Adult T-cell leukemia-lymphoma (ATL) is divided into two types for decision of therapeutic strategy; aggressive variant includes acute, lymphoma and chronic type with poor prognostic factor, and indolent variant includes smoldering and chronic type without any factors. Prognosis of aggressive ATL by chemotherapy alone is very poor. We have reported the possibility of improvement of prognosis in aggressive ATL by allogeneic hematopoietic stem cell transplantation  for the first time in 2001, however transplant-related mortality (TRM) was very high. In order to reduce TRM and improve outcomes of aggressive ATL, we conducted prospective clinical trials of allo-HSCT using the reduced intensity conditioning (RIC). The first clinical trial (NST-1) was performed for evaluating allo-HSCT with RIC using HLA matched sibling donor in the patients of aggressive ATL aged over 50, and subsequently NST-2 trial was applied for the same eligibility. Conditioning regimen in NST-1 included busulfan, fludarabine and antithymocyte globulin (ATG), but omitted ATG in NST-2 because of early relapse. In all 29 patients registered in NST-1 and -2, 3-year and 5-year overall survival rates (OS) were 36% and 34% respectively. Eight patients were died of TRM. Ten of the 29 patients obtained long-term survival with good performance status. Subsequently, NST-3 trial was conducted with similar eligibility with NST-2 to confirm the efficacy of the therapeutic strategy of RIC. Twenty patients were transplanted and 2-year OS was 53%. The availability of sibling donors is becoming difficult mainly due to the aging of not only patients but also donors. Therefore, NST-4 trial was performed with similar eligibility using unrelated bone marrow (UBM) as alternative stem cell source. Fifteen patients were transplanted, we have confirmed the feasibility of UBM and 2-year OS was 67%. For the further improvement of allo-HSCT for aggressive ATL, the study using cord blood (NST-5) is on the way.


Zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, produced anti-tumor effects through apoptosis induction or S-phase arrest depending on human mesothelioma cells tested. An addition of isoprenoid, geranylgeraniol but not farnesol, negated these ZOL-induced effects, indicating that the ZOL-mediated effects were attributable to depletion of geranylgeranyl pyrophosphates which were substrates for prenylation processes of small guanine-nucleotide-binding regulatory proteins . ZOL-treated cells decreased a ratio of membrane to cytoplasmic fractions in RhoA, Cdc42 and Rab6 but less significantly Rac1 proteins, indicating that these proteins were possible targets for ZOL-induced actions. We further analyzed which small G proteins were responsible for the three ZOL-induced effects, caspase-mediated apoptosis, S-phase arrest and morphological changes, using inhibitors for respective small G proteins and siRNA for Cdc42. ZOL-induced apoptosis is due to insufficient prenylation of Rab proteins because an inhibitor of geranlygeranyl transferase II that was specific for Rab family proteins prenylation, but not others inhibitors, activated the same apoptotic pathways that ZOL did. ZOL suppressed an endogenous topoisomerase II activity, which was associated with apoptosis and S-phase arrest in respective cells because we detected the same cell cycle changes in etoposide-treated cells. Inhibitors for geranlygeranyl transferase I and for RhoA produced morphological changes and disrupted actin fiber structures, both of which were similar to those by ZOL treatments. These data demonstrated that anti-tumor effects by ZOL were attributable to inhibited functions of respective small G proteins and topoisomerase II activity, and suggested that cellular factors were involved in the differential cell cycle changes. BPs administered roteins) .46 Ras family proteins are either farnesylated by farnsyl transferase or geranylgeranylated by geranylgeranyl transferase I. In contrast, the majority of Rho family proteins and Rab family proteins are geranylgeranylated by geranylgeranyl transferase I and II, respectively. These lipid modifications are essential for most of small G proteins to bind to cytoplasmic and organelle membranes where prenylated small G proteins become functional, whereas unprenylated small G proteins remain in the cytoplasm and non-functional.5Isoprenoid lipids, farnesyl pyrophosphate and geranylgeranyl pyrophosphate, are substrates for prenylation processes that mediate farnesylation and geranylgeranylation of small G proteins, respectively.8 These cytotoxic actions are attributable to a number of mechanisms including apoptosis induction and anti-angiogenesis,10 but it is not well investigated as to which small G proteins produce the cytotoxic effects.The nitrogen-containing BPs (N-BPs) also induce cytotoxicity to osteoclasts, which is favorable for enhanced bone mineralization, and recent studies also showed that N-BPs had cytotoxic activities on tumors such as breast and prostate cancer.11 ZOL treatments induced apoptotic cell death or S-phase arrest in cell cycle, and moreover caused morphological changes from fibroblast-like to spherical shapes. In the present study, we examined what kinds of small G proteins are responsible to these ZOL-mediated effects using inhibitors or small interfering RNA (siRNA) for the respective small G proteins and for prenylating enzymes.We recently showed that zoledronic acid (ZOL), which is one of the N-BPs to inhibit farnesyl pyrophosphate synthetase, produced cytotoxic activities to human mesothelioma.We examined ZOL-mediated anti-tumor effects in human mesothelioma cells . ProlifeWe investigated the involvement of farnesylation or geranylgeranylation in the ZOL-induced apoptosis and S-phase arrest. We examined an effect caused by addition of farnesol (FOH) and geranylgeraniol (GGOH), which were converted into farnesyl pyrophosphate and geranylgeranyl pyrophosphate, respectively, to determine ZOL's targets among small G proteins. A supplementary use of GGOH in MSTO-211H cells reduced ZOL-mediated downregulation of Mcl-1 expression, dephosphorylation levels of pRb and Akt, and cleavages of caspase-9, -3 and PARP . MoreoveWe examined what kinds of small G proteins were unprenylated with ZOL treatments by testing the cellular distributions because some of antibodies specific to the unprenylated form was unavailable. Fractionated cytoplasmic and membrane portions were examined for the expressions of RhoA, Rac1 and Cdc42, belonging to the Rho family, and Rab6, one of the Rab family proteins . ZOL treWe used inhibitors or siRNA for respective small G proteins to identify possible small G proteins that were involved in ZOL-induced apoptosis or S-phase arrest . C3 tranWe further investigated the effects of inhibitors for geranylgeranylation, GGTI-298 and NE10790, which inhibit geranylgeranyl transferase I and II, respectively . GGTI-29We investigated a possible mechanism of apoptosis induction by the inhibitors that increased sub-G1 fractions in MSTO-211H cells, C3 transferase, GGTI-298 and NE10790 . C3 tranWe investigated a possible mechanism of ZOL-induced S-phase arrest in EHMES-10 cells. We firstly examined topoisomerase (Topo) II activities in ZOL-treated cells . Cell exCells treated with ZOL showed morphological changes, altering fibroblastic into round-shaped configurations and being less adhesive to a culture plate . ZOL treIn the present study, we investigated possible molecules responsible for ZOL-induced apoptosis, S-phase arrest and morphological changes in mesothelioma. We examined the target molecules with supplementary isoprenoids, specific inhibitors and siRNA for small G proteins because ZOL-mediated unprenylation of small G proteins resulted in the loss of functions. These experiments collectively indicated that the ZOL-mediated apoptosis and morphological changes were due to inhibition of Rab family and Rho family proteins, respectively. The S-phase arrest observed in ZOL-treated cells was not attributed to unprenylation of small G proteins but to inhibition of Topo II activity.12 We did not determine in the present study what kinds of Rab family proteins contributed to ZOL-induced apoptosis, but the experiments with NE10790 narrowed down the targeted Rab family proteins. Rab family proteins possess a one- or two-cysteine-containing motif at the C terminal, and NE10790 inhibits geranylgeranylation of only the second of the two-cysteines residues.15 The targets of NE10790 are consequently Rab family proteins with two-cysteine-containing motifs such as Rab1, Rab5 and Rab6. Some Rab family proteins are highly expressed in tumors and can be involved in progression and invasion of the tumors.17 Rab family proteins are also involved in intracellular transport among organelles, which is crucial for cell survival. We thereby presume that the Rab functions in tumors cannot be substituted by other small G proteins.N-BPs can produce cytotoxic effects, but the precise mechanism remains uncharacterized in particular as for which small G proteins are involved. We examined differential effects of FOH and GGOH and demonstrated that geranylgeranylation was crucial for the cytotoxicity. Treatments with GGTI-298 or NE10790 confirmed that deficient geranylgeranylation induced cell death and we further demonstrated that C3 transferase but not NSC23766 or siRNA for Cdc42 induced cell death. Expression patterns of apoptosis-related molecules indicated that NE10790 but not GGTI-298 or C3 transferase activated the same apoptotic pathways that ZOL did. We thereby concluded that ZOL-induced apoptotic cell death was attributable to the loss of Rab family proteins' functions. A previous study also demonstrated that NE10790 induced apoptosis without cell cycle arrest but did not investigate precise mechanisms as to which apoptosis signals were involved.18 ZOL-induced depletion of isoprenoid can stimulate a feedback mechanism in the mevalonate pathway and enhance the hydroxymethylglutaryl-CoA reductase activity accordingly , and EHMES-1, EHMES-10 and JMN-1B cells were provided by Dr Hamada . ZOL were purchased from Novartis , and FOH, GGOH, GGTI-298, ETO, topotecan and clodronate were from Sigma-Aldrich . C3 transferase and NSC23766 were from Cytoskeleton  and Merck Millipore , respectively. NE10790 were provided by Dr Ebetino FH . We purchased siRNA duplex targeting Cdc42 and non-specific control siRNA from Invitrogen . Transfection of cells with the siRNA was conducted with Lipofectamine RNAiMAX (Invitrogen).μg/ml) and stained with propidium iodide (50 μg/ml). The fluorescence intensity was analyzed with FACSCalibur and CellQuest software .Cells were fixed in 100% ethanol, treated with RNase  (WST assay). The amount of formazan produced was determined with the absorbance at 450 nm and the relative viability was calculated based on the absorbance without any treatments.http://rsbweb.nih.gov/ij). GTP-binding RhoA and Rac1 proteins were isolated with a pull-down assay using a Rho activation assay biochem kit and a Rac1 activation assay biochem kit (Cytoskeleton) according to the manufacturer's protocol.Cells lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and the proteins were transferred to a nitrocellulose membrane. The membrane was hybridized with antibodies against pRb, phosphorylated pRb at Ser 795, Akt, phosphorylated Akt at Ser 473, caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, PARP , Mcl-1, unprenylated Rap1A, Rab6 , RhoA, Cdc42, Rac1 (Cytoskeleton), Ras (BD Biosciences) or actin (Sigma-Aldrich). The membranes were developed with the ECL system . Membrane and cytoplasm fractions were separated with a native membrane extraction kit (Merck Millipore) according to the manufacturer's protocol. Intensity of hybridized bands was determined with the public domain Image J program . The relative activity was calculated based on luminescence intensity of cells without any treatment.Topo I and Topo II activities were determined with corresponding kits according to the manufacturer's protocol . Cell extract and substrate DNA, supercoiled DNA for Topo I or catenated kinetoplast DNA for Topo II, were incubated with (for Topo II activity) and without ATP (Topo I). The reaction mixtures were digested with proteinase K and then subjected to agarose gel electrophoresis.Cells cultured on a Matrigel (BD biosciences)-coated cover glass were fixed with 3.7% formaldehyde and treated with Alexa Fluor 488 phalloidin (Invitrogen) followed by ProLong Gold antifade reagent (Invitrogen). The fluorescence images were taken with a confocal microscope, Leica TCS-SPE .


There is compelling clinical and experimental evidence to suggest that natural killer (NK) cells play a critical role in the recognition and eradication of tumors. Efforts at using NK cells as antitumor agents began over two decades ago, but recent advances in elucidating NK cell biology have accelerated the development of NK cell-targeting therapeutics. NK cell activation and the triggering of effector functions is governed by a complex set of activating and inhibitory receptors. In the early phases of cancer immune surveillance, NK cells directly identify and lyse cancer cells. Nascent transformed cells elicit NK cell activation and are eliminated. However, as tumors progress, cancerous cells develop immunosuppressive mechanisms that circumvent NK cell-mediated killing, allowing for tumor escape and proliferation. Therapeutic intervention aims to reverse tumor-induced NK cell suppression and sustain NK cells’ tumorlytic capacities. Here, we review tumor–NK cell interactions, discuss the mechanisms by which NK cells generate an antitumor immune response, and discuss NK cell-based therapeutic strategies targeting activating, inhibitory, and co-stimulatory receptors. The recent FDA approvals of the programmed cell death protein 1 (PD-1)-targeted checkpoint inhibitors pembrolizumab and nivolumab mark the latest successes in the rapidly expanding field of cancer immunotherapies. Immunotherapy represents a paradigm shift in cancer treatment; instead of targeting tumor cells, the goal of immunotherapy is to augment and expand the immune system’s intrinsic antitumor response. To date, diverse immunotherapeutic modalities have been accepted as viable strategies for eliminating cancerous cells. Cytokines, cancer vaccines, adoptive cell transfers, and especially checkpoint inhibitors constitute valuable elements in the immunotherapeutic armamentarium. However, a class of important immune-modulators is conspicuously absent: agents that utilize the power of innate immune cells to eradicate tumors. An important class of innate immune cells that play a critical role in mediating the antitumor immune response is the natural killer (NK) cell.bright population and a CD56dim population. The CD56bright population produces immunoregulatory cytokines, including interferon-γ (IFN-γ), tumor necrosis factor-beta (TNF-B), tumor necrosis factor-α (TNF-α), granulocyte macrophage-colony stimulating factor (GMCSF), IL-10, and IL-13  class I molecules would render target cells susceptible to NK cell attack . The “miet cells , 16. Howet cells . While NNatural killer cell cytotoxicity can be demonstrated in several related ways. The primary mechanism of cytotoxicity is based on granule exocytosis upon formation of an immunological synapse. NK cells contain preformed cytoplasmic granules that resemble secretory lysosomes and contain perforin and granzymes . Perfori+ T cells to polarize toward a Th1 subset and accelerates the development of activated macrophages and cytotoxic, tumor-targeting CD8+ T cells . ADCC is thought to play an important role in mediating the antitumor effects of many of the monoclonal antibody (mAb) therapies used today as standard of care treatments for both solid tumors and hematologic malignancies . In ADCCDeath receptor-induced apoptosis is a perforin-independent mechanism by which NK cells lyse target cells . This cyin vitro and in vivo  and nivolumab and pembrolizumab (anti-PD-1 mAbs) demonstrate the potential of this therapeutic strategy. Ipilimumab  was approved in 2011 for the treatment of unresectable or metastatic melanoma, and blocks the CTLA-4-mediated signaling in T cells . CTLA-4 Within the signaling pathways that govern NK lytic capacity, the killer cell immunoglobulin-like receptor (KIR) family is a dominant group of negative regulators. KIR receptors bind to the self-MHC class I ligands  and upon ligation transmit signals that abrogate the effects of activating receptors . The preThe development of a candidate anti-KIR antibody had to overcome significant challenges. The KIR gene content varies substantially from individual to individual depending on the inherited KIR haplotype and the KIR family is composed of several structurally different proteins, necessitating an antibody that has cross-reactivity between different KIRs . DespiteIn addition to KIRs, the CD94/NKG2A heterodimer is another target for NK cell checkpoint blockade. The natural ligand of CD94/NKG2A is HLA-E, a non-classical HLA class I molecule that is expressed on the cell surface of most leukocytes and on transformed cells, including virus-infected cells and tumor cells , 86. LigActivating co-stimulatory pathways to potentiate antitumor immune responses is a promising approach for augmenting NK-mediated tumor clearance. Members of the tumor necrosis factor receptor superfamily (TNFRsf) include several co-stimulatory proteins with key roles in the regulation of the activation, proliferation, and apoptosis of lymphocytes, including NK cells.First identified in 1989, CD137 (or 4-1BB) is a co-stimulatory receptor and member of the TNF receptor superfamily . CD137 iAnti-CD137 mAb therapy has also entered clinical testing. The anti-CD137 antibody, urelumab, is currently in clinical trials with rituximab for patients with non-Hodgkin’s lymphoma (NCT01775631) and with cetuximab in patients with colorectal cancer or head and neck cancer (NCT02110082). In addition to urelumab, clinical trials of Pfizer’s anti-CD137 mAb, PF–05082566, are also ongoing (NCT01307267). A recent presentation of the preliminary findings reports that 27 patients with mixed tumor types have been treated with PF-05082566; disease stabilization was the best overall response, observed in 22% (6/27) patients .in vivo in tumor-bearing mice enhanced antitumor immunity, resulting in increased survival in four separate murine tumor models of diverse histology and immunogenicity  cells and NKs . In NK cgenicity . The inigenicity . Immunolgenicity . Additio+ NK have lower levels of perforin and granzyme B and demonstrate lower levels of cytotoxicity relative to CD27- NK cells (In addition to its co-stimulatory role on T cells, the expression of CD27, or TNFRSF7, differentiates the NK cell compartment into two functionally distinct subsets. Circulating CD27NK cells . The absNK cells . SignaliNK cells .The potential for CD27 ligation to generate an antitumor response has been confirmed in preclinical models. In a xenograft models of lymphoma, administration of the humanized anti-CD27 antibody, 1F5, significantly prolonged survival . The fulIn the future, immunotherapeutic agents that directly enhance NK cell-mediated tumor eradication will play a leading role in cancer treatment strategies. NK cells have novel mechanisms of participating in immune defense, making them uniquely appealing for cancer immunotherapy. Enhancing NK cell tumorlytic capacity is also a compelling combinatorial treatment strategy and would complement current standard of care treatments based on mAb therapy. The potential for NK-targeted agents to augment the antitumor effects of T cell checkpoint blockade is actively under consideration. As NK cell-based therapies move into the clinic, identifying prognostic biomarkers in the treatment populations will be crucial to the rational design of clinical studies. Concurrently, a greater effort must be made to profile the effects of novel immunotherapeutic agents, like checkpoint inhibitors, on NK cell function. The NK cell is now accepted as an integral part of the immunologic antitumor response. A number of promising NK-targeting therapeutics are in early-phase trials, and the results are eagerly awaited.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Participant non-response to postal questionnaires in randomised controlled trials (RCT) can jeopardise trial results.We evaluated two interventions aimed at increasing postal questionnaire response rates in a 2x2 partial factorial RCT nested within a single, large, UK-wide RCT of medical expulsive therapy for ureteric stones. The interventions were: questionnaire pre-notification via SMS text prior to posting the trial questionnaires versus no pre-notification; for non-responders to the initial questionnaire mailing, use of an e-mail reminder (containing a hyperlink for online questionnaire completion) versus a postal reminder with paper questionnaire. Participants were randomised to the pre-notification comparison, the reminder comparison or both, depending on whether they supplied mobile telephone number and/or email address. The primary outcome was response to 4 and 12 week questionnaires.418 participants were randomised to the SMS pre-notification comparison: the intervention had no effect on response rates at either questionnaire time point. In sub-group analyses, SMS pre-notification increased response rates in women (p=0.038), but only at the first time-point. 119 participants were randomised to the reminder comparison. There was no difference in response rates between those randomised to e-mail or postal reminders.SMS text pre-notification of questionnaire delivery and email delivery of reminders did not improve response rates. There was some evidence to suggest SMS text pre-notification may be effective in women. While e-mail reminders had no impact on response rates, their use could result in postage cost savings. Further studies of these interventions, nested in different RCTS, are warranted, with subsequent meta-analysis.


Hepatitis C virus (HCV) is the leading cause of hepatocellular carcinoma (HCC), and several antiviral agents are available for the treatment of chronic HCV infection. However, the impact of antiviral therapy on the long-term outcomes of HCV-related HCC patients remains inconclusive. We aimed to examine the impact of antiviral therapy on the long-term outcomes of HCV-related HCC patients. We conducted a systematic review using PRISMA guidelines to identify trials and English-language literature from PubMed, Ovid MEDLINE, Scopus and the Cochrane Library database till August 2014. Randomized trials of antiviral treatments examining the effects of antiviral therapy on CHC patients and HCV-related HCC patients were screened and selected. We identified 6 trials evaluated the effectiveness of interferon (IFN)-alfa treatment, 3 studies examined pegylated interferon-alfa treatment, and 2 studies examined IFN-beta treatment. IFN-based therapy may decrease HCC incidence in HCV cirrhotic patients after a >5-year follow-up, improve liver reserve, decrease HCC recurrence rate, and increase survival rate in HCV-related HCC patients after curative HCC therapy. In conclusion, IFN-based therapy is beneficial and may be recommended in the management of HCV-related HCC patients who are IFN eligible. Hepatitis C virus (HCV) infection is the leading cause of chronic liver diseases, leading to the progression of liver disease from chronic hepatitis to cirrhosis, with the complications of liver failure and hepatocellular carcinoma (HCC)14Because understanding the impact of antiviral regimens on HCV-related HCC is essential for the treatment plan designing of CHC patients with or without HCC, several trials have examined the impact of antiviral agents on HCV-related HCC patients. However, most of these trials differ significantly in the study population, protocols, antiviral regimens, and definitions of outcomes7891011To this end, we conducted this systematic review to examine the impact of antiviral therapy on the long-term outcomes of HCV-related HCC patients. As the effect of antiviral therapy on the HCC risk of CHC patients remain unconcluded, and antiviral therapy is not likely to affect the long-term outcomes of HCV-related HCC patients when it does not decrease the HCC risk in CHC patients. We investigated the impact of antiviral regimens on the HCC incidence of patients with HCV infection first. Once positive findings were shown in the first part, we then examined the long-term outcomes of HCV-related HCC patients by searching relative references and examining the benefits as well as harms of antiviral agents for HCV-related HCC patients.What is the effect of antiviral treatment on the HCC incidence of patients with chronic HCV infection?What is the effect of antiviral treatment on HCC outcomes of patients with HCV-related HCC (including the recurrence or progression of HCC)?What is the effect of antiviral treatment on clinical outcomes  of patients with HCV-related HCC?What are the comparative harms associated with antiviral treatments in patients with HCV-related HCC?A review protocol and an analytic framework were developed with input and discussion from the authors to set clear questions, ensure a standardized and repeatable review process between reviewers , and incWe followed the PRISMA recommendations for systematic literature analysis, which is an evidence-based set of items for reporting in systematic reviews and meta-analyses, and focuses on ways to ensure the transparent and complete reportingTwo reviewers independently evaluated studies for inclusion. We included randomized trials on HCV-related HCC patients that investigated IFN-based therapy, including mono-therapy with IFN or PEG-IFN alfa; dual therapy with IFN plus ribavirin or PEG-IFN alfa plus ribavirin; and triple therapy with PEG-IFN , ribavirin, and either telaprevir or boceprevir or newer DAAs. Clinical outcomes were mortality, cirrhosis, hepatic decompensation, hepatocellular carcinoma, and need for transplantation. HCC outcomes were the recurrence and progression of HCC. Sustained virological response (SVR) was defined as the absence of detectable HCV RNA in the serum 6 months after the end of a course of therapy. Biological response (BR) was defined as a decrease in serum ALT to within the normal range after treatment. Harms included withdrawals due to adverse events, serious adverse events, neutropenia, anemia, psychological adverse events, influenza-like symptoms, and rash.We restricted the inclusion to English-language articles. Article selection was determined a priori using the following inclusion criteria: (i) only adults were included in the trial; (ii) patients with chronic HCV infection were included in the study examining effects of antiviral treatments on HCC incidence; and patients with HCV-related HCC were included in the study examining effects of antiviral treatments on HCC outcomes; (iii) the study used IFN- or PEG-IFN-based therapy combined with or without other antiviral agents. We excluded: (i) review articles, (ii) editorials; (iii) letters to the editor (iv) case reports; (v) research protocols; (vi) duplicated records; (vii) sub-analyses of the same clinical trial; if the final study results had been published; (ix) interim reports if the final results had been published; (x) regimens with antiviral drugs not approved by the U.S. Food and Drug Administration for HCV infection.One investigator abstracted details of the study, and a second investigator reviewed data for accuracy. The methodological quality of each study was assessed using the Cochrane Collaboration’s tool for assessing risk of bias in randomized trials 14. Two iThe flow chart of the systematic review is shown in Five RCTs, including 1926 patients, examining the impact of antiviral agents on patients with HCV infection were identified, and summarized in Table 245151617417et al. did not identify a significant difference in the normalization of ALT level between groups, they found that ALT deceased to <80 IU/L in 19/45(42.2%) IFN-alfa treated patients [9/45 (20%) in controls]. Moreover, they also found lower AFP and higher serum albumin levels in IFN-treated patients. For PEG-IFN-alfa 2a treatment, HALT-C study showed a 0.45–fold decline of serum ALT levels among treated patients at 1.5 years (0.21 times among control patients), a 0.47-fold decline among treated patients at 3.5 years (0.19 times among control patients), and a significant difference of 0.28 times the upper limit of the normal range .Four studies reported the improvement of serum alanine aminotransferase (ALT) levels, and decreased serum HCV RNA level in treated subjects. A biochemical end-of–therapy response was observed in 5/38 (13%) IFN-beta treated patients [2/23 (9%) in controls], and 6/47 (12.8%) IFN-alfa treated patients [0/39 in controls]16et al. & HALT-C)4et al. & HALT-C)155,1516Two studies 154Compared to untreated controls, 8 studies showed a biochemical response with serum ALT decline, and 2 showed a better liver reserve in CHC patients after receiving IFN-based therapy11Our study had a few limitations. First, a positive “publication” bias that tends to publish research with a positive outcome more frequently may be introduced into this study2829This systematic review supports the beneficial effects of IFN-based treatment for decreasing HCC incidence in HCV-related cirrhotic patients, and HCC recurrence rate in HCV-related HCC patients after curative HCC therapy. Moreover, IFN-based therapy is associated with the improvement of serum ALT levels and better liver reserve in HCV-related HCC patients after curative HCC therapy. However, future studies are required to clarify the impact of DAAs on HCC risk and cirrhotic complication in CHC patients and HCV-related HCC patients after curative HCC therapy.How to cite this article: Hsu, C.-S. et al. Systemic Review: Impact of Interferon-based Therapy on HCV-related Hepatocellular Carcinoma. Sci. Rep.5, 9954; doi: 10.1038/srep09954 (2015).


The Hepatitis C virus (HCV) infection exhibits a high global prevalence frequently associated with hepatocellular carcinoma, taking years to develop. Despite the standardization of highly sensitive HCV quantitative RT-PCR (qRT-PCR) detection methods, false-negative diagnoses may be generated with current methods, mainly due to the presence of PCR inhibitors and/or low viral loads in the patient’s sample. These false-negative diagnoses impact both public health systems, in developing countries, and an in lesser extent, in developed countries, including both the risk of virus transmission during organ transplantation and/or blood transfusion and the quality of the antiviral treatment monitoring. To adopt an appropriate therapeutic strategy to improve the patient’s prognosis, it is urgent to increase the HCV detection sensitivity. Based upon previous studies on HBV, we worked on the capacity of the scavenger acute phase protein, Apolipoprotein H (ApoH) to interact with HCV. Using different approaches, including immunoassays, antibody-inhibition, oxidation, ultracentrifugation, electron microscopy and RT-PCR analyses, we demonstrated specific interactions between HCV particles and ApoH. Moreover, when using a two-step HCV detection process, including capture of HCV by ApoH-coated nanomagnetic beads and a home-made real-time HCV-RT-PCR, we confirmed the presence of HCV for all samples from a clinical collection of HCV-seropositive patients exhibiting an RT-PCR COBAS® TaqMan® HCV Test, v2.0 (COBAS)-positive result. In contrast, for HCV-seropositive patients with either low HCV-load as determined with COBAS or exhibiting HCV-negative COBAS results, the addition of the two-step ApoH-HCV-capture and HCV-detection process was able to increase the sensitivity of HCV detection or more interestingly, detect in a genotype sequence-independent manner, a high-proportion (44%) of HCV/RNA-positive among the COBAS HCV-negative patients. Thus, the immune interaction between ApoH and HCV could be used as a sample preparation tool to enrich and/or cleanse HCV patient’s samples to enhance the detection sensitivity of HCV and therefore significantly reduce the numbers of false-negative HCV diagnosis results. Until the recent introduction of hepatitis C virus (HCV) screening tests, this viral infection has represented the major cause of blood transfusion-associated hepatitis . Near 17Hepacivirus genus from the Flaviviridae family  and centrifuged for 18 h at 300,000 x g at 4°C.Duplicate samples of freshly-collected EDTA-plasma were used to separate the three major lipoprotein classes by sequential, isopycnic centrifugation in a TLA-120.2 fixed angle rotor , as described previously . DensitySamples of HC-VP (Hepatitis C virus particles) isolated from sucrose gradient were incubated in 2% glutaraldehyde in PBS and applied to an ApoH-coated400-mesh Formvar-carbon-coated copper grid for approximately 2 min. Excess fluid was drawn away with filter paper. The sample was then negatively stained with 1% (w/v) filtered aqueous uranyl acetate for 2 min and, after washing in 1% uranyl acetate, examined using a Zeiss EM 10C/CR transmission electron microscope, as described previously .Bland-Altman plot analysis was performed to assess the agreement level between the quantification of HCV/RNA with and without ApoH-sample pretreatment method. ANOVA multiparametric tests have been done using the GraphPad PRISM 5 software™.An archived collection of sera from HCV-seropositive human patients with or without a viral load detection determined by a quantitative real-time PCR technology using the COBAS® TaqMan® HCV Test, v2.0 were used for this study (Metropolitan French USDEP project) and were part of an already-established collection "HCV-LO-2005-2006" of anonymized leftover samples at the Virology Lab of The University Hospital Center (CHU) “La Tronche”, Grenoble, France. The shipping of these clinical samples for research purposes to the Laboratoire d’Immuno-Physiopathologie Moléculaire Comparée  during 2005 and 2006, were perfectly in line with the French ethical regulations. Healthy donors samples and human albumin was purchased at the Centre Regional de Transfusion Sanguine de Lille, France.Two different approaches were used to evidence the binding between ApoH and HCV-related particles: an RT-PCR assay, in order to detect viral RNA after the virus capture with ApoH-coated beads and an immuno-enzymatic assay in order to detect ApoH-captured HCV antigens, using ApoH-coated plates.In the experiment shown in In the experiment shown in To assess the involvement and specificity of coated ApoH to capture HCV from patients’ sera, plates and nanomagnetic beads were coated with another serum acute phase protein, the α1-acid glycoprotein, the binding was measured by both immunoassay (with an anti-HCV/E2 MAb) and HCV RT-PCR. Both In order to assess the specificity of interaction between ApoH and HCV, we used ApoH-coated plates to capture HCV and we assessed the inhibition of viral capture using an anti-ApoH MAb . An irreTo ascertain the specificity of these results as well as the efficiency of the HCV-capture by ApoH, a broad range of sera coming from either HCV-seropositive or HCV-seronegative patients, exhibiting different pathologies, was assayed for the detection of HCV by the ApoH immunoassay. Sera of 43 hospital-archived clinical samples from HCV seropositive patients, harboring the HCV genotypes 1 to 5, as well as 10 sera from healthy blood donors were diluted in a Tris buffer and then added to ApoH-coated microtitration plates. Once captured, the HCV envelope antigen was detected using an anti-HCV/E2 MAb. As shown in HCV-positive sera submitted to buoyant and sedimentation processes exhibited a high viral heterogeneity in their gradient location. This heterogeneity may be due to the existence of defective particles or the association of viral particles to different serum components, including antibodies, low-density lipoprotein (LDL), very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL).In order to assess whether the binding of ApoH to HCV was able to retain whole HCV/RNA particles, a pool of HCV/RNA-positive sera from untreated patients or HCV-negative blood donors sera were spun by ultra-centrifugation at 436,000 x g. The resulting pellets, supernatants and floating lipids were tested for their capacity to bind to ApoH-coated microtitration plates and subsequently tested for their RNA content by RT-PCR. Because all the reported studies on the HCV density have been done on the floating lipids, we decided to characterize pelleted HCV particles. Thus, after 1 h of ultracentrifugation, the pellet was further purified on CsCl gradient [10–60% (w/w)]. Ten-fold diluted fractions of this gradient were tested with the ApoH-HCV ELISA immunoassay. Thereafter, we also tried to identify lipoproteic fractions, from HCV/RNA-positive but untreated patients, exhibiting an affinity for ApoH. Thus, a pool (n = 10) of plasma previously used for density analysis was fractionated into four parts, corresponding to the floating densities of VLDL (<1.006 g/L), LDL (1.006 to 1.063 g/L), HDL (1.063 < d < 1.210 g/L) as well as to the bulk plasma proteins. 2+, Fe3+, Ca2+, Mg2+, Mn2+, Zn2+ or Cu2+. 2+, Mg2+, Mn2+ or Zn2+ into the capture medium. However, the presence of Fe2+, Fe3+ or Cu2+ induced respectively significant three-, two- and five-fold increases of the captured HCV copy numbers.In a previous work , we haveIron and copper are redox-active metals and involved in oxidative injury mechanisms, including the lipid peroxidation. To assess whether anionic lipid peroxidation mechanisms are involved in the HCV recognition by ApoH, polymyxin B was added at a concentration of 100 μg/mL. 3 IU/mL were processed in parallel using (i) the Real-Time COBAS® TaqMan® HCV Test, v2.0 without the ApoH-sample pretreatment step and (ii) a home-made HCV RT-PCR assay done after the ApoH-sample pretreatment step. A significant correlation was found for both methods  for a viral load concentration varying between 103 and 107 IU/mL. The Bland–Altman plot shows the differences between the two assays  suggesting that the antigen recognized by ApoH belongs to a large macromolecular complex. High-density molecular complexes containing HCV/RNA have been reported but not always characterized . Some ofThe HCV-ApoH capture applied to sequentially ultra-centrifuged plasmas confirmed the presence of HCV as a lipoviral particle, in both VLDL and HDL fractions, but not in the LDL one, thus confirming the association of HCV with different lipoprotein fractions that have been already described . The Polvia the phospholipids in function of their configuration and their oxidative status.ApoH binding to phospholipids is reported to depend on their oxidation state . Oxidizevia several lipoprotein receptors. In favour of the inhibition hypothesis, it has been reported that ApoH is able to activate the lipoprotein lipase [in vivo [Altogether, these data lead us to hypothesize that ApoH could play a pivotal role in the evolution of HCV infection, either favouring or inhibiting the infection. In favour of the facilitating hypothesis it has been reported that lipoprotein receptors such as SR-BI or LDL-R  are repon lipase , which in lipase . Further[in vivo  suggesti[in vivo .3 IU/mL, a significant correlation was found between both methods. In contrast, for viral loads below 103 IU/mL or negative COBAS HCV RT-PCR samples from hospital-diagnosed patients, it appeared that the ApoH-sample pretreatment significantly improved the sensitivity of HCV detection in a genotype-independent manner. In particular, this ApoH sample pretreatment permits to easily detect HCV in those cases where the sole application of current methods generates erroneous false-negative HCV diagnoses. Thus 11 out of 25 (44%) seropositive samples that were COBAS HCV-PCR-negative were found HCV-positive with the open home-made HCV-PCR following the sample preparation with the ApoH-viral capture method. The presence of HCV/RNA was confirmed by DNA sequencing. The Blast nucleic acid sequences similarities with different annotated data banks showed that these sequences belonged to different HCV genotypes. In order to explain these differences between the presence of HCV in the presence or in the absence of ApoH beads, we have hypothesized that ApoH-coated nanomagnetic beads permitted HCV sensitive detection by a prior viral cleansing and elimination of PCR inhibitors. Different studies have reported the presence of PCR inhibitors in nucleic acids extracts, while others underline the adequacy of sampling and nucleic acids extraction in yields of microbial nucleic acids [Here, we have shown that the ApoH-coated beads can be used as an HCV-capture method to enhance the sensitivity of virus detection, resulting either from an endpoint or a quantitative RT-PCR as shown in ic acids , 82. Con


Dynamic PET provides temporal information about tracer uptake. However, each PET frame has usually low statistics, resulting in noisy images. The goal is to study effects of prior regularisation on dynamic PET data. Quantification and noise in image-domain and time-domain as well as impact on parametric images is assessed.8 and scatter and random fractions were both 35%. The data consists of 23 frames as applied in our clinical protocol. For reconstruction, the Ordinary Poisson Ordered Subset Expectation Maximisation (OP-OSEM) method was used in PRESTO [Dynamic PET data for the Siemens 3T MR-BrainPET was simulated with time-activity curves (TAC) of  F-FDG obn PRESTO , which aFigure Figure This study shows improvement on PET image quality in terms of noise (up to 50% reduction) as well as in parametric images when using prior regularisation in dynamic PET data. Both OP-OSEM and MRP OP-OSEM show similar quantification, with higher RCs for MRP OP-OSEM in low-count frames.


Mem-ADSVM: a two-layer multi-label predictor for identifying multi-functional types of membrane proteins” Identifying membrane proteins and their multi-functional types is an indispensable yet challenging topic in proteomics and bioinformatics. In this article, we provide data that are used for training and testing Mem-ADSVM (Wan et al., 2016. “ Specifications TableValue of the data•Knowing the functional types of membrane proteins can be helpful to elucidate the biological functions of membrane proteins.•This article presents the first comprehensive dataset that contains non-membrane proteins, single-functional-type membrane proteins and multi-functional-type membrane proteins.•The dataset presented here can be used as an important benchmark dataset to evaluate the performance of membrane-protein predictors.1http://bioinfo.eie.polyu.edu.hk/MemADSVMServer/datasets.html.Using benchmark datasets for evaluating the performance of predictors are of great significance in various domains of bioinformatics 2The dataset (we named as ‘Dataset II(C)’) here is a benchmark dataset to evaluate Mem-ADSVM


Dietary restriction (DR) is known to be one of the most effective interventions to increase stress resistance, yet the mechanisms remain elusive. One of the most obvious DR-induced changes in phenotype is an increase in locomotor activity. Although it is conceptually perceivable that nutritional scarcity should prompt enhanced foraging behavior to garner additional dietary resources, the significance of enhanced movement activity has not been associated with the DR-dependent increase of stress resistance. In this study, we confirmed that flies raised on DR exhibited enhanced locomotive activity and increased stress resistance. Excision of fly wings minimized the DR-induced increase in locomotive activity, which resulted in attenuation of the DR-dependent increase of stress resistance. The possibility that wing clipping counteracts the DR by coercing flies to have more intake was ruled out since it did not induce any weight gain. Rather it was found that elimination of reactive oxygen species (ROS) that is enhanced by DR-induced upregulation of expression of antioxidant genes was significantly reduced by wing clipping. Collectively, our data suggests that DR increased stress resistance by increasing the locomotor activity, which upregulated expression of protective genes including, but not limited to, ROS scavenger system. Aging is a multifaceted process that alters organism's structural architecture and functional capacity over the course of life. Unfortunately, aging-associated phenotypical and cellular changes often lead to a decrease in capability for daily tasks while susceptibility to many diseases such as cardiovascular disorders, cancer, and dementia is increased –3. Over DR reduces nutrient intake without inducing malnutrition . However Drosophila as an in vivo animal model to explore the importance of DR-induced increased locomotor activity in the organism's ability to cope with environmental stress. Apart from the ease of culturing in laboratory settings and the proven track record of providing insight into human diseases [ Drosophila's relatively short lifespan let us monitor the significance of locomotor activity over the entire course of life. Using the wing excision technique, which successfully attenuated the DR-induced increase in locomotor activity [We useddiseases , Drosophactivity , we demo w1118 strain obtained from Bloomington Drosophila Stock Center  was used for the experiments performed in this study. As described previously [ Drosophila diet  in a light (12-hour light/dark cycle), temperature (25°C), and humidity (60%) controlled fly incubator. Flies were transferred to fresh bottles approximately every 3 weeks to refresh the stock. Unless otherwise specified, all experiments were performed in the fly incubator.Adulteviously , the fli Drosophila food for 4 days, which allows female flies to have a sufficient time to mate and lay eggs. After that, parents were discarded and eggs were hatched to larvae, which metamorphose into the adult fly through the pupae stage. Although most flies eclosed 10 days after crossings in our experimental condition, we sorted out female flies 14 days after crossings in order to collect only age-matched mated female flies for this study. Female flies were used throughout experiments since phenotype changes induced by dietary restriction have been shown to be much greater in female flies than in male files [2 was used to temporary anesthetize flies for sorting and wing clipping. Sorted female flies were fed on either rich diet  or restricted diet . The recipes for the preparation of AL and DR are the same as the standard Drosophila diet with the exception that 1.5% live yeast was replaced with 5% or 0.5% yeast extract for AL or DR, respectively. For wing clipping, both sides of fly wings were manually removed with fine tweezers. Sorting and wing clipping were completed within 3 min to minimize CO2 toxicity.10 virgin female flies and 3–5 male flies were kept together inside bottles containing a standardle files . CO2 was Drosophila LAM25 Locomotor Activity Monitor  and data were acquired and processed with DAMSystem 308 software .Flies at the age of 10 days were transferred to new polystyrene vials (25 mm diameter) that contain freshly made DR or AL diets. Then, the vials were placed into theTo examine flies' ability to resist environmental stresses, starvation and reactive oxygen species (ROS) stress assays were performed. For the starvation stress assay, flies at the age of 10 days were transferred to starvation media made of 1% agar. Survivorship curves were constructed after scoring dead flies every 4 hours. Flies were transferred to fresh starvation media every day and dead flies were eliminated when possible. For ROS stress assay, 10-day-old flies were transferred to either AL or DR diet supplemented with 0.2 mM paraquat. Survivorship curves were generated by scoring dead flies every day. The flies were transferred to fresh paraquat supplemented diets every other day.β-tubulin (CG9277) F, ACA-TCC-CGC-CCC-GTG-GTC, R, AGA-AAG-CCT-TGC-GCC-TGA-ACA-TAG; superoxide dismutase 1 , F, CAA-GGG-CAC-GGT-TTT-CTT-C, R, CCT-CAC-CGG-AGA-CCT-TCA-C; catalase (CG6871), F, TGA-CTA-CAA-AAA-CTC-CCA-AAC-G, R, TTG-ATT-CCA-ATG-GGT-GCT-C; and phospholipid hydroperoxide glutathione peroxidase , F, GAC-ATC-GGC-GAG-GTG-TTC, R, ACT-TGG-TGA-AGT-TCC-ACT-TGA-TT. The specificity of amplicons was verified with a melting curve analysis and the messenger levels were normalized using β-tubulin as an internal control and calculated according to the ΔΔCt method [To compare gene transcription levels, qPCR was performed. According to the manufacturer's protocols total RNAs were isolated from five flies using RNeasy Mini Kit  and cDNA was prepared from the isolated total RNA with QuantiTect Reverse Transcription Kit . To quantify the amount of transcripts, SYBR Green based qPCR was performed with SensiFast SYBR NO-Rox Kit  using a Rotor-Gene Q thermocycler  under the following conditions: 95°C, 5 seconds for denaturation, 55°C, and 20 seconds for annealing and extension. The following gene specific primer sets were used: t method .Drosophila diets  were purchased from Hansol Tech Inc. . Propionic acid  and paraquat  were obtained from Junsei Chemical Co. Ltd.  and Sigma-Aldrich , respectively.t-test and ANOVA with Bonferroni post hoc test were used for the statistical comparison between two independent groups and more than two independent groups, respectively.Log-rank test was used for testing significance between survivorship curves. Unpaired two-tailed Student'  Drosophila Locomotor Activity Monitor and their spontaneous activities were measured in 10-minute bins for 24 hours. As reported, DR drastically increased flies' total locomotor activity , phospholipid hydroperoxide glutathione peroxidase (PHGPx), and catalase  in fly's hemolymph (unpublished data), which may alter the excitability of the contractile unit composed of muscular cells and neurons. In agreement with our preliminary observations, low calorie diet has been reported to increase neurotransmission at neuromuscular junction, which may contribute to enhanced motor function [Another intriguing question is, what is the driving force that keeps the high level of locomotor activity despite the DR-induced reduction in total calorie intake? In this regard, Katewa et al.  recentlyfunction . FurtherFrom clinical viewpoint, the importance of maintaining a higher level of locomotor activity for improved stress resistance should be mentioned. Accumulated body of literature unambiguously concludes that sarcopenia is closely related to increased morbidity and mortality both in healthy individuals and in patients . This imFinally, oxidative stress has been regarded as the main culprit that accelerates aging. Apart from directly causing damage to macromolecules such as nucleic acids, proteins, and lipids, ROS can initiate cellular signaling pathways that contribute to disease. For instance, growth factors trigger cellular proliferative cascades via mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K) pathways, both of which are further activated by ROS, resulting in sustained growth signals. This effect is postulated to underlie aberrant proliferation in tumorigenesis . HoweverIn conclusion, we anticipate that our study would provide mechanistic insight into the DR effects that would help develop practical interventions to improve the senile quality of life by mimicking the DR-induced cellular changes.Dietary restriction (DR) strengthens the organism against environmental stresses through poorly characterized mechanisms. In our study, DR-enhanced locomotor activity was experimentally reduced by wing clipping. This manipulation did not affect the pattern of food intake, which was indirectly measured by weight gain. However, attenuation of the DR-enhanced locomotor activity decreased the flies' resistance against environmental stresses. Furthermore, we found that the level of antioxidant gene transcripts was significantly reduced in wing-clipped flies reared on DR. Therefore, we suggest that upregulation of the ROS scavenger system by a DR-induced increase in locomotor activity may serve as a molecular basis to exert the beneficial effect of DR.


De novo genome assembly can be challenging due to inherent properties of the reads, even when using current state-of-the-art assembly tools based on de Bruijn graphs. Often users are not bio-informaticians and, in a black box approach, utilise assembly parameters such as contig length and N50 to generate whole genome sequences, potentially resulting in mis-assemblies.k-mer approaches of SPAdes and IDBA. We demonstrate that whole genome mapping can be used to identify these mis-assemblies and can guide the selection of the best k-mer size which yields the highest N50 without mis-assemblies.Utilising several assembly tools based on de Bruijn graphs like Velvet, SPAdes and IDBA, we demonstrate that at the optimal N50, mis-assemblies do occur, even when using the multi-k-mer selection and higher quality de novo genome assembly of bacterial genomes.We demonstrate the utility of whole genome mapping (WGM) as a tool to identify mis-assemblies and to guide  Such assembly tools based on de Bruijn graph algorithms, like Velvet [k-mers as building blocks, but as most users are not bio-informaticians, these tools are often considered as an encrypted black box with the quality of the assembly usually determined by statistics parameters such as the N50 and the size and number of contigs or scaffolds produced by the assemblers [k-mer size is crucial as too low or too high k-mer sizes lead to sub-optimal assemblies. Indeed, low quality reads might produce false positive vertices, repeats lead to branching, while an uneven distribution of the reads results in gaps. The use of smaller k-mers reduces the problem associated with low quality reads and their uneven distribution, while larger k-mer sizes help to bridge repeat regions decreasing the branching problem [k-mer sizes are usually selected, evaluating optimization by aiming for high N50 values and long, but fewer contigs. Whole Genome Mapping  is a relatively novel technique that generates high-resolution restriction maps of a genome based on the alignment of single DNA molecules cut with restriction enzymes and ordered with high resolution and accuracy [de novo assembled contigs against previously sequenced related genomes [Genome assembly is often a primary step in the process of yielding results that lead to interpretation of biological data and hence sub-optimally assembled genomes might lead to faulty conclusions . Factorse Velvet  and SPAde Velvet  use k-mesemblers . However problem . In a baaccuracy . WGM was genomes .k-mer size selection and for optimization of parameters for de novo genome assembly. The whole genome sequence of a methicillin-resistant Staphylococcus aureus (MRSA) strain (E-MRSA15-CC22-SCCmecIV) was generated on an Illumina HiSeq-2000 via 2X150b paired end sequencing [de novo assembled using Velvet, SPAdes and IDBA-UD employing a range of k-mers. Velvet, using a single k-mer approach ranging from k-mer size 81 to 123 showed an initial increase in N50 (until k-mer size 115) and longest contig size and a concomitant decrease in the total number of contigs with increasing k-mer size with both these parameters positively influencing the assembly outcome  was identified for the mapped contigs (>40 kb) for higher k-mer sizes  and Mu50-CC5-SCCmecII . In addition, the sequence of Klebsiella pneumoniae ST258 was similarly generated using Illumina HiSeq-2000 via 2X150b paired end sequencing and was assembled using all three assembly tools. In this case, apart from mis-assemblies seen for Velvet, also SPAdes and IDBA were shown to produce mis-assemblies for certain k-mer sizes  strain E-MRSA15-CC22-SCCmecIV and assembly statistics of Velvet for Klebsiella pneumoniae showing an initial increase in contig size and N50 when using higher k-mer sizes, but revealing mis-assemblies associated with higher k-mer sizes in some cases.Assembly statistics of Velvet, SPAdes and IDBA-UD applied on Click here for file


Roux-en-Y gastric bypass (RYGB) surgery is a very effective bariatric procedure to achieve significant and sustained weight loss, yet little is known about the procedure’s impact on the brain. This study examined the effects of RYGB on the brain’s response to the anticipation of highly palatable versus regular food.High fat diet-induced obese rats underwent RYGB or sham operation and were then tested for conditioned place preference (CPP) for the bacon-paired chamber, relative to the chow-paired chamber. After CPP, animals were placed in either chamber without the food stimulus, and brain-glucose metabolism (BGluM) was measured using positron emission tomography (μPET).Bacon CPP was only observed in RYGB rats that had stable weight loss following surgery. BGluM assessment revealed that RYGB selectively activated regions of the right and midline cerebellum (Lob 8) involved in subjective processes related to reward or expectation. Also, bacon anticipation led to significant activation in the medial parabrachial nuclei (important in gustatory processing) and dorsomedial tegmental area  in RYGB rats; and activation in the retrosplenial cortex (default mode network), and the primary visual cortex in control rats.RYGB alters brain activity in areas involved in reward expectation and sensory (taste) processing when anticipating a palatable fatty food. Thus, RYGB may lead to changes in brain activity in regions that process reward and taste-related behaviors. Specific cerebellar regions with altered metabolism following RYGB may help identify novel therapeutic targets for treatment of obesity. Obesity is a behavioral and metabolic disorder with devastating health consequences . More thObesity is associated with impulsivity and impaired self-control for consumption of palatable foods. These behaviors are also implicated in the over-consumption of drugs and alcohol. The brain’s reward circuitry is involved in regulating normal and pathological eating and its dysfunction could contribute to obesity ,5. Thus,The dopamine (DA) pathway plays an important role in reward, expectation of reward, and conditioning. Neurons originating in the ventral tegmental area (VTA) project to the nucleus accumbens (NAcc), prefrontal cortex (PFC), amygdala and hippocampus. In rodents, expectations for feeding and drinking are associated with accumbal DA increase ,7. In faHere we examined the relationship between brain activity and food-reward anticipation, and how this relationship is impacted by Roux-en Y gastric bypass surgery (RYGB), following a chronic high-fat diet. Using a conditioned-place preference (CPP) procedure, we paired a familiar, high-fat food (bacon) with salient contextual cues (chamber attributes such as color and flooring). After the conditioning phase, animals were tested for CPP. Following this test, each animal’s brain-glucose metabolism (BGluM) was measured while exposed to the chamber without food (cues without stimulus). Scans were performed twice per animal, once in each chamber in counterbalanced order. Thereby, BGluM measurements captured each animal’s anticipation for food while exposed to each food-paired environment. Since bacon is a highly palatable food, a positive correlation between BGluM and preference for the bacon-paired chamber was expected, especially with regions associated with hunger and reward, notably the hypothalamus (homeostatic consumption) and NAcc (non-homeostatic consumption), respectively. We also hypothesized that RYGB influenced regional brain activation to food cues. Decreased food consumption following RYGB has been suggested to be in response to reduced reactivity to food cues –26. Howead-libitum high-fat (HF) group (Sham-AL), 12 were assigned to the pair-fed HF group (Sham-PF), and 9 were assigned to the ad-libitum normal diet group (Sham-ND). The ad-libitum group had unrestricted access to food and water. The pair-fed group had unlimited access to water, but their food was limited to the average intake of the RYGB group 24-hours earlier. At seven weeks of age, the animals’ body-weights were measured daily (1200-1400h), along with food and water intakes. The pair-fed group received 75% of their daily caloric intake (900-1100h) in the morning and the remaining 25% (1700-1900h) in the evening. The Sham-ND group received standard rat chow . All other animals  were given a HF-enriched diet starting at 6 weeks of age  . Animals were originally on 12-hour light cycle , then one week prior to the start of the experiment, animals were switched to a reverse 12-hour light cycle . This was done so that all testing and handling were performed during the dark cycle when rodents are typically awake rather than the light cycle when they typically sleep. One week after light dark reversal was previously shown to be enough to restore baseline levels of locomotor activity as well as heart rate and blood pressure . This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Brookhaven National Lab (#: 212). All surgery was performed under ketamine xylazine anesthesia, and all efforts were made to minimize suffering.Fifty-eight Sprague-Dawley male rats  were used. Twenty-six rats underwent RYGB surgery or Sham surgery  at 17 weeks of age. Eleven of the control rats were assigned to the All surgical procedures (sham or RYGB) were performed at Pennsylvania State University as previously described ; S1 Mate. BrieflyD-glucose (FDG) during PET scans (details below), animals were injected with saline immediately prior to each conditioning session. Test days 1 and 2 lasted 15 minutes, and during these sessions, animals had unrestricted access to the two chambers and no food stimulus was provided (cues only).Beginning at 20-week age , animals were conditioned in a three-chamber CPP apparatus . Sc. Sc30]. During the FDG uptake period, rats were placed in either the bacon- or chow-paired chamber immediately after the injection . The placement was randomized so that half of the rats from each group were placed first in the bacon-paired chamber and then in the chow-paired chamber and the other half vice-versa. This paradigm was designed to mimic the conditioning session of the CPP procedure with the exception that chow or bacon was not administered.Images were spatially normalized and co-registered to a magnetic resonance image (MRI) atlas  using th3 = 16.78, p< 0.001; p <0.05). There were no differences amongst the three HF groups. After surgery, immediate weight decreases were observed in all groups, with the greatest decreases observed in the RYGB group . The RYGB animals with the greatest weight-loss stability  likely responded best to the surgery, compared to the remaining 75% of this group . In fact, weight-loss differed significantly between these two sub-groups .The health of the RYGB animals was variable , as were their weights and survivals. To control for the effects of RYGB on health and weight loss, animals were objectively stratified into sub-groups that were analyzed separately. To accomplish stratification, percent weight loss was calculated relative to each subject’s pre-operative weight. These values, which assessed the stability of each subject’s body weight, varied within the RYGB animals (range: 11.3–48.9%) greatly exceeding the Sham-AL animals , yet across the five groups, body weights differed significantly . Based on pair-wise comparisons (Holm-Sidak method), the body weight of the Q2/3/4 group was significantly less than for Sham-PF ,-AL ,-ND  groups and the Q1 group .Intra-group body weight was stable the week before and during CPP . Pair-wise comparisons (Holm-Sidak method) revealed that Q2/3/4’s intake was greater than that of Sham-AL ,-PF  and-ND groups . Intake did not differ from that of Q1’s . As a consequence, Sham-PF did not differ from Sham–AL.Food intake was temporally stable during the two CPP weeks . Holm-Sidak post-hoc tests revealed that Q2/3/4’s water consumption was significantly greater than Q1 , Sham-AL  and-PF  groups, while all other comparisons were not significant .Intra-group water intake was also temporally stable during the CPP experiment . Post-hoc comparisons (Holm-Sidak method) revealed differences only in Q1 group, who showed significantly greater CPP after conditioning , we compared CPP between test-sessions and across groups. Bacon-cue preference was significantly different between pre- and post-conditioning trials , group  and the interaction ; There was a trend for increased bacon consumption throughout conditioning relative to pre-conditioning (Days -2 and -1), which was driven by the Sham-ND group. For cumulative food intake throughout conditioning, there were significant effects for food , 7 animals were excluded . SPM analysis revealed significant differences for the contrast bacon > chow for RYGB and AL (high-fat) groups  but no dAs expected, RYGB animals lost weight, while sham-operated HF-fed animals continued to gain weight ,34. HoweOnly the healthy (Q1) RYGB group showed increased preference for the bacon-paired chamber . This suPrevious sham-controlled studies reported that RYGB decreased preference for high concentrations of sucrose but increased preference for lower concentrations ,37. LiteRYGB may affect both taste preferences and actual preferential intake of different macronutrients, and it is important to distinguish between an increase in liking or disliking a taste despite the absence in increases in intake, due to the restrictive nature of the RYGB . In obesPrior studies reported that animals fed only normal chow ignore high-fat diets . We showIn this study, the RYGB rats with the least weight-loss  responded to the surgery with the least weight loss compared to the remaining 75% of rats in this group. Following RYGB, whereas most patients lose a substantial amount of 60–70%) excess body weight and voluntarily restrict consumption of calorie-dense, highly palatable foods, and report fewer cravings ,46–49; a% excess Bacon anticipation activated distinct parts of the cerebellum in both RYGB and AL rats. The cerebellum is involved with reward-based reversal learning  expectedThe cerebellum receives dopaminergic, serotonergic, noradrenergic, and cholinergic inputs that perform functional modulation of the brain . SensoriIn addition, bacon anticipation in RYGB rats also activated an area approximating the medial parabrachial nuclei (MPB) and the dorsomedial tegmental area (DMTg). The MPB is involved in sensory representation related to gustatory processing ,94 whereThese findings may hold important clinical implications. In fact, these results, together with recent reports of increased susceptibility to non-food rewards, such as increased use of alcohol ,75 suggeIn summary, RYGB alters reactivity of areas involved in reward expectation and sensory processing when subjects anticipate a fatty highly palatable food such as bacon. Moreover cerebellum response to food cues in the RYGB rats predicted their CPP responses; identifying the cerebellum as a region that may mediate some of the changes in eating behaviors that follow RYGB. Overall, these observations suggest that despite reduced food intake and improved food choices after RYGB, brain areas that are involved in reward anticipatory functions may be more active.S1 Materials(DOCX)Click here for additional data file.


A vaccine against malaria is urgently needed for control and eventual eradication. Different approaches are pursued to induce either sterile immunity directed against pre-erythrocytic parasites or to mimic naturally acquired immunity by controlling blood-stage parasite densities and disease severity. Pre-erythrocytic and blood-stage malaria vaccines are often seen as opposing tactics, but it is likely that they have to be combined into a multi-stage malaria vaccine to be optimally safe and effective.Since many antigenic targets are shared between liver- and blood-stage parasites, malaria vaccines have the potential to elicit cross-stage protection with immune mechanisms against both stages complementing and enhancing each other. Here we discuss evidence from pre-erythrocytic and blood-stage subunit and whole parasite vaccination approaches that show that protection against malaria is not necessarily stage-specific. Parasites arresting at late liver-stages especially, can induce powerful blood-stage immunity, and similarly exposure to blood-stage parasites can afford pre-erythrocytic immunity.The incorporation of a blood-stage component into a multi-stage malaria vaccine would hence not only combat breakthrough infections in the blood should the pre-erythrocytic component fail to induce sterile protection, but would also actively enhance the pre-erythrocytic potency of this vaccine. We therefore advocate that future studies should concentrate on the identification of cross-stage protective malaria antigens, which can empower multi-stage malaria vaccine development. In malaria endemic areas, immunity against severe disease caused by blood-stage parasites can be acquired after only one or two infections, while infections with high parasite densities still occur There is only very limited evidence for protection against pre-erythrocytic malaria parasites (sporozoites and liver-stage parasites) in naturally exposed populations 2Given that there are shared antigens between the different life cycle stages of the malaria parasite 3Plasmodium berghei irradiated sporozoites they were exposed to blood-stage parasites P. berghei infection model in mice, multiple booster immunizations with high numbers of irradiated sporozoites resulted in delayed patency and reduced peak blood-stage parasitemia after sporozoite challenge , suggesting that cross-stage protective responses targeting blood-stage parasites may have developed. It has however also been observed that a fulminant blood-stage infection can suppress protective immune responses elicited by irradiated sporozoites against liver-stage antigens The proteome of liver-stage parasites becomes increasingly similar to blood-stage parasites as liver development proceeds uis 3 uis 4 p36psap-1p52/p36P. yoelii fabb/f knockout sporozoites can control and clear blood-stage parasitemia following challenge with blood-stage parasites, possibly by inducing an effective T cell response Targeted deletion of parasite genes important for liver-stage development is an alternative strategy to arrest parasite development in hepatocytes. Similar to irradiated and chemically attenuated Cross-stage immunity therefore appears to be more efficient if liver-stage parasites arrest late in development as their antigenic profile becomes similar to blood-stage parasites 4Plasmodium replicates massively in the liver such that one infected hepatocyte can release up to up to 40,000 blood-stage parasites purine nucleoside phoshphorylase gene (pnp) nucleoside transporter 1 (nt1) gene P. yoelii gives rise to severely attenuated blood-stage infections, and mice that had undergone an infection with these knockout parasites did not develop detectable patent blood-stage parasitemia after infectious mosquito bite or sporozoite challenge P. yoelii blood-stage parasites. Liver parasite burden is significantly reduced in these mice following sporozoite challenge Disruption of the It is presumed that the main purpose of blood-stage components in a multi-stage vaccine is to protect against breakthrough blood-stage infection 5Plasmodium life cycle stages including sporozoites, infected hepatocytes and blood-stage parasites. CPS immunization was first described in rodents P. chabaudi blood-stage parasites derived from a donor mouse infected by mosquito bite have a substantially reduced liver-parasite burden following mosquito bite challenge The induction of cross-stage immunity should be facilitated by exposure to both pre-erythrocytic and blood-stage parasites during immunization. Chemoprophylaxis with sporozoites (CPS) immunization, which uses infectious wild-type sporozoites combined with prophylactic antimalarial drug treatment (often chloroquine), allows the immune system to experience all vertebrate 6Plasmodium matures in the liver, it is potentially capable of inducing immunity not only against pre-erythrocytic but also against blood-stage parasites Cross-stage immunity would be a powerful means to improve protective efficacy of malaria vaccines, however data to support this hypothesis are sparse. During liver-stage development both the amount of antigen and the antigenic similarity to blood-stage parasites increases . Thus asFuture studies should therefore investigate the potential of cross-stage immunity directly. In our opinion whole parasite immunization approaches are particularly useful to answer the question whether cross-stage immunity contributes to protection and to identify protective cross-stage antigens for subunit malaria vaccine development. If however a whole parasite immunization strategy were proven to be safe, effective and practical first, there would be no reason not to implement it. Pre-erythrocytic vaccines should evaluate blood-stage protection after direct injection of blood-stage parasites, while blood-stage vaccines should demonstrate whether liver-parasite burden is reduced following sporozoite challenge. If cross-stage immune responses can be elicited, target antigens and crucial immunological mechanisms mediating it should be characterized using proteome-wide screening approaches (immunomics) of antibody and T cell reactivity comparing protected and unprotected individuals The authors declare no conflict of interest.


Tremelimumab is a selective human IgG2 mAb inhibitor of CTLA-4 which promotes T-cell activity through CTLA-4 inhibition. Both durvalumab and tremelimumab have demonstrated acceptable safety profiles and promising clinical activity as single agents in multiple tumor types. Recent data suggest that combined checkpoint inhibition may generate superior antitumor activity versus monotherapy, resulting in higher, deeper, and more durable response rates, which suggests that dual inhibition may overcome barriers to immune response in historically non-immune responsive tumors. We describe an ongoing study assessing the safety and antitumor activity of durvalumab in combination with tremelimumab in patients with advanced solid tumors.The immune system is able to control the growth of many types of cancers. Most tumors show infiltration by tumor infiltrating lymphocytes, but tumors modulate the local microenvironment by expressing immune-inhibitory molecules. Blockade of immune checkpoints such as cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), programmed cell death-1 (PD-1), and programmed cell death ligand-1 (PD-L1) has shown clinical activity in immune-responsive tumors as well as those historically deemed unresponsive to treatment. Durvalumab (MEDI4736) is a selective, high affinity human IgG1 monoclonal antibody (mAb) that blocks PD-L1 binding to PD-1 . Eligible patients (≥18 years) will have recurrent or metastatic (R/M) disease and may have been previously treated in the R/M setting. The primary objective of this study is to assess the safety and tolerability, and maximum tolerated dose of durvalumab in combination with tremelimumab. The secondary objectives are to assess antitumor activity , pharmacokinetics, pharmacodynamics, and immunogenicity of the combination. Exploratory objectives will include an assessment of biomarkers that may correlate with clinical activity of durvalumab in combination with tremelimumab. Recruitment is ongoing, with an estimated enrollment of 225–240 patients.ClinicalTrials.gov identifier NCT02261220.


Like plants, the mammalian LCO biological activity depended upon the presence and type of terminal substitutions. Un-substituted oligo-chitins of similar chain lengths were unable to modulate angiogenesis indicating that mammalian cells, like plant cells, can distinguish between LCOs and un-substituted oligo-chitins. The cellular mode-of-action of the biologically active LCOs in mammals was determined. The stimulation or inhibition of endothelial cell adhesion to vitronectin or fibronectin correlated with their pro- or anti-angiogenic activity. Importantly, novel and more easily synthesised LCO-like disaccharide molecules were also biologically active and de-acetylated chitobiose was shown to be the primary structural basis of recognition. Given this, simpler chitin disaccharides derivatives based on the structure of biologically active LCOs were synthesised and purified and these showed biological activity in mammalian cells. Since important chronic disease states are linked to either insufficient or excessive angiogenesis, LCO and LCO-like molecules may have the potential to be a new, carbohydrate-based class of therapeutics for modulating angiogenesis.Lipochitin oligosaccharides (LCOs) are signaling molecules required by ecologically and agronomically important bacteria and fungi to establish symbioses with diverse land plants. In plants, oligo-chitins and LCOs can differentially interact with different lysin motif (LysM) receptors and affect innate immunity responses or symbiosis-related pathways. In animals, oligo-chitins also induce innate immunity and other physiological responses but LCO recognition has not been demonstrated. Here LCO and LCO-like compounds are shown to be biologically active in mammals in a structure dependent way through the modulation of angiogenesis, a tightly-regulated process involving the induction and growth of new blood vessels from existing vessels. The testing of 24 LCO, LCO-like or oligo-chitin compounds resulted in structure-dependent effects on angiogenesis  These naturally occurring molecules act as microbial signals that are required to establish nitrogen-fixing nodule formation and mycorrhization respectively. These symbiotic interactions are of paramount ecological and agronomic importance and contribute a significant proportion of biologically-available nitrogen and phosphorous to agronomic systems respectively N-acetylglucosamine residues. In addition, they have an N-linked acyl substitution at the non-reducing end and can often have modifications to the C6 position of the reducing end N-acetly glucosamine residue. Rhizobia synthesise LCOs by first making a de-acetylated chitin-oligomer by the combined action of an oligo-chitin synthase (NODC) and an LCO de-acetylase (NODB), which removes an acetyl group from the non-reducing end residue N-linked fatty acids (C16–C18) and reducing-end, O-linked sugars or sulphate e.g., LYK3 and LYK4) preferentially recognise chitin oligomers as microbe-associated molecular patterns (MAMPs) and this induces an innate, ‘general-immune’ response Rhizobial and fungal LCOs comprise of three to five β-1,4-linked e.g., DG42 e.g., GH18) de novo, but chitooligosacharides also appear to play a role in their early development Although LCO and, in particular, chitin-like molecules are widespread in nature, mammals are not known to make chitins or chitin-like molecules although they encode chitin synthase genes  and has essential roles in development, reproduction and repair. In addition, a plethora of important diseases are associated with excessive or insufficient angiogenesis including solid tumour formation, age-related macular degeneration, diabetic retinopathy, atherosclerosis, peptic ulcers, rheumatoid arthritis, hypertension and ischemic conditions in vivo angiogenesis, the ECs that form new blood vessels from existing vessels re-activate, migrate, proliferate and reorganise into tubes. Several biological assays have been developed to assess candidate angiogenesis therapeutics ex vivo it is considered to more closely mimic in vivo conditions and it has been used to successfully identify the anti-angiogenic therapeutic, Muparfostat (PI-88), which is already in phase III cancer trials in vitro assays examine the individual component steps of angiogenesis and are used to define the cellular mode-of-action of a particular compound over a shorter time frame During ex vivo rat aorta ring angiogenesis assay was used to assess this possibility. Using this assay, we discovered that certain naturally-occurring LCOs, but not chitin oligomers, affected angiogenesis in a structure-dependent manner in vitro. Therefore, we synthesised or assessed a broader range of naturally occurring LCOs or ‘LCO-like’-molecules for angiogenesis-regulating activity to determine the structural requirement for this activity. Structure-activity assays identified the molecular constraints for LCO- or LCO-like molecules to have effects on angiogenesis. The cellular mode-of-action was determined using in vitro biological assays that tested individual cellular components of angiogenesis . Finally, since a de-acetylated chitobiose (but not chitobiose) was found to be biologically active, a synthesis was devised to generate novel derivatives of this disaccharide that have LCO-like substitutions as a route to develop a pipeline of potential therapeutics. The results showed that these disaccharides may provide cheaper and more-readily-synthesised candidates for future angiogenesis therapeutics based on the structures of LCOs.Since plants and animals share the capacity to recognise chitin oligomers as MAMPs The experiments conducted in this manuscript were performed under the Australian National University Animal Experimentation Ethics Committee stipulation A2011/35. The Institutional ethics committee specifically approved this study.Aortic segments  were cultured in a gel-fibrin matrix using six replicates per treatment 4 cells/well) were added to 48-well plates in 300 µl/well of M199 medium, supplemented with 20% HIFBS, 0.24 µg/ml gentamycin, 2 mM L-glutamine, 0.04 mg/ml ECGS  and 0.135 mg/ml heparin and incubated at 37°C in 5% CO2 until confluent. The HUVEC monolayers were wounded, rinsed twice with 300 µl/well of culture medium before 300 µl/well of medium was added with, or without, the test compound. Wound recovery was photographed several times daily using an Olympus IX81 CellR microscope and quantified using NIH Image J software. Compounds were tested at 25 µg/ml in these in vitro assays.Human microvessel endothelial cells (HMECs) and human umbilical vein endothelial cells (HUVECs) were assessed for enhanced or inhibited attachment to fibronectin or vitronectin using the Rose Bengal assay E. coliLCO and LCO-like compounds were generated in transgenic 29   anoside) . SelectiP values less than 0.05 were considered statistically significant.Data and results are reported as means ± SE. Statistical significance was measured using two tailed t-test for in vitro for pro- or anti-angiogenic effects on microvessel growth from rat aorta segments Escherichia coli harbouring rhizobial LCO genes  Medicago (compound 24). The artificial LCO-like molecules used included de-acetylated chitin pentasaccharide derivatives each with non-naturally occurring m-benzamide- or phenyl-acetyl-substitutions to the non-reducing terminus Twenty-four LCO, LCO-like or chitin oligomer molecules were synthesised or obtained and quantitatively and qualitatively screened segments . The come.g., compound 1–2) N-linked non-reducing end acylation and a reducing end fucose or methyl-fucose. Compounds 1 and 2 were shown to possess anti-angiogenic activity akin to the control anti-angiogenic and cancer therapeutic compound Muparfostat  devoid of the N-acetyl group on the non-reducing terminal residue. NODZ, which is encoded by a transferase gene N-acetylglucosamine  were chemically synthesised . Compounds 14–16 contained synthetic lipophilic side chains designed to impart a longer retention time in vivo than that expected for compound 11. As expected, compounds 14 and 15 were strongly anti-angiogenic  components Click here for additional data file.


Helicobacter hepaticus at three-months-of-age putting them at increased risk for mammary tumor development. Tumorigenesis was multifocal and characterized by extensive infiltrates of myeloperoxidase-positive neutrophils otherwise implicated in cancer progression in humans and animal models. To test whether neutrophils were important in etiopathogenesis in this bacteria-triggered model system, we next systemically depleted mice of neutrophils using thrice weekly intraperitoneal injections with anti-Ly-6G antibody. We found that antibody depletion entirely inhibited tumor development in this H. hepaticus-infected model. These data demonstrate that host neutrophil-associated immune responses to intestinal tract microbes significantly impact cancer progression in distal tissues such as mammary glands, and identify gut microbes as novel targets for extra-intestinal cancer therapy.Recent studies suggest that gastrointestinal tract microbiota modulate cancer development in distant non-intestinal tissues. Here we tested mechanistic hypotheses using a targeted pathogenic gut microbial infection animal model with a predilection to breast cancer. FVB-Tg(C3-1-TAg)cJeg/JegJ female mice were infected by gastric gavage with  Helicobacter hepaticus, a murine bacterium frequently colonizing the gut of mice  mice [H. hepaticus in the FVB-Tg(C3-1-TAg)cJeg/JegJ mouse model [H. hepaticus , with H. hepaticus-infected mice having a higher percentage of abnormal glands with mammary intraepithelial neoplasia (MIN) when compared to controls  were previously shown to enhance tumorigenesis in mouse models [H. hepaticus-infected and uninfected mice. We found that the H. hepaticus infection status correlated with higher numbers of MIN-associated MPO-positive cells in statistically significant levels (P = 0.002)  Figure .H. hepaticus-induced inflammation [H. hepaticus-infected mice had multifocal well-sized mammary adenocarcinoma tumors , H. hepaticus-infected mice that were treated with anti-Ly6G antibody had no evidence of mammary adenocarcinoma tumors   in three mice , hyperplasia with marked atypia in two , hyperplasia with atypia, intermediate grade in one  hyperplasia without atypia, low grade in three mice .Finally, based upon earlier findings showing that neutrophils are a consistent feature of ammation  and cancammation , 11, 20,ammation , 37, 38,ammation . While H) Figure . The neu) Figure . SpecifiIn the present study we use the well-characterized C3-1-TAg mouse model of mammary cancer to show that bacteria residing in the gut mucosa affect the evolution of neoplastic lesions in mammary glands. We find here that the neutrophil is an important cellular element of this mechanism, which links innate immune and neoplastic events occurring in topographically distant epithelia. This highlights the neutrophil as an important mediator of the remote effect of gut microbiota on mammary epithelial carcinogenesis. Further, these findings build upon the pivotal role of systemic immune balance in emerging pre-neoplastic lesions throughout the body not obviously linked with chronic inflammation.H. hepaticus-infected C57BL/6 ApcMin Rag2-deficient female mice develop mammary tumors with increased frequency [H. hepaticus develop prostate tumors transplantable to uninfected mice using purified lymph node cells obtained from H. hepaticus-infected mice alone [adenomatosis polyposis coli gene status [H. hepaticus infection per se or to microbe-increased multiplicity of intestinal polyps. Taken together, these facts raise doubts about roles of gut microbiota in mammary epithelial carcinogenesis beyond ApcMin mice. The results of the present paper contribute towards showing that H. hepaticus accelerates mammary carcinogenesis in other animal systems, in this case the C3-1-TAg female mouse that is a widely utilized mouse model for hormonally-dependent cancer [Previously we have shown that ce alone , implicace alone . Howevere status  and accee status . Given me status , it is dt cancer . Thus thFurther, these data provide additional evidence that breast cancer is associated with microbial dysbiosis in the gut. Altering gut microbes can regulate the immune system and lower the risk of breast cancer; in particular, overgrowth or lack of certain types of bacteria in the gut have been associated with many diseases ranging from obesity, to digestive disorders, to cancers . DiffereAPC gene, which is central in the β-catenin/WNT pathway of carcinogenesis in human breast cancer is emerging [APC mutant mice nor humans carrying germline mutations in APC develop spontaneously mammary cancer in a high percentage [Interestingly, our current results suggest that the remote effect of gut microbiota in mammary carcinogenesis does not specifically depend on the molecular pathway of mammary carcinogenesis involved. C3-1-TAg mice develop mammary cancer due to hormonally-dependent functional inactivation of the P53 and Rb tumor suppressor genes, which leads mammary epithelia to cell cycle regulation defects, uncontrolled proliferation and resistance to apoptosis . The inaemerging , neitherrcentage , 63. By rcentage . This faH. hepaticus on mammary carcinogenesis does not connect specifically with the histological type of the mammary tumor. Previously we found H. hepaticus promotes formation of mammary adenosquamous carcinoma in ApcMin mice [Our compiled results also show that the effect of intestinal Min mice . Here, wMin mice , 64, 65.Importantly, the C3-1-TAg mice on a FVB background develop neither IBD nor intestinal tumors . This suH. hepaticus is not unique among gut bacteria exerting effects on mammary gland tissue. Our other recent findings show that the probiotic bacterium Lactobacillus reuteri, when introduced in the GI tract flora of mice, has the opposite effect of suppressing mammary tumor formation in genetically-susceptible HER2 mutant mice [H. hepaticus or suppression of mammary tumorigenesis by probiotic L. reuteri, we discover differences in the immune cell composition of the mammary gland microenvironment. An interesting aspect is that these gut-mammary connections may be covert; even in the absence of demonstrable inflammatory disease in the gut or elsewhere, we find that H. hepaticus-infected mice have not only higher risk for cancer but also more frequent inflammatory cells associating with mammary glands at early neoplastic stages.It appears that ant mice . In bothH. hepaticus-infected mice when compared to their uninfected controls. Neutrophils are a consistent feature of H. hepaticus-induced inflammation [In the present study we found that neutrophils associate with mammary lesions in higher numbers in the ammation  and cancammation , 11, 20.ammation . Studiesammation . Also, tammation , pancreaammation , colonicammation .H. hepaticus infection [H. hepaticus-infected C3-1-TAg mice depleted of Ly-6G+ neutrophils had significantly fewer mammary lesions, and in those cases only preneoplastic and early neoplastic lesions, in contrast to their non-depleted matching controls which exhibited palpable mammary tumors. The dependence of mammary tumorigenesis upon the presence of neutrophils highlights the neutrophil as an important mediator of the remote effect of gut microbiota on mammary epithelial carcinogenesis. Adoptive transfer experiments [Taken together with our earlier data these facts led us to test whether the depletion of neutrophils could affect mammary carcinogenesis in this mouse model, as well. Recognizing the etiopathogenic potential of myeloid immune precursor trafficking in lymph nodes, spleen, and target tissues after nfection , it's ponfection . We founeriments  using hiIn conclusion, the results of the present study indicate that gut microbiota bacterial elements, in the absence of overt inflammatory disease, contribute to the character of the subclinical, systemic inflammatory tone of the mammalian organism. Based on these findings it appears that systemic inflammatory tone, which is mediated, at least in part, by neutrophils, affects the evolution of preneoplastic lesions to cancer in epithelia locating distantly from the gut, such as those of the mammary glands.Helicobacter hepaticus infection by gastric gavage at the age of twelve weeks, and then monitor for three weeks until euthanasia using carbon dioxide overdose. Control mice of the same age underwent gastric gavage with media alone. Subsets of infected mice were injected intraperitoneally with anti-Ly-6G antibody to deplete neutrophils, or with sham isotype-matched antibody alone, thrice weekly for 3.5–4 weeks starting three days before infection. Each experimental group included 5–10 animals per group with one replicate experiment with statistically similar outcomes to validate results. Mammary tissues were collected upon necropsy and then examined histologically.Genetically-inbred FVB-Tg(C3-1-TAg)cJeg/JegJ female mice , were housed and handled in Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited facilities with diets, experimental methods, and housing as specifically approved by the Institutional Animal Care and Use Committee. Experimental design was to expose mice to H. hepaticus and then housed separately in a bio-containment area within the same animal facility. H. hepaticus  [H. hepaticus-specific primers to confirm bowel colonization.A total of 51 experimental mice were dosed at 12 weeks of age with H. hepaticus. Treatment with antibody continued concurrent with H. hepaticus infection for a duration of 3 weeks, with euthanasia occurring at age 15 weeks. Treated mice were then compared with mice receiving a comparable dose of sham isotype antibody alone. Depletion of neutrophils was confirmed by undetectably low levels of MPO+ cells in spleens of mice treated with anti-Ly-6G antibody compared to sham-treated controls.Mice were treated with anti-Ly6G antibody  at 200 ug per mouse intraperitoneally 3X weekly for 3–4 weeks starting 3 days before infection with U test was used for analyzing histomorphometry data. Mammary tumor multiplicity was evaluated using the Kruskal-Wallis analysis followed by the Dunn's multiple comparison test. Mammary gland lesion staging comparison between groups was done with the Chi-square test. Replicate experiments were not significantly different. A p-value < 0.05 was statistically significant.The Mann-Whitney For histologic evaluation, formalin-fixed tissues were embedded in paraffin, cut at 5 μm, and stained with hematoxylin and eosin. Hyperplastic and preneoplastic lesions in the mammary gland were staged based on previously published consensus criteria  as previH. hepaticus-infected and non-infected mice was determined in 50 abnormal mammary duct profiles from each group. For that, histological sections from non-tumoral areas of 5 randomly selected mice per experimental group were used. Starting from the upper left corner of each histological section and moving towards its lower right, the first 10 abnormal glands (hyperplastic or with early neoplasia) found were staged and the result was recorded.For quantitative histomorphometry, MPO-positive cells were counted in 20 randomly-selected images of x20 representative high power fields using the ImageJ processing and analysis program  as previously described  and resu


The requirement of IVIg-induced iTreg and their antigen-specificity in attenuation of AHR and airway inflammation remains unknown.IVIg is a polyclonal IgG preparation with potent immune-modulating properties. We demonstrated that IVIg protects against airway hyperreactivity (AHR) and airway inflammation in mouse models of allergic airway disease, accompanied by peripheral induction of Foxp3+Treg by the application of diphtheria toxin (DT). Mice were sensitized and challenged with ovalbumin (OVA) and treated with IVIg. AHR was measured using a FlexiVent small animal ventilator. Total and antigen-specific IgE, as well as pro-inflammatory cytokines levels were determined in serum and alveolar lavage, using ELISA.We utilized DEREG mice, carrying a transgenic diphtheria toxin receptor under the control of the Foxp3 promoter, allowing for selective depletion of Foxp3reg cells (nTreg) were depleted before but not following IVIg treatment demonstrated an induction of Foxp3+Treg to IVIg therapy and did not develop AHR and airway inflammation to allergen-challenge. Adoptive transfer of enriched IVIg-induced iTreg from OVA-IVIg treated mice failed to transfer protection to mice exposed to ragweed, but was protective in OVA-sensitized and challenged mice.In the absence of Treg, due to multiple DT doses before and after the treatment, IVIg was not able to attenuate AHR, diminish IgE levels and Th-2 type cytokine production, nor alleviate airway inflammation. However, mice in which the pre-established Treg can be induced from effector CD4+T-cells in the absence of nTreg. IVIg-induced antigen specific Treg are capable of suppressing all aspects of antigen-driven airway inflammation in an antigen-specific manner.T


Binge eating disorder (BED) is characterized by the ingestion of a large amount of food in a short period of time and the preferential choice of high palatable food. BED is the most common eating disorder, which is associated with other medical and psychiatric complications, most notably obesity and depression . The effWe adapted a recently developed training paradigm that effectively induces a sustained binge-like eating behavior in mice, which mimics many characteristics of binge eating in BED patients . We firsSelective serotonin reuptake inhibitors, which enhance brain 5-hydroxytryptamine (5-HT) signals, showed some anti-binge efficacy in clinical trials . These fGiven the fact that estrogen replacement therapy is associated with increased risk of breast cancer, our collaborator, Dr. DiMarchi from Indiana University, developed an estrogen-based conjugate named glucagon-like peptide-1-estrogen (GLP-1-estrogen). This conjugate uses GLP-1 as a “carrier” to deliver estrogens preferentially to GLP-1 receptor-enriched regions, which can produce profound body weight lowering effects but with minimal uptake by the breast tissue . We founFuture directions include delineating the intracellular mechanisms by which estrogen regulates 5-HT neurons, and identifying the neural circuits downstream of 5-HT neurons that are important for the regulation of binge-like eating behavior. We hope these efforts will identify more rational targets for the development of novel therapies that can treat BED and other related eating disorders.