Abstract: The accumulation of high levels of adenosine in tumors activates adenosine A2A and A2B receptors on immune cells to suppress tumor rejection We evaluated the effects of intratumor injection of a n

Abstract: Hyperimmune activation is a strong predictor of disease progression during pathogenic immunodeficiency virus infections and is mediated in part by sustained type I IFN signaling in response to adventitious microbial infection. The immune inhibitory receptor programmed death-1 (PD-1) regulates functional exhaustion of virus-specific CD8(+) T cells during chronic infections, and in vivo PD-1 blockade has been shown to improve viral control of SIV. Here, we show that PD-1 blockade during chronic SIV infection markedly reduced the expression of transcripts associated with type I IFN signaling in the blood and colorectal tissue of rhesus macaques (RMs). The effect of PD-1 blockade on type I IFN signaling was durable and persisted even under conditions of high viremia. Reduced type I IFN signaling was associated with enhanced expression of some of the junction-associated genes in colorectal tissue and with a profound decrease in plasma LPS levels, suggesting a possible repair of gut-associated junctions and decreased microbial translocation into the blood. PD-1 blockade enhanced immunity to gut-resident pathogenic bacteria, control of gut-associated opportunistic infections, and survival of SIV-infected RMs. Our results suggest PD-1 blockade as a potential novel therapeutic approach to enhance combination antiretroviral therapy by suppressing hyperimmune activation in HIV-infected individuals.

Abstract: Anti-angiogenic therapy is an anti-cancer strategy that targets the new vessels that grow to provide oxygen and nutrients to actively proliferating tumor cells. Most of the current anti-cancer reagents used in the clinical setting indiscriminately target all rapidly dividing cells, resulting in severe adverse effects such as immunosuppression, intestinal problems and hair loss. In comparison, anti-angiogenic reagents theoretically have fewer side effects because, except in the uterine endometrium, neoangiogenesis rarely occurs in healthy adults. Currently, the most established approach for limiting tumor angiogenesis is blockade of the vascular endothelial growth factor (VEGF) pathway. In line with the results of preclinical studies, significant therapeutic effects of VEGF blockers have been reported in various types of human cancers, even in patients with progressive/recurrent cancer who could not otherwise be treated. However, some patients are refractory to this treatment or acquire resistance to VEGF inhibitors. Moreover, several studies have shown that VEGF blockade damages healthy vessels and results in adverse effects such as hemorrhagic and thrombotic events. In recent research that indicated possible ways to overcome these problems, several VEGF-independent and tumor-selective pro-angiogenic mechanisms were discovered that could be targeted in combination with or without conventional VEGF blockade. These findings offer opportunities to greatly improve current anti-angiogenic treatment for cancer.

Abstract: Objectives To establish if changes in Th1/Th17 cell populations previously reported in experimental arthritis occur in patients with rheumatoid arthritis (RA) treated with anti-tumour necrosis factor α (TNFα) agents, and whether the therapeutic response to anti-TNFα is compromised in patients and mice because of elevated Th17/IL-17 levels Finally, to assess the efficacy of combined blockade of anti-TNFα and anti-IL-17 in experimental arthritis Methods A longitudinal study of two independent cohorts (cohort 1, n=24; cohort 2, n=19) of patients with RA treated with anti-TNFα biological agents was carried out to assess their Th17/IL-17 levels before and after the start of anti-TNFα therapy IL-12/23p40 production was assessed in plasma Peripheral blood lymphocytes (PBLs) and monocytes Mice with collagen-induced arthritis (CIA) were treated with anti-TNFα alone, anti-IL17 alone or a combination of the two Efficacy of treatment and response was assessed from changes in Disease Activity Score 28–erythrocyte sedimentation rate scores in patients, and in clinical scores and histological analysis in CIA Results Significant increases in circulating Th17 cells were observed in patients after anti-TNFα therapy and this was accompanied by increased production of IL-12/23p40 There was an inverse relationship between baseline Th17 levels and the subsequent response of patients with RA to anti-TNFα therapy In addition, PBLs from non-responder patients showed evidence of increased IL-17 production Similarly, in anti-TNFα-treated mice, there was a strong correlation between IL-17 production and clinical score Finally combined blockade of TNFα and IL-17 in CIA was more effective than monotherapy, particularly with respect to the duration of the therapeutic effect Conclusions These findings, which need to be confirmed in a larger cohort, suggest that a Th17-targeted therapeutic approach may be useful for anti-TNFα non-responder patients or as an adjunct to anti-TNFα therapy, provided that safety concerns can be addressed

Abstract: Deep neuromuscular blockade during certain surgical procedures may improve operating conditions. Sugammadex can be used to reverse deep neuromuscular blockade without waiting for spontaneous recovery. This randomised study compared recovery times from neuromuscular blockade induced by rocuronium 0.6 mg.kg(-1), using sugammadex 4 mg.kg(-1) administered at 1-2 post-tetanic count (deep blockade) or neostigmine 50 μg.kg(-1) (plus atropine 10 μg.kg(-1)) administered at the re-appearance of the second twitch of a train-of-four stimulation (moderate blockade), in patients undergoing laparoscopic surgery. The primary efficacy variable was the time from the start of sugammadex/neostigmine administration to recovery of the train-of-four ratio to 0.9. Patients receiving sugammadex recovered 3.4 times faster than patients receiving neostigmine (geometric mean (95% CI) recovery times of 2.4 (2.1-2.7) and 8.4 (7.2-9.8) min, respectively, p<0.0001). Moreover, 94% (62/66) of sugammadex-treated patients recovered within 5 min, vs 20% (13/65) of neostigmine-treated patients, despite the difference in the depth of neuromuscular blockade at the time of administration of both drugs. The ability to provide deep neuromuscular blockade throughout the procedure but still permit reversal at the end of surgery may enable improved surgical access and an enhanced visual field.

Abstract: Objective:To compare two drug regimens to treat resistant hypertension.Methods:In a prospective, randomized, open blinded endpoint study, 167 patients with mean baseline daytime ambulatory blood pressure 135 mmHg or more and/or 85 mmHg or more, despite 4 weeks’ treatment with irbesartan 300 mg/day,

Abstract: Pancreatic cancer is a largely incurable disease, and increasing evidence supports strategies targeting multiple molecular mediators of critical functions of pancreatic ductal adenocarcinoma cells. Intracellular redox state modulates the activity of various signal transduction pathways and biological processes, including cell survival, drug resistance and responsiveness to microenvironmental factors. Recently, it has been shown that the transcription factor STAT3 is under redox control, but the mechanisms involved in its regulation are unknown. Here, we demonstrate for the first time that STAT3 DNA binding and transcriptional activity is directly regulated by the redox function of the APE1/Ref-1 endonuclease, using overexpression and redox-specific mutational strategies, and gene knockdown. Also, pharmacological blockade of APE1/Ref-1 by the redox-selective inhibitor E3330 abrogates STAT3 DNA binding. Since APE1/Ref-1 also exerts redox control on other cancer-associated transcription factors, we assessed the impact of dual-targeting of STAT3 signaling and APE1/Ref-1 redox on pancreatic cancer cell functions. We observed that disruption of APE1/Ref-1 redox activity synergizes with STAT3 blockade to potently inhibit the proliferation and viability of human PDAC cells. Mechanistically, we show that STAT3–APE1/Ref-1 dual targeting promotes marked tumor cell apoptosis, with engagement of caspase-3 signaling, which are significantly increased in comparison to the effects triggered by single target blockade. Also, we show that STAT3–APE1/Ref-1 dual blockade results in significant inhibition of tumor cell migration. Overall, this work demonstrates that the transcriptional activity of STAT3 is directly regulated by the redox function of APE1/Ref-1, and that concurrent blockade of STAT3 and APE1/Ref-1 redox synergize effectively inhibit critical PDAC cell functions.

Abstract: Blockade of JAK2-mediated extrinsic survival signals restores sensitivity of CML cells to ABL inhibitors

Abstract: Angiogenesis is essential for the growth of primary tumors and for their metastasis. This process is induced by factors, such as vascular endothelial growth factors (VEGFs), that bind to transmembrane VEGF receptors (VEGFRs). VEGF-A is the primary factor involved with angiogenesis; it binds to both VEGFR-1 and VEGFR-2. The inhibition of angiogenesis by obstructing VEGF-A signaling has been investigated as a method to treat solid tumors, but the development of resistance to this blockade has complicated treatment. The major mechanisms of this resistance to VEGF-A blockade include signaling by redundant receptors, such as the fibroblast growth factors, angiopoietin-1, ephrins, and other forms of VEGF. Other major mechanisms of resistance are increased metastasis of hypoxia-resistant tumor cells, recruitment of cell types capable of promoting VEGF-independent angiogenesis, and increased circulation of nontumor proangiogenic factors. Additional mechanisms of resistance to VEGF-A blockade include heterogeneity of responsiveness among tumor cells, use of anti-VEGF-A agents at insufficient doses or for insufficient duration, altered sensitivity to anti-VEGF-A agents by mutations in endothelial cells or vascular remodeling, maintenance of vascular sleeves that allow for easy regrowth of tumor vasculature upon discontinuation of therapy, vascular cooption, and intussusceptive angiogenesis. An understanding of these mechanisms may lead to the development of targeted therapies that overcome this resistance. Some of these approaches include the combined inhibition of redundant angiogenic pathways, proper patient selection for various therapies based on gene expression profiles, blockade of cellular migration by inhibition of colony-stimulating factor, or the use of agents to disrupt vascular architecture. Cancer 2012;3455–3467. © 2011 American Cancer Society.

Abstract: Cancer immunotherapy has shown promising results when combined with chemotherapy. Blocking CTLA-4 signaling by monoclonal antibody between cycles of chemotherapy may inhibit cancer cell repopulation and enhance the antitumoral immune reaction, thus improve the efficacy of chemotherapy in mesothelioma. The impact of CTLA-4 blockade on the early stage of tumor development was evaluated in a subcutaneous murine mesothelioma model. CTLA-4 blocking antibody was administered following each cycle of chemotherapy, and monotherapy was included as controls. Antitumor effect was evaluated by tumor growth delay and survival of the animals. Tumor cell repopulation was quantified by bromodeoxyuridine incorporation and Ki67 by immunohistochemistry and/or flow cytometry. In vitro cell killing was determined by classic chromium-released assay, and reverse transcription PCR (RT-PCR) was carried out to determine the gene expression of associated cytokines. Anti-CTLA-4 monoclonal antibody was able to inhibit tumor growth at early stage of tumor development. Antitumor effect was achieved by administration of CTLA-4 blockade between cycles of chemotherapy. Tumor cell repopulation during the intervals of cisplatin was inhibited by CTLA-4 blockade. Anti-CTLA-4 therapy gave rise to an increased number of CD4 and CD8 T cells infiltrating the tumor. RT-PCR showed that the gene expression of interleukin IL-2, IFN-γ, granzyme B, and perforin increased in the tumor milieu. Blockade of CTLA-4 signaling showed effective anticancer effect, correlating with inhibiting cancer cell repopulation between cycles of chemotherapy and upregulating tumor-infiltrating T lymphocytes, cytokines, and cytolytic enzymes in a murine mesothelioma model.

Abstract: CTLA-4 is a surface receptor on activated T cells that delivers an inhibitory signal, serving as an immune checkpoint. Treatment with anti-CTLA-4 Abs can induce clinical responses to different malignancies, but the nature of the induced Ag-specific recognition is largely unknown. Using microarrays spotted with >8000 human proteins, we assessed the diversity of Ab responses modulated by treatment with CTLA-4 blockade and GM-CSF. We find that advanced prostate cancer patients who clinically respond to treatment also develop enhanced Ab responses to a higher number of Ags than nonresponders. These induced Ab responses targeted Ags to which preexisting Abs are more likely to be present in the clinical responders compared with nonresponders. The majority of Ab responses are patient-specific, but immune responses against Ags shared among clinical responders are also detected. One of these shared Ags is PAK6, which is expressed in prostate cancer and to which CD4(+) T cell responses were also induced. Moreover, immunization with PAK6 can be both immunogenic and protective in mouse tumor models. These results demonstrate that immune checkpoint blockade modulates Ag-specific responses to both individualized and shared Ags, some of which can mediate anti-tumor responses.

Abstract: Renin-angiotensin-aldosterone system (RAAS) blockade is the most effective and widely used treatment for attenuating CKD. Enthusiasm must be tempered, however, by recognition that CKD progresses in many patients despite this blockade, and therapeutic trials with alternative agents have proven

Abstract: We investigated the effects of dual renin-angiotensin system (RAS) blockade on angiotensin-converting enzyme-2 (Ace2) expression, hypertension, and renal proximal tubular cell (RPTC) apoptosis in t...

Abstract: Aberrant neuronal activity in injured peripheral nerves is believed to be an important factor in the development of neuropathic pain. Pharmacological blockade of that activity has been shown to mitigate the onset of associated molecular events in the nervous system. However, results in preventing onset of pain behaviors by providing prolonged nerve blockade have been mixed. Furthermore, the experimental techniques used to date to provide that blockade were limited in clinical potential in that they would require surgical implantation. To address these issues, we have used liposomes (SDLs) containing saxitoxin (STX), a site 1 sodium channel blocker, and the glucocorticoid agonist dexamethasone to provide nerve blocks lasting ∼1 wk from a single injection. This formulation is easily injected percutaneously. Animals undergoing spared nerve injury (SNI) developed mechanical allodynia in 1 wk; nerve blockade with a single dose of SDLs (duration of block 6.9 ± 1.2 d) delayed the onset of allodynia by 2 d. Treatment with three sequential SDL injections resulting in a nerve block duration of 18.1 ± 3.4 d delayed the onset of allodynia by 1 mo. This very prolonged blockade decreased activation of astrocytes in the lumbar dorsal horn of the spinal cord due to SNI. Changes in expression of injury-related genes due to SNI in the dorsal root ganglia were not affected by SDLs. These findings suggest that formulations of this kind, which could be easy to apply clinically, can mitigate the development of neuropathic pain.

Abstract: Renin-angiotensin-aldosterone system (RAAS) blockade is currently the best-documented treatment strategy to delay the progression of chronic proteinuric nephropathies. Several large randomized controlled trials have shown the renoprotective potential of angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs) in nephropathies of almost any etiology. Mineralocorticoid receptor antagonists and the direct renin inhibitor aliskiren as add-on treatments to standard therapy including the optimal dose of ACEIs or ARBs reduce albuminuria or proteinuria and retard development of renal dysfunction more than placebo. No clinical evidence is available, however, about whether these strategies may influence long-term kidney disease outcomes. Combined RAAS blockade may be offered only to patients with proteinuric chronic nephropathies who do not achieve full and persistent remission of proteinuria with ACEI or ARB alone. They need to be carefully monitored for hyperkalemia and worsening of kidney function. This article reviews an evidence-based approach to use of RAAS-inhibiting agents in kidney diseases, considers combination RAAS blockade treatment strategies and discusses some perspectives related to the implementation of RAAS blockade in renal protection.

Abstract: Background and purpose Blockade of adenosine A2A receptors (A2AR) affords robust neuroprotection in a number of brain conditions, although the mechanisms are still unknown. A likely candidate mechanism for this neuroprotection is the control of neuroinflammation, which contributes to the amplification of neurodegeneration, mainly through the abnormal release of pro-inflammatory cytokines such as interleukin(IL)-1β. We investigated whether A2AR controls the signaling of IL-1β and its deleterious effects in cultured hippocampal neurons.

Abstract: Summary So-called RAS-dependent pathways, such as those signalling via mitogen-activated protein kinase kinase (MEK)/mitogen-activated protein kinase (MAPK) and phosphoinositide-3 kinase (PI3K)/Akt, are implicated in proliferation and survival of multiple myeloma (MM) cells. However, the effects of their combined blockade and its potential therapeutic utility for the treatment of RAS-mutated MM have not systematically been analysed. Here, we tested the functional consequences of single versus combined inhibition of the MEK/MAPK and PI3K/Akt pathways in a large series of primary MM samples (n = 55) and MM cell lines (n = 11). Additionally, the anti-myeloma activity of different treatments was analysed with respect to the RAS mutation status. PI3K/Akt blockade was generally more pro-apoptotic than blockade of MEK/MAPK both in cell lines and in primary MM samples. Simultaneous blockade of both pathways led to significantly enhanced anti-myeloma activity in 75% of primary MM samples, whereas the remainder was largely resistant. Resistance to combination blockade was exclusively observed in RAS wildtype cases, whereas sensitivity was noted in RAS wildtype and in RAS mutated MM. These results suggest that oncogenic RAS is a predictor of sensitivity to combination treatment with PI3K/Akt and MEK/MAPK inhibitors and that such an approach might therefore be beneficial for this genetically well-defined subgroup of MM patients.

Abstract: Immune regulation has been shown to be involved in the progressive growth of some murine tumours. Interruption of immune regulatory pathways via activation of 4-1BB or cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) blockade appears to be a promising strategy for cancer immunotherapy. In this study, we examined the effectiveness of 4-1BBL-expressing tumor cell vaccine in combination with CTLA-4 blockade on rejection of murine prostate cancer RM-1. We found that the combination of both a vaccine consisting of 4-1BBL-expressing RM-1 cells and CTLA-4 blockade resulted in regression of RM-1 tumors and a significant increase in survival of the tumour cell recipients, compared to that of either treatment alone. The combined vaccination resulted in higher CTL against RM-1 cells and increased secretion of IFN-γ, TNF-α, and IL-2 in the mix-cultured supernatant. These results suggest that combining activation of 4-1BB and blockade of CTLA-4 may offer a new strategy for prostate cancer immunotherapy.

Abstract: Blockade of the CD40/CD154 pathway remains one of the most effective means of promoting graft survival following transplantation. However, the effects of CD40/CD154 antagonism on dendritic cell (DC) phenotype and functionality following transplantation remain incompletely understood. To dissect the effects of CD154/CD40 blockade on DC activation in vivo, we generated hematopoietic chimeras in mice that expressed a surrogate minor Ag (OVA). Adoptive transfer of OVA-specific CD4(+) and CD8(+) T cells led to chimerism rejection, which was inhibited by treatment with CD154 blockade. Surprisingly, CD154 antagonism did not alter the expression of MHC and costimulatory molecules on CD11c(+) DCs compared with untreated controls. However, DCs isolated from anti-CD154-treated animals exhibited a significant reduction in inflammatory cytokine secretion. Combined blockade of inflammatory cytokines IL-6 and IL-12p40 attenuated the expansion of Ag-specific CD4(+) and CD8(+) T cells and transiently inhibited the rejection of OVA-expressing cells. These results suggest that a major effect of CD154 antagonism in vivo is an impairment in the provision of signal three during donor-reactive T cell programming, as opposed to an impact on the provision of signal two. We conclude that therapies designed to target inflammatory cytokines during donor-reactive T cell activation may be beneficial in attenuating these responses and prolonging graft survival.

Abstract: Acute resolving viral infections are often associated with a strong and multi-specific T-cell response, whereas in persistent viral infections T-cell responses are often impaired. It has been suggested that the resuscitation of the antiviral T-cell response could be a powerful tool to target persisting viruses. Several immunoregulatory pathways, such as IL-10 and TGF-β, have been shown to be involved in the induction of T-cell exhaustion and viral persistence. In this study, we sought to investigate whether TGF-β signaling is also relevant in the maintenance of T-cell exhaustion after viral persistence has been established, and whether blockade of TGF-β signaling could improve control of viral replication in a mouse model of persistent virus infection. Using the LCMV clone 13 model, we analyzed the frequency, function, and phenotype of virus-specific CD4 and CD8 T cells following therapeutic TGF-β signaling blockade. We show that in vivo blockade of the TGF-β receptor failed to substantially enhance the antiviral T-cell response, and was insufficient to mediate a therapeutically-relevant reduction of viral titers in different tissues. Thus, although TGF-β signaling has the ability to hamper antiviral immunity, its pharmacological blockade may not be sufficient to tackle persistent viruses.

Abstract: BACKGROUND AND PURPOSE Combination therapies are becoming increasingly important for the treatment of high blood pressure. Little is known about whether double blockade of angiotensin II (AT1) receptors and angiotensin-converting enzyme (ACE) exert synergistic metabolic effects.

Abstract: To the Editor: Ipilimumab is a novel fully human immunostimulatory monoclonal antibody (mAb) that abrogates the function of cytotoxic T-lymphocyte antigen-4 (CTLA-4). CTLA-4 is an immune-inhibitory surface molecule expressed on activated T cells that exerts a dampening effect on the immune system. CTLA-4 is highly expressed on regulatory T cells that control T-cell-related immune responses and inflammation. A recent study documented the overall survival benefit for patients with metastatic melanoma treated with ipilimumab plus dacarbazine. However, one of the most frequent drug-related adverse events profile with ipilimumab is primarily immune-related and includes rash, colitis, hypophysitis, thyroiditis, and hepatitis. The mechanism by which these immune-related adverse events (IRAEs), which can be sometimes severe and life-threatening, occurs remains largely unknown. Here we document a patient treated with ipilimumab who developed a severe enterocolitis of the respective proportion of circulating T-lymphocyte subsets and propose, based on these results, that ipilimumab could induce a sustained dysbalance between regulatory and effector T cells, favoring the occurrence of gut inflammation and lesions. A 56-year-old male was referred to our department after a 3-month history of severe diarrhea. His medical history was notable for thoracic melanoma, for which he underwent a complete surgical resection followed by the systemic administration of ipilimumab (10 mg/kg every 3 weeks for eight courses) as an adjuvant immunotherapy regimen. Within a few days of the last infusion, he experienced the onset of profuse watery diarrhea associated with progressive body weight loss, hypoalbuminemia, high levels of fecal calprotectin, and serum C-reactive protein (CRP). Ipilimumab was stopped and the patient was initially treated as an outpatient with stable doses of steroids (intravenous methylprednisolone 2 mg/kg/d). Repeated microbiologic stool and blood samples were negative, including a search for cytomegalovirus (CMV) infection and toxins for Clostridium difficile. A colonoscopy revealed a severe pancolitis featured by extensive, deep, and confluent ulcerations covered with mucopurulent exudates and spontaneous bleeding surrounded by diffusely inflamed mucosa. In addition, lesions were not confined to the colon, since superficial ulcerations were also observed in the terminal ileum. Intestinal exam showed a dense inflammatory infiltrate and the absence of granuloma. Despite longstanding steroid therapy (3 months after onset of diarrhea) and total parenteral nutrition, the diarrhea progressed with up to 10–15 bloody bowel movements per day with persistent abdominal pain. Hence, the patient was considered refractory to steroids and a blood sample was collected and peripheral blood (PB) lymphocytes isolated to characterize the frequency of circulating regulatory Foxp3þ T cells as well as the proportion of ‘‘effector’’ T cells (including interferon-gamma [IFN-c]or interleukin [IL]-17-producing CD4þ T cells and cytotoxic granzyme B expressing CD8þ T cells) before changing the line of therapy. Strikingly, our flow cytometric analysis revealed an unusually low frequency of PB regulatory Foxp3þ CD4þT cells (0.2% of total cells gated on CD3þ cells) that contrasted with that of PB cytotoxic granzyme Bþ T cells (81.0% of total cells gated on CD3þCD8þ cells) and also those of PB IFN-cand IL-17-expressing CD4þ T cells (27.3% and 1.3% of total cells gated on CD3þCD4þ cells, respectively) (Fig. 1). An abdominal CT scan revealed a severe colitis located especially on the transverse and left colon without complications such as intestinal perforation or abscess. The patient received two infusions of infliximab (5 mg/kg at weeks 0 and 2) as second-line therapy and his diarrhea progressively improved to grade 1 with a drop of fecal calprotectin (from 3265 to 1780 lg/g stool) and was completely resolved by 2 months. During the course of enterocolitis, the patient developed also a hypophysitis, a thyroiditis, and a moderate hepatitis; the outcome of all these IRAEs was favorable. Manipulation of the immune system by abrogating CTLA-4, an immune regulatory molecule, represents a new and promising antitumoral strategy to induce better disease control and to prolong survival in patients with advanced melanoma. Rash, colitis, and hepatitis so far represent the most frequent induced IRAEs even if various other organs may be affected, and our observation represents a caricatural picture of the side effects that may occur under ipilimumab. In a recent clinical trial, gastrointestinal IRAEs were reported in 41 (35%) out of 115 patients who experienced diarrhea and/or colitis during treatment with ipilimumab. An onset of gastrointestinal IRAEs usually We thank SFR Biosciences Gerland-Lyon Sud (UMS344/US8) for the contribution of the platform of cytometry. Copyright VC 2011 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1002/ibd.21927 Published online inWiley Online Library (wileyonlinelibrary.com).