Intravenous Challenge Research Articles

Site-Dependent Immune Escape Due to Impaired Dendritic Cell Cross-Priming

T-cell recognition of tumor neoantigens is critical for cancer immune surveillance and the efficacy of immunotherapy. Tumors can evade host immunity by altering their antigenicity or orchestrating an immunosuppressive microenvironment, leading to outgrowth of poorly immunogenic tumors through the well-established process of cancer immunoediting. Whether cancer immune surveillance and immunoediting depend on the tissue site of origin, however, is poorly understood. Herein, we studied T-cell-mediated surveillance of antigenic, clonal murine pancreatic adenocarcinoma cells expressing neoantigen. Whereas such tumors are robustly eliminated after subcutaneous or intravenous challenge, we observed selective immune escape within the pancreas and peritoneum. Tumor outgrowth occurred in the absence of immunoediting, and antitumor immunity could not be rescued by PD-1 or CTLA-4 checkpoint blockade. Instead, tumor escape was associated with diminished CD8+ T-cell priming by type I conventional dendritic cells (cDC1). Enhancing cDC1 cross-presentation by CD40 agonist treatment restored immunologic control by promoting T-cell priming and broadening T-cell responses through epitope spread. These findings demonstrate that immune escape of highly antigenic tumors can occur without immunoediting in a tissue-restricted manner and highlight barriers to cDC1-mediated T-cell priming imposed by certain microenvironments that must be addressed for successful combination immunotherapies.

Sustained viremia suppression by SHIVSF162P3CN-recalled effector-memory CD8+ T cells after PD1-based vaccination.

HIV-1 functional cure requires sustained viral suppression without antiretroviral therapy. While effector-memory CD8+ T lymphocytes are essential for viremia control, few vaccines elicit such cellular immunity that could be potently recalled upon viral infection. Here, we investigated a program death-1 (PD1)-based vaccine by fusion of simian immunodeficiency virus capsid antigen to soluble PD1. Homologous vaccinations suppressed setpoint viremia to undetectable levels in vaccinated macaques following a high-dose intravenous challenge by the pathogenic SHIVSF162P3CN. Poly-functional effector-memory CD8+ T cells were not only induced after vaccination, but were also recalled upon viral challenge for viremia control as determined by CD8 depletion. Vaccine-induced effector memory CD8+ subsets displayed high cytotoxicity-related genes by single-cell analysis. Vaccinees with sustained viremia suppression for over two years responded to boost vaccination without viral rebound. These results demonstrated that PD1-based vaccine-induced effector-memory CD8+ T cells were recalled by AIDS virus infection, providing a potential immunotherapy for functional cure.

Efficient Treatment and Pre-Exposure Prophylaxis in Rhesus Macaques by an HIV Fusion-Inhibitory Lipopeptide

Two new HIV fusion-inhibitory lipopeptides (LP-97 and LP-98) were designed with extremely potent and long-acting antiviral activity, and monotherapy with a low-dose LP-98 sharply reduced viral load and maintained long-term viral suppression in SHIVSF162P3-infected 21 rhesus macaques. Promisingly, we found that five treated monkeys achieved posttreatment control (PTC) efficacy and they hided lower viral DNA in deep lymph nodes, whereas monkeys with stable viral rebound had higher viral DNA in superficial lymph nodes. Tissues of the PTC monkeys also exhibited significantly decreased quantitative viral outgrowth (QVOA) and fewer PD-1+ central memory CD4+ T cells, and CD8+ T cells critically contributed the PTC efficacy. Moreover, LP-98 when administrated as pre-exposure prophylaxis (PrEP) provided complete protection in 46 monkeys against SHIVSF162P3 and SIVmac239 infections via intrarectal, intravaginal or intravenous challenge. In conclusion, such lipopeptides possess high potential to achieve functional HIV cure and prevention.

Broadly neutralizing antibody-mediated protection of macaques against repeated intravenous exposures to simian-human immunodeficiency virus.

The opioid epidemic has increased parentally acquired HIV infection. To inform the development of a long-acting prevention strategy, we evaluated the protective efficacy of broadly neutralizing antibodies (bNAbs) against intravenous simian-human immunodeficiency virus (SHIV) infection in macaques. Five cynomolgus macaques were injected once subcutaneously with 10-1074 and 3BNC117 (10 mg each kg-1) and were repeatedly challenged intravenously once weekly with SHIVAD8-EO (130 TCID50), until infection was confirmed via plasma viral load assay. Two control macaques, which received no antibody, were challenged identically. Plasma viremia was monitored via RT-qPCR assay. bNAb concentrations were determined longitudinally in plasma samples via TZM-bl neutralization assays using virions pseudotyped with 10-1074-sensitive (X2088_c9) or 3BNC117-sensitive (Q769.d22) HIV envelope proteins. Passively immunized macaques were protected against a median of five weekly intravenous SHIV challenges, as compared to untreated controls, which were infected following a single challenge. Of the two bNAbs, 10-1074 exhibited relatively longer persistence in vivo. The median plasma level of 10-1074 at SHIV breakthrough was 1.1 μg ml-1 (range: 0.6-1.6 μg ml-1), whereas 3BNC117 was undetectable. Probit modeling estimated that 6.6 μg ml-1 of 10-1074 in plasma corresponded to a 99% reduction in per-challenge infection probability, as compared to controls. Significant protection against repeated intravenous SHIV challenges was observed following administration of 10-1074 and 3BNC117 and was due primarily to 10-1074. Our findings extend preclinical studies of bNAb-mediated protection against mucosal SHIV acquisition and support the possibility that intermittent subcutaneous injections of 10-1074 could serve as long-acting preexposure prophylaxis for persons who inject drugs.

AcidoCEST MRI Evaluates the Bone Microenvironment in Multiple Myeloma.

Multiple myeloma (MM) is an incurable disease of malignant plasma cells in the bone marrow (BM). Adaptive responses to hypoxia may be an essential element in MM progression and drug resistance. This metabolic adaptation involves a decrease in extracellular pH (pHe), and it depends on the upregulation of glucose transporters (GLUTs) that is common in hypoxia and in cancer cells. CEST MRI is an imaging technique that assesses pHe indirectly by the exchange rate of magnetic saturation transfer between labile protons on a solute and water. Thus, this study aimed to determine the feasibility of acidoCEST MRI for pHe measurement using an orthotopic mouse model of MM compared with GLUT1 immunofluorescence staining as a reference. Orthotopic BM engrafted MM xenografts were established in NSG/NOD mice using the human RPMI8226 myeloma cell line. AcidoCEST MRI was performed approximately 6 weeks after intravenous challenge, before and after intravenous administration of iopamidol. BM pHe values were generated via fitting the CEST spectrum with the Bloch-McConnell equations. Samples were decalcified, sectioned, and immunostained for GLUT1 expression. Pearson's correlation was used to assess the relationship between pHe and [H3O+] versus GLUT1 expression. Ten mice underwent acidoCEST MRI followed by immunofluorescent histologic analysis. A strong negative correlation was seen between pHe versus GLUT1 expression (r = - 0.75, p < 0.001). After transformation of pH to [H3O+], a strong positive correlation between [H3O+] and GLUT1 expression was observed (r = 0.8, p < 0.001). AcidoCEST MRI can measure the extracellular pH of bone marrow affected by multiple myeloma. In this MM orthotopic mouse model, pHe measured by acidoCEST MRI showed strong correlations with the metabolic phenotype of BM tumor assessed by immunofluorescent histological assessment of GLUT1 overexpression.

Immunotherapy-induced antibodies to endogenous retroviral envelope glycoprotein confer tumor protection in mice.

Following curative immunotherapy of B16F10 tumors, ~60% of mice develop a strong antibody response against cell-surface tumor antigens. Their antisera confer prophylactic protection against intravenous challenge with B16F10 cells, and also cross-react with syngeneic and allogeneic tumor cell lines MC38, EL.4, 4T1, and CT26. We identified the envelope glycoprotein (env) of a murine endogenous retrovirus (ERV) as the antigen accounting for the majority of this humoral response. A systemically administered anti-env monoclonal antibody cloned from such a response protects against tumor challenge, and prophylactic vaccination against the env protein protects a majority of naive mice from tumor establishment following subcutaneous inoculation with B16F10 cells. These results suggest the potential for effective prophylactic vaccination against analogous HERV-K env expressed in numerous human cancers.

Effect of Ketamine on Human Neurochemistry in Posterior Cingulate Cortex: A Pilot Magnetic Resonance Spectroscopy Study at 3 Tesla.

Ketamine is a powerful glutamatergic long-lasting antidepressant, efficient in intractable major depression. Whereas ketamine’s immediate psychomimetic side-effects were linked to glutamate changes, proton MRS (1H-MRS) showed an association between the ratio of glutamate and glutamine and delayed antidepressant effect emerging ∼2 h after ketamine administration. While most 1H-MRS studies focused on anterior cingulate, recent functional MRI connectivity studies revealed an association between ketamine’s antidepressant effect and disturbed connectivity patterns to the posterior cingulate cortex (PCC), and related PCC dysfunction to rumination and memory impairment involved in depressive pathophysiology. The current study utilized the state-of-the-art single-voxel 3T sLASER 1H-MRS methodology optimized for reproducible measurements. Ketamine’s effects on neurochemicals were assessed before and ∼3 h after intravenous ketamine challenge in PCC. Concentrations of 11 neurochemicals, including glutamate (CRLB ∼ 4%) and glutamine (CRLB ∼ 13%), were reliably quantified with the LCModel in 12 healthy young men with between-session coefficients of variation (SD/mean) <8%. Also, ratios of glutamate/glutamine and glutamate/aspartate were assessed as markers of synaptic function and activated glucose metabolism, respectively. Pairwise comparison of metabolite profiles at baseline and 193 ± 4 min after ketamine challenge yielded no differences. Minimal detectable concentration differences estimated with post hoc power analysis (power = 80%, alpha = 0.05) were below 0.5 μmol/g, namely 0.39 μmol/g (∼4%) for glutamate, 0.28 μmol/g (∼10%) for Gln, ∼14% for glutamate/glutamine and ∼8% for glutamate/aspartate. Despite the high sensitivity to detect between-session differences in glutamate and glutamine concentrations, our study did not detect delayed glutamatergic responses to subanesthetic ketamine doses in PCC.

A Mouse Model of Candidiasis.

The intravenous challenge model of Candida albicans infection in mice is a well-established procedure that mirrors disseminated candidiasis in humans. In this model, in which the fungus is delivered into the bloodstream causing a systemic infection, the kidneys are the primary target organs. Mice develop renal failure and septic shock that recapitulates the progressive sepsis seen in humans during severe clinical cases. This model is used to study inflammation and the host immune response against fungal infection. This chapter describes the intravenous candidiasis infection protocol, detailing different steps from the preparation of the inoculum, injection of Candida, monitoring of animals, collection of tissue from infected mice, sample preparation and analysis of several parameters related to infection and the inflammatory response.

Role of astroglial Connexin 43 in pneumolysin cytotoxicity and during pneumococcal meningitis.

Streptococcus pneumoniae or pneumococcus (PN) is a major causative agent of bacterial meningitis with high mortality in young infants and elderly people worldwide. The mechanism underlying PN crossing of the blood brain barrier (BBB) and specifically, the role of non-endothelial cells of the neurovascular unit that control the BBB function, remains poorly understood. Here, we show that the astroglial connexin 43 (aCx43), a major gap junctional component expressed in astrocytes, plays a predominant role during PN meningitis. Following intravenous PN challenge, mice deficient for aCx43 developed milder symptoms and showed severely reduced bacterial counts in the brain. Immunofluorescence analysis of brain slices indicated that PN induces the aCx43–dependent destruction of the network of glial fibrillary acid protein (GFAP), an intermediate filament protein specifically expressed in astrocytes and up-regulated in response to brain injury. PN also induced nuclear shrinkage in astrocytes associated with the loss of BBB integrity, bacterial translocation across endothelial vessels and replication in the brain cortex. We found that aCx4-dependent astrocyte damages could be recapitulated using in vitro cultured cells upon challenge with wild-type PN but not with a ply mutant deficient for the pore-forming toxin pneumolysin (Ply). Consistently, we showed that purified Ply requires Cx43 to promote host cell plasma membrane permeabilization in a process involving the Cx43-dependent release of extracellular ATP and prolonged increase of cytosolic Ca2+ in host cells. These results point to a critical role for astrocytes during PN meningitis and suggest that the cytolytic activity of the major virulence factor Ply at concentrations relevant to bacterial infection requires co-opting of connexin plasma membrane channels.

Humoral and Cell-Mediated Immune Response Validation in Calves after a Live Attenuated Vaccine of Babesia bigemina.

The current vaccines to control bovine Babesia bigemina (B. bigemina) infection are not fully protective and vaccination failures incur heavy losses to the cattle industry around the world. Using modified micro-aerophilous stationary phase, we developed a culture-derived attenuated live vaccine against B. bigemina and tested a single subcutaneous inoculation of 2 × 108 infected erythrocytes in calves. The protection was measured after a lethal intravenous challenge with 5 × 108 virulent calf-derived B. bigemina. Our results demonstrated that a single shot of attenuated vaccine was capable of inducing robust humoral and cell-mediated immune responses in calves. We found a significant increase in the IgG antibody titers post-challenge and a strong proliferation of both CD4+ and CD8+ T cells contributing towards the protection. Our vaccine provided complete protection and parasitic clearance, which was followed for more than 100 days post-challenge. This immunity against babesiosis was directly linked to strong humoral responses; however, the parasitic clearance was attributed to significant T cells effector responses in vaccinated calves as compared to the infected control calves. We anticipate that these results will be helpful in the development of more efficient culture-derived vaccines against Babesia infections, thus reducing significant global economic losses to farmers and the cattle industry.

Microvascular Stasis Inhibition By Hemopexin in the Townes Mouse Model of Sickle Cell Disease

Polymerization of hemoglobin-S (HbS) in the deoxy conformation shortens the lifespan of sickle red blood cells and promotes intravascular and extravascular hemolysis. When sickle red blood cells are lysed intravascularly, HbS is released into the vascular space where it can consume nitric oxide and be oxidized to higher oxidative forms. During these reactions, ferric (Fe3+) HbS is formed, which readily releases heme. The released heme can activate the innate immune pattern recognition receptor toll-like receptor 4 (TLR4) on endothelium, leading to P-selectin expression, NF-ĸB activation, and microvascular stasis in sickle cell disease (SCD) mice with implanted dorsal skin-fold chambers (DSFCs). Heme-induced TLR4 signalling and stasis are blocked by administering hemopexin with the heme. Plasma hemopexin has the highest binding affinity for free heme, rendering it relatively nonreactive and delivering it safely to the liver for endocytosis and degradation by heme oxygenase-1 (HO-1). Due to chronic hemolysis, hemopexin levels are depleted in SCD patients and mice. We previously reported that intravenous hemopexin induces hepatic HO-1 activity and inhibits ongoing, spontaneous stasis in the venules of SCD mice for up to 48 hours. Inhibition of HO-1 activity with tin protoporphyrin (SnPP) and the administration of carbon monoxide (CO) to SnPP-treated mice demonstrated that HO-1 and its reaction product CO play important roles in the protection against stasis. In the current studies, we asked the question how much of the hemopexin-mediated protection is due to preventing heme activation of TLR4 signalling versus induction of HO-1 and CO production. We wondered if hemopexin complexed with heme would be as effective as hemopexin alone in treating a vaso-occlusive crisis (VOC) induced with hemoglobin heme. First, in a stasis prevention model, we intravenously administered different doses of hemopexin in Townes SCD mice with a DSFC one hour prior to a hemoglobin challenge. Briefly, anesthetized mice were placed on an intravital microscopy stage, and 20-24 flowing subcutaneous venules were selected and mapped. After baseline selection of flowing venules and one hour after administration of different doses of hemopexin (0 - 160 mg/kg body weight), microvascular stasis was triggered by infusing hemoglobin (1 µmol/kg). The same vessels were re-examined for stasis (no flow) and percent stasis was calculated. Our results show that hemopexin effectively reduced stasis in a dose responsive manner when delivered one hour prior to the hemoglobin challenge (Figure 1A). Second, we evaluated hemopexin in a VOC acute treatment model by infusing various doses of hemopexin 30 minutes after the intravenous hemoglobin challenge. We found a dose-dependent effect of hemopexin to reduce and resolve microvascular stasis during an acute VOC one hour after hemopexin infusion (Figure 1B). We obtained similar results after inducing stasis with lipopolysaccharide (LPS) or hypoxia-reoxygenation in place of hemoglobin challenge. Third, we evaluated equimolar hemopexin-heme complexes in a VOC acute treatment model by infusing the same doses of hemopexin-heme 40 minutes after the intravenous hemoglobin challenge (1 µmol/kg) to further elucidate the effects of heme scavenging versus HO-1 induction. Our results showed a striking, dose-dependent reduction in stasis, albeit to a detectably lower degree than hemopexin free of heme (Figure 1C). In summary, hemopexin has a dose-dependent effect to prevent and treat vaso-occlusion induced in a mouse model of SCD by hemoglobin, LPS, and hypoxia-reoxygenation. This effect is partially replicated by hemopexin pre-complexed with heme, suggesting that the beneficial effect is not limited to clearance of circulating heme and prevention of TLR4 signalling. The partial effectiveness of hemopexin-heme complex is consistent with our prior published data implicating CO generation by HO-1 as playing a role in the relief of vaso-occlusion by hemopexin. We conclude that hemopexin shows promise for the treatment of VOC. Additional experiments are needed to clarify the activity of hemopexin in clearance-dependent and -independent mechanisms of resolution of vaso-occlusion. Clinical trials of hemopexin are warranted to evaluate its safety, tolerability and potential efficacy in relieving vaso-occlusive pain crisis in patients with SCD. Disclosures Gentinetta: CSL Behring: Current Employment. Vercellotti:CSL Behring: Research Funding. Kato:CSL Behring AG: Current Employment. Brinkman:CSL Behring: Current Employment. Zuercher:CSL Behring AG: Current Employment. Belcher:Mitobridge-Astellas: Consultancy, Research Funding; CSL Behring: Consultancy, Research Funding; Hillhurst Biopharmaceuticals: Consultancy.

Gut-educated IgA plasma cells defend the meningeal venous sinuses.

The central nervous system has historically been viewed as an immune-privileged site, but recent data have shown that the meninges-the membranes that surround the brain and spinal cord-contain a diverse population of immune cells1. So far, studies have focused on macrophages and T cells, but have not included a detailed analysis of meningeal humoral immunity. Here we show that, during homeostasis, the mouse and human meninges contain IgA-secreting plasma cells. These cells are positioned adjacent to dural venous sinuses: regions of slow blood flow with fenestrations that can potentially permit blood-borne pathogens to access the brain2. Peri-sinus IgA plasma cells increased with age and following a breach of the intestinal barrier. Conversely, they were scarce in germ-free mice, but their presence was restored by gut re-colonization. B cell receptor sequencing confirmed that meningeal IgA+ cells originated in the intestine. Specific depletion of meningeal plasma cells or IgAdeficiency resulted in reduced fungal entrapment in the peri-sinus region and increased spread into the brain following intravenous challenge, showing that meningeal IgA is essential for defending the central nervous system at this vulnerable venous barrier surface.

Predicting Antidepressant Citalopram Treatment Response via Changes in Brain Functional Connectivity After Acute Intravenous Challenge.

Introduction: The early and therapy-specific prediction of treatment success in major depressive disorder is of paramount importance due to high lifetime prevalence, and heterogeneity of response to standard medication and symptom expression. Hence, this study assessed the predictability of long-term antidepressant effects of escitalopram based on the short-term influence of citalopram on functional connectivity.Methods: Twenty nine subjects suffering from major depression were scanned twice with resting-state functional magnetic resonance imaging under the influence of intravenous citalopram and placebo in a randomized, double-blinded cross-over fashion. Symptom factors were identified for the Hamilton depression rating scale (HAM-D) and Beck's depression inventory (BDI) taken before and after a median of seven weeks of escitalopram therapy. Predictors were calculated from whole-brain functional connectivity, fed into robust regression models, and cross-validated.Results: Significant predictive power could be demonstrated for one HAM-D factor describing insomnia and the total score (r = 0.45–0.55). Remission and response could furthermore be predicted with an area under the receiver operating characteristic curve of 0.73 and 0.68, respectively. Functional regions with high influence on the predictor were located especially in the ventral attention, fronto-parietal, and default mode networks.Conclusion: It was shown that medication-specific antidepressant symptom improvements can be predicted using functional connectivity measured during acute pharmacological challenge as an easily assessable imaging marker. The regions with high influence have previously been related to major depression as well as the response to selective serotonin reuptake inhibitors, corroborating the advantages of the current approach of focusing on treatment-specific symptom improvements.

Effect of calfhood nutrition on metabolic hormones, gonadotropins, and estradiol concentrations and on reproductive organ development in beef heifer calves.

This study examined the effect of plane of nutrition on the endocrinological regulation of the hypothalamic-pituitary-ovarian (HPO) axis in beef heifer calves during a critical sexual developmental window early in calf hood. Forty Holstein-Friesian × Angus heifers (mean age 19 d, SEM = 0.63) were assigned to a high (HI; ADG 1.2 kg) or moderate (MOD; ADG 0.50 kg) nutritional level from 3 to 21 wk of life. Intake was recorded using an electronic calf feeding system, BW was recorded weekly, and blood samples were collected on the week of age 5, 10, 15, and 20 for metabolite, reproductive, and metabolic hormone determination. At 19 wk of age, on sequential days, an 8-h window bleed was carried out for luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estradiol analysis. To characterize anterior pituitary gland function, an intravenous GnRH challenge was conducted (19 wk of age). Blood was collected via a jugular catheter every 15 min for 135 min for the analysis of LH, FSH, and estradiol. Calves were subsequently euthanized at 21 wk of age; the anterior pituitary, metabolic organs, and reproductive tract were weighed, and ovarian surface follicular numbers and oocytes recovered were recorded. Mean ADG was 1.18 and 0.50 kg for HI and MOD, respectively, resulting in a 76.6-kg difference in BW (P < 0.001). Blood insulin, glucose, and IGF-1 concentrations were greater (P < 0.001) for HI compared with MOD. There was a diet × time interaction for leptin (P < 0.01); concentrations were greater in HI compared with MOD at 20 wk of age with no difference between treatments before this. Dietary treatment did not alter the concentrations of adiponectin or anti-mullerian hormone. There was a diet × time interaction for FSH, whereby MOD had greater concentrations than HI at 10, 15, and 20, but not at 5 wk of age. Over the duration of an 8-h window bleed (19 wk of age), serum concentrations of LH, LH pulse frequency, and LH pulse amplitude were unaffected by treatment, whereas FSH (0.23 vs. 0.43 ng/mL) and estradiol (0.53 vs. 0.38 ng/mL) concentrations were less than and greater, respectively, for HI than MOD (P < 0.05). Likewise, following a GnRH challenge, the area under the curve analysis revealed greater (P < 0.01) estradiol and lesser (P < 0.01) FSH concentrations in calves on the HI relative to MOD diet, whereas concentrations of LH were unaffected (P = 0.26) between treatments. Ovarian surface follicle numbers were greater (P < 0.05) in HI compared with MOD. Total reproductive tract, uterus, and ovarian tissue expressed relative to BW were greater (P < 0.05) for HI compared with MOD. In conclusion, enhanced nutrition in early calfhood advances the ontogeny development of the HPO axis.

HIV-1 acquisition in a man with ulcerative colitis on anti-α4β7 mAb vedolizumab treatment.

HIV-1 acquisition in a man with ulcerative colitis on anti-α4β7 mAb vedolizumab treatment.

Trans-ocular brain impedance index for assessment of cerebral autoregulation in a porcine model of cerebral hemodynamic perturbation.

Cerebrovascular autoregulation (CA) is often impaired following traumatic brain injury. Established technologies and metrics used to assess CA are invasive and conducive for measurement, but not for continuous monitoring. We developed a trans-ocular brain impedance (TOBI) method that may provide non-invasive and continuous indices to assess CA. In this study, we monitored impedance metrics such as respiratory-induced impedance amplitude changes (dz) as well as a novel impedance index (DZx), which is a moving Pearson correlation between mean arterial pressure (MAP) and dz. Yorkshire swine were instrumented to continuously record ICP, MAP, and cerebral blood flow (CBF). TOBI was recorded by placement of standard ECG electrodes on closed eyelids and connected to a data acquisition system. MAP, ICP and CBF were manipulated utilizing an intravenous vasopressor challenge. TOBI indices (dz and DZx) were compared to the hemodynamic indicators as well as pressure reactivity index (PRx). During the vasopressor challenge, dz was highly correlated with ICP, CPP, and CBF (r = < -0.49, p < 0.0001). ICP, CPP, and CBF had a mean percent increase (standard deviation) from baseline of 29(23.2)%, 70(25)%, and 37(72.6)% respectively while dz decreased by 31(15.6)%. Receiver operator curve test showed high predictive performance of DZx when compared to PRx with area under the curve above 0.86, with high sensitivity and specificity. Impedance indices appear to track changes in PRx and hemodynamics that affect cerebral autoregulation. TOBI may be a suitable less invasive surrogate to PRx and capable of tracking cerebral autoregulation.

Exercise and fluid challenge during right heart catheterisation for evaluation of dyspnoea.

This prospective study compared exercise test and intravenous fluid challenge in a single right heart catheter procedure to detect latent diastolic heart failure in patients with echocardiographic heart failure with preserved ejection function. We included 49 patients (73% female) with heart failure with preserved ejection function and pulmonary artery wedge pressure ≤15 mmHg. A subgroup of 26 patients had precapillary pulmonary hypertension. Invasive haemodynamic and gas exchange parameters were measured at rest, 45° upright position, during exercise, after complete haemodynamic and respiratory recovery in lying position, and after rapid infusion of 500 mL isotonic solution. Most haemodynamic parameters increased at both exercise and intravenous fluid challenge, with the higher increase at exercise. Pulmonary vascular resistance decreased by –0.21 wood units at exercise and –0.56 wood units at intravenous fluid challenge (p = 0.3); 20% (10 of 49) of patients had an increase in pulmonary artery wedge pressure above the upper limit of 20 mmHg at exercise, and 20% above the respective limit of 18 mmHg after intravenous fluid challenge. However, only three patients exceeded the upper limit of pulmonary artery wedge pressure in both tests, i.e. seven patients only at exercise and seven other patients only after intravenous fluid challenge. In the subgroup of pulmonary hypertension patients, only two patients exceeded pulmonary artery wedge pressure limits in both tests, further five patients at exercise and four patients after intravenous fluid challenge. A sequential protocol in the same patient showed a significantly higher increase in haemodynamic parameters at exercise compared to intravenous fluid challenge. Both methods can unmask diastolic dysfunction at right heart catheter procedure, but in different patient groups.

FRI0016 R835, A NOVEL IRAK1/4 DUAL INHIBITOR IN CLINICAL DEVELOPMENT, BLOCKS TOLL-LIKE RECEPTOR 4 (TLR4) SIGNALING IN HUMAN AND MOUSE

Background:Toll-Like Receptors (TLR) and Interleukin-1 Receptors (IL-1R) play a critical role in the innate immune response as microbial sensors, providing a bridge between the innate and adaptive immunity (1). Interleukin receptor associated kinases (IRAK) 1 and 4 are serine/threonine kinases that are essential for signaling downstream of most TLRs and IL-1Rs and the resulting production of pro-inflammatory cytokines (2). Suppression of TLR and IL-1R signaling through inhibition of IRAK1/4 kinases is a promising therapeutic approach for the treatment of inflammatory and autoimmune diseases.Objectives:The aim of the study was to characterize the effects of R835, a novel small molecule inhibitor of IRAK1/4, on TLR4 signaling.Methods:R835 was identified in cell-based assays measuring cytokine production induced by LPS (TLR4 ligand). Inhibition of IRAK1 and IRAK4 kinases by R835 was confirmed in biochemical assays and cell lysates. The ability of R835 to inhibit TLR4 signaling was further evaluated in human and mouse whole blood assays. R835 was tested in a mouse model of LPS-induced cytokine release. Mice were pre-treated orally with vehicle or R835 prior to challenge with LPS and serum cytokine levels were monitored over a 24-hour period.Results:We have identified R835, a selective small molecule inhibitor of IRAK1 and IRAK4. R835 blocked LPS/TLR4 signaling and the resulting production of proinflammatory cytokines in both human and mouse cells and whole blood. R835 suppressed serum cytokine elevation in mice challenged with LPS.Conclusion:Our study demonstrates that R835, through inhibition of IRAK1/4 kinase activity, blocks LPS-induced cytokine production in vitro and in vivo. In a recent phase 1 study, R835 substantially reduced the increase of serum cytokines after an intravenous LPS challenge in healthy volunteers. Importantly, this shows that the pharmacological inhibition of IRAK1/4 pathway by R835 in humans mirrors the results obtained in mice. To our knowledge, R835 is the first dual IRAK1/4 inhibitor to enter clinical development and demonstrate inhibition of TLR4-induced cytokines in both mice and humans. R835 is a promising clinical candidate that will allow the exploration of IRAK1/4 inhibition in the treatment of cytokine-driven rheumatic and autoimmune diseases.

Neutralizing Antibodies Protect against Oral Transmission of Lymphocryptovirus

SummaryEpstein-Barr virus (EBV) is a cancer-associated pathogen for which there is no vaccine. Successful anti-viral vaccines elicit antibodies that neutralize infectivity; however, it is unknown whether neutralizing antibodies prevent EBV acquisition. Here we assessed whether passively delivered AMMO1, a monoclonal antibody that neutralizes EBV in a cell-type-independent manner, could protect against experimental EBV challenge in two animal infection models. When present prior to a high-dose intravenous EBV challenge, AMMO1 prevented viremia and reduced viral loads to nearly undetectable levels in humanized mice. AMMO1 conferred sterilizing immunity to three of four macaques challenged orally with rhesus lymphocryptovirus, the EBV ortholog that infects rhesus macaques. The infected macaque had lower plasma neutralizing activity than the protected animals. These results indicate that a vaccine capable of eliciting adequate titers of neutralizing antibodies targeting the AMMO1 epitope may protect against EBV acquisition and are therefore highly relevant to the design of an effective EBV vaccine.

CD122-selective IL-2 complexes target γδ T and NK cells to reduce tumor-promoting Th17 effects and synergize with αPD-L1 to treat primary and metastatic bladder cancer

Abstract αPD-L1 bladder cancer (BC) immunotherapy is effective in &amp;lt;30% of cases. To address the large αPD-L1-unresponsive subset of patients, we tested αIL-2/IL-2 complexes (IL-2c) that block IL-2 from binding high-affinity IL-2Rα (CD25) for preferential IL-2Rβ (CD122) binding. Regulatory T cells (Tregs) capture IL-2 by CD25 whereas CD8+T, γδ T, and NK cells use CD122. We hypothesized that the tumor microenvironment, including local immune cells in primary versus metastatic BC, differentially affects immunotherapy responses. We used PD-L1+ mouse BC cell lines MB49 and MBT-2, for intravesical ([IVe] in bladder) and intravenous (IV) challenge studies of local versus metastatic BC. αPD-L1 or IL-2c alone reduced tumor burden and extended survival in IVe MB49 and MBT-2. Treg depletion using FOXP3DTR mice further enhanced IVe IL-2c effects, consistent with the known tumor-promoting role of Tregs in human BC. Using in vivo cell depletion approaches, we found that γδ T cells and NK cells, but not CD8+ T cells, were necessary for IL-2c efficacy in bladder. γδ T cells also reduced intratumoral Th17 cells that promote MB49 growth and are elevated in human BC. We confirmed γδ T cell effects in δ TCR KO mice, which abrogated IL-2c efficacy but not αPD-L1 efficacy. Neither αPD-L1 nor IL-2c alone treated metastatic MB49 and MBT-2 BC but the combination improved survival in both. These data are consistent with our recent findings in human BC patients in whom γδ T cell and NK cell cytotoxicity improved BCG immunotherapy. Thus, IL-2c is a promising novel BC immunotherapy that can improve bladder-specific immunity in primary BC. In metastatic BC, combination with αPD-L1 may also be a successful BC treatment strategy due to engagement of innate and adaptive immune responses.