Increased Bone Marrow Uptake and Accumulation of Very-Late Antigen-4 Targeted Lipid Nanoparticles.

Lipid nanoparticles (LNPs) have evolved rapidly as promising delivery systems for oligonucleotides, including siRNAs. However, current clinical LNP formulations show high liver accumulation after systemic administration, which is unfavorable for the treatment of extrahepatic diseases, such as hematological disorders. Here we describe the specific targeting of LNPs to hematopoietic progenitor cells in the bone marrow. Functionalization of the LNPs with a modified Leu-Asp-Val tripeptide, a specific ligand for the very-late antigen 4 resulted in an improved uptake and functional siRNA delivery in patient-derived leukemia cells when compared to their non-targeted counterparts. Moreover, surface-modified LNPs displayed significantly improved bone-marrow accumulation and retention. These were associated with increased LNP uptake by immature hematopoietic progenitor cells, also suggesting similarly improved uptake by leukemic stem cells. In summary, we describe an LNP formulation that successfully targets the bone marrow including leukemic stem cells. Our results thereby support the further development of LNPs for targeted therapeutic interventions for leukemia and other hematological disorders.

Activation-induced colocalisation of SCAMP5 with IFNα in human plasmacytoid dendritic cells.

INTRODUCTION: Plasmacytoid dendritic cells (pDCs) are the main producers of type I interferon (IFN) in SLE. pDCs express high secretory carrier membrane protein 5 (SCAMP5). Recent work in transfected HEK cells connects SCAMP5 to the type I IFN secretory pathway. To further study the role of SCAMP5 in IFNα secretion by pDCs, we focused on the subcellular distribution of SCAMP5 in human pDCs freshly isolated from peripheral blood. METHODS: We measured SCAMP5 expression by flow cytometry in peripheral blood mononuclear cells of healthy subjects (n=8). Next, we assessed the colocalisation of SCAMP5 with IFNα in pDCs of healthy subjects (n=4) by evaluating bright detail similarity (BDS) scores using ImageStream technology. RESULTS: We confirm that SCAMP5 is highly expressed by pDCs derived from peripheral blood. In activated pDCs, we show that SCAMP5 colocalises with IFNα (mean BDS 2.0±0.1; BDS >2.0 in 44% of pDCs). CONCLUSION: SCAMP5 colocalises with IFNα in activated human pDCs, in support of a role of this trafficking protein in the secretion of type I IFN by pDCs.

Analysis of the glyco-code in pancreatic ductal adenocarcinoma identifies glycan-mediated immune regulatory circuits.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive malignancies with a 5-year survival rate of only 9%. Despite the fact that changes in glycosylation patterns during tumour progression have been reported, no systematic approach has been conducted to evaluate its potential for patient stratification. By analysing publicly available transcriptomic data of patient samples and cell lines, we identified here two specific glycan profiles in PDAC that correlated with progression, clinical outcome and epithelial to mesenchymal transition (EMT) status. These different glycan profiles, confirmed by glycomics, can be distinguished by the expression of O-glycan fucosylated structures, present only in epithelial cells and regulated by the expression of GALNT3. Moreover, these fucosylated glycans can serve as ligands for DC-SIGN positive tumour-associated macrophages, modulating their activation and inducing the production of IL-10. Our results show mechanisms by which the glyco-code contributes to the tolerogenic microenvironment in PDAC.

A yeast FRET biosensor enlightens cAMP signaling.

The cAMP-PKA signaling cascade in budding yeast regulates adaptation to changing environments. We developed yEPAC, a FRET-based biosensor for cAMP measurements in yeast. We used this sensor with flow cytometry for high-throughput single cell-level quantification during dynamic changes in response to sudden nutrient transitions. We found that the characteristic cAMP peak differentiates between different carbon source transitions and is rather homogenous among single cells, especially for transitions to glucose. The peaks are mediated by a combination of extracellular sensing and intracellular metabolism. Moreover, the cAMP peak follows the Weber-Fechner law; its height scales with the relative, and not the absolute, change in glucose. Last, our results suggest that the cAMP peak height conveys information about prospective growth rates. In conclusion, our yEPAC-sensor makes possible new avenues for understanding yeast physiology, signaling, and metabolic adaptation.

Selective tumor antigen vaccine delivery to human CD169+ antigen-presenting cells using ganglioside-liposomes.

Priming of CD8+ T cells by dendritic cells (DCs) is crucial for the generation of effective antitumor immune responses. Here, we describe a liposomal vaccine carrier that delivers tumor antigens to human CD169/Siglec-1+ antigen-presenting cells using gangliosides as targeting ligands. Ganglioside-liposomes specifically bound to CD169 and were internalized by in vitro-generated monocyte-derived DCs (moDCs) and macrophages and by ex vivo-isolated splenic macrophages in a CD169-dependent manner. In blood, high-dimensional reduction analysis revealed that ganglioside-liposomes specifically targeted CD14+ CD169+ monocytes and Axl+ CD169+ DCs. Liposomal codelivery of tumor antigen and Toll-like receptor ligand to CD169+ moDCs and Axl+ CD169+ DCs led to cytokine production and robust cross-presentation and activation of tumor antigen-specific CD8+ T cells. Finally, Axl+ CD169+ DCs were present in cancer patients and efficiently captured ganglioside-liposomes. Our findings demonstrate a nanovaccine platform targeting CD169+ DCs to drive antitumor T cell responses.

Tn Antigen Expression Contributes to an Immune Suppressive Microenvironment and Drives Tumor Growth in Colorectal Cancer.

Expression of the tumor-associated glycan Tn antigen (αGalNAc-Ser/Thr) has been correlated to poor prognosis and metastasis in multiple cancer types. However, the exact mechanisms exerted by Tn antigen to support tumor growth are still lacking. One emerging hallmark of cancer is evasion of immune destruction. Although tumor cells often exploit the glycosylation machinery to interact with the immune system, the contribution of Tn antigen to an immunosuppressive tumor microenvironment has scarcely been studied. Here, we explored how Tn antigen influences the tumor immune cell composition in a colorectal cancer (CRC) mouse model. CRISPR/Cas9-mediated knock out of the C1galt1c1 gene resulted in elevated Tn antigen levels on the cell surface of the CRC cell line MC38 (MC38-Tnhigh). RNA sequencing and subsequent GO term enrichment analysis of our Tnhigh glycovariant not only revealed differences in MAPK signaling and cell migration, but also in antigen processing and presentation as well as in cytotoxic T cell responses. Indeed, MC38-Tnhigh tumors displayed increased tumor growth in vivo, which was correlated with an altered tumor immune cell infiltration, characterized by reduced levels of cytotoxic CD8+ T cells and enhanced accumulation of myeloid-derived suppressor cells. Interestingly, no systemic differences in T cell subsets were observed. Together, our data demonstrate for the first time that Tn antigen expression in the CRC tumor microenvironment affects the tumor-associated immune cell repertoire.

BOB.1 controls memory B-cell fate in the germinal center reaction.

During T cell-dependent (TD) germinal center (GC) responses, naïve B cells are instructed to differentiate towards GC B cells (GCBC), high-affinity long-lived plasma cells (LLPC) or memory B cells (Bmem). Alterations in the B cell-fate choice could contribute to immune dysregulation leading to the loss of self-tolerance and the initiation of autoimmune disease. Here we show that mRNA levels of the transcription regulator BOB.1 are increased in the lymph node compartment of patients with rheumatoid arthritis (RA), a prototypical autoimmune disease caused by the loss of immunological tolerance. Investigating to what extent levels of BOB.1 impact B cells during TD immune responses we found that BOB.1 has a crucial role in determining the B cell-fate decision. High BOB.1 levels promote the generation of cells with phenotypic and functional characteristics of Bmem. Mechanistically, overexpression of BOB.1 drives ABF1 and suppresses BCL6, favouring Bmem over LLPC or recycling GCBC. Low levels of BOB.1 are sufficient for LLPC but not for Bmem differentiation. Our findings demonstrate a novel role for BOB.1 in B cells during TD GC responses and suggest that its dysregulation may contribute to the pathogenesis of RA by disturbing the B cell-fate determination.

Disruption of sialic acid metabolism drives tumor growth by augmenting CD8+ T cell apoptosis.

Sialylated glycan structures are known for their immunomodulatory capacities and their contribution to tumor immune evasion. However, the role of aberrant sialylation in colorectal cancer and the consequences of complete tumor desialylation on anti-tumor immunity remain unstudied. Here, we report that CRISPR/Cas9-mediated knock out of the CMAS gene, encoding a key enzyme in the sialylation pathway, in the mouse colorectal cancer MC38 cell line completely abrogated cell surface expression of sialic acids (MC38-Sianull ) and, unexpectedly, significantly increased in vivo tumor growth compared to the control MC38-MOCK cells. This enhanced tumor growth of MC38-Sianull cells could be attributed to decreased CD8+ T cell frequencies in the tumor microenvironment only, as immune cell frequencies in tumor-draining lymph nodes remained unaffected. In addition, MC38-Sianull cells were able to induce CD8+ T cell apoptosis in an antigen-independent manner. Moreover, low CMAS gene expression correlated with reduced recurrence-free survival in a human colorectal cancer cohort, supporting the clinical relevance of our work. Together, these results demonstrate for the first time a detrimental effect of complete tumor desialylation on colorectal cancer tumor growth, which greatly impacts the design of novel cancer therapeutics aimed at altering the tumor glycosylation profile.

A Hybrid III stepped wedge cluster randomized trial testing an implementation strategy to facilitate the use of an evidence-based practice in VA Homeless Primary Care Treatment Programs.

BACKGROUND: Homeless veterans often have multiple health care and psychosocial needs, including assistance with access to housing and health care, as well as support for ongoing treatment engagement. The Department of Veterans Affairs (VA) developed specialized Homeless Patient Alignment Care Teams (HPACT) with the goal of offering an integrated, "one-stop program" to address housing and health care needs of homeless veterans. However, while 70% of HPACT's veteran enrollees have co-occurring mental health and substance use disorders, HPACT does not have a uniform, embedded treatment protocol for this subpopulation. One wraparound intervention designed to address the needs of homeless veterans with co-occurring mental health and substance use disorders which is suitable to be integrated into HPACT clinic sites is the evidence-based practice called Maintaining Independence and Sobriety through Systems Integration, Outreach, and Networking-Veterans Edition, or MISSION-Vet. Despite the promise of MISSION-Vet within HPACT clinics, implementation of an evidence-based intervention within a busy program like HPACT can be difficult. The current study is being undertaken to identify an appropriate implementation strategy for MISSION-Vet within HPACT. The study will test the implementation platform called Facilitation and compared to implementation as usual (IU). The aims of this study are as follows: (1) Compare the extent to which IU or Facilitation strategies achieve fidelity to the MISSION-Vet intervention as delivered by HPACT homeless provider staff. (2) Compare the effects of Facilitation and IU strategies on the National HPACT Performance Measures. (3) Compare the effects of IU and Facilitation on the permanent housing status. (4) Identify and describe key stakeholders' (patients, providers, staff) experiences with, and perspectives on, the barriers to, and facilitators of implementing MISSION. DESIGN: Type III Hybrid modified stepped wedge implementation comparing IU to Facilitation across seven HPACT teams in three sites in the greater Los Angeles VA system. This is a cluster randomized trial. DISCUSSION: Integrating MISSION-Vet within HPACT has the potential to improve the health of thousands of veterans, but it is crucial to implement the intervention appropriately in order for it to succeed. The lessons learned in this protocol could assist with a larger roll-out of MISSION within HPACT. This protocol is registered with clinicaltrials.gov and was assigned the number NCT 02942979.

Feasibility of alcohol screening among patients receiving opioid treatment in primary care.

BACKGROUND: Identifying and treating problem alcohol use among people who also use illicit drugs is a challenge. Primary care is well placed to address this challenge but there are several barriers which may prevent this occurring. The objective of this study was to determine if a complex intervention designed to support screening and brief intervention for problem alcohol use among people receiving opioid agonist treatment is feasible and acceptable to healthcare providers and their patients in a primary care setting. METHODS: A randomised, controlled, pre-and-post design measured feasibility and acceptability of alcohol screening based on recruitment and retention rates among patients and practices. Efficacy was measured by screening and brief intervention rates and the proportion of patients with problem alcohol use. RESULTS: Of 149 practices that were invited, 19 (12.8 %) agreed to participate. At follow up, 13 (81.3 %) practices with 81 (62.8 %) patients were retained. Alcohol screening rates in the intervention group were higher at follow up than in the control group (53 % versus 26 %) as were brief intervention rates (47 % versus 19 %). Four (18 %) people reduced their problem drinking (measured by AUDIT-C), compared to two (7 %) in the control group. CONCLUSIONS: Alcohol screening among people receiving opioid agonist treatment in primary care seems feasible. A definitive trial is needed. Such a trial would require over sampling and greater support for participating practices to allow for challenges in recruitment of patients and practices.

Nestin-Expressing Precursors Give Rise to Both Endothelial as well as Nonendothelial Lymph Node Stromal Cells.

During embryogenesis, lymph nodes form through intimate interaction between lymphoid tissue inducer and lymphoid tissue organizer (LTo) cells. Shortly after birth in mice, specialized stromal cell subsets arise that organize microenvironments within the lymph nodes; however, their direct precursors have not yet been identified. In the bone marrow, mesenchymal stem cells are labeled with GFP in nestin-GFP mice, and we show that during all stages of development, nestin(+) cells are present within lymph nodes of these mice. At day of birth, both mesenchymal CD31(-) and endothelial CD31(+) LTo cells were GFP(+), and only the population of CD31(-) LTo cells contained mesenchymal precursors. These CD31(-)nestin(+) cells are found in the T and B cell zones or in close association with high endothelial venules in adult lymph nodes. Fate mapping of nestin(+) cells unambiguously revealed the contribution of nestin(+) precursor cells to the mesenchymal as well as the endothelial stromal populations within lymph nodes. However, postnatal tamoxifen induced targeting of nestin(+) cells in nes-creER mice showed that most endothelial cells and only a minority of the nonendothelial cells were labeled. Overall our data show that nestin(+) cells contribute to all subsets of the complex stromal populations that can be found in lymph nodes.

Internalization and presentation of myelin antigens by the brain endothelium guides antigen-specific T cell migration.

Trafficking of myelin-reactive CD4(+) T-cells across the brain endothelium, an essential step in the pathogenesis of multiple sclerosis (MS), is suggested to be an antigen-specific process, yet which cells provide this signal is unknown. Here we provide direct evidence that under inflammatory conditions, brain endothelial cells (BECs) stimulate the migration of myelin-reactive CD4(+) T-cells by acting as non-professional antigen presenting cells through the processing and presentation of myelin-derived antigens in MHC-II. Inflamed BECs internalized myelin, which was routed to endo-lysosomal compartment for processing in a time-dependent manner. Moreover, myelin/MHC-II complexes on inflamed BECs stimulated the trans-endothelial migration of myelin-reactive Th1 and Th17 2D2 cells, while control antigen loaded BECs did not stimulate T-cell migration. Furthermore, blocking the interaction between myelin/MHC-II complexes and myelin-reactive T-cells prevented T-cell transmigration. These results demonstrate that endothelial cells derived from the brain are capable of enhancing antigen-specific T cell recruitment.

Multifactorial resistance to aminopeptidase inhibitor prodrug CHR2863 in myeloid leukemia cells: down-regulation of carboxylesterase 1, drug sequestration in lipid droplets and pro-survival activation ERK/Akt/mTOR.

Aminopeptidase inhibitors are receiving attention as combination chemotherapeutic agents for the treatment of refractory acute myeloid leukemia. However, the factors determining therapeutic efficacy remain elusive. Here we identified the molecular basis of acquired resistance to CHR2863, an orally available hydrophobic aminopeptidase inhibitor prodrug with an esterase-sensitive motif, in myeloid leukemia cells. CHR2863 enters cells by diffusion and is retained therein upon esterase activity-mediated conversion to its hydrophilic active metabolite drug CHR6768, thereby exerting amino acid depletion. Carboxylesterases (CES) serve as candidate prodrug activating enzymes given CES1 expression in acute myeloid leukemia specimens. We established two novel myeloid leukemia sublines U937/CHR2863(200) and U937/CHR2863(5uM), with low (14-fold) and high level (270-fold) CHR2863 resistance. The latter drug resistant cells displayed: (i) complete loss of CES1-mediated drug activation associated with down-regulation of CES1 mRNA and protein, (ii) marked retention/sequestration of the prodrug, (iii) a substantial increase in intracellular lipid droplets, and (iv) a dominant activation of the pro-survival Akt/mTOR pathway. Remarkably, the latter feature coincided with a gain of sensitivity to the mTOR inhibitor rapamycin. These finding delineate the molecular basis of CHR2863 resistance and offer a novel modality to overcome this drug resistance in myeloid leukemia cells.

A Human Cell Line Model for Interferon-α Driven Dendritic Cell Differentiation.

The CD34+ MUTZ-3 acute myeloid leukemia cell line has been used as a dendritic cell (DC) differentiation model. This cell line can be cultured into Langerhans cell (LC) or interstitial DC-like cells using the same cytokine cocktails used for the differentiation of their primary counterparts. Currently, there is an increasing interest in the study and clinical application of DC generated in the presence of IFNα, as these IFNα-DC produce high levels of inflammatory cytokines and have been suggested to be more potent in their ability to cross-present protein antigens, as compared to the more commonly used IL-4-DC. Here, we report on the generation of IFNα-induced MUTZ-DC. We show that IFNα MUTZ-DC morphologically and phenotypically display characteristic DC features and are functionally equivalent to "classic" IL-4 MUTZ-DC. IFNα MUTZ-DC ingest exogenous antigens and can subsequently cross-present HLA class-I restricted epitopes to specific CD8+ T cells. Importantly, mature IFNα MUTZ-DC express CCR7, migrate in response to CCL21, and are capable of priming naïve antigen-specific CD8+ T cells. In conclusion, we show that the MUTZ-3 cell line offers a viable and sustainable model system to study IFNα driven DC development and functionality.

Replication of Plasmodium in reticulocytes can occur without hemozoin formation, resulting in chloroquine resistance.

Most studies on malaria-parasite digestion of hemoglobin (Hb) have been performed using P. falciparum maintained in mature erythrocytes, in vitro. In this study, we examine Plasmodium Hb degradation in vivo in mice, using the parasite P. berghei, and show that it is possible to create mutant parasites lacking enzymes involved in the initial steps of Hb proteolysis. These mutants only complete development in reticulocytes and mature into both schizonts and gametocytes. Hb degradation is severely impaired and large amounts of undigested Hb remains in the reticulocyte cytoplasm and in vesicles in the parasite. The mutants produce little or no hemozoin (Hz), the detoxification by-product of Hb degradation. Further, they are resistant to chloroquine, an antimalarial drug that interferes with Hz formation, but their sensitivity to artesunate, also thought to be dependent on Hb degradation, is retained. Survival in reticulocytes with reduced or absent Hb digestion may imply a novel mechanism of drug resistance. These findings have implications for drug development against human-malaria parasites, such as P. vivax and P. ovale, which develop inside reticulocytes.

CD1d-restricted antigen presentation by Vγ9Vδ2-T cells requires trogocytosis.

CD1d-restricted invariant natural killer T cells (iNKT) constitute an important immunoregulatory T-cell subset that can be activated by the synthetic glycolipid α-galactosylceramide (α-GalCer) and play a dominant role in antitumor immunity. Clinical trials with α-GalCer-pulsed monocyte-derived dendritic cells (moDC) have shown anecdotal antitumor activity in advanced cancer. It was reported that phosphoantigen (pAg)-activated Vγ9Vδ2-T cells can acquire characteristics of professional antigen-presenting cells (APC). Considering the clinical immunotherapeutic applications, Vγ9Vδ2-T APC can offer important advantages over moDC, potentially constituting an attractive novel APC platform. Here, we demonstrate that Vγ9Vδ2-T APC can present antigens to iNKT. However, this does not result from de novo synthesis of CD1d by Vγ9Vδ2-T, but critically depends on trogocytosis of CD1d-containing membrane fragments from pAg-expressing cells. CD1d-expressing Vγ9Vδ2-T cells were able to activate iNKT in a CD1d-restricted and α-GalCer-dependent fashion. Although α-GalCer-loaded moDC outperformed Vγ9Vδ2-T APC on a per cell basis, Vγ9Vδ2-T APC possess unique features with respect to clinical immunotherapeutic application that make them an interesting platform for consideration in future clinical trials.

Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity.

The impact of nutritional status during fetal life on the overall health of adults has been recognized; however, dietary effects on the developing immune system are largely unknown. Development of secondary lymphoid organs occurs during embryogenesis and is considered to be developmentally programmed. Secondary lymphoid organ formation depends on a subset of type 3 innate lymphoid cells (ILC3) named lymphoid tissue inducer (LTi) cells. Here we show that mouse fetal ILC3s are controlled by cell-autonomous retinoic acid (RA) signalling in utero, which pre-sets the immune fitness in adulthood. We found that embryonic lymphoid organs contain ILC progenitors that differentiate locally into mature LTi cells. Local LTi cell differentiation was controlled by maternal retinoid intake and fetal RA signalling acting in a haematopoietic cell-autonomous manner. RA controlled LTi cell maturation upstream of the transcription factor RORγt. Accordingly, enforced expression of Rorgt restored maturation of LTi cells with impaired RA signalling, whereas RA receptors directly regulated the Rorgt locus. Finally, we established that maternal levels of dietary retinoids control the size of secondary lymphoid organs and the efficiency of immune responses in the adult offspring. Our results reveal a molecular link between maternal nutrients and the formation of immune structures required for resistance to infection in the offspring.

Lost in transition: start-up of glycolysis yields subpopulations of nongrowing cells.

Cells need to adapt to dynamic environments. Yeast that fail to cope with dynamic changes in the abundance of glucose can undergo growth arrest. We show that this failure is caused by imbalanced reactions in glycolysis, the essential pathway in energy metabolism in most organisms. The imbalance arises largely from the fundamental design of glycolysis, making this state of glycolysis a generic risk. Cells with unbalanced glycolysis coexisted with vital cells. Spontaneous, nongenetic metabolic variability among individual cells determines which state is reached and, consequently, which cells survive. Transient ATP (adenosine 5'-triphosphate) hydrolysis through futile cycling reduces the probability of reaching the imbalanced state. Our results reveal dynamic behavior of glycolysis and indicate that cell fate can be determined by heterogeneity purely at the metabolic level.

Fatal attraction in glycolysis: how Saccharomyces cerevisiae manages sudden transitions to high glucose.

In the model eukaryote Saccharomyces cerevisiae, it has long been known that a functional trehalose pathway is indispensable for transitions to high glucose conditions. Upon addition of glucose, cells with a defect in trehalose 6-phosphate synthase (Tps1), the first committed step in the trehalose pathway, display what we have termed an imbalanced glycolytic state; in this state the flux through the upper part of glycolysis outpaces that through the lower part of glycolysis. As a consequence, the intermediate fructose 1,6-bisphosphate (FBP) accumulates at low concentrations of ATP and inorganic phosphate (Pi). Despite significant research efforts, a satisfactory understanding of the regulatory role that trehalose metabolism plays during such transitions has remained infamously unresolved. In a recent study, we demonstrate that the startup of glycolysis exhibits two dynamic fates: a proper, functional, steady state or the imbalanced state described above. Both states are stable, attracting states, and the probability distribution of initial states determines the fate of a yeast cell exposed to glucose. Trehalose metabolism steers the dynamics of glycolysis towards the proper functional state through its ATP hydrolysis activity; a mechanism that ensures that the demand and supply of ATP is balanced with Pi availability under dynamic conditions. [van Heerden et al. Science (2014), DOI: 10.1126/science.1245114.].

Myeloid cells as effector cells for monoclonal antibody therapy of cancer.

Monoclonal antibodies (mAbs) have become an important addition to chemo- and/or radiotherapy for the treatment of cancer. They have multiple effector functions that can lead to eradication of tumor, including induction of apoptosis, growth inhibition, and initiation of complement-dependent lysis. Furthermore, mAbs can recruit immune effector cells. Traditionally, natural killer cells have been considered as the main effector cell population in mAb-mediated tumor killing. Myeloid cells have potent cytotoxic ability, as well. Monocytes and macrophages have been shown to induce antibody-dependent cytotoxicity and phagocytosis of tumor cells in the presence of IgG anti-tumor mAb. Furthermore, neutrophils are the most abundant population of circulating white blood cells, and as such may constitute a formidable source of effector cells. However, when targeting neutrophils for tumor therapy, antibodies of the IgA subclass may be more effective. This article focuses on enlisting myeloid effector cells for mAb-based immunotherapy of cancer. Additionally, methods to study mAb-dependent phagocytosis of tumor cells by macrophages are compared.