlled and ideal dosing determined, combinatorial immunotherapy may dramatically improve the clinical outcomes of ovarian cancer patients.Early clinical successes have validated immunotherapeutic treatment strategies and immunotherapies hold immense potential to improve outcomes for patients with ovarian cancer. In future research, it will be important to identify the dominant immunosuppressive pathways within ovarian tumors. A better understanding of the relevant immuno-oncologic pathways and their corresponding biomarkers will allow patients to be optimally matched with therapies. In conjunction with biomarker research, combination strategies should continue to be explored. Combination approaches are uniquely appealing because different classes of immunotherapies target potentially synergistic stages of tumor immunity. Within the tumor microenvironment, immunotherapies may increase antigen release or antigen presentation, induce cytotoxicity via effector immune subsets, or remove immunosuppression. Identifying the optimal combination of drugs to provoke a concerted antitumor response will translate to substantial improvements in long-term clinical benefit.


Recent studies have shown that C-reactive protein is not just an indicator of cardiovascular disease (CVD) incidence and mortality. Thus, early detection of a continous increase in CRP concentrations may be useful in predicting subsequent development of CVD or metabolic syndrome.The objective of this study was to analyze high sensitivity C-reactive protein (hs-CRP) in apparently healthy young Korean adolescents and determine confounding factors for high hs-CRP in this population.We enrolled 197 middle school students (93 boys and 104 girls) who participated in a general health check-up at a tertiary hospital in Seoul. We measured height, weight, waist circumference and blood pressure and investigated hs-CRP concentrations, insulin levels, insulin resistance and lipid profiles. Hs-CRP levels were measured using the Behring BN II nephelometer  and log-transformed for analysis.hs-CRP concentration was significantly higher in boys than in girls (P=0.012). Pearson’s correlation coefficients revealed a significant correlation between log- transformed hs-CRP and BMI , waist circumference , systolic  and diastolic blood pressure , and ALT . In stepwise multivariate linear regression analysis, sex , waist circumference, and diastolic blood pressure were positively and fasting serum HDL-cholesterol level was negatively associated with log-transformed hs-CRP.We found that there exists a gender difference in hs-CRP concentrations in apparently healthy adolescents and that log-transformed hs-CRP concentrations were positively associated with male, waist circumference and diastolic blood pressure and negatively associated with HDL-cholesterol level. The gender difference and the contibuting factors for hs-CRP found in these healthy adolescents suggests a possibly relevant pathophysiological mechanism involved in the increase of cardiovascular risk associated with childhood obesity.


Antiseptic agents such as chlorhexidine digluconate, polyhexamethylene biguanide, benzalkonium chloride and octenidine dihydrochloride (octenidine) has been used widely in clinical settings to prevent bacterial infection, especially for antisepsis of skin and mucous membranes. However, irritation and cytotoxic effects of these compounds on human cell has been reported.This study compared the biocompatibility of newly-synthesized bis-quaternary ammonium compounds and biocides that have been commonly used as antiseptics, examining their antimicrobial activity, cytotoxic effect and expression of inflammatory cytokine-related genes.3-(3-Hydroxy-2-(hydroxymethyl)-2-{[(1-dodecylpyridinium-3-yl)oxy]methyl}propoxy)-1- dodecylpyridinium dibromide (3HHDMP) was synthesized.The antimicrobial activity of newly-synthesized compounds and commonly-used antiseptics were assessed by minimum bactericidal concentrations (MBC) and cytotoxic effect on normal human epidermal keratinocytes.The expression of inflammatory cytokines in normal human epidermal keratinocytes was quantified using real-time reverse transcription PCR (RT-PCR).A series of new bis-quaternary ammonium compounds, including Against Gram-negative and Gram-positive bacteria, 3HHDMP showed potent antimicrobial activity comparable to that of the octenidine. In a cytotoxic test using human epidermal keratinocytes, toxicity of 3HHDMP was equal or lower compared to that of quaternary ammonium compounds, although 3HHDMP showed higher toxicity than biguanide-based compounds. The comparison of the biocompatibility index as defined by antimicrobial activity and cytotoxic effect on human cell revealed that 3HHDMP had equal or greater biocompatibility compared with the biocides tested. Moreover, in the expression analysis of cytokine-related genes by cell culture method, increase of expression of cytokine-related genes in the cells stimulated with 3HHDMP was slower than that of existing quaternary ammonium compounds.From these results, it was concluded that the series of new bis-quaternary ammonium compounds including 3HHDMP had excellent biocompatibility, and thus the availability for application to the skin surface was suggested.None declared.


A 40-year-old woman, previously healthy, presented with a 6-month history of foreign body sensation of the pharynx with recent progressive dysphagia when swallowing both solid and liquid foods. A lateral cervical radiograph showed severe anterior osteophytosis on C3-C4, which was confirmed on computed tomography scan . The posterior pharyngeal wall was compressed by the anterior spurs at the C3-C4 level. Anterior resection of the ventral spinal osteophytes was performed via an antero-lateral extrapharyngeal approach. After the operation, the dysphagia resolved. Cervical osteophytes are common but osteophytes causing dysphagia due to compression of pharynx and oesophagus are unusual. The most common aetiologies are diffuse idiopathic skeletal hyperostosis (Forestier's disease) and ankylosing spondylitis. Initial treatment includes diet modifications, non-steroidal anti-inflammatory and muscle relaxants medications. Osteophytectomy may be considered in certain patients where conservative management fails. Stabilization of the spine is not advocated.


RANKL/RANK/OPG pathway modifications and the crosstalk between metastatic cells and bone have been indicated as potential drivers of the process. Interactions between tumor and bone cells have been studied We directly co-cultured bone metastatic human breast cancer cells (BOKL) with osteo-differentiated human mesenchymal cells (BMSCs) from 3 different donors. BMSCs and BOKL were then enzymatically separated and FACS sorted. We found a significant increase in the RANKL/OPG ratio as compared to control, which was not observed in BOKL cultured in medium conditioned by BMSCs, neither in BOKL directly cultured with fibroblasts or medium conditioned by fibroblasts. Direct co-culture with osteo-differentiated BMSCs caused BOKL aggregation while proliferation was not affected by co-culture. To more specifically associate RANKL expression to osteogenic differentiation degree of BMSCs, we determined their osteogenic markers expression and matrix calcification relative to osteoblasts and fibroblasts.In conclusion, our co-culture model allowed to demonstrate for the first time that direct contact but not paracrine interactions between human metastatic breast cancer cells and bone cells has a significant effect on RANKL/OPG expression in bone metastatic cells. Furthermore, only direct contact with the bone microenvironment induced BOKL clustering without however significantly influencing their proliferation and migration.The online version of this article (doi:10.1186/1476-4598-13-238) contains supplementary material, which is available to authorized users. Bone metastases arise in nearly 70% of patients with advanced breast cancer, indicating bone as a favorable microenvironment for metastases, which onset dramatically alters physiological bone turnover, leading to bone lysis. It has been reported  that thiin vitro co-culture models have been established [Recently, interactions between metastatic cells and metastatic niche cells have been indicated as potential drivers of the metastatic process . Thus, tablished –10. The ablished , 6 involablished  and mechablished . Moreoveablished , 8, onlyablished , 10 implablished .Our work was thus aimed at investigating, by means of direct co-culture, the interactions between a line of fluorescently tagged bone metastatic human breast cancer cells, MDA-MB231-BO-KL (BOKL) and a bone microenvironment represented by osteo-differentiated primary human bone marrow stromal cells (BMSCs), in terms of modifications on RANKL/RANK/OPG expression, cell proliferation and migration.To investigate whether specific direct contact between bone and tumor cells is involved in RANKL/OPG ratio modifications, we compared gene expression of BOKL after 3 days of direct co-culture with osteo-differentiated BMSCs (co-culture BMSCs) with that of BOKL cultured in conditioned medium from osteo-differentiated BMSCs (CM BMSCs) and that of BOKL in direct or indirect co-culture with fibroblasts, non osteogenic cells . Based on literature data showing that cell populations with equivalent capacity to form bone metastasis also share equal expression of genes related to bone invasion and metastasis , we chosResults of PCR analyses showed a strong and statistically significant upregulation  of RANKL in BOKL co-culture BMSCs as compared to control 1 but not in BOKL CM BMSCs and BOKL CM MRC-5 Additional file 1:Additional file 2: Figure S1: Confirmation of osteo-differentiation of BMSCs. A) Quantification of calcium deposits for BMSCs from three different patients. ***: p < 0.001, compared to fibroblasts. B) Quantification of ALP activity, normalized to the total protein content. All error bars represent standard deviations of at least three different experiments. (PPT 188 KB)


Injection drug users (IDUs) face limited access to HIV testing and experience delayed HIV diagnoses. New York City (NYC) pharmacies play an important role in HIV prevention among IDUs through the Expanded Syringe Access Program (ESAP), which provides access to sterile syringes without a prescription. We examined in-pharmacy HIV testing among syringe/non-syringe customers in ESAP pharmacies in NYC.Syringe/non-syringe pharmacy customers were recruited in two ESAP community pharmacies in high HIV prevalence neighborhoods to complete a 30-minute survey. In-pharmacy HIV testing was offered to all HIV-negative participants. Descriptive statistics, chi-square and Fisher’s exact tests were performed for categorical outcomes and t-tests for continuous outcomes to determine significant differences in individual characteristics between patrons who accepted in-pharmacy HIV testing compared with those who declined. Stepwise multivariable logistic regression was used to assess the relationship between receiving in-pharmacy HIV testing and customer characteristics identified from bivariate analysesOf 327 participants, most were male (57%), black (80%), had ever used hard drugs (88%); 217 (66.4%) were HIV-negative, and 39.6% received in-pharmacy HIV testing. After adjustment, being female , having multiple sex partners , having an HIV test more than 12 months ago , injecting drugs , and seeing the same regular medical provider  were associated with receiving in-pharmacy HIV test.This study presents evidence for a potential new venue for HIV testing that could provide access for hard-to-reach populations that have limited access to HIV testing. The approach we used was feasible to pharmacists. Given that pharmacies are located everywhere in NYC and have access to new point-of-care and home-based HIV testing technologies, the provision of HIV testing in pharmacies could provide easy access to HIV testing for IDUs as well as other populations.


Novel developments in tumor-immunotherapy show promising results in cancer therapy and might provide new opportunities for neuroblastoma treatment. To define suitable immunological targets, it is crucial to understand the role of the immune system and the mechanisms underlying neuroblastoma-induced immune suppression. We previously demonstrated accumulation of pro-inflammatory immune cells within tumor tissues and effective anti-inflammatory treatment with low-dose aspirin in the TH-MYCN mouse model. This model can be further used to dissect detailed mechanisms of neuroblastoma-induced immune suppression relevant for tumor development and tumor progression with the overall aim to define suitable targets for neuroblastoma immunotherapy.in vitro coculture assays with splenocytes of neuroblastoma-bearing TH-MYCN mice. Immune-cell status and functions were compared to wildtype controls. Reagents targeting immune suppressive mechanisms were tested in vitro. Coculture-assays using tumor supernatant-treated bone marrow cells were established to define factors responsible for the induction of suppressive immune cells.Flow-cytometric analysis of tumor tissues and spleens were performed, as well as +Ly6c+Ly6g+ myeloid-derived suppressor cells (MDSC) in tumor-bearing mice compared to controls. The myeloid cell populations found in tumor-bearing animals expressed high levels of PD1-binding ligand, PD-L1, suggesting a mechanism of T-cell inhibition and a possible target for immunotherapy. In spleens, total T cell numbers were reduced, while the CD4/CD8 ratio and expression of activation markers remained unchanged. RegulatoryT cells were present in tumor tissues, but their frequency was not enhanced in spleens of cancer-bearing animals, indicating that MDSCs play a major role in peripheral immune suppression and tumor progression. Sorted from spleens of tumor-bearing mice, these MDSCs were capable of suppressing T cell proliferation in vitro through the production of indolamine-2,3-dioxygenase (IDO) and inducible nictric oxide synthase (iNOS). To dissect the mechanisms of MDSC-induction in neuroblastoma, naïve bone marrow cells were cultured with neuroblastoma tumor cell line supernatants. This led to the development of suppressive cells with an "MDSC-like" phenotype. This system will be further exploited to define key factors enhancing the accumulation of MDSCs in this malignancy.Neuroblastoma-bearing animals develop splenomegaly, indicating an active role of the peripheral immune system during MYCN-driven neuroblastoma development. Flow-cytometric analysis of spleens showed accumulation of CD11bTumor-promoting inflammation contributes to neuroblastoma tumor development with active involvement of the peripheral immune system. Further studies are needed to define factors of the tumor microenvironment inducing direct or indirect immune-suppression. Immunological interventions by targeting various suppressive mechanisms will be evaluated aiming at future clinical therapeutic application.


Recent work by Rosenberg, et. al. demonstrates the role of mutated epitopes in natural anti-cancer adaptive immunity. This has led some to hypothesize that cancer vaccines may need to induce or augment immunity against mutated and potentially patient-specific epitopes to achieve therapeutic efficacy. In the case of whole-cell cancer vaccines, this could happen either indirectly through epitope spreading, or directly against 'hotspot' mutation sites on oncogenes. The purpose of this study is to determine whether patients who received an irradiated GM-CSF-secreting whole-cell prostate cancer vaccine, GVAX, preferentially developed humoral immune responses against mutated proteins. Archived pre-treatment and week 11 serum from patients enrolled on an investigator-initiated combination immunotherapy trial were assessed using protein arrays . Antibody responses that were increased in the week 11 sera were considered increased as a result of immunotherapy. Next we utilized reported liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics and whole-exome sequencing data for LNCaP and PC3, the two cell lines contained in prostate GVAX, to assess whether the targets of elevated antibody responses detected by protein array were directed preferentially against the mutated proteins contained in the vaccine. Of the 7554 different human proteins included in our analysis, a mean of 63 candidate responses were identified for each patient (SD = 43). These candidate responses were then compared to published non-synonymous mutations in LNCaP and PC3, published LC-MS/MS proteomics on the protein content of LNCaP and PC3, and randomized lists of proteins on the array [Supported by The Prostate Cancer Foundation, DAMD PC02009, Robert W. Franz, the Chiles Foundation, ARCS Foundation: Portland, and the OCTRI, OSLER TL1 award.


Dasatinib is a potent second-generation tyrosine kinase inhibitor for the treatment of chronic myeloid leukemia. The most common adverse event associated with dasatinib therapy is fluid retention, including pleural effusion. Dasatinib-related chylothorax has rarely been reported. The clinical manifestations, pathophysiology, management, and prognosis are not fully understood. Here we report a 40-year-old woman presenting with chylothorax following dasatinib use. We propose the hypothesis of its mechanism as well as offering a review of the relevant literature. Dasatinib (Sprycel®) is a highly potent small-molecule inhibitor of BCR-ABL and Src family tyrosine kinases, which is indicated for the treatment of adults with newly diagnosed chronic-phase chronic myeloid leukemia (CML), resistance or intolerance to prior CML therapy, or Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). Pleural effusion was a common complication in 14%-32% of all patients in clinical trials [A 40-year-old female, a housewife, had been diagnosed with CML with b2a2 BCR-ABL fusion 8 years ago. The initial presentation was peripheral leukocytosis with splenomegaly. She received 400 mg imatinib daily after the diagnosis and was titrated to 300 mg twice a day due to slow log reduction of BCR-ABL after 2 years. The log reduction achieved major molecular response half a year later. Four years after the diagnosis, the treatment was shifted to 50 mg of dasatinib twice a day because of intolerable knee pain. Her knee pain improved soon and the log reduction achieved complete molecular response (CMR) after 3 years of dasatinib treatment. Her liver and renal functions were monitored regularly and were within normal ranges.Forty months after dasatinib use, the patient complained of progressive dyspnea and aggressive cough. Chest radiography revealed elevation of the right hemidiaphragm and small pleural effusion of the left . ComputeDasatinib (Sprycel®) is a second-generation BCR-ABL tyrosine kinase inhibitor that targets most imatinib-resistant BCR-ABL mutations (except the T315I and F317V mutants) by distinctly binding to active and inactive ABL-kinase . Later, Chylothorax is caused by chyle leakage from the thoracic duct into the pleural space, which results from disruption or obstruction of the thoracic duct. Chyle typically contains high levels of triglycerides and often has a turbid and milky appearance. Triglyceride concentrations of pleural effusion greater than 110 mg/dL (1.24 mmol/L) strongly support the diagnosis . In thisDasatinib-related pleural effusions are generally lymphocyte-predominant exudates . These fSrc kinase inhibition by dasatinib is also possibly related to changes of vascular endothelial growth factor-mediated vascular permeability and stability of the pleural epithelium ,24. The The management of dasatinib-related pleural effusion including diuretics and a short course of prednisone (40 mg daily for 4 days) was suggested . Once-daIn conclusion, in patients under dasatinib treatment who develop chylothorax, dasatinib-related chylothorax should be considered. Dasatinib should be discontinued if the chylothorax is intractable. Further investigation to define the pathogenesis of dasatinib-related chylothorax is warranted since dasatinib is increasingly being used.


Breast cancer is a heterogeneous disease that is clinically classified into three groups with different therapeutic strategies: estrogen receptor (ER)-positive group receiving endocrine therapy, HER2-amplified group with benefit from HER2-targeted therapy, and triple- negative breast cancer  having only chemotherapy option due to lack of therapeutic targets (reviewed in ). AlthouIn recent years, accumulating evidence indicates that the loss of the PcG protein MEL-18 is associated with many aggressive behaviors independent of PcG complex functions in breast cancer. MEL-18 has a tumor suppressive function by inhibiting breast tumor growth and angiogenesis as a negative regulator of PI3K-AKT pathway in a BMI-1-independent manner . These rJ Clin Invest, we have focused more on understanding the mechanisms underlying the role of MEL-18 in ER- mediated hormonal regulation of breast cancer [ESR1 encoding ER and conferred hormone independence and tamoxifen resistance on ER-positive breast cancer [The loss of ER function is one of the key signatures for EMT and CSC, as well as aggressive behavior in human breast cancer. Thus in the latest work published in t cancer . Among tt cancer . Interest cancer . TogetheESR1 may lead to block the antagonistic effect of tamoxifen on ER in ER-positive breast cancer. It is also notable that MEL-18 regulates ESR1 expression via modulating the SUMOylation of ESR1 transcription factors, p53 and SP1, suggesting a strategy for SUMOylation-mediated ER restoration to recover tamoxifen sensitivity. Second, constitutive activation of AKT pathway by MEL-18 loss may overcome the tamoxifen-mediated growth inhibitory effect. Hyperactivation of PI3K/AKT signaling has been implied for hormone-independent breast cancer growth. This may also explain why MEL-18 loss could accelerate ER-positive breast cancer growth despite leading ER loss. Additionally, we assume that expansion of breast CSC accompanied by EMT promotion due to MEL-18 loss may also contribute to tamoxifen resistance in breast cancer.Insofar as tamoxifen resistance due to the loss of MEL-18, we suggest two molecular mechanisms, target alteration and bypass of estrogen signaling via ER-independent growth signaling . First, In summary, we have demonstrated that MEL-18 has a tumor suppressive function in breast cancer via multiple mechanisms, including regulation of signal cascade, miRNA-mediated epigenetic alteration, and SUMOylation-dependent transcriptional regulation in a PcG-independent manner. Loss of MEL-18 might be a critical feature in acquiring aggressive behaviors, including EMT and CSC promotion, hormone independence, and endocrine therapy resistance in breast cancer, suggesting the emerging role of MEL-18 as a new determinant of breast cancer aggressiveness.


Pollen-food allergy syndrome (PFAS) is a relatively rare form of food allergy which develops in individuals who are sensitized to pollen. Tree pollens, especially birch pollen, frequently induce PFAS; however, the incidence of PFAS due to grass or weed pollens such as ragweed or mugwort is relatively rare. Mugwort-mustard allergy syndrome (MMAS) is an example of a PFAS in which individuals sensitized to mugwort may develop an allergy to mustard and experience severe reactions. We herein describe a case of MMAS due to broccoli consumption. Pollen-food allergy syndrome (PFAS) is a relatively rare form of food allergy which develops in individuals who are sensitized to pollen . Tree poA 73-year-old Japanese male presented to our hospital with dyspnea and swelling of the lips and eyelids which occurred 30 minutes after eating boiled broccoli. He had no history of allergic reactions to latex or fruits. The patient's clinical findings suggested he was experiencing an immediate-type hypersensitivity reaction to broccoli, such as oral allergy syndrome (OAS) or PFAS. Therefore, tests for allergic reactions including the CAP fluorescent enzyme immunoassay RAST (CAP-FEIA RAST) test and a skin-prick test (SPT) were performed. A blood test revealed an elevated level of serum immunoglobulin E  and IgE antibodies specific for mugwort  were detected in the CAP-FEIA RAST test, although no IgE antibodies specific for almond or mustard were detected. An SPT was then performed using the prick-to-prick technique with samples of mugwort, broccoli, cauliflower, cabbage, mustard, almond, and peanut. The results of the SPT were assessed as false positive (+), positive (2+), strongly positive (3+), and most positive (4+) when the average wheal diameter was more than 25%, 50%, 100%, and 200%, respectively, of a positive control induced by 10 mg/mL of histamine dihydrochloride 30 minutes after administration [Mugwort pollens are known to cross-react with some fruits and vegetables. Therefore, mugwort-related PFAS is classified as celery-mugwort-spice syndrome (CMSS), MMAS, mugwort-peach association (MPA), or mugwort-chamomile association (MCA) . PatientPollen allergens of MMAS are believed to include Art v4 (a profilin), Art v3 (an LTP), and Art v60 kDa  . We specFurther studies are needed to identify the causative allergens of MMAS because the cross-reactivity between Art v3 and LTP in broccoli remains a possibility. However, the present case will help expand our knowledge of MMAS.


Esophageal cancer is the malignant tumor with very poor prognosis and increasing incidence often diagnosed at very late stage, so the prognosis of affected patients is unsatisfactory, despite the development of therapeutic option such as surgery, chemotherapy and radiotherapy. Consequently, there is a great need for biomarkers to allow a tailored multimodality approach with increased efficiency. Altered expression of microRNAs has been reported in wide range of malignancies, including esophageal cancer. The aim of this study was to examine the expression levels of candidate microRNAs in esophageal cancer and evaluate their diagnostic and prognostic potential.Using quantitative real-time PCR, expression levels of 9 candidate microRNAs were examined in 62 tissue samples, 23 esophageal adenocarcinomas, 22 esophageal squamous cell carcinomas and 17 adjacent esophageal mucosa samples. MicroRNA expression levels were further analyzed in regards to clinico-pathological features of esophageal cancer patients. We observed significantly decreased levels of miR-203 and increased levels of miR-21 in adenocarcinoma tissues when compared to normal mucosa. MiR-29c and miR-148 indicated good ability to distinguish between histological subtypes of esophageal cancer. MiR-203 and miR-148 were linked to disease-free survival and overall survival in esophageal adenocarcinoma patients, and miR-148 also in esophageal squamous cell carcinoma patients.Our data suggest that altered expression of miR-21, miR-29c, miR-148 and miR-203 are related to neoplastic transformation and progression of the disease and these microRNAs could serve as a potential diagnostic and prognostic biomarkers in esophageal cancer.http://www.diagnosticpathology.diagnomx.eu/vs/4646922201567057The virtual slide(s) for this article can be found here:  Esophageal cancer (EC) is the seventh most common cancer worldwide and the sixth most common cause of cancer death . There aEsophageal adenocarcinoma (EAC) is one of the fastest rising cancers in Western society. Incidence has increased by 600% within the last 30 years . The reaBoth EAC and ESCC are usually detected at an advanced stage, requiring a multimodal concept of therapy, however the overall survival of esophageal cancer remains lower than other solid tumors . AlthougMicroRNA (miRNA) are short non-coding RNA with ability to regulate important cellular processes as differentiation, cell cycle, proliferation or apoptosis. They are known to be deregulated in most tumor types , in whicA total of 62 tissue samples from two esophageal cancer cohorts were included in the study. First cohort  consisted of 17 EAC and 5 ESCC cases, whereas for 17 EAC cases paired esophageal mucosa samples were available. Second cohort  consisted of 6 EAC and 17 ESCC cases. All subjects were of the same ethnicity (Caucasian). Clinico-pathological features including age, sex, histology and treatment were recorded and summarized in Table Forty-five samples of tumor tissue and seventeen samples of non-cancerous esophageal mucosa were collected and stored in RNAlater . Afterward, tissue samples were homogenized , and total RNA enriched for small RNA fraction was isolated using fenol-chloroform extraction with TRIzol reagent . Concentration and purity of RNA were determined using a Nanodrop ND-1000 , and RNA integrity was measured by Agilent 2100 Bioanalyzer using Agilent RNA 6000 Nano Kit . Samples were either stored at -80°C or further processed.cDNA was synthesized from total RNA using miRNA-specific primers according to the Taq-Man MicroRNA Assay protocol . Real-time polymerase chain reaction (PCR) was performed according to manufacturing protocol  using the Applied Biosystems 7500 Instrument as described previously [-ΔCt method. Differences between expression levels in paired samples were evaluated by non-parametric Wilcoxon test. Differences between miRNA levels in EAC and ESCC samples were evaluated by non-parametric Mann–Whitney U-test. Survival analyses were carried out using the log-rank test and Kaplan-Meier survival analysis. All calculations were performed using Prism version 6.00 . P-values of less than 0.05 were considered statistically significant.The threshold cycle data were calculated by SDS 2.0.1 software . The average expression levels of measured miRNAs were normalized using RNU44 as reference gene and subsequently analyzed by the 2To determine whether the selected miRNAs  have a diagnostic potential, expression levels were analyzed for each miRNA in 17 EAC samples and 17 paired adjacent mucosa. There were 2 miRNAs differentially expressed in tumor tissue and adjacent mucosa. MiR-203 levels were significantly down-regulated in tumor tissue , miR-21 levels were significantly up-regulated in tumor tissue   in 22 ESCC samples and 22 EAC samples. We found that miR-148 and miR-29c were decreased in ESCC when compared to EAC and have ability to significantly distinguish ESCC and EAC tissues , respectively . Down-regulation of miR-148 was associated with significantly shorter overall survival  in ESCC patients. Interestingly, opposite observation was reached in EAC patients, where cases with up-regulation of miR-148 showed significantly shorter disease free survival  and overall survival , indicating opposite functioning of miR-148 in EAC and ESCC (Figure et al., where up-regulation of miR-21 could predict unfavorable prognosis in esophageal cancer [Our results indicate that miR-203 plays role of tumor suppressor in EAC since the expression levels of this miRNA were significantly decreased in tumor tissue of EAC patients. Those results are in compliance with another studies, where miR-203 displayed stepwise decrease in expression during malignant progression from healthy tissue through Barrett’s esophagus to EAC -11. Morel cancer .The second aim of our study was to discriminate EAC from ESCC tissue. For the first time, we found that the expression levels of miR-148a and miR-29c are significantly higher in EAC when compared to ESCC tissue. Moreover up-regulation of miR-148 could predict unfavorable prognosis in EAC cancer and surprisingly favorable prognosis in ESCC patients. It seems that miR-148 can play the role of tumor suppressor or tumor oncogene depending on histological subtype of esophageal cancer.et al. has reported the definition of miR-148a as a novel prognostic oncomiR in glioblastoma with high levels considered as a risk factor for glioblastoma patients survival [MiR-148 was not yet studied in association with esophageal cancer, but it is known to act as oncogene and also as tumor suppressor in different tumor types. Thus, Kim survival . When ansurvival . On the survival . Low expOur data indicate, that miR-203 and miR-21 could serve as diagnostic biomarkers. Further, miR-29c and miR-148 have ability to distinguish between EAC and ESCC histological subtypes of esophageal cancer. In future, the diagnosis could be assessed for instance from non-invasive brush biopsy of esophagus . Moreove


The dorsal striatum integrates inputs from multiple brain areas to coordinate voluntary movements, associative plasticity, and reinforcement learning. Its projection neurons consist of the GABAergic medium spiny neurons (MSNs) that express dopamine receptor type 1 (D1) or dopamine receptor type 2 (D2). Cholinergic interneurons account for a small portion of striatal neuron populations, but they play important roles in striatal functions by synapsing onto the MSNs and other local interneurons. By combining the modified rabies virus with specific Cre- mouse lines, a recent study mapped the monosynaptic input patterns to MSNs. Because only a small number of extrastriatal neurons were labeled in the prior study, it is important to reexamine the input patterns of MSNs with higher labeling efficiency. Additionally, the whole-brain innervation pattern of cholinergic interneurons remains unknown. Using the rabies virus-based transsynaptic tracing method in this study, we comprehensively charted the brain areas that provide direct inputs to D1-MSNs, D2-MSNs, and cholinergic interneurons in the dorsal striatum. We found that both types of projection neurons and the cholinergic interneurons receive extensive inputs from discrete brain areas in the cortex, thalamus, amygdala, and other subcortical areas, several of which were not reported in the previous study. The MSNs and cholinergic interneurons share largely common inputs from areas outside the striatum. However, innervations within the dorsal striatum represent a significantly larger proportion of total inputs for cholinergic interneurons than for the MSNs. The comprehensive maps of direct inputs to striatal MSNs and cholinergic interneurons shall assist future functional dissection of the striatal circuits. As the key input station in the basal ganglia, the dorsal striatum coordinates neuronal signals from multiple cortical and subcortical regions and plays a critical role in voluntary movements, motor learning, cognition, and emotion ,2,3,4. DCholinergic interneurons consist of approximately 2% of total neurons in the dorsal striatum . AlthougIdentification of the inputs to various types of striatal neurons is essential for understanding how neuronal activity patterns are shaped. Using the traditional method of tract tracing, numerous studies have shown that the dorsal striatum receives converging inputs from the cortex, thalamus, SNc, and other subcortical nuclei ,25,26,27In this study, we studied the whole-brain input patterns of D1- and D2- MSNs and cholinergic interneurons by selectively infecting these neurons with the modified rabies virus. Our results reveal that the two types of MSNs and the cholinergic interneurons receive extensive inputs from discrete areas in the cortex, thalamus, amygdala, globus pallidus, and SNc. Moreover, cholinergic interneurons receive particularly dense local innervations from non-cholinergic neurons within the dorsal striatum.All procedures were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) of National Institute of Biological Sciences (NIBS), Beijing, China and the Ethics Committee of Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences.ad libitum. Adult mice of either sex (8–16 weeks old) were used for all experiments, whereas age-matched wild-type C57BL/6J mice were used as the control.The BAC-transgenic Chat-Cre [MMRRC Tg(Chat-cre)24Gsat], D1-Cre [MMRRC Tg(Drd1a-cre)EY262Gsat], and D2-Cre [MMRRC Tg(Drd2-cre)ER44Gsat] mouse lines were acquired from MMRRC  . These m8 infecting unit per milliliter. The helper viruses included AAV with a DIO sequence carrying TVA (AAV-DIO-EGFP–TVA) or rabies glycoprotein (AAV-DIO-RG). The AAV-DIO-EGFP-TVA plasmid was constructed by subcloning the CAG promoter from the AAV-CAG-GFP-IRES-CRE plasmid (Addgene Plasmid 48201) and the coding region of EGFP:2A:TVA from the pAAV-EF1a-FLEX-GT plasmid  into the DIO cassette of the plasmid pAAV-EF1a-DIO-hChR2(H134R)-EYFP  .,35.34,35We injected helper AAV viral vectors and then modified rabies virus into the center of the unilateral dorsal striatum of all three Cre-expressing mouse lines (n = 4 mice for each line). We quantified the number of mCherry-expressing neurons in individual nuclei within the entire brain of each mouse and then normalized the cell numbers by the total number of input neurons within each animal to correct for the variability in the numbers of starter cells . In both–16, two-tailed t-test), which further suggests that the intrastriatal input neurons were non-cholinergic.Due to the strong intrastriatal innervation of dorsal striatal cholinergic neurons, we asked whether the striatal input neurons were cholinergic. Following virus injections into ChAT-Cre mice, we labeled dorsal striatal cholinergic neurons by immunostaining against ChAT . We did vs. 300 nL in this study) and/or variability in effective titers of rabies virus. The labeling density in this study is more consistent with that revealed by many previous retrograde tracings. Most likely, striatal MSNs receive much more extensive inputs than what can be inferred from the number of ~200 of retrograde labeled neurons.Second, we observed substantial retrograde labeling in the secondary visual cortex, cingulate cortex, centrolateral/paracentral thalamic nucleus, GPe, and SNc, whereas the labeling within these areas was very modest or absent in the prior study . Our datWe found that the striatal cholinergic interneurons received much stronger intrastriatal innervation than the MSNs, which is consistent with the role of cholinergic interneurons in intrastriatal processing ,52,53,54Comprehensive maps of monosynaptic input patterns for MSNs and cholinergic neurons should facilitate dissection of physiological and behavioral functions of various afferents of the dorsal striatum. The dorsal striatum contains additional interneurons, including the GABA/parvalbumin fast spiking interneurons and GABA/somatostatin/nitric oxide interneurons . The inpIn this study, we mapped the distribution of input neurons to D1- and D2-expressing projection neurons and ChAT-expressing interneurons in the dorsal striatum using transsynaptic retrograde tracing with a modified rabies virus. All three types of neurons receive extensive inputs from cortical and thalamic areas that are involved in sensory and motor processing. They also receive input from limbic areas such as the cingulate cortex and amygdala. Additional notable input areas include the GPe and the SNc. Distal innervation patterns are similar for the two types of MSNs and the cholinergic interneurons. However, cholinergic interneurons receive denser intrastriatal inputs from the non-cholinergic neurons within the dorsal striatum.