First trimester human umbilical cord perivascular cells (HUCPVC) modulate the kynurenine pathway and glutamate neurotransmission in an LPS-induced mouse model of neuroinflammation.

BACKGROUND: The Kynurenine Pathway (KP) of tryptophan degradation and glutamate toxicity is implicated in several neurological disorders, including depression. The therapeutic potential of mesenchymal stromal cells (MSC), owing to their well documented phagocytosis-driven mechanism of immunomodulation and neuroprotection, has been tested in many neurological disorders. However, their potential to influence KP and the glutamatergic system has not yet been investigated. Hence, this study sought to investigate the effect of HUCPVC, a rich and potent source of MSC, on Lipopolysaccharide (LPS)-activated KP metabolites, KP enzymes, and key components of glutamate neurotransmission. METHODS: The immunomodulatory effect of peripherally administered HUCPVC on the expression profile of kynurenine pathway metabolites and enzymes was assessed in the plasma and brain of mice treated with LPS using LCMS and QPCR. An assessment of the glutamatergic system, including selected receptors, transporters and related proteins was also conducted by QPCR, immunohistochemistry and Western blot. RESULTS: HUCPVC were found to modulate LPS-induced activation of KP enzymes and metabolites in the brain associated with neurotoxicity. Moreover, the reduced expression of the glutamatergic components due to LPS was also found to be significantly improved by HUCPVC. CONCLUSIONS: The immunomodulatory properties of HUCPVC appear to confer neuroprotection, at least in part, through their ability to modulate the KP in the brain. This KP modulation enhances neuroprotective regulators and downregulates neurotoxic consequences, including glutamate neurotoxicity, which is associated with neuroinflammation and depressive behavior.

Time course and mechanistic analysis of human umbilical cord perivascular cell mitigation of lipopolysaccharide-induced systemic and neurological inflammation.

BACKGROUND AIMS: Because of their potent immunomodulatory and anti-inflammatory properties, mesenchymal stromal cells are a major focus in the field of stem cell therapy. However, the precise mechanisms underlying this are not entirely understood. Human umbilical cord perivascular cells (HUCPVCs) are a promising cell therapy candidate. This study was designed to evaluate the time course and mechanisms by which HUCPVCs mitigate lipopolysaccharide (LPS)-induced systemic and neurological inflammation in immunocompetent mice. To explore the underlying mechanisms, the authors investigated the biodistribution and fate of HUCPVCs. METHODS: Male C57BL/6 mice were randomly allocated to four groups: control, LPS, HUCPVCs or LPS + HUCPVCs. Quantitative polymerase chain reaction, enzyme-linked immunosorbent assay and cytokine arrays were used to assess changes in pro-inflammatory mediators systemically and in the brain. Depressive-like behavioral changes were evaluated via a forced swim test. Quantum dot (qDot) labeling and immunohistochemistry were used to assess the biodistribution and fate of HUCPVCs and interactions with recipient innate immune cells. RESULTS: A single intravenously delivered dose of HUCPVCs significantly reduced the systemic inflammation induced by LPS within the first 24 h after administration. HUCPVC treatment abrogated the upregulated expression of pro-inflammatory genes in the hippocampus and cortex and attenuated depressive-like behavior induced by LPS treatment. Biodistribution analysis revealed that HUCPVC-derived qDots rapidly accumulated in the lungs and demonstrated limited in vivo persistence. Furthermore, qDot signals were associated with major recipient innate immune cells and promoted a shift in macrophages toward a regulatory phenotype in the lungs. CONCLUSIONS: Overall, this study demonstrates that HUCPVCs can successfully reduce systemic and neurological inflammation induced by LPS within the first 24 h after administration. Biodistribution and fate analyses suggest a critical role for the innate immune system in the HUCPVC-based immunomodulation mechanism.

Vascular Dysfunction after Modeled Traumatic Brain Injury Is Preserved with Administration of Umbilical Cord Derived Mesenchymal Stromal Cells and Is Associated with Modulation of the Angiogenic Response.

Vascular dysfunction arising from blood-brain barrier (BBB) breakdown after traumatic brain injury (TBI) can adversely affect neuronal health and behavioral outcome. Pericytes and endothelial cells of the neurovascular unit (NVU) function collectively to maintain strict regulation of the BBB through tight junctions. Secondary injury mechanisms, such as pro-angiogenic signals that contribute to pericyte loss, can prolong and exacerbate primary vascular injury. Human umbilical cord perivascular cells (HUCPVCs) are a source of mesenchymal stromal cells (MSCs) that have been shown to reduce vascular dysfunction after neurotrauma. We hypothesized that the perivascular properties of HUCPVCs can reduce vascular dysfunction after modeled TBI by preserving the pericyte-endothelial interactions. Rats were subjected to a moderate fluid percussion injury (FPI) and intravenously infused with 1,500,000 HUCPVCs post-injury. At acute time points (24 h and 48 h) quantitative polymerase chain reaction (qPCR) analysis demonstrated that the gene expression of angiopoietin-2 was increased with FPI and reduced with HUCPVCs. Immunofluorescent assessment of RECA-1 (endothelial cells) and platelet-derived growth factor receptors (PDGFR-ß) (pericytes) revealed that capillary and pericyte densities as well as the co-localization of the two cells were decreased with FPI and preserved with HUCPVC administration. These acute HUCPVC-mediated protective effects were associated with less permeability to Evan's blue dye and increased expression of the tight junction occludin, suggesting less vascular leakage. Further, at 4 weeks post-injury, HUCPVC administration was associated with reduced anxiety and decreased ß-amyloid precursor protein (ß-APP) accumulation. In summary, HUCPVCs promoted pericyte-endothelial barrier function that was associated with improved long-term outcome.

Axon Degeneration Is Rescued with Human Umbilical Cord Perivascular Cells: A Potential Candidate for Neuroprotection After Traumatic Brain Injury.

Traumatic brain injury (TBI) leads to delayed secondary injury events consisting of cellular and molecular cascades that exacerbate the initial injury. Human umbilical cord perivascular cells (HUCPVCs) secrete neurotrophic and prosurvival factors. In this study, we examined the effects of HUCPVC in sympathetic axon and cortical axon survival models and sought to determine whether HUCPVC provide axonal survival cues. We then examined the effects of the HUCPVC in an in vivo fluid percussion injury model of TBI. Our data indicate that HUCPVCs express neurotrophic and neural survival factors. They also express and secrete relevant growth and survival proteins when cultured alone, or in the presence of injured axons. Coculture experiments indicate that HUCPVCs interact preferentially with axons when cocultured with sympathetic neurons and reduce axonal degeneration. Nerve growth factor withdrawal in axonal compartments resulted in 66 ± 3% axon degeneration, whereas HUCPVC coculture rescued axon degeneration to 35 ± 3%. Inhibition of Akt (LY294002) resulted in a significant increase in degeneration compared with HUCPVC cocultures (48 ± 7% degeneration). Under normoxic conditions, control cultures showed 39 ± 5% degeneration. Oxygen glucose deprivation (OGD) resulted in 58 ± 3% degeneration and OGD HUCPVC cocultures reduced degeneration to 34 ± 5% (p < 0.05). In an in vivo model of TBI, immunohistochemical analysis of NF200 showed improved axon morphology in HUCPVC-treated animals compared with injured animals. These data presented in this study indicate an important role for perivascular cells in protecting axons from injury and a potential cell-based therapy to treat secondary injury after TBI.

Combinatorial Treatment Using Umbilical Cord Perivascular Cells and Aß Clearance Rescues Vascular Function Following Transient Hypertension in a Rat Model of Alzheimer Disease.

Transient hypertension is a risk factor for Alzheimer disease (AD), but the effects of this interaction on brain vasculature are understudied. Addressing vascular pathology is a promising avenue to potentiate the efficacy of treatments for AD. We used arterial spin labeling magnetic resonance imaging to longitudinally assess brain vascular function and immunohistopathology to examine cerebrovascular remodeling and amyloid load. Hypertension was induced for 1 month by administration of l-NG-nitroarginine-methyl-ester in TgF344-AD rats at the prodromal stage. Following hypertension, nontransgenic rats showed transient cerebrovascular changes, whereas TgF344-AD animals exhibited sustained alterations in cerebrovascular function. Human umbilical cord perivascular cells in combination with scyllo-inositol, an inhibitor of Aß oligomerization, resulted in normalization of hippocampal vascular function and remodeling, in contrast to either treatment alone. Prodromal stage hypertension exacerbates latter AD pathology, and the combination of human umbilical cord perivascular cells with amyloid clearance promotes cerebrovascular functional recovery.

Mesenchymal Stromal Cells Modulate Peripheral Stress-Induced Innate Immune Activation Indirectly Limiting the Emergence of Neuroinflammation-Driven Depressive and Anxiety-like Behaviors.

BACKGROUND: Hyperactivation of innate immunity has been implicated in the etiology of mood disorders, including major depressive disorder (MDD). Mesenchymal stromal cells (MSCs) have demonstrated potent immunomodulatory capabilities in the context of chronic inflammatory disease and injury but have yet to be evaluated in stress-based preclinical models of MDD. We sought to test the ability of intravenous MSCs to modulate innate immune activation and behavioral patterns associated with repeated social defeat (RSD). METHODS: Murine RSD-induced innate immune activation as well as depressive and anxiety-like behaviors were assessed in unstressed, RSD, and RSD + human MSC groups. Biodistribution and fate studies were performed to inform potential mechanisms of action. RESULTS: MSCs reduced stress-induced circulating proinflammatory cytokines, monocytes, neuroinflammation, and depressive and anxiety-like behaviors. Biodistribution analyses indicated that infused MSCs distributed within peripheral organs without homing to the brain. Murine neutrophils targeted MSCs in the lungs within hours of administration. MSCs and recipient neutrophils were cleared by recipient macrophages promoting a switch toward a regulatory phenotype and systemic resolution of inflammation. CONCLUSIONS: Peripheral delivery of MSCs modulates central nervous system inflammatory processes and aberrant behavioral patterns in a stress-based rodent model of MDD and anxiety. Recent studies suggest that host immune cell-mediated phagocytosis of MSCs in vivo can trigger an immunomodulatory cascade, resulting in resolution of inflammation. Our data suggest that similar mechanisms may protect distal organs, including the brain, from systemic, stress-induced proinflammatory spikes and may uncover unexpected targets in the periphery for novel or adjunct treatment for a subset of patients with MDD.

Interleukin-6 Regulates Adult Neural Stem Cell Numbers during Normal and Abnormal Post-natal Development.

Circulating systemic factors can regulate adult neural stem cell (NSC) biology, but the identity of these circulating cues is still being defined. Here, we have focused on the cytokine interleukin-6 (IL-6), since increased circulating levels of IL-6 are associated with neural pathologies such as autism and bipolar disorder. We show that IL-6 promotes proliferation of post-natal murine forebrain NSCs and that, when the IL-6 receptor is inducibly knocked out in post-natal or adult neural precursors, this causes a long-term decrease in forebrain NSCs. Moreover, a transient circulating surge of IL-6 in perinatal or adult mice causes an acute increase in neural precursor proliferation followed by long-term depletion of adult NSC pools. Thus, IL-6 signaling is both necessary and sufficient for adult NSC self-renewal, and acute perturbations in circulating IL-6, as observed in many pathological situations, have long-lasting effects on the size of adult NSC pools.

A Glo1-Methylglyoxal Pathway that Is Perturbed in Maternal Diabetes Regulates Embryonic and Adult Neural Stem Cell Pools in Murine Offspring.

Maternal diabetes is known to adversely influence brain development in offspring. Here, we provide evidence that this involves the circulating metabolite methylglyoxal, which is increased in diabetes, and its detoxifying enzyme, glyoxalase 1 (Glo1), which when mutated is associated with neurodevelopmental disorders. Specifically, when Glo1 levels were decreased in embryonic mouse cortical neural precursor cells (NPCs), this led to premature neurogenesis and NPC depletion embryonically and long-term alterations in cortical neurons postnatally. Increased circulating maternal methylglyoxal caused similar changes in embryonic cortical precursors and neurons and long-lasting changes in cortical neurons and NPCs in adult offspring. Depletion of embryonic and adult NPCs was also observed in murine offspring exposed to a maternal diabetic environment. Thus, the Glo1-methylglyoxal pathway integrates maternal and NPC metabolism to regulate neural development, and perturbations in this pathway lead to long-lasting alterations in adult neurons and NPC pools.

In vitro characterization of neurite extension using induced pluripotent stem cells derived from lissencephaly patients with TUBA1A missense mutations.

BACKGROUND: Lissencephaly, or smooth brain, is a severe congenital brain malformation that is thought to be associated with impaired neuronal migration during corticogenesis. However, the exact etiology of lissencephaly in humans remains unknown. Research on congenital diseases is limited by the shortage of clinically derived resources, especially for rare pediatric diseases. The research on lissencephaly is further limited because gyration in humans is more evolved than that in model animals such as mice. To overcome these limitations, we generated induced pluripotent stem cells (iPSCs) from the umbilical cord and peripheral blood of two lissencephaly patients with different clinical severities carrying alpha tubulin (TUBA1A) missense mutations (Patient A, p.N329S; Patient B, p.R264C). RESULTS: Neural progenitor cells were generated from these iPSCs (iPSC-NPCs) using SMAD signaling inhibitors. These iPSC-NPCs expressed TUBA1A at much higher levels than undifferentiated iPSCs and, like fetal NPCs, readily differentiated into neurons. Using these lissencephaly iPSC-NPCs, we showed that the neurons derived from the iPSCs obtained from Patient A but not those obtained from Patient B showed abnormal neurite extension, which correlated with the pathological severity in the brains of the patients. CONCLUSION: We established iPSCs derived from lissencephaly patients and successfully modeled one aspect of the pathogenesis of lissencephaly in vitro using iPSC-NPCs and iPSC-derived neurons. The iPSCs from patients with brain malformation diseases helped us understand the mechanism underlying rare diseases and human corticogenesis without the use of postmortem brains.

Ankrd11 is a chromatin regulator involved in autism that is essential for neural development.

Ankrd11 is a potential chromatin regulator implicated in neural development and autism spectrum disorder (ASD) with no known function in the brain. Here, we show that knockdown of Ankrd11 in developing murine or human cortical neural precursors caused decreased proliferation, reduced neurogenesis, and aberrant neuronal positioning. Similar cellular phenotypes and aberrant ASD-like behaviors were observed in Yoda mice carrying a point mutation in the Ankrd11 HDAC-binding domain. Consistent with a role for Ankrd11 in histone acetylation, Ankrd11 was associated with chromatin and colocalized with HDAC3, and expression and histone acetylation of Ankrd11 target genes were altered in Yoda neural precursors. Moreover, the Ankrd11 knockdown-mediated decrease in precursor proliferation was rescued by inhibiting histone acetyltransferase activity or expressing HDAC3. Thus, Ankrd11 is a crucial chromatin regulator that controls histone acetylation and gene expression during neural development, thereby providing a likely explanation for its association with cognitive dysfunction and ASD.

Temporal and visual source memory deficits among ecstasy/polydrug users.

OBJECTIVES: We wished to investigate whether source memory judgements are adversely affected by recreational illicit drug use. METHOD: Sixty-two ecstasy/polydrug users and 75 non ecstasy users completed a source memory task, in which they tried to determine whether or not a word had been previously presented and if so, attempted to recall the format, location and temporal position in which the word had occurred. RESULTS: While not differing in terms of the number of hits and false positive responses, ecstasy/polydrug users adopted a more liberal decision criterion when judging if a word had been presented previously. With regard to source memory, users were less able to determine the format in which words had been presented (upper versus lower case). Female users did worse than female nonusers in determining which list (first or second) a word was from. Unexpectedly, the current frequency of cocaine use was negative associated with list and case source memory performance. CONCLUSIONS: Given the role that source memory plays in everyday cognition, those who use cocaine more frequently might have more difficulty in everyday tasks such as recalling the sources of crucial information or making use of contextual information as an aid to learning.

Prospective memory deficits in illicit polydrug users are associated with the average long-term typical dose of ecstasy typically consumed in a single session.

RATIONALE: Neuroimaging evidence suggests that ecstasy-related reductions in SERT densities relate more closely to the number of tablets typically consumed per session rather than estimated total lifetime use. To better understand the basis of drug related deficits in prospective memory (p.m.) we explored the association between p.m. and average long-term typical dose and long-term frequency of use. METHOD: Study 1: Sixty-five ecstasy/polydrug users and 85 nonecstasy users completed an event-based, a short-term and a long-term time-based p.m. task. Study 2: Study 1 data were merged with outcomes on the same p.m. measures from a previous study creating a combined sample of 103 ecstasy/polydrug users, 38 cannabis-only users, and 65 nonusers of illicit drugs. RESULTS: Study 1: Ecstasy/polydrug users had significant impairments on all p.m. outcomes compared with nonecstasy users. Study 2: Ecstasy/polydrug users were impaired in event-based p.m. compared with both other groups and in long-term time-based p.m. compared with nonillicit drug users. Both drug using groups did worse on the short-term time-based p.m. task compared with nonusers. Higher long-term average typical dose of ecstasy was associated with poorer performance on the event and short-term time-based p.m. tasks and accounted for unique variance in the two p.m. measures over and above the variance associated with cannabis and cocaine use. CONCLUSIONS: The typical ecstasy dose consumed in a single session is an important predictor of p.m. impairments with higher doses reflecting increasing tolerance giving rise to greater p.m. impairment.

CBP regulates the differentiation of interneurons from ventral forebrain neural precursors during murine development.

The mechanisms that regulate appropriate genesis and differentiation of interneurons in the developing mammalian brain are of significant interest not only because interneurons play key roles in the establishment of neural circuitry, but also because when they are deficient, this can cause epilepsy. In this regard, one genetic syndrome that is associated with deficits in neural development and epilepsy is Rubinstein-Taybi Syndrome (RTS), where the transcriptional activator and histone acetyltransferase CBP is mutated and haploinsufficient. Here, we have asked whether CBP is necessary for the appropriate genesis and differentiation of interneurons in the murine forebrain, since this could provide an explanation for the epilepsy that is associated with RTS. We show that CBP is expressed in neural precursors within the embryonic medial ganglionic eminence (MGE), an area that generates the vast majority of interneurons for the cortex. Using primary cultures of MGE precursors, we show that knockdown of CBP causes deficits in differentiation of these precursors into interneurons and oligodendrocytes, and that overexpression of CBP is by itself sufficient to enhance interneuron genesis. Moreover, we show that levels of the neurotransmitter synthesis enzyme GAD67, which is expressed in inhibitory interneurons, are decreased in the dorsal and ventral forebrain of neonatal CBP(+/-) mice, indicating that CBP plays a role in regulating interneuron development in vivo. Thus, CBP normally acts to ensure the differentiation of appropriate numbers of forebrain interneurons, and when its levels are decreased, this causes deficits in interneuron development, providing a potential explanation for the epilepsy seen in individuals with RTS.

Transient maternal IL-6 mediates long-lasting changes in neural stem cell pools by deregulating an endogenous self-renewal pathway.

The mechanisms that regulate the establishment of adult stem cell pools during normal and perturbed mammalian development are still largely unknown. Here, we asked whether a maternal cytokine surge, which occurs during human maternal infections and has been implicated in cognitive disorders, might have long-lasting consequences for neural stem cell pools in adult progeny. We show that transient, maternally administered interleukin-6 (IL-6) resulted in an expanded adult forebrain neural precursor pool and perturbed olfactory neurogenesis in offspring months after fetal exposure. This increase is likely the long-term consequence of acute hyperactivation of an endogenous autocrine/paracrine IL-6-dependent self-renewal pathway that normally regulates the number of forebrain neural precursors. These studies therefore identify an IL-6-dependent neural stem cell self-renewal pathway in vivo, and support a model in which transiently increased maternal cytokines can act through this pathway in offspring to deregulate neural precursor biology from embryogenesis throughout life.

Methylation of the TERT promoter and risk stratification of childhood brain tumours: an integrative genomic and molecular study.

BACKGROUND: Identification of robust biomarkers of malignancy and methods to establish disease progression is a major goal in paediatric neuro-oncology. We investigated whether methylation of the TERT promoter can be a biomarker for malignancy and patient outcome in paediatric brain tumours. METHODS: For the discovery cohort, we used samples obtained from patients with paediatric brain tumours and individuals with normal brain tissues stored at the German Cancer Research Center (Heidelberg, Germany). We used methylation arrays for genome-wide assessment of DNA. For the validation cohort, we used samples obtained from several tissues for which full clinical and follow-up data were available from two hospitals in Toronto (ON, Canada). We did methylation analysis using quantitative Sequenom and pyrosequencing of an identified region of the TERT promoter. We assessed TERT expression by real-time PCR. To establish whether the biomarker could be used to assess and predict progression, we analysed methylation in paired samples of tumours that transformed from low to high grade and from localised to metastatic, and in choroid plexus tumours of different grades. Finally, we investigated overall survival in patients with posterior fossa ependymomas in which the identified region was hypermethylated or not. All individuals responsible for assays were masked to the outcome of the patients. FINDINGS: Analysis of 280 samples in the discovery cohort identified one CpG site (cg11625005) in which 78 (99%) of 79 samples from normal brain tissues and low-grade tumours were not hypermethylated, but 145 (72%) of 201 samples from malignant tumours were hypermethylated (>15% methylated; p<0.0001). Analysis of 68 samples in the validation cohort identified a subset of five CpG sites (henceforth, upstream of the transcription start site [UTSS]) that was hypermethylated in all malignant paediatric brain tumours that expressed TERT but not in normal tissues that did not express TERT (p<0.0001). UTSS had a positive predictive value of 1.00 (95% CI 0.95-1.00) and a negative predictive value of 0.95 (0.87-0.99). In two paired samples of paediatric gliomas, UTSS methylation increased during transformation from low to high grade; it also increased in two paired samples that progressed from localised to metastatic disease. Two of eight atypical papillomas that had high UTSS methylation progressed to carcinomas, while the other six assessed did not progress or require additional treatment. 5-year overall survival was 51% (95% CI 31-71) for 25 patients with hypermethylated UTSS posterior fossa ependymomas and 95% (86-100) for 20 with non-hypermethylated tumours (p=0.0008). 5-year progression-free survival was 86% (68-100) for the 25 patients with non-hypermethylated UTSS tumours and 30% (10-50) for those with hypermethylated tumours (p=0.0008). INTERPRETATION: Hypermethylation of the UTSS region in the TERT promoter is associated with TERT expression in cancers. In paediatric brain tumours, UTSS hypermethylation is associated with tumour progression and poor prognosis. This region is easy to amplify, and the assay to establish hypermethylation can be done on most tissues in most clinical laboratories. Therefore the UTSS region is a potentially accessible biomarker for various cancers. FUNDING: The Canadian Institute of Health Research and the Terry Fox Foundation.

Metformin activates an atypical PKC-CBP pathway to promote neurogenesis and enhance spatial memory formation.

VIDEO ABSTRACT: Although endogenous recruitment of adult neural stem cells has been proposed as a therapeutic strategy, clinical approaches for achieving this are lacking. Here, we show that metformin, a widely used drug, promotes neurogenesis and enhances spatial memory formation. Specifically, we show that an atypical PKC-CBP pathway is essential for the normal genesis of neurons from neural precursors and that metformin activates this pathway to promote rodent and human neurogenesis in culture. Metformin also enhances neurogenesis in the adult mouse brain in a CBP-dependent fashion, and in so doing enhances spatial reversal learning in the water maze. Thus, metformin, by activating an aPKC-CBP pathway, recruits neural stem cells and enhances neural function, thereby providing a candidate pharmacological approach for nervous system therapy.

Effects of ecstasy/polydrug use on memory for associative information.

RATIONALE: Associative learning underpins behaviours that are fundamental to the everyday functioning of the individual. Evidence pointing to learning deficits in recreational drug users merits further examination. OBJECTIVES: A word pair learning task was administered to examine associative learning processes in ecstasy/polydrug users. METHODS: After assignment to either single or divided attention conditions, 44 ecstasy/polydrug users and 48 non-users were presented with 80 word pairs at encoding. Following this, four types of stimuli were presented at the recognition phase: the words as originally paired (old pairs), previously presented words in different pairings (conjunction pairs), old words paired with new words, and pairs of new words (not presented previously). The task was to identify which of the stimuli were intact old pairs. RESULTS: Ecstasy/ploydrug users produced significantly more false-positive responses overall compared to non-users. Increased long-term frequency of ecstasy use was positively associated with the propensity to produce false-positive responses. It was also associated with a more liberal signal detection theory decision criterion value. Measures of long term and recent cannabis use were also associated with these same word pair learning outcome measures. Conjunction word pairs, irrespective of drug use, generated the highest level of false-positive responses and significantly more false-positive responses were made in the divided attention condition compared to the single attention condition. CONCLUSIONS: Overall, the results suggest that long-term ecstasy exposure may induce a deficit in associative learning and this may be in part a consequence of users adopting a more liberal decision criterion value.