Drosophila melanogaster, UDP-galactose 4′-epimerase (GALE) catalyzes two distinct reactions, interconverting UDP-galactose  and UDP-glucose (UDP-glc) in the final step of the Leloir pathway of galactose metabolism, and also interconverting UDP-N-acetylgalactosamine  and UDP-N-acetylglucosamine (UDP-glcNAc). All four of these UDP-sugars serve as vital substrates for glycosylation in metazoans. Partial loss of GALE in humans results in the spectrum disorder epimerase deficiency galactosemia; partial loss of GALE in Drosophila melanogaster also results in galactose-sensitivity, and complete loss in Drosophila is embryonic lethal. However, whether these outcomes in both humans and flies result from loss of one GALE activity, the other, or both has remained unknown. To address this question, we uncoupled the two activities in a Drosophila model, effectively replacing the endogenous dGALE with prokaryotic transgenes, one of which (Escherichia coli GALE) efficiently interconverts only UDP-gal/UDP-glc, and the other of which (Plesiomonas shigelloides wbgU) efficiently interconverts only UDP-galNAc/UDP-glcNAc. Our results demonstrate that both UDP-gal and UDP-galNAc activities of dGALE are required for Drosophila survival, although distinct roles for each activity can be seen in specific windows of developmental time or in response to a galactose challenge. By extension, these data also suggest that both activities might play distinct and essential roles in humans.In both humans and  In this manuscript we apply a fruit fly model to explore the relative contributions of each of two different activities attributed to a single enzyme—UDP-galactose 4′-epimerase (GALE); partial impairment of human GALE results in the potentially severe metabolic disorder epimerase deficiency galactosemia. One GALE activity involves interconverting UDP-galactose and UDP-glucose in the Leloir pathway of galactose metabolism; the other activity involves interconverting UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. We have previously demonstrated that complete loss of GALE is embryonic lethal in fruit flies, but it was unclear which GALE activity loss was responsible for the outcome. Using genetically modified fruit flies, we were able to remove or give back each GALE activity individually at different times in development and observe the consequences. Our results demonstrate that both GALE activities are essential, although they play different roles at different times in development. These results provide insight into the normal functions of GALE and also have implications for diagnosis and intervention in epimerase deficiency galactosemia. Galactose is an essential component of glycoproteins and glycolipids in metazoans, and as a constituent monosaccharide of the milk sugar, lactose, also serves as a key nutrient for mammalian infants. Galactose is also found in notable quantities in some fruits, vegetables, and legumes. Galactose is both synthesized and catabolized in all species via the Leloir pathway, which is highly conserved across branches of the evolutionary tree The reactions of the Leloir pathway are catalyzed by the sequential activities of three enzymes: (1) galactokinase (GALK) which phosphorylates alpha-D-galactose to form galactose-1-phosphate , (2) galactose-1-phosphate uridylyltransferase (GALT), which transfers uridine monophosphate (UMP) from uridine diphosphoglucose (UDP-glc) to gal-1P, forming UDP-galactose  and releasing glucose-1-phosphate (glc-1P), which can proceed to phosphoglucomutase and the glycolytic pathway, and (3) UDP-galactose 4′-epimerase (GALE) which interconverts UDP-gal and UDP-glc hGALE allele V94M, which leads to severe epimerase-deficiency galactosemia in the homozygous state, encodes an enzyme that retains ∼5% residual activity toward UDP-gal but ∼25% residual activity toward UDP-galNAc Deficiency in any of the three Leloir enzymes in humans results in a form of the metabolic disorder galactosemia, although the symptoms and clinical severity differ according to which enzyme is impaired and the extent of the impairment. Profound loss of hGALE results in generalized epimerase-deficiency galactosemia, an autosomal recessive and potentially severe disorder. To date, however, no patient has been reported with complete loss of GALE, and even the most severely affected demonstrate at least 5% residual enzyme activity Drosophila melanogaster model of GALE deficiency GALE is essential in Drosophila; animals completely lacking endogenous dGALE succumb as embryos, and conditional loss of dGALE in larvae results in death within two to four days of knockdown. Finally, partial loss of dGALE leads to galactose sensitivity in larvae, and transgenic expression of human GALE (hGALE) rescues each of these negative outcomes To address this question, we applied a Drosophila model to uncouple and examine the individual roles of GALE separately. Toward that end, we generated flies that lacked endogenous dGALE and expressed either of two prokaryotic transgenes, one encoding E. coli GALE (eGALE) which exhibits an approximately 8,000-fold substrate preference for UDP-gal/UDP-glc over UDP-galNAc/UDP-glcNAc P. shigelloides wbgU, which exhibits an approximately 2,000-fold substrate preference for UDP-galNAc/UDP-glcNAc over UDP-gal/UDP-glc dGALE-deficient Drosophila we determined that both GALE activities are required for survival of embryos and larvae. We also found that restoration of one activity or the other in later development rescued some phenotypes. Combined, these results provide insight into the varied roles of dGALE in Drosophila development and homeostasis, and by extension, suggest that hGALE may play similarly complex and essential roles in humans.Here we have applied our transgenic Drosophila GALE interconverts the first of these substrate pairs  Human and other mammalian GALE enzymes efficiently interconvert both UDP-gal/UDP-glc and UDP-galNAc/UDP-glcNAc e.g. . PrevioueGALE (UAS-eGALE) or wbgU (UAS-wbgU), respectively, each in a conditionally dGALE-impaired background. Each of these prokaryotic GALE genes has been demonstrated previously to encode epimerase activity toward only one of the two sets of epimer pairs . eGALE alone nor wbgU alone was sufficient to rescue survival of the dGALE-deficient animals; however, expression of both eGALE and wbgU, in combination, was sufficient. These results demonstrate that GALE activities toward both UDP-gal and UDP-galNAc are essential for survival of D. melanogaster. To rule out the possibility that rescue with eGALE plus wbgU in combination occurred not because both GALE activities are essential but rather because neither individual transgene expressed sufficient enzyme, we also tested additional eGALE and wbgU transgenes that individually demonstrated higher levels of expression; none was sufficient to rescue (data not shown). Of note, there also was no apparent over-expression phenotype; for example, animals expressing either eGALE or wbgU in addition to endogenous dGALE, and animals dramatically over-expressing human GALE, remained viable, fertile, and appeared morphologically normal (data not shown).Previously, we created and characterized two dGALE in Drosophila using a dGALEUAS-RNAi transgene  in combination with a temperature sensitive allele of yeast GAL80 (tsGAL80)  . Using ttsGAL80conditional dGALE knockdown system to include different GAL4-dependent GALE transgenes and have applied this expanded system to test the ability of each transgene, or pair of transgenes, to compensate for the loss of endogenous dGALE. By using age-synchronized cohorts of animals and shifting from the permissive (18°C) to the restrictive temperature (28–29°C) at different times we also were able to test the ability of each GALE transgene, or pair of transgenes, to sustain survival and fecundity at different stages of development. At 18°C these animals expressed endogenous dGALE, but not their transgenes, and at 28–29°C these animals expressed their transgenes but not dGALE .Of note, while elopment , these smparable . As expedGALE knockdown was initiated during early to mid-stage pupal development, animals of both sexes displayed diminished fecundity. Expression of eGALE alone, but not wbgU alone, rescued the male defect, whereas expression of both prokaryotic transgenes in combination, or hGALE alone, was required to rescue the female defect. These results indicate that GALE activity toward UDP-gal is both necessary and sufficient for male fecundity, but that GALE activities toward both UDP-gal and UDP-galNAc are required for female fecundity.To examine fecundity, we collected and sequestered newly eclosed virgin female and male flies from each surviving cohort, crossed them to an equal number of wild-type flies of the opposite sex, and counted the numbers of viable offspring resulting from each cross. Crosses resulting in large numbers of viable offspring (>50) were scored as “normal fecundity”. Crosses resulting in fewer than 10 viable offspring were scored as “reduced fecundity,” and crosses resulting in no viable offspring were scored as “loss of fecundity” . For exaDrosophila expressing a hypomorphic allele of dGALE are viable but sensitive to galactose exposure GALE activities in coping with environmental galactose, we collected adult flies in which dGALE knockdown coupled with hGALE, eGALE, wbgU, or eGALE plus wbgU transgene expression was initiated using the tsGAL80 system during late larval or early-to-mid-pupal development. These animals were allowed to develop on a standard molasses-based food, and were then transferred as newly eclosed adults to food containing either 555 mM glucose as the sole sugar, or 555 mM glucose plus 175 mM galactose.We have previously demonstrated that GALE transgene were also monitored  is an essential enzyme in Our results demonstrate that developing animals require at least some GALE activity toward both epimer pairs, even in the absence of dietary galactose. Complete loss of either activity in embryos is lethal . In animHowever, knockdown of either GALE activity in early development, or knockdown of both activities in later development has consequences. For example, loss of activity toward UDP-gal in larvae results in impaired fecundity of both males and females, while loss of activity toward UDP-galNAc in larvae results in impaired fecundity of females but not males. Individual loss of one activity or the other later in development also results in differential sensitivity to galactose. Specifically, both male and female flies deficient in GALE activity toward UDP-gal exhibit a markedly reduced lifespan when exposed to galactose; this effect is not seen in wild-type flies or in flies uniquely deficient in GALE activity toward UDP-galNAc.Drosophila extends beyond the Leloir pathway. Whether GALE activity toward UDP-galNAc is essential because of its presumed role in establishing and maintaining substrate pools for glycosylation, or for some other reason, remains unknown. Prior studies in GALE-deficient mammalian cells These results support two important conclusions. First, the essential role of GALE in development and homeostasis of Drosophila deficient in GALE activity toward UDP-galNAc, e.g. compromised survival in embryos and compromised fecundity in adult females, do not result from gal-1P accumulation. This is an important point because it challenges the common supposition that gal-1P underlies pathophysiology in both classic and epimerase deficiency galactosemias. Clearly there must be another basis for the negative outcomes observed in these animals. It is also interesting to note that while loss of GALE activity toward UDP-galNAc in developing animals has phenotypic consequences, at least for female fecundity, it does not appear to negatively impact the “global” level of UDP-galNAc in animals exposed to galactose. The explanation for this apparent disparity might involve subtle or tissue-specific differences below the threshold of detection of our experimental approach.The disparate metabolic profiles observed in GALE-impaired flies exposed to galactose provide a window of insight into potential mechanisms behind the outcomes observed. For example, gal-1P accumulates to abnormal levels in animals missing GALE activity toward UDP-gal but not UDP-galNAc, and only those animals demonstrate substantially reduced lifespan when exposed to galactose as adults. This metabolic result is expected, since only GALE activity toward UDP-gal should impact the Leloir pathway, and this outcome result implies that gal-1P might contribute to the early demise of these animals. However, the gal-1P result also implies that the negative outcomes observed in The implications of this work for patients with epimerase deficiency galactosemia are two-fold. First, these results demonstrate that both GALE activities are essential for health of flies, and possibly also people. To our knowledge clinical laboratories that test patient samples for GALE activity only test activity toward UDP-gal. While this practice is certainly understandable, given that mutations may impact the two GALE activities differently The Drosophila stocks used in this study are listed in UAS-eGALE and UAS-wbgU transgenes were generated by subcloning the eGALE and wbgU coding sequences, respectively, as EcoRI/XhoI fragments, into pUASTEcoRI and XhoI sites in the pUAST polylinker region. The wbgU sequence was amplified from a plasmid generously provided by Peng George Wang (Ohio State University). Resulting plasmids were confirmed by sequence analysis. UAS-eGALE stocks were generated using standard transgenic techniques following injection of the transgene into embryos by the fly core of the Massachusetts General Hospital, Charlestown, MA. UAS-wbgU stocks were generated using standard transgenic techniques following injection of the transgene into embryos by Genetic Services, Inc., Cambridge, MA. Transformants were selected by the presence of the white gene within pUAST. Expression of functional eGALE or wbgU was confirmed by enzymatic assay of lysates from transformants.Lysates were prepared and assays for GALK, GALT and GALE with UDP-gal as the substrate were performed (n≥3) as described previously hGALE or wbgU transgenes, which were diluted to a greater extent. Lysates from 22CAct5C>hGALE animals were diluted 1∶60. Lysates from 19AAct5C>wbgU animals were diluted 1∶20.Activity was calculated from the conversion of UDP-galNAc to UDP-glcNAc. The initial reaction mixture concentrations were: 100 mM glycine pH 8.7, 1.6 mM UDP-galNAc and 0.5 mM NAD. Enzyme assays were performed as described in Sanders et al. GALE knockdown was initiated at 24-hour intervals throughout development was achieved as described previously ts}10P{tubP-GAL80 and 12030R-2 was used in all crosses. These flies were then crossed to the appropriate genotypes to obtain offspring expressing various transgenes; for: no transgene, P{Act5C-GAL4}25FO1 ; +/TTSTL, Tb, Hu; eGALE only, 62A/CyOP{Act5C-GAL4}25FO1, UAS-eGALE; wbgU only, 19AP{Act5C-GAL4}25FO1/CyO; P{Act5C-GAL4}25FO1/CyO ; UAS-wbgU/TM6B; eGALE plus wbgU, 62A/CyOP{Act5C-GAL4}25FO1, UAS-eGALE ; 19AUAS-wbgU/TM6B; hGALE, 22C/TM6BP{Act5C-GAL4}25FO1/CyO ; UAS-hGALE. Adult flies eclosing from the vials were scored for the presence or absence of humeral and/or curly, as appropriate for each cross.Generation of animals in which dGALE knockdown with concurrent transgene expression was achieved throughout development were obtained as described above. These animals were maintained on standard molasses medium until eclosion. Within 24 hours after eclosion, approximately 20 virgin male or female flies were placed in fresh vials of food containing 555 mM glucose only or 555 mM glucose plus 175 mM galactose. Flies were transferred to fresh food every 2–3 days, and the number of dead flies in each vial was recorded every other day. Log rank and Wilcoxon tests were used for statistical analysis using the program JMP (http://www.jmp.com/).Animals in which Cohorts of newly hatched larvae raised at 18°C were transferred to vials of food containing either 555 mM glucose only or 555 mM glucose plus 175 mM galactose. The larvae were maintained at 18°C for one additional day, then transferred to 28°–29°C and allowed to develop for another four days prior to harvest. Metabolites were extracted and quantified as described previously Table S1D. melanogaster stocks and alleles used in this study.(DOCX)Click here for additional data file.


Secondary obstructive cholangiopathy is characterized by intra- or extrahepatic bile tract obstruction. Liver inflammation and structural alterations develop due to progressive bile stagnation. Most frequent etiologies are biliary atresia in children, and hepatolithiasis, postcholecystectomy bile duct injury, and biliary primary cirrhosis in adults, which causes chronic biliary cholangitis. Bile ectasia predisposes to multiple pathogens: viral infections in biliary atresia; Gram-positive and/or Gram-negative bacteria cholangitis found in hepatolithiasis and postcholecystectomy bile duct injury. Transmembrane toll-like receptors (TLRs) are activated by virus, bacteria, fungi, and parasite stimuli. Even though TLR-2 and TLR-4 are the most studied receptors related to liver infectious diseases, other TLRs play an important role in response to microorganism damage. Acquired immune response is not vertically transmitted and reflects the infectious diseases history of individuals; in contrast, innate immunity is based on antigen recognition by specific receptors designated as pattern recognition receptors and is transmitted vertically through the germ cells. Understanding the mechanisms for bile duct inflammation is essential for the future development of therapeutic alternatives in order to avoid immune-mediated destruction on secondary obstructive cholangiopathy. The role of TLRs in biliary atresia, hepatolithiasis, bile duct injury, and primary biliary cirrhosis is described in this paper. Biliary epithelial cells secrete immunoglobulin A in relation to pathogen-associated molecular patterns (PAMPs). When biliary epithelium gets in touch with a pathogen, it is then activated by PAMPs and induces different mechanisms to prevent or limit tissue damage by means of Toll-like receptor-3 (TLR-3), which in turn stimulates transcriptional nuclear factor-κB (NF-κB) after production of antiviral interferon-1β (IFN-1β) [Innate immunity acts, responds, modulates, and provides defense against viral and bacterial infections through trans-membrane cell receptors encoded in the germ line, among them are the Toll-like receptors (TLRs). As part of the innate immune response, there are several endogenous antibiotics against (IFN-1β) .β2 integrin. Pathogen microorganisms by themselves can release bacterial peptides such as N-formate, potent neutrophil chemoattractant in transepithelial migration [Many symptomatic infectious diseases of the biliary tract are characterized by neutrophil migration across the epithelium and accumulation within the lumen. Transepithelial migration of neutrophils represents an important factor in mucous epithelium defense of inflammatory diseases. When inflammatory reaction starts on biliary epithelium, neutrophils traverse vascular endothelium to lamina propria and reach epithelium. Transepithelial migration of neutrophils can be initiated by stimulation of interleukin-8 (IL-8) as a consequence to host-pathogen interaction and regulated by early events in response to transepithelial migration of CD11b/CD18-igration . β, tumor necrosis factor-alpha (TNF-α), and IL-6), chemotactic cytokines (monocyte chemotactic protein (MCP-1), inhibitory factor macrophage migration (MIF)), proinflammatory proteins, reactive oxygen species, and adhesion molecules and expression of receptors that recognize microorganisms, such as TLRs in biliary epithelium cells, which provides protection against bacterial and viral infections. On the other hand, biliary epithelium is constantly exposed to PAMPs that do not trigger inflammatory responses, denominated as immune tolerance, which maintains homeostasis between the body and commensal bacterial flora, thus avoiding excessive damage to the epithelium [Biliary innate immunity is actively involved in inflammation by secreting cytokines  , 21, andSalmonella typhimurium. Flagellin is a potent activator of systemic inflammation in humans, and its serum levels are positively correlated with clinical severity in presence of bacteremic shock by playing an important role in the immunopathogenesis of inflammatory bowel disease. Flagellin is also capable of activating apoptotic signaling pathways and proinflammation and can lead to activation of innate immunity [TLR-5 recognizes the bacterial component flagellin protein of immunity .TLR-7 is involved in recognition of imidazoquinolines. These receptors induce type I IFN production during viral infections and are expressed on endosome of dendritic cells, human plasmacytoid cells, myeloid cells, and monocyte-derived cells; thereby, its expression on different body systems depends on the number of dendritic cells held by each organ.TLR-8 is activated in viral infections producing type I IFN. It belongs to a family of endosomic recognition pattern receptors capable of producing type I IFN MyD88-dependent and detect pathogen-derived nucleic acids. Human monocytes constitutively express TLR-8 .β because its effects can be blocked by TLR-9 antagonists, suggesting activation of the innate immune response within the HSC. The TLR-9 responds to viral infections by producing type I IFN [TLR-9 recognizes bacterial DNA with unmethylated motifs. Apoptotic bodies containing microkernels are a source of DNA. The unmethylated motifs of cytosine-phosphate-guanosine- (CpG-) DNA are recognized by TLR-9. TLR-9-deficient mice manifest less liver injury in response to poisoning by carbon tetrachloride, bile duct ligation, acetaminophen and fatty liver hepatitis model. TLR-9 activates different signaling cascades resulting in generation of myofibroblasts by hepatic stellate cells (HSCs), collagen type 1, and overexpression of the profibrogenic cytokine TGF-pe I IFN .locus on chromosome 4p14 [κB and IFN regulatory factor [TLR-10 is an orphan members of human TLRs. It shares a common ome 4p14 . In expeome 4p14 . In co-iy factor .Different cholangiopathies result from biliary tract dysfunction, and they can be divided into autoimmune, genetic, infectious, drug-induced, or ischemic, and on the other hand by complete biliary obstruction with intra- and extrahepatic bile stasis; therefore the pathogenesis of some cholangiopathies remains unclear, which results in limited treatments. TLRs are expressed on the liver, an organ that receives blood from the portal circulation of lower extremities and bowel with frequent exposure to PAMPs of invading microorganisms and antigenic components of the diet; therefore, healthy liver can develop immunological tolerance . PAMPs aκB into the nucleus with transcriptional activation of chemokines-encoding genes and pro- and anti-inflammatory cytokines [Liver immune response is complex due to competition of many cell populations, including lymphocytes binding with natural killer (NK) and CD8+ T cells which may be unusually abundant. Dendritic cells (DCs) and activated hepatic stellate cells (HSCs) show TLR-4 signals which participate in hepatic inflammation and fibrogenesis. These cells can respond to TLRs signals and act as antigen-presenting cells for T cells. TLRs have been found in the liver when infected with viral hepatitis B or C, and also in animal models of bile duct injury induced by ligation, partial hepatectomy, drug toxicity (acetaminophen), and ischemia-reperfusion. TLR-4 is specific for LPS by transferring the signal inside the cell and promoting translocation of NF-ytokines .α) with subsequent activation of HSCs, promoting fibrogenesis (excessive deposit extracellular matrix proteins) and TLRs participation, which results in cirrhosis and liver failure. The activation of HSCs is controlled by many cytokines and growth factors, including TGF-β which is considered the main inductor on transformation of HSCs, while growth factor platelet-derived (PDGF) plays a critical role in stimulating the proliferation of HSCs. Recent studies suggest that TLR-4 participates in the production of hepatic fibrosis by activation of HSCs by TGF-β [Any immune or inflammatory liver disease activates Kupffer cells; these can trigger production of inflammatory cytokines , even though biopsy results obtained during Kasai procedure are contradictory [Biliary atresia is characterized by a progressive inflammatory sclerosing obstructive cholangiopathy which appears within the first afterbirth months. The precise etiology and pathogenesis remains unknown; it could be caused by viral infection with adictory . Immunohadictory , 57. It adictory . A previous study published by Huang et al. found significantly increased expression of TLR-5 and MxA mRNA in liver biopsy from early biliary atresia. These results imply the participation of TLR-7 and type 1 interferon signaling in the pathogenesis of biliary atresia, specifically on early stages and its association with overproduction of IL-8 . DespiteEscherichia coli (β-glucuronidase) is the most frequently isolated bacteria in bile ducts. On the other hand, DNA of Campylobacter species has been reported by PCR in bile and bladder biopsies from patients with hepatolithiasis [It is considered a bile duct disease histologically characterized by formation of gallstones and chronic proliferative cholangitis. Reported incidence in western countries is about 1%, although it is not rare in Asia (including Japan). Its natural history can lead to development of biliary cirrhosis, portal hypertension, and liver failure, with risk of cholangiocarcinoma in approximately 10% of patients. Bacterial infections of biliary tract and presence of cholestasis have been implicated as the main etiopathogenic factor of lithogenesis in patients with calcium bilirubinate stones, and ithiasis . It is sTLRs role in patients with bile duct injury after cholecystectomy has not been elucidated. Experimental studies on animals submitted to bile duct ligation have been reported; however, human reactions are not analogous to those on experimental models. Secondary obstructive congenital or acquired cholangiopathies generate significant morbidity and mortality in both children and adults because cholangiocytes proliferate at the final stage of the disease, resulting in bile duct destruction, liver fibrosis, following cirrhosis and finally liver failure. We have to look into the behavior of TLRs in serum and liver tissue biopsies from patients with biliary atresia, hepatolithiasis, biliary injury previously undergone cholecystectomy, and primary biliary cirrhosis in order to describe the role of TLRs for planning alternative surgical or biomolecular treatment strategies.α, IL-1β, and IL-6 due to activation of Kupffer cells, and high baseline levels of LPS-induced TLR-4 expression. However, regulation of TLR-4 is altered in monocytes from cirrhotic patients which produce frequent bacterial infections [Patients with liver cirrhosis have chronic endotoxemia, elevated serum TNF-fections .Primary biliary cirrhosis has an immune etiology and produces biochemical and structural alterations of the liver acting as an obstructive cholangiopathy. Patients with chronic liver diseases regardless of etiology are likely to develop liver fibrosis that can lead to cirrhosis with consequent complications such as portal hypertension and/or liver failure. All chronic liver diseases including cholangiopathy generate a constant process of fibrosis where the innate immune system and activation of monocytes and NK cells induce chronic inflammation . Primaryβ can be blocked by TLR-9 antagonists [Specific activation of TLR-2 in hepatitis C and homo- or heterodimerization with TLR-1 or TLR-6 has been shown; also, when these are blocked a significant decrease on production of proinflammatory cytokines appears, suggesting that some proteins produced by hepatitis C activate innate immune . TLR-9-dagonists . TLR-2 dagonists . TLR-2 aagonists . TLR-3 iagonists . Therefoβ-defensins induced by inflammatory stimuli and produced specifically on infection site. β-defensins interact on bacteria membrane through exchange of hydrophobic amino acids. They are expressed mainly on epithelial cells and have a broad activity spectrum against Gram-positive and Gram-negative bacteria, fungi, and certain viruses [β-defensins behaviour in secondary obstructive cholangiopathy in patients at hospital admission and at hospital discharge considering as variable the administration of antibiotic therapy. Agonists of TLR-7 and TLR9 are under consideration in the management of hepatitis C [TLR-1, TLR-2, TLR-3, TLR-7, TLR-8, TLR-9, and TLR-10 induce type 1 IFN production during viral infections . TLR-3 p viruses . All of atitis C . The bet


We have generated two RA models, human T-cell leukemia virus type I (HTLV-I) transgenic mice and IL-1 receptor antagonist (Ra)-deficient (KO) mice, to elucidate the pathogenic mechanisms of the disease. Both models spontaneously developed arthritis closely resembling that of RA in humans. We found that TNF-, but not IL-6-, deficiency suppressed development of arthritis in IL-1Ra KO mice, while IL-6 but not TNF was involved in the HTLV-I transgenic mouse model [Dcir gene is linked to several autoimmune diseases including RA and SLE, we have generated Dcir KO mice to examine the roles of this gene in the immune system. We found that aged Dcir KO mice spontaneously developed sialadenitis and enthesitis associated with elevated serum autoantibodies [Dcir KO mice after aging. Dcir KO mouse-derived bone marrow cells (BMCs) differentiated into DCs more efficiently than did wild-type BMCs upon treatment with GM-CSF, owing to enhanced STAT-5 phosphorylation. These findings indicate that DCIR is crucial for maintaining the homeostasis of the immune system, suggesting that Dcir is one of novel targets for the treatment of RA.We found that the expression of C-type lectin receptor (CLR) genes was augmented in the affected joints of these models using DNA microarrays. Dendritic cell immunoreceptor (DCIR) is one of such CLRs with a carbohydrate recognition domain in their extracellular carboxy terminus and an ITIM in its intracellular amino terminus. Because human shared syntenic locus containing the tibodies . DCs werMuratin1, which encodes uncharacterized and secreted protein, is specifically up-regulated in affected joins of both models. Interestingly, the development of collagen-induced arthritis was markedly exacerbated in Muratin1 KO mice. I would like to discuss the roles of Muratin-1 in the development of arthritis.We have also found that the expression of


Obesity is reaching epidemic worldwide and is risk factor for cardiovascular disease and type 2 diabetes. Although plasma high density lipoprotein (HDL) and apolipoprotein A-I (apoA-I) are inversely correlated to obesity, whether HDLs have anti-obesity effect remains unclear until a recent study reporting the direct anti-obesity effect of apoA-I and its mimetic peptide. However, the mechanism is not fully understood. Increasing adipose energy expenditure through attainment of brown adipocyte phenotype in white adipose tissue is considered a potential strategy to combat obesity. Specific inhibition of autophagy in adipose tissue is associated with reduced adiposity which is attributed to the attainment of brown adipocyte phenotype in white adipose tissue and the increased energy expenditure. HDL and apoA-I could activate PI3K-Akt-mTORC1 signaling which negatively regulates autophagy. The links between HDL/apoA-I and autophagy brings a new understanding on the anti-obesity effect of HDL and apoA-I. Obesity is the most common nutritional disorder and is associated with important comorbidities such as dyslipidemia, atherosclerosis, type 2 diabetes and insulin resistance . Anti-obThe well-established cardioprotective nature of high-density lipoproteins (HDLs) has made them and their main protein apolipoprotein A-I (apoA-I) popular targets for potential cardiovascular therapies. Current HDL-based therapies including direct infusion of rHDL and apoA-I mimetic peptides have exhibted abroad beneficial effects aside from mediating cholesterol efflux such as anti-inflammatory, anti-oxidative actions and shown potential usefulness as therapy for diseases involving chronic inflammation and oxidative stress .One of the proatherogenic effects of obesity is attributable to its accompanying dyslipidemia. The prominent dyslipidemia in obesity is low high density lipoprotein cholesterol (HDL-C) levels and apoA-I. Epidemiological studies have shown a strong inverse correlation between HDL-C, apoA-I and obesity, especially in individuals with visceral obesity . This inObesity is defined medically as a state of increased body weight, more specifically adipose tissue, or sufficient magnitude to produce adverse health consequence . As the HDL is generally considered as a protective factor for cardiovascular disease. In addition to the well known effect of mediating reverse cholesterol transport, it also exerts other beneficial functions such as anti-oxidative, anti-inflammatory and anti-thrombotic actions . It’s wePrevious findings that several polymorphrism in apolipoprotein A1(ApoA-I) gene have been associated with obesity in Brazilian population and that body fat content was increased in apoA-I null mice model lead to query about the role apoA-I played in obesity development ,14. SeveUp to date, there are several strategies for pharmacotherapy of obesity, including: (1) appetite suppression through central stimulation of anorexigenic signals or blocking orexigenic signals; (2) inhibition of nutrient digestion and absorption through gastrointestinal mechanism; (3) stimulation of fat mobilization and decreasing triacylglycerol synthesis and deposition in fat depots; (4) increase lipid oxidation or thermogenesis by uncoupling fuel metabolism, thereby enhancing energy expenditure . In the Adipose tissue contains two distinct types of fat cells, white and brown. The balance between white and brown adipose tissue (BAT) is a factor that determines obesity. White fat cells are specialized for the storage of chemical energy as triglycerides, while brown fat cells have very limited ability to store lipid and serve the primary function of dissipating chemical energy through adaptive thermogenesis . Since bAmple evidences support some plasticity exists between white and brown adipocyte. In mice and rats, exposure to cold or β-adrenergic agonists induces the appearance of brown adipocyte in traditional white fat pats, which originate differently from the classic brown adipocyte that develop before birth because these newly formed brown adipocyte did not express the YFP reporter gene, which was conspicuously expressed in the interscapular brown fat cells from the same mice -35. SimiAutophagy, defined as a highly regulated process involving the bulk degradation of cytoplasmic macromolecules and organelles in mammalian cells via the lysosomal system, plays a housekeeping role in removing misfolded or aggregated proteins, clearing damaged organelles, eliminating intracellular pathogens, and balancing sources of energy at critical times in development and in response to nutrient stress ,39. As aTo further elucidate the role of autophagy in the state of obesity, recent studies used knockout mice model with adipose tissue specific deletion of autophagy-related gene Atg7 and Atg5 respectively. The Autophagy-related gene knockout mice exhibited a metabolic favorable phenotype. The mutant mice were slimmer with increased insulin sensitivity and showed resistance to high-fat-diet induced obesity compared with wide-type mice. The mutant mice contain only 20% of the mass of WAT found in wild-type mice ,45,46. COne of the major roles of autophagy is to serve as a cellular adaptive reaction in order to sustain the internal organization in the case of insufficient external nutrient supply or augmented energy demands through digesting their own interior. On the one hand, autophagy needs to be upregulated under nutrient restriction. On the other hand, cells have to avoid excessive and enduring self-digestion. In order to keep the delicate balance between external energy and nutrient supply and internal consumption, complex regulatory network that sense the environmental change is required.The nutrient sensing kinase mTORC1 is well known to negatively regulate the autophagic machinery. Diverse external signals such as growth factor, amino acids, normoxia, or high energy levels all activate mTORC1 and result in the inhibition of autophagy -53. mTORAMP-activated protein kinase (AMPK) is regarded as the main energy-sensing enzyme that promotes all varieties of catabolic pathways and blocks several anabolic pathways . AMPK haSterol depletion, a selective form of nutrient depletion, has been reported to induce autophagy. Recently, it was suggested that in the situation of sterol limitation, apart from the de novo synthesis and receptor-mediated uptake pathway, cell could recycle cytoplasmic lipid droplet as a source of cholesterol through the process of autophagy . Sterol Taken that HDL and apoA-I are inversely correlated to obesity and that apoA-I exhibits a direct anti-obesity action, it is of great interest to answer the question whether HDL/apoA-I could modulate adipocyte autophagy. Under conditions of excessive nutrition such as obesity, ER becomes stressed and results in activation of the copying system, which is termed unfolding protein response (UPR), in many metabolic tissues such as adipose tissue. Autophagy is activated by UPR so that unfolded and misfolded proteins could be degraded . Recent A major upstream signaling of mTORC1 is the phosphatidylinositol 3-kinase (PI3K) pathway. The binding of growth factor or insulin to cell surface receptors activates PI3K, which converts the plasma membrane lipid PIP2 to PIP3, subsequently recruits PDK1 and Akt to the plasma. Following being phosphorylated by PDK1, activated Akt positively regulates mTORC1 through phosphorylation-dependent inhibition of TSC2 with consequent autophagy inhibition .Although numerous evidences showed HDL/apoA-I actived PI3K-Akt pathway in different cell lines, the underlying molecule mechanism still remains unclear. Rising evidences suggested that SR-BI, which mediated multiple antiatherogenic functions of HDL, was involved in PI3K-Akt signaling activation in response to HDL stimuli. Knock-down of SR-B1 by siRNA significantly attenuated HDL induced PI3K-Akt-eNOS signaling and prostacyclin production in endothelial cells . ConsistObesity, especially central obesity, is risk factor of cardiovascular disease and type 2 diabetes mellitus. Attainment of phenotype resembling brown adipocyte in WAT could reduce adipose deposit through enhanced β-oxidation. Inhibiting autophagy in adipose tissue leads white adipocyte to acquire features of brown adipocyte, therefore exerting an anti-obesity effect and improving obesity associated insulin sensitivity. Taken that HDL and apoA-I has direct anti-obesity effect in rat and that HDL activate PI3K-Akt signaling which negatively regulate autophagy through mTORC1, we propose that HDL/apoA-I inhibit autophagy through PI3K-Akt signaling, leading to phenotype transition of white adipocyte. This new link between HDL/apoA-I and autophagy provide a new understanding of the possible mechanism underline the antiobesity effect of HDL/apoA-I.AMPK: AMP-activated protein kinase; apoA-I: Apolipoprotein A-I; apoA-I-Tg: apoA-I transgenic; BAT: Brown adipose tissue; C/EBP: CCAAT-enhancer-binding proteins; FFAs: Free fatty acid; HDL: High-density lipoprotein; LC3: Microtuble-associated protein light chain 3; mTORC1: Mammalian target of rapamycin complex 1; MEFs: Mouse embryonic fibroblast; oxLDLs: Oxidised low density lipoproteins; PDK: Phosphoinositide-dependent protein kinase; PI3K: Phosphoinositide 3-kinase; PPAR-γ: Peroxisome proliferator-activated receptor gamma; S1P: Sphingosine 1-phosphate; SR-B1: Scavenger receptor class B type I; SREBP: Sterol regulatory element-binding protein; TG: Triglyceride; TSC: Tuberous sclerosis complex; UCP: Uncoupling protein; UIK1 (unc-51-like kinase) WAT: White adipose tissue.The authors declare that they have no competing interests.SW and DQP conceived the study, its design and drafted the manuscript. All authors read and approved the final manuscript.


To the Editor: Kala-azar  is a fatal disease caused by a protozoan parasite Leishmania donovani and transmitted by the female sandfly, Phlebotomus argentipes. In the state of Assam, India, kala-azar epidemics occurred during 1875–1950 and resulted in thousands of deaths in the districts of Kamrup, Garo Hills, Goalpara, and Nagaon . During the survey we recorded 4 suspected cases of post–kala-azar dermal leishmaniasis (PKDL).At bimonthly intervals during 2012, we conducted house-to-house surveys in 4 villages in the district of Kamrup, 845 households and 4,376 persons. Residents are socioeconomically poor and depend on agriculture and nearby brick kiln industries for their livelihood; persons involved in these industries generally keep cattle, and areas of cow manure provide breeding sites for sandflies. Persons reported with fever for >2 weeks, anemia, weight loss, and palpable spleen or liver and who were negative for malaria were tested for kala-azar by using the rK39 diagnostic kit . We obtained bone marrow biopsy samples from selected persons who exhibited the symptoms listed above. A total of 162 persons had positive kala-azar results according to rK39 testing during 2008–2012; of these, 44 (27%) were children. Microscopic examination of bone marrow biopsy samples from 5 persons showed Case-patient 1, a 16-year-old boy . We microscopically examined Giemsa-stained slides and found L. donovani parasite in 1 sample. Using QIAamp DNA Mini Kit (QIAGEN), we isolated parasite DNA from the samples and used it for the first round of PCR with primers 5′-AAATCGGCTCCGAGGCGGGAAAC-3′ and 5′-GGTACACTCTATCAGTAGCAC-3′ as described by Salotra et al. . Ecologic conditions of the areas where kala-azar outbreaks occurred are conducive to sandfly breeding; thus, regular spraying of DDT is needed. Preventive measures to control spread of kala-azar to other areas of Assam would be an effective step for the kala-azar control program.In India, PKDL develops in 5%–15% of treated kala-azar case-patients (Skin lesions of patients with post–kala-azar dermal leishmaniasis, Assam, India.


In contrast, RNAi depletion of capt, tsr and ssh in Drosophila S2 cells all affect actin-based lamella formation differently. Whether loss of these three related genes might cause the same effect in the same tissue remains unclear.CAP/Capulet (Capt), Slingshot (Ssh) and Cofilin/Twinstar (Tsr) are actin-binding proteins that restrict actin polymerization. Previously, it was shown that low resolution analyses of loss-of-function mutations in Loss-of-function mutant clones were generated using the MARCM or EGUF system whereas overexpression clones were generated using the Flip-out system. Immunostaining were then performed in eye imaginal discs with clones. FRAP was performed in cultured eye discs.Drosophila eye imaginal disc as a model system. Surprisingly, we found that capt and ssh, but not tsr, mutant cells within and posterior to the morphogenetic furrow (MF) shared similar phenotypes. The capt/ssh mutant cells possessed: (1) hexagonal cell packing with discontinuous adherens junctions; and (2) largely complementary accumulation of excessive phosphorylated myosin light chain (p-MLC) and F-actin rings at the apical cortex. We further showed that the capt/ssh mutant phenotypes depended on the inactivation of protein kinase A (PKA) and activation of Rho.Here, we compared their loss-of-function phenotype at single-cell resolution, using a sheet of epithelial cells in the Although Capt, Ssh and Tsr were reported to negatively regulate actin polymerization, we found that Capt and Ssh, but not Tsr, share overlapping functions during eye morphogenesis. Remodeling of the actin cytoskeleton is controlled by various groups of actin-binding proteins that function at different steps to promote dynamic F-actin assembly and disassembly. Cyclase-associated protein (CAP) homologs have been suggested to act as actin monomer sequestering proteins through their C-terminal actin-binding domains to suppress the spontaneous polymerization of actin ,2. Cofilcapt and tsr encode the Drosophila CAP and cofilin orthologues, Capulet (Capt) and Twinstar (Tsr), respectively. Loss-of-function mutations in capt, ssh and tsr all cause increased ectopic accumulation of F-actin in various Drosophila tissues that leads to defects in epithelial morphogenesis  P[UAS-GFP.S65T]/CyO[Loss-of-function mosaic clones were generated using the MARCM system . Entirelbination . Flip-ouS65T]/CyO.For immunostaining, third instar larval eye imaginal discs were dissected and fixed in 4% paraformaldehyde. Antibodies used were: rat anti-DE-cad , mouse anti-Arm , rabbit anti-pSer19-MLC , rat anti-Ci155 , rabbit anti-Egfr , Alexa ubi-DE-cad-GFP were dissected and placed in a drop of serum-free M3 medium at room temperature. A small area (1500 nm2) was photobleached. Fluorescence intensity in the bleached region or tricellular junction was measured at each time point using Zeiss LSM software.Images were acquired using a 63× NA1.4 Oil Plan-Apochromat objective lens on a confocal microscope . The fluorescence intensity and apical surface area were quantified using Zeiss LSM software. The percentage of cells with x-sided polygons was quantified manually by measuring the number of neighboring cells. For fluorescence recovery after photobleaching (FRAP), third instar larval eye discs with capt, ssh and tsr in the Drosophila eye epithelia, at low resolution, all caused similar accumulation of F-actin and enlarged apical area [capt, tsr and ssh might differentially affect epithelial morphogenesis, we compared the phenotypes of the loss-of-function null alleles  at high magnification using the MARCM system (marked by the presence of CD8-GFP expression) in third instar eye imaginal discs [capt/ssh[captE636 mutant ICs often had enlarged surface areas . Importantly, this strong p-MLC staining outlined the cell morphology and partially overlapped with the F-actin ring  of the fragmented AJs in captE636 mutant cells, we found no significant recovery of fluorescence signal for over 55 s  of ubi-DE-cad-GFP discs  of a tricellular junction did not significantly change for up to 3 minutes . Together, our results demonstrate that capt plays an important role in cells within and posterior to the MF, and loss of Capt not only affects F-actin polymerization, as reported, but also MLC phosphorylation/activation, the continuity of AJs, and changes cell shape.The F Figure A,B, but,s Figure A′. Howevs Figure A as wells Figure B in captssh encodes Slingshot, a cofilin phosphatase, and ssh mutant clones in the eye showed a strong accumulation of F-actin and an enlarged apical surface [ssh1-63 mutant clones within and posterior to the MF exhibited similar phenotypes to captE636 mutant clones. These included (1) adoption of a hexagonal cell shape and discontinuous AJs , similar to ADF/cofilin, sequesters actin monomers and negatively regulates F-actin formation . Howevercapt/ssh in F-actin/p-MLC accumulation were only confined to cells within and posterior to the MF  phosphorylates Ci155 to promote its proteolysis into the Ci75 repressor in cells anterior to the MF and this proteolysis is blocked by the activation of Hh signaling within the MF. We first generated ectopic clones, anterior to the MF, overexpressing the regulatory subunit of PKA (R*) to sequester the catalytic subunit  in captE636 mutant clones using MARCM. These clones tended to be small, however, we found that inhibition of Rho activity abolished capt-mediated accumulation of F-actin and p-MLC within and posterior to the MF  is not completely the same as disrupting cofilin and p-cofilin recycling (as in ssh mutant cells). The reason for this is unclear. One possibility is that cofilin activity is regulated by multiple additional mechanisms, including dephosphorylation of phosphorylated cofilin by chronophin and general phosphatase [Cofilin is activated by Ssh, the phenotypic differences between ferently . Recent ing disc ,28. Thussphatase . Alternacapt/ssh mutant cells, fragmented AJs were stable and they assembled at regions where cadherin and high levels of F-actin overlapped. Thus, fragmented AJs and spot AJs might have a similar function but they differ in underlying F-actin organization. We showed that the fragmented AJs can also non-autonomously assemble along the interface between wild-type cells when they were surrounded by the captE636 mutant cells. One possibility is that when there is a fragmented AJ formed at one side of a wild-type cell , this fragmented AJ cannot link to the adjacent AJ  at the tricellular junction, and therefore this adjacent AJ also becomes fragmented once the associated actomyosin contracts.Spot AJs in early embryonic epithelia contain homophilic DE-cad clusters in a stable microdomain and are bona fide sites of adhesion . Small bcapt is required to prevent premature photoreceptor differentiation ahead of the MF [capt/ssh mutant phenotypes, including the apical surface area, cannot be detected anterior to the MF. By using the enrichment of EGF receptor to mark MF cells [capt mutant cells with enrichment of EGF receptor in a capt mutant clone spanning the MF , EGFR (red) and GFP (blue). Bracket indicates 8-10 rows of captE636 mutant MF cells (dots) with elevated levels of EGFR. Bars, 2 μm.Click here for file


N-methyl-D-aspartate receptor (NMDAR) to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), and the number of functional release sites at proximal inputs reached a maximum during the critical period for the sensory-dependent survival of newborn cells, well before the completion of dendritic arborization. EM analysis showed an accompanying change in postsynaptic density shape during the same period of time. Interestingly, the latter morphological changes disappeared in more mature newly-formed neurons, when the NMDAR to AMPAR ratio had decreased and functional presynaptic terminals expressed only single release sites. Together, these findings show that the first glutamatergic inputs to adult-generated OB interneurons undergo a unique sequence of maturation stages.The olfactory bulb (OB) receives and integrates newborn interneurons throughout life. This process is important for the proper functioning of the OB circuit and consequently, for the sense of smell. Although we know how these new interneurons are produced, the way in which they integrate into the pre-existing ongoing circuits remains poorly documented. Bearing in mind that glutamatergic inputs onto local OB interneurons are crucial for adjusting the level of bulbar inhibition, it is important to characterize when and how these inputs from excitatory synapses develop on newborn OB interneurons. We studied early synaptic events that lead to the formation and maturation of the first glutamatergic synapses on adult-born granule cells (GCs), the most abundant subtype of OB interneuron. Patch-clamp recordings and electron microscopy (EM) analysis were performed on adult-born interneurons shortly after their arrival in the adult OB circuits. We found that both the ratio of  Adult neurogenesis, a process encompassing the generation, maturation and synaptic integration of new neurons in the adult brain, represents a striking form of structural adult neural plasticity -3. In thGCs are the largest population of interneurons in the bulb, outnumbering principal neurons by 100 to one . StrikinIn this study, we focused on the excitatory proximal inputs to newborn GCs. Pioneering studies on embryonal and postnatal development have shown that the first contacts to be formed are MC-GC dendrodendritic synapses at embryonic day (E)17. The formation of these first synaptic contacts is followed by the appearance of MC-GC axodendritic synapses at E18 ,28. ThusPrevious morphological studies have shown that adult-born GCs can be categorized into five distinct classes . To charTo examine early glutamatergic inputs contacting newborn GCs, we used pre-embedding immunolabeling for GFP combined with electron microscopy (EM). This ultrastructural analysis was performed in the deep portion of the GC layer, thus excluding TC axons located in the internal plexiform layer. At 7 dpi, EM analysis showed GFP-positive cells in the GC layer receiving asymmetric contacts from axon terminals. Serial sectioning revealed that some synapses at 7 dpi had a simple shape with a compact and homogeneous post-synaptic density (PSD) (7 over 13 sections) Figure , whereas+ currents and input membrane resistance (Rin). Previous studies showed that inward Na+ currents appeared mostly on class 5 newborn GCs; that is, one week after viral injection quinoxaline-7-sulfonamide (NBQX) was applied to obtain NMDAR-mediated excitatory postsynaptic currents (EPSCs). Traces were derived from the subtraction of NMDAR-mediated EPSCs from AMPAR-mediated and NMDAR-mediated currents. Scale bar = 1 ms and 1 pA. (B) The amplitude of AMPAR-mediated EPSCs was much higher in class 5 than in class 3 cells   .Click here for file2+-permeable AMPARs in class 3 GCsSupplementary Figure 4: Ca. (A) (Black) AMPAR-mediated EPSCs at the holding potential of -70 mV (red). This trace shows predicted data, obtained by inversion of the trace shown in black and multiplication by four-sevenths. (B) (Green) AMPAR-mediated and NMDAR-mediated EPSCs at the holding potential of +40 mV. (Pink) NMDAR-mediated EPSCs at the same holding potential. This current was recorded in the presence of NBQX. (Blue) AMPAR-mediated current at the holding potential of +40 mV. This experimental trace was generated by subtracting the NMDAR-mediated component from both receptor-mediated currents (Green minus pink). (C) Comparison between the predicted and experimental traces. (A-C) Scale bar = 1 ms and 2 pA. (D) The maximum amplitude of the trace obtained from dividing experimental values by predicted values. If AMPAR-mediated EPSCs in class 3 GCs are mediated by Ca2+-impermeable AMPARs, this ratio would be equal to 1 (dashed line), given the linear current-voltage relationship of Ca2+-impermeable AMPAR. The experimental values obtained were significantly lower than the predicted values .Click here for fileSupplementary Figure 5: Axon terminals make contacts with both adult-born and pre-existing GCs. A large axon terminal (Ax: red) makes two synapses with a GFP-positive dendrite (GC at 7 days post-injection: green) and a GFP-negative dendrite (a presumptive pre-existing GC: blue). Arrows point to the postsynaptic density. Note that the synapse onto the GFP-positive profile has a complex morphology and shows clear perforation. Scale bar = 50 nm.Click here for file


SapC-DOPS has a high fusogenic affinity for phosphatidylserine-enriched microdomains on surfaces of target cell membranes. Incorporation of a far-red fluorophore, CellVue Maroon (CVM), into the nanovesicles allows for in vivo non-invasive visualization of the agent in targeted tissue. Given that phosphatidylserine is present only on the inner leaflet of healthy plasma membranes but is “flipped” to the outer leaflet upon cell damage, we hypothesized that SapC-DOPS would target tissue damage associated with inflammatory arthritis due to local surface-exposure of phosphatidylserine. Optical imaging with SapC-DOPS-CVM in two distinct models of arthritis, serum-transfer arthritis  and collagen-induced arthritis (CIA) revealed robust SapC-DOPS-CVM specific localization to arthritic paws and joints in live animals. Importantly, intensity of localized fluorescent signal correlated with macroscopic arthritic disease severity and increased with disease progression. Flow cytometry of cells extracted from arthritic joints demonstrated that SapC-DOPS-CVM localized to an average of 7–8% of total joint cells and primarily to CD11b+Gr-1+ cells. Results from the current studies strongly support the application of SapC-DOPS-CVM for advanced clinical and research applications including: detecting early arthritis onset, assessing disease progression real-time in live subjects, and providing novel information regarding cell types that may mediate arthritis progression within joints.Rheumatoid arthritis is a chronic inflammatory disease affecting approximately 1% of the population and is characterized by cartilage and bone destruction ultimately leading to loss of joint function. Early detection and intervention of disease provides the best hope for successful treatment and preservation of joint mobility and function. Reliable and non-invasive techniques that accurately measure arthritic disease onset and progression are lacking. We recently developed a novel agent, SapC-DOPS, which is composed of the membrane-associated lysosomal protein saposin C (SapC) incorporated into 1,2-dioleoyl- Rheumatoid arthritis (RA) is a chronic inflammatory disease that affects 1% of the population. RA is manifested as chronic inflammation leading to severe pain and loss of mobility. If unchecked, disease culminates in destruction of joint tissue, loss of function, and a profound reduction in quality of life. Several effective biologics have been developed over the last decade that limit arthritis progression for a large percentage of RA patients, and in a number of cases, achieved remission. These therapies are most effective when used to intervene early in the RA disease process; however, reliable and manageable tools for evaluation of early joint inflammation and disease remission in response to therapeutics are lacking. Recently, new guidelines put forth by the American College of Rheumatology (ACR) and The European League Against Rheumatism (EULAR) outlined standard assessments of measuring clinical outcomes, which are needed both for clinical practice and clinical trials Joint counts are part of the ACR and EULAR proposed criteria, and an essential part of the clinical component of assessing rheumatic disease. However, several studies have suggested that differences in joint counts do not necessarily correlate with clinical outcomes  the decrease in exposure to radiation as compared to X-ray (2) the clear targeting and localization of molecular probes associated with disease pathogenesis and (3) minimal acquisition time.Optical imaging is a relatively new imaging modality and provides great promise for analyzing events occurring early in the pathogenesis of arthritis (reviewed in sn-Glycero-3-Phospho-L-Serine (DOPS) and a fusogenic protein, saposin C (SapC) to image arthritic joints. SapC associates with phosphatidylserine (PS) on cell membranes by embedding amino and carboxyl-end amphipathic helices into the outer leaflet of membranes Targeting of damaged tissue to enhance imaging of inflammation in arthritis could provide a valuable tool for the early assessment of disease and for the progression of disease. In the current study, we have characterized the targeting of novel nanovesicles composed of 1,2-Dioleoyl-The current study determines the signal distribution in mice of SapC-DOPS labeled with a far-red fluorophore, CellVue® Maroon (CVM), as assessed by optical imaging in two arthritis mouse models that each exhibit different disease courses and etiology. Furthermore, the intensity of SapC-DOPS signal as compared with arthritic severity over the course of disease was determined and cell types within joints that are targeted by SapC-DOPS were investigated. Our results suggest that SapC-DOPS localizes specifically to cells involved in early inflammatory stages of arthritis and that this novel agent has potential use as an imaging agent for inflammatory arthritis.All experiments involving mice complied with National Institutes of Health guidelines. The protocol was approved by the Institutional Animal Care and Use Committee of the Cincinnati Children's Hospital Research Foundation .DBA/1J male mice eight weeks of age were injected intradermally at the base of the tail with 100 µg of bovine type II collagen (CII)  in complete Freund's adjuvant (CFA) on day 1 and given a similar challenge once again 21 days following the initial CII injection, as previously described Sera were collected from KRN×NOD F1 mice, and 150 µl was given by intraperitoneal injection to C57Bl/6J mice at day 0 of disease. Calipers were used to measure paw thickness and mouse ankle thickness. For ankle thickness, elliptical area of mouse ankles was calculated using intermalleolar measurements and measurement of the area between the dorsal talus and calcaneus.An aliquot of CellVue® Maroon  in ethanol was mixed with phospholipid solvent for bath sonication preparation by the procedure as previously described 6 cells were stained in 100 ul of FACS buffer  containing 10 ul of 2.4G2 (American Type Culture Collection) hybridoma supernatant. Three antibody panels were assessed for cells from CIA treated mice as follows: panel 1 = anti-CD4-FITC , anti-CD8-Pacific Blue  and anti-CD19-PE ; Panel 2 = anti-CD11b-FITC , anti-CD11c-PE , anti-Gr-1-AF700 , and anti-F4/80-E450 ; Panel 3 = anti-CD31-FITC  and anti-CD55-PE . Three antibody panels were assessed for cells from K/BxN treated mice as follows (clones as above): panel 1 = anti-CD4-FITC, anti-CD8-Pacific Blue and anti-CD19-PE; Panel 2 = anti-CD11b-FITC, anti-CD11c-PE, and anti-Gr-1-E450; Panel 3 = anti-CD31-FITC, anti-CD55-PE and anti-F4/80-E450. Cells were acquired using a FACSCanto I RUO analytical cytometer and analyzed using FACSDiva software .For joint cell analysis, mice were sacrificed, paws were removed above the ankle joint, skin was dissected away and the remaining tissue was placed in 0.5 ml of RPMI. Using a number 10 scalpel blade and forceps, tissue was separated from bones and joints and dissected/shredded as previously described Correlation was analyzed using Pearson Product Moment Correlation and Spearman Rank Order Correlation. Unpaired Students t-test was used to compare normally distributed parametric data between groups. Mann Whitney was used to compare nonparametric data between groups.To test the hypothesis that the PS targeting agent SapC-DOPS localizes to joint tissue following local destructive inflammatory events, the uptake of SapC-DOPS labeled with CVM (SapC-DOPS-CVM) was measured by optical imaging of mice challenged with two established models of inflammatory joint disease, each with distinct etiologies. First, mice challenged with K/BxN serum-transfer arthritis were examined seven days following sera injection. Mice were given SapC-DOPS-CVM and exhibited robust CVM fluorescence in macroscopically arthritic paws as compared to arthritic paws in mice not given SapC-DOPS-CVM . Mice noSapC-DOPS-CVM targeting of arthritic tissue was further assessed by examining localization in mice challenged with CIA, one of the most well-characterized of the murine models of inflammatory joint disease. A cohort of DBA/1J mice was challenged, and as expected, macroscopic disease was apparent at 28 days following primary CII injection. Mice exhibiting macroscopically apparent arthritis also displayed localization of SapC-DOPS-CVM accumulation as determined by IVIS® imaging specifically in macroscopically arthritic, but not non-arthritic paws 28 days following primary CII injection . In miceGiven that SapC-DOPS targets macroscopically apparent arthritic joints, it was of great interest to determine the kinetics of SapC-DOPS accumulation during the course of arthritic disease. Determination of SapC-DOPS-CVM localization over time provides information as to whether the intensity of signal correlates with the severity of disease. Since both the K/BxN and CIA models of inflammatory joint disease exhibit distinct etiologies and unique time courses of disease initiation and progression, SapC-DOPS uptake and accumulation was assessed over time in both models beginning with disease initiation and continuing through the peak of disease severity. First, K/BxN sera were administered to C57Bl/6 mice and animals were imaged by IVIS® six hours later and then daily. SapC-DOPS-CVM was administered every other day, beginning at the initial sera administration. Average radiance values (IVIS® intensity) of designated elliptical areas of the hind paws were determined and the mean values of groups for each day are indicated in In parallel analyses, CII-challenged mice were given SapC-DOPS-CVM at day 23 and every other day thereafter, and imaged daily from days 24–34 following CII primary immunization . Similarhigh) Since SapC-DOPS-CVM localizes specifically to arthritic joints, it was of great interest to determine cell types targeted by SapC-DOPS-CVM in both the K/BxN and CIA arthritis models. Cells extracted from joints were stained with labeled antibodies to cell-surface markers on lymphocytes , myeloid cells , endothelial cells (CD31), and synovial fibroblasts  with overt macroscopic disease, respectively. Similar to the K/BxN mice, cells extracted from joints were stained with labeled antibodies for lymphoid, myeloid, endothelial and synovial fibroblast cells as described above. In The present studies investigate the utility and underlying mechanism(s) of SapC-DOPS nanovesicles as an optical imaging agent to detect tissue damage in inflammatory arthritic joints. In two separate animal models of inflammatory arthritis, SapC-DOPS labeled with the fluorophore CVM was visualized locally in arthritic inflammatory joints. Furthermore, throughout the course of both K/BxN and CIA inflammatory arthritis SapC-DOPS intensity increased and correlated with increases in multiple independent parameters of arthritic disease assessment. Joint cells targeted by SapC-DOPS-CVM were primarily positive for both CD11b and Gr-1—cell markers indicative of neutrophils—that have previously been shown to be critical mediators of inflammatory events in arthritis Optical imaging has great promise for use in inflammatory arthritis, particularly in RA, in which the peripheral joints are the most highly affected. Assessment of joint damage in RA has been measured primarily through x-ray imaging analysis, which does not readily provide information regarding the earliest local pathogenic events , which occur prior to joint damage. MRI and ultrasound are beginning to be used as modalities to assess soft tissue damage; however, each of these modalities has significant factors hindering their routine use in the clinic. Optical imaging in humans has primarily involved imaging of vasculature and lymphatics using the non-specific, near-infrared dye indocyanine green Using optical imaging our studies indicate that SapC-DOPS-CVM appears to localize to arthritic joints. Our prior optical imaging studies regarding tissue-specific distribution of SapC-DOPS-CVM signal demonstrated fluorescent signal in only liver and spleen of otherwise unchallenged nude mice that diminished over time and was not detectable 48 hours following injection SapC-DOPS-CVM localization as assessed by optical imaging may be useful as a marker for both the detection of early pre-clinical disease and for closely monitoring disease progression. Analysis of SapC-DOPS-CVM accumulation in joints over the course of both K/BxN and CIA arthritis indicated an increase in CVM signal as arthritis progressed. A significant finding of the present study is that clinical parameters, such as paw thickness and ankle circumference in the K/BxN model showed significant correlations with CVM intensity . Non-sera treated mice that received SapC-DOPS-CVM every other day, accumulated significant low-level signal that plateaued early in disease. This result indicates that such low level of fluorescent signal may accumulate non-specifically due to the repeated administration of the fluorescent agent but would not hinder detection of early arthritic disease. While our studies focus on proof-of-principle for assessing joint damage in a mouse model over time, in clinical practice low level signal accumulation would not be expected since multiple injections over a short period of time would not be necessary. In parallel CIA analysis, signal intensity of SapC-DOPS-CVM localization to paw joints also increased in CII-challenged mice. In CII-treated mice, macroscopically arthritic paws showed a significant correlation with increased CVM signal intensity. Interestingly and specifically pertinent to early detection of inflammation and disease, in CII challenged mice a limited number of paw joints that were not overtly arthritic showed an increase in CVM signal intensity, which we believe may represent the earliest stages of arthritis. Future studies will histologically assess microscopic arthritis over the time course of disease to directly assess the histological changes that might be occurring sub-clinically in these paws as related to SapC-DOPS-CVM accumulation. Our results did not indicate any affects on arthritic disease progression and severity as both K/BxN and CIA arthritic disease progressed similarly to previous experiments performed in our laboratory; however, the results of the current study do not absolutely exclude the possibility that SapC-DOPS alone alters the kinetics and severity of arthritic disease.Analysis of SapC-DOPS-CVM targeted cells extracted from arthritic joints indicates that the main cell type targeted is CD11b and Gr-1 positive. Mature granulocytes, particularly neutrophils that reside in tissues, express Gr-1 on their cell surface A significant finding from these studies was that SapC-DOPS is able to target arthritic joints in multiple disease models that exhibit distinct etiologies The striking observation on arthritis-selective targeting of the novel compound SapC-DOPS suggests that this molecule may be used to detect inflammation in a variety of settings and contexts. Studies directed at evaluating whether SapC-DOPS can detect subclinical inflammation; other arthritides; and/or other inflammatory disorders remain to be performed; however, the results presented here argue that SapC-DOPS facilitated imaging may be used as a clinical parameter that could be highly valuable to assess inflammatory joint disease. One of the advantages to SapC-DOPS is its flexibility in incorporating other agents within the nanovesicles and targeting inflammatory regions. This technology also has the promising potential for specific delivery of agents to joints, which would be beneficial particularly with respect to therapies that when administered globally have undesirable side effects.


BRCA1 or BRCA2 mutation carriers are characterized by deficient homologous recombination (HR) of DNA, that makes them particularly sensitive to platinum compounds or inhibitors of poly (ADP-ribose) polymerase (PARP). Outstandingly durable complete responses to high dose chemotherapy have been observed in several cases of BRCA-related metastatic breast cancer (BC). Multiple lines of evidence indicate that women with BRCA1-related BC may derive less benefit from taxane-based treatment than other categories of BC patients. There is virtually no reports directly assessing drug response in hereditary colorectal cancer (CRC) patients; studies involving non-selected  CRC with high-level microsatellite instability (MSI-H) suggest therapeutic advantage of irinotecan. Celecoxib has been approved for the treatment of familial adenomatous polyposis (FAP). Hereditary medullary thyroid cancers (MTC) have been shown to be highly responsive to a multitargeted tyrosine kinase inhibitor vandetanib, which exerts specific activity towards mutated RET receptor. Given the rapidly improving accessibility of DNA analysis, it is foreseen that the potential predictive value of cancer-associated germ-line mutations will be increasingly considered in the future studies.Tumors arising in patients with hereditary cancer syndromes may have distinct drug sensitivity as compared to their sporadic counterparts. Breast and ovarian neoplasms from  Search for hereditary cancer genes was always regarded as a high priority translational research with immediate health impact. It was foreseen, that the discovery of tumor predisposing mutations and the development of appropriate genetic tests will allow identifying yet healthy subjects, who are at nearly fatal risk of specific type of cancer and thus may benefit from a timely medical intervention. Since the discovery of major cancer genes in the mid 1990s, thousands of mutation carriers have been subjected to intensive surveillance programs in order to secure early diagnosis of the disease . While tBRCA1 and BRCA2, contribute to substantial share of breast and ovarian tumor incidence around the globe, and have been studied with significant level of comprehension [CHEK2, PALB2 (FANCN), ATM, NBN (NBS1), BRIP1 , BLM, are less frequent and have not been subjected yet to systematic clinical studies.The best-known hereditary cancer genes, ehension . MutatioBRCA1 and BRCA2 genes play a central role in the repair of double-strand DNA breaks by homologous recombination (HR). Cancers in BRCA heterozygous individuals arise due to somatic inactivation of the remaining wild-type allele of the gene. This provides a critical biological difference between cancerous and normal cells: while BRCA-driven tumors are characterized by HR deficiency, normal tissues from the same individuals retain non-affected BRCA allele and the ability to cope with DNA damage [BRCA1- and BRCA2-related cancers demonstrate somewhat distinct picture of genetic abnormalities [p53 inactivation is an absolute prerequisite for the propagation of BRCA-dysfunctional tumors cells [A damage ,5. Althomalities ,7, they malities -11. Cellrs cells -15.BRCA1 has a wider spectrum of functions than BRCA2 [BRCA1 is involved in breast cell differentiation and transcriptional regulation of the estrogen receptor (ER) [BRCA1-mutated breast carcinomas (BC) do not express ER, while the hormonal receptor pattern in BRCA2-associated BC is similar to sporadic cases [BRCA1 is also essential for the mitotic spindle checkpoint as it triggers cellular suicide in response to microtubule damage [an BRCA2 ,17. In ator (ER) . It has ic cases . BRCA1 ie damage .BRCA1- and BRCA2-defective cells to various anticancer agents. Surprisingly, while the effect of individual compounds has been repeatedly evaluated in diverse model systems  cell line to paclitaxel as compared to isogenic cells with reconstituted BRCA1 function. Tassone et al. [BRCA1-deficient breast cancer cells to vinorelbine and argued that the differences in the mechanism of action between various microtubule interfering drugs have to be considered while interpreting the results of BRCA1 studies. DeLigio and Zorio [BRCA1-mutated cells may be essential in determining the response to taxanes and vinca alkaloids. BRCA2 preclinical studies suggested little impact of the status this gene in determining the response to microtubule interfering agents [Analysis of microtubule poisons produced even more complicated picture. It has been repeatedly demonstrated that function ,26,49-51e damage , one cane damage  reportede et al.  showed hnd Zorio  commenteg agents ,33,46,47BRCA-deficient model systems. Single-agent cyclophosphamide showed only slight antitumor activity against BRCA1-mutated human breast cancer xenografts growing in nude mice [BRCA2-deficent vs. BRCA2-proficient mouse mammary tumor cell lines identified alkylating agents  as the most potent and specific inhibitors of cell growth; furthermore, high efficacy of these drugs was confirmed in animal experiments [Alkylating agents are almost always included in the standard schemes for the treatment of breast and ovarian cancers. Surprisingly, this class of drugs has not been subjected to systematic studies in ude mice . At the eriments .BRCA1- [BRCA2-defective cells [Topoisomerase I inhibitors are rarely used for the treatment of breast cancer, but included in some therapeutic schemes for ovarian cancer. High sensitivity to these drugs was suggested for both BRCA1- ,54 and Bve cells ,46,55, ave cells .BRCA-deficient tumors [BRCA2-mutated cells [There is a good agreement in the literature that single-agent antimetabolites, 5-fluorouracil and gemcitabine, do not exert specific action against t tumors ,33,46. IBRCA-deficient cancers [BRCA1 or BRCA2, they cannot eliminate double-strand breaks by error-free mechanism. As result, cancers arising in BRCA carriers are selectively sensitive to PARP inhibitors, while the normal tissues from the same individuals retain a non-affected BRCA allele and are therefore capable to compensate the consequences of decreased PARP activity [There is a growing number of studies demonstrating pronounced efficacy of specific inhibitors of poly (ADP-ribose) polymerase (PARP) against  cancers -40,56-60activity ,62. PARPactivity ,38,39,59BRCA2- and a certain fraction of BRCA1-related BC express estrogen and/or progesterone receptors and are therefore expected to benefit from endocrine therapy. While a couple of studies examined the chemopreventive impact of tamoxifen or oophorectomy in BRCA carriers [BRCA status in determining the effect of endocrine intervention in neoadjuvant or metastatic setting.The majority of carriers  and somecarriers , there iBRCA-related vs. sporadic breast cancers are summarized in the Table BRCA1/2-related cancers to the neoadjuvant anthracycline-containing regimens. Interestingly, Hubert et al. [BRCA2 vs. BRCA1 carriers. The data of Petit et al. [BRCA1 carriers receiving 5-fluorouracil, epirubicin and cyclophosphamide achieved pathologic complete response (pCR), while pCR was detected in 21/43 (49%) sporadic triple-negative BC.Data on the efficacy of conventional chemotherapeutic schemes in t et al.  and Wongt et al.  observedt et al.  are in sBRCA2 but not BRCA1 carriers. Low efficacy of CMF therapy in BRCA1-related BC was also described by Byrski et al. [The only available study of metastatic BC included patients treated by either anthracycline-based or CMF (CMF-like) therapy . In conti et al. .BRCA1-mutated BC receiving neoadjuvant combination of doxorubicin and docetaxel. Moiseyenko et al. [BRCA1-linked BC to the epirubicin-docetaxel doublet. Wysocki et al. [BRCA1 carriers. Kriege et al. [BRCA1 carriers, while BRCA2-related BC did not fare worse as compared to sporadic controls. It appears, that the preclinical and clinical evidence warning against the early use of taxanes for the treatment of BRCA1-related subtype of BC has already achieved a critical threshold; probably, specifically designed retrospective studies assessing BRCA1 status in distinct categories of taxane users may accelerate further understanding of this issue [Several investigators analyzed the use of taxane-containing schemes. Byrski et al.  observedo et al.  reportedi et al.  genotypee et al.  confirmeis issue .BRCA1 carriers. Byrski et al. [BRCA1 carriers included in the study demonstrated pCR. Moiseyenko et al. [BRCA1 reading frame and therefore function of the corresponding protein [BRCA1 proficiency will result in sensitization of the cancer cells to estrogen antagonists and taxanes [The experience of the use of single-agent cisplatin for the treatment of hereditary BC is still limited to i et al.  reportedi et al.  used ciso et al.  observed protein ,79. One  taxanes .BRCA-related BC. Since BRCA-deficient BC are particularly sensitive to DNA damaging agents, use of intensive cytotoxic treatment may provide additional benefit to this category of patients. Furthermore, high dose therapy is likely to counteract tumor adaptation to the drugs, e.g. to induce rapid killing of cancer cells and therefore decrease the probability of developing secondary BRCA1-restoring mutations. It is also important to consider that BRCA-driven cancers are often characterized by young age at onset, i.e. the majority of these patients would retain sufficient health conditions to qualify for a risky medical intervention. Huang et al. [BRCA2-related BC, who was treated by high dose chemotherapy and remains disease-free for more than 11 years. Vollebergh et al. [BRCA1-related cancers. Complete long term responders included 1 out of 2 BRCA1- and 1 out of 2 BRCA2-carriers.There are a few case reports on the extremely successful use of high dose chemotherapy in metastatic g et al.  describeh et al.  presenteBRCA carriers evaluated the efficacy of the PARP inhibitor olaparib  [The only prospective clinical trial specifically designed for 0059436) . The stuBRCA1-driven BC cases. However, both these drugs have limited duration of response, so their use may require the addition of other anticancer agents [In agreement with preclinical findings, cisplatin and olaparib clearly outperform conventional treatment schemes when administered to r agents ,38,39,59BRCA-deficiency in cancer cell can be caused either by germ-line mutation followed by the "second hit", or by somatic inactivation of the BRCA1 gene. BRCA-inactive tumors constitute the minority of breast cancers (up to 10-15%), and are usually accumulated among family history positive or triple-negative cases. In contrast to BC, the majority of ovarian carcinomas have signs of BRCA inactivation, commonly defined in the literature as "BRCAness" [BRCA-deficiency in OC appears to be a plausible explanation of the clinical success of platinum-based schemes in the treatment of this disease.RCAness" ,15. FreqBRCA1/2- mutated vs. sporadic OC cases [BRCA-driven OC to platinum-containing treatments as compared to the mutation-negative tumors. Interestingly, prolonged tumor responses were documented both for taxane-free schemes and for the combination of platinating drugs with paclitaxel [Three studies compared response to the standard chemotherapeutic regimens in OC cases -85. Thesclitaxel -86.BRCA-mutated OC patients, who experienced prior chemotherapy [Two independent large trials evaluated the efficacy of olaparib in otherapy ,88. Audeotherapy  observedBRCA1 and BRCA2. CHEK2 appears to be the most studied gene of this class. It confers elevated risk of breast cancer, while its heterozygous occurrence among ovarian cancer patients is not elevated [CHEK2-mutated BC frequently express estrogen receptor [CHEK2 by RNA-interference increased cell sensitivity to PARP inhibition [CHEK2 carriers, who were treated by neoadjuvant single-agent epirubicin, while this outcome was rare  in the non-carriers [Hereditary BC research led to identification of several genes other than elevated ,90. CHEKreceptor ,92. Inachibition . The onlcarriers .PALB2  has been proven to cause breast and pancreatic hereditary cancer [BRCA2-related function of the PALB2, pancreatic cancer xenografts obtained from a PALB2 carrier demonstrated pronounced sensitivity to cisplatin and mitomycin C [BRCA2 carriers was described in several other case reports [BRCA2-related advanced lung cancer, which was treated by mitomycin C, cisplatin, and vincristine [BRCA2 carrier, who suffered from castration-resistant prostate cancer, showed durable marker response and resolution of bone metastases after the administration of olaparib [BRCA2-related prostate cancer, which was treated by cisplatin after becoming insensitive to androgen ablation. This therapy resulted in normalization of prostate-specific antigen level and symptomatic relief for period of 8 months; docetaxel was administered after the disease progression, and also led to an evident tumor response.y cancer ,96. In aomycin C . Importaomycin C . Increas reports -100. Thecristine . Anotherolaparib . Vesprinolaparib  have desBLM gene mutations in hereditary predisposition to breast cancer. This study included 5 patients treated by conventional neoadjuvant therapy; nearly complete pathological response was observed in 3 cases, while the remaining 2 women showed partial reduction of the tumor mass.Sokolenko et al.  have recNBN (NBS1) and BRIP1  genes [Preclinical data suggest specific drug sensitivity pattern for the cells with inactivated 1) genes ,93. It mMLH1, MSH2, PMS2 and MSH6 genes. Virtually all tumors from HNPCC mutation carriers are characterized by the defect of mismatch repair (MMR), which is manifested by so-called high-level microsatellite instability (MSI-H). MSI-H occurs in up to 15% of colorectal cancers (CRC), however the majority of the microsatellite-unstable carcinomas are sporadic; hereditary CRC constitute approximately one fifth of MSI-H cases and account for only 2-3% of the total CRC incidence. Mutations in HNPCC-related genes may also predispose to a number of non-colonic tumors, including endometrial, gastric, urothelial, ovarian and some other neoplasms [Hereditary non-polyposis colorectal cancer (HNPCC) is caused by germ-line mutations in eoplasms ,106.MLH1, MSH2, PMS2 or MSH6; sporadic MSI-H tumors are usually driven by methylation of the MLH1 gene promoter that may be a consequence of wide-spread abnormalities of epigenetic regulation ("methylator phenotype"). For unknown reason, BRAF mutations occur only in sporadic but not in hereditary MSI-H tumors [Given the rarity of hereditary CRC and the requirement of expensive multigene test for its definite diagnosis, the collection of clinical series for this disease represents a challenge. Instead, there is an intensive research focusing on MSI-H tumors as a distinct CRC entity; although sporadic and hereditary MSI-H CRC tumors share essential bioclinical features, many experts warn against combined analysis of these two tumor subsets. It is emphasized, that while hereditary CRC affect relatively young subjects, sporadic MSI-H cases are accumulated among elderly patients. Hereditary CRC arise due to mutational inactivation of the H tumors ,107.BAT26) allow to differentiate between true MSI-H and irrelevant mutational noise; hence, consideration of dinucleotide loci may increase the frequency of false-positive MSI-H detection and further compromise the conclusions of clinical trials [Although MSI-H tumors tend to be poorly differentiated, they are usually characterized by favorable disease course. In particular, MSI-H tumors show relatively low relapse rates after potentially curative surgery ,109; in l trials .MSH2 and MLH1, may result in distinct patterns of chemosensitivity [Sensitivity of MMR-deficient cells to various anticancer drugs has been a subject of multiple laboratory studies. It is important to acknowledge, that naturally occurring MSI-H cancer cells have highly increased mutation rate and therefore accumulate significant number of "secondary" genetic lesions; depending of the spectrum of the target genes, these secondary lesions may substantially modify the response to treatment modalities ,113. Fursitivity .Most of preclinical studies point at resistance of MSI-H cells to 5-fluorouracil (5-FU) ,115. MSIThere is a good consensus in the literature that MSI-H CRC patients do not benefit from 5-fluorouracil based adjuvant therapy . Some reData on the use of chemotherapy for advanced MSI-H CRC are limited by a few small studies. Liang et al.  and BrueMLH1, MSH2 or MSH6 mutation carriers. Despite encouraging preclinical and epidemiological evidence, neither of these compounds influenced the risk of adenoma formation during the four years of the study.Burn et al.  analyzedAPC gene. Some patients bear an attenuated form of this disease; mild manifestation of FAP may indicate the involvement of another genetic lesion, i.e. homozygous inactivation of MUTYH gene [Familial adenomatous polyposis (FAP) is manifested by multiple polyps, which cause severe gastrointestinal symptoms and frequently progress into cancer lesions. Classical FAP is caused by a dominant germ-line mutation of the TYH gene . DevelopTYH gene . Based oTYH gene . EarlierTYH gene .RET tyrosine kinase. It can be a part of multiple endocrine neoplasia (MEN) type 2A (MEN2A) or type 2B (MEN2B) syndromes, or manifest as a single-organ lesion  [RET-transformed cancer cells [Hereditary medullary thyroid cancer (MTC) is caused by germ-line mutation in r, FMTC) . A noveler cells . A clinier cells . Preciseer cells . Comparaer cells .BRCA1-related and triple-negative BC [BRCA1 may be inactivated not only by germ-line but also by somatic alterations, several investigations suggested to use BRCA1 expression as predictive marker of response to platinum-based and taxane-based therapy [BRCA deficiency or other critical defects of homologous recombination; in particular, it has been observed that tumors with presumably impaired repair of DNA double-strand breaks show characteristic pattern of acquired mutations [BRCA1, the mutations of RET oncogene are observed not only in hereditary, but also in sporadic medullary thyroid carcinomas; it is expected, that at least a subset of RET-driven non-hereditary MTC should respond to vandetanib therapy [Hereditary neoplasms make relatively little contribution in the total cancer incidence. Nevertheless, advances in the treatment of this category of tumors may have broader practical implications, as many sporadic tumors develop phenotype similar to hereditary cancers. This issue was particularly intensively discussed in breast cancer research, owing to substantial overlap between ative BC . Given t therapy ,148-152.utations ,154. Sim therapy . While f therapy ,107.Patients with hereditary tumors often benefit from distinct drugs as compared to sporadic cases. The detection of cancer-predisposing germ-line mutations among the participants of clinical trials has rarely been considered, due to significant cost of genetic testing. Given the rapidly increasing accessibility of DNA analysis, it is foreseen that a large number of germ-line mutation carriers will be included in forthcoming trials and/or identified within retrospective collections of biological material. The analysis of correlations between genotype and drug response may substantially improve treatment outcomes, both for hereditary cancer patients and for subjects bearing phenocopies of familial tumors.AC: doxorubicin and cyclophosphamide; BC: breast cancer; CMF: cyclophosphamide, methotrexate and fluorouracil; cCR: clinical complete response; CR: complete response; CRC: colorectal cancer; FAC: 5-fluorouracil, doxorubicin and cyclophosphamide; FAP: familial adenomatous polyposis; FEC: 5-fluorouracil, epirubicin and cyclophosphamide; HNPCC: hereditary non-polyposis colorectal cancer; HR: homologous recombination; MMR: mismatch repair; MTC: medullary thyroid cancer; MSI-H: high-level microsatellite instability; OC: ovarian cancer; OR: objective response; OS: overall survival; PARP: poly (ADP-ribose) polymerase; pCR: pathological complete response; PD: progressive disease; PFS: progression-free survival; PR: partial response; SD: stable disease.The authors declare that they have no competing interests.Both authors contributed to the literature search, data analysis and manuscript preparation. Both authors read and approved the final manuscript.


RV217/ECHO aims at capturing HIV-1 incident cases during the very earliest stages of acute HIV-1 infection(AHI). Limitations in single genome amplification(SGA) sampling depth have the potential to confound the study of multiplicity of infection and viral evolution during AHI. Here we combined SGA and deep, next-generation sequencing(NGS) to investigate the prevalence of minor viral variants during pre-peak viremia and characterize subsequent viral evolution.A male CSW from Thailand with documented nucleic acid testing(NAT)-conversion and seroconversion, and with measured viremia peak, was studied. Plasma viruses from pre-peak viremia, immediate post-peak viremia, and 6 months post-infection were characterized by SGA and targeted NGS. A library prepared from 1,500 PCR-equivalent copies was analyzed using IonTorrent(LifeTechnologies).Reading coverage was>20K and the experimentally-measured error rate was<0.0028/base.Based on 47 env SGA sequences from pre-peak viremia, the infection was initiated by a single T/F virus (mean env pair-wise nt diversity:0.047%).A second variant, highly similar but distinguishable from the T/F , was detected during immediate post-peak viremia(18/56 SGA sequences). By 6 months post-infection, the viral population was dominated by the second variant along with numerous recombinants.NGS analysis of multiple, independent subgenomic areas with concentrated sequence variation, revealed that the second variant was already established at 1% at pre-peak viremia. Outgrowth of the second variant, along with its derivatives, was confirmed by longitudinal NGS.Supplementing SGA analysis with NGS allowed us to demonstrate the presence of two T/F viruses at pre-peak viremia with substantial frequency differences, in the study participant. Due to its implications, the prevalence of this phenomenon needs to be further explored. The application of NGS to the study of HIV-1 evolution during AHI promises to foster the advancement of vaccine development.


In recent years MRI-guided interventional electrophysiology (EP) has rapidly emerged as a promising alternative to conventional X-ray guided ablation. We present a novel MRI-compatible electrophysiologic ablation and recording system, performing realistic biatrial simulated procedures.To date, preclinical studies evaluating MRI-guided ablation have used non-irrigated catheters limited to test lesions in the right heart. Modern complex EP procedures require externally irrigated catheters to produce adequate lesion depth. We present the first feasibility study of an externally-irrigated MRI-compatible ablation catheter, and integrated EP pacing and recording system. We demonstrate the feasibility of pulmonary vein (PV) and cavotricuspid isthmus (CTI) ablation, the targets of ablation in atrial fibrillation and typical atrial flutter respectively.An externally irrigated MRI-compatible ablation and diagnostic electrophysiology catheters and integrated EP recording system  were tested in n=11 sheep, in a 1.5T MRI scanner. The diagnostic catheters were placed in the coronary sinus, and right ventricular apex. Power controlled  lesions were formed at the pulmonary veins and cavotricuspid isthmus with the ablation catheter. Real-time intracardiac electrograms were recorded during MR imaging. Steady state free precession (SSFP) non-breath-hold images were repeatedly acquired to guide the catheter to the target region using passive catheter tracking. Markers in the catheter were used to ensure adequate visualization of the catheter. Lesion visualization was performed using both non-contrast (T2-weighted turbo spin echo pulse sequence) and gadolinium-DTPA enhanced T1-weighted imaging (inversion-recovery gradient echo pulse sequence).The catheters were able to be visualised and navigated throughout the heart. An average of 8±2.5 lesions were formed near the ostia of the pulmonary veins, and 6.5±1.3 lesions were formed at the cavotricuspid isthmus. The electrophysiological endpoint of bidirectional block was achieved in all cases. Mean procedure duration was 150±55 minutes. Histologic examination demonstrated transmurality of lesion formation at both the pulmonary vein ostia, and cavotricuspid isthmus. Lesion visualisation with both T2W imaging and contrast-enhanced imaging correlated with sites of injury at autopsy (see image). Non-contrast agent-enhanced techniques were suitable for repetitive lesion visualization during interventions, thus allowing for intra-procedural monitoring of ablation success.These data represent the most realistic simulated interventional MRI-guided electrophysiology procedures in the published literature, demonstrating the use of multiple catheters, an integrated EP pacing and recording system, and the use of an externally-irrigated ablation catheter. For the first time, the feasibility of CMR guided pulmonary vein ablation is shown, opening the possibility of MR-guided complex electrophysiology procedures, including pulmonary vein isolation for atrial fibrillation.Dr Ganesan is funded by a Cardiovascular Lipid Award from Pfizer Australia.


Left ventricular outflow tract tachycardia (LVOT-VT) is an uncommon type of idiopathic left ventricular tachycardia (ILVT) thought to be due to cyclic adenosine mono phosphate (c-AMP) mediated triggered activity. The arrhythmia can be terminated pharmacologically with calcium channel blockers or beta-blockers . LVOT-VTYet another classification of LVOT-VT  was intoTachycardiomyopathy, though much more common with supraventricular arrhythmias, has also been described with ventricular arrhythmias. Most of these reports were in case of right ventricular out flow tract tachycardias (RVOT-VT). In this issue of the journal, Mora G et al  describeVarious approaches have been described for the ablation of LVOT-VT. Shimoike E, et al  mapped tAdvanced technologies like intracardiac echocardiography , three d