Glyburide confers neuroprotection against age-related macular degeneration (AMD).

Glyburide, a sulfonylurea drug used to treat type 2 diabetes, boasts neuroprotective effects by targeting the sulfonylurea receptor 1 (SUR1) and associated ion channels in various cell types, including those in the central nervous system and the retina. Previously, we demonstrated that glyburide therapy improved retinal function and structure in a rat model of diabetic retinopathy. In the present study, we explore the application of glyburide in non-neovascular ("dry") age-related macular degeneration (AMD), another progressive disease characterized by oxidative stress-induced damage and neuroinflammation that trigger cell death in the retina. We show that glyburide administration to a human cone cell line confers protection against oxidative stress, inflammasome activation, and apoptosis. To corroborate our in vitro results, we also conducted a case-control study, controlling for AMD risk factors and other diabetes medications. It showed that glyburide use in patients reduces the odds of new-onset dry AMD. A positive dose-response relationship is observed from this analysis, in which higher cumulative doses of glyburide further reduce the odds of new-onset dry AMD. In the quest for novel therapies for AMD, glyburide emerges as a promising repurposable drug given its known safety profile. The results from this study provide insights into the multifaceted actions of glyburide and its potential as a neuroprotective agent for retinal diseases; however, further preclinical and clinical studies are needed to validate its therapeutic potential in the context of degenerative retinal disorders such as AMD.

Phototoxicity of low doses of light and influence of the spectral composition on human RPE cells.

Light is known to induce retinal damage affecting photoreceptors and retinal pigment epithelium. For polychromatic light, the blue part of the spectrum is thought to be the only responsible for photochemical damage, leading to the establishment of a phototoxicity threshold for blue light (445 nm). For humans it corresponds to a retinal dose of 22 J/cm2. Recent studies on rodents and non-human primates suggested that this value is overestimated. In this study, we aim at investigating the relevance of the current phototoxicity threshold and at providing new hints on the role of the different components of the white light spectrum on phototoxicity. We use an in vitro model of human induced pluripotent stem cells (hiPSC)-derived retinal pigment epithelial (iRPE) cells and exposed them to white, blue and red lights from LED devices at doses below 22 J/cm2. We show that exposure to white light at a dose of 3.6 J/cm2 induces an alteration of the global cellular structure, DNA damage and an activation of cellular stress pathways. The exposure to blue light triggers DNA damage and the activation of autophagy, while exposure to red light modulates the inflammatory response and inhibits autophagy.

Neuropsychological Functioning in Sexual Offenders With and Without Pedophilic Disorder.

This study sought to examine neuropsychological functioning in men with pedophilic disorder (PD), in order to assess whether findings from prior neuropsychological studies are replicated in a diverse sample including men with non-contact sexual offenses. It was hypothesized that when non-contact offenders are included in the study, a slowed processing speed will emerge as the only finding unique to men with PD. A comprehensive neuropsychological battery was administered to 58 men convicted of a sexual offense, 20 of whom were classified as having PD. The sample included men with contact sexual offenses (n = 33), non-contact sexual offenses (n = 5), and child sexual abuse material (CSAM) offenses (n = 20). Test performance was compared by PD status. Participants with PD performed significantly better on verbal memory and visual discrimination than those without PD. Men with PD made more errors on a set-shifting task but no significant differences were seen in domains of attention, intellectual functioning, visual learning and memory, visuospatial ability, or language ability. Effect sizes were generally small, although some medium effects were seen (visual discrimination and verbal learning and memory). Scores in both groups (with and without PD) were generally in the average range across tasks. Within the subgroup of CSAM offenders, minimal differences emerged between those with and without PD, although those with PD were slower on visuomotor set-shifting but made fewer errors (d = - 0.89). CSAM offenders with PD were in the high average range on many tasks of intellectual functioning; however, a potential trend was identified such that CSAM offenders without PD had lower scores on a task of verbal learning and memory, with medium effect sizes observed. As few differences in neuropsychological functioning emerged when comparing offenders with and without PD, differences demonstrated in prior research may be better attributed to contact offending status rather than sexual interest.

In Vitro and In Vivo Safety of Hyaluronic Acid-Decorated Microparticles for Intravitreal Injection of Palmitoylethanolamide, Citicoline, or Glial-Cell-Derived Neurotrophic Factor.

The treatment of posterior eye segment diseases through intravitreal injection requires repeated injections of an active molecule, which may be associated with serious side effects and poor patient compliance. One brilliant strategy to overcome these issues is the use of drug-loaded microparticles for sustained release, aiming at reducing the frequency of injections. Therefore, the aim of this work was to assess the safety features of poly(lactic-co-glycolic acid) (PLGA)-based, hyaluronic acid-decorated microparticles loaded with palmitoylethanolamide (PEA), citicoline (CIT), or glial-cell-derived neurotrophic factor (GDNF). Microparticles were prepared by double emulsion-solvent evaporation and fully characterized for their technological features. Microparticles possessed a satisfactory safety profile in vitro on human retinal pigment epithelial (ARPE-19) cells. Interestingly, the administration of free GDNF led to a loss of cell viability, while GDNF sustained release displayed a positive effect in that regard. In vivo results confirmed the safety profile of both empty and loaded microparticles. Overall, the outcomes suggest that the produced microparticles are promising for improving the local administration of neuroprotective molecules. Further studies will be devoted to assess the therapeutic ability of microparticles.

Comparing the Ability of MMPI-2 and MMPI-2-RF Validity Scales to Detect Feigning: A Meta-Analysis.

Several meta-analyses of the Minnesota Multiphasic Personality Inventory-2 (MMPI-2) and Minnesota Multiphasic Personality Inventory-2 Restructured Form (MMPI-2-RF) have examined these instruments' ability to detect symptom exaggeration or feigning. However, limited research has directly compared whether the scales across these two instruments are equally effective. This study used a moderated meta-analysis to compare 109 MMPI-2 and 41 MMPI-2-RF feigning studies, 83 (56.46%) of which were not included in previous meta-analyses. Although there were differences between the two test versions, with most MMPI-2 validity scales generating larger effect sizes than the corresponding MMPI-2-RF scales, these differences were not significant after controlling for study design and type of symptoms being feigned. Additional analyses showed that the F and Fp-r scales generated the largest effect sizes in identifying feigned psychiatric symptoms, while the FBS and RBS were better at detecting exaggerated medical symptoms. The findings indicate that the MMPI-2 validity scales and their MMPI-2-RF counterparts were similarly effective in differentiating genuine responders from those exaggerating or feigning psychiatric and medical symptoms. These results provide reassurance for the use of both the MMPI-2 and MMPI-2-RF in settings where symptom exaggeration or feigning is likely. Findings are discussed in the context of the recently released MMPI-3.

Identifying positive impression management using the MMPI-2 and the MMPI-2-RF: A meta-analysis.

Objective:Individuals may be motivated to conceal or minimize psychological symptoms and engage in positive impression management (PIM) in order to achieve desired outcomes across high stakes contexts (e.g. fitness for duty evaluations, pre-sentencing assessments, medical procedure evaluations, civil commitment). Despite the importance of this topic, the most recent meta-analysis of the MMPI-2, a widely used instrument for detecting PIM, was conducted more than two decades ago. Method:Using a Robust Variance Estimation method, this meta-analysis synthesized the results of 27 studies that examined the MMPI-2 (k = 22) and MMPI-2-RF (k = 5) validity scales' ability to discriminate individuals who engage in PIM from genuine responders, with a particular focus on the L, K, and S scales. Results:The MMPI-2 L scale produced the largest effect size (g = 1.30), whereas the MMPI-2-RF L-r scale effect size was moderate (g = 1.16). Moderate effect sizes were also found for the K (g = 1.01) and K-r (g = 1.21) scales, and for the MMPI-2 S scale (g = 1.23). Conclusions: Effect sizes did not significantly vary between the two versions of the MMPI. Findings suggest that both versions of the MMPI have demonstrated utility in identifying PIM, but clinicians should interpret T scores conservatively to account for the modest elevations associated with defensiveness. Findings are discussed in the context of the recently released MMPI-3.

Exploiting the DNA Damaging Activity of Liposomal Low Dose Cytarabine for Cancer Immunotherapy.

Perhaps the greatest limitation for the continually advancing developments in cancer immunotherapy remains the immunosuppressive tumor microenvironment (TME). The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) axis is an emerging immunotherapy target, with the resulting type I interferons and transcription factors acting at several levels in both tumor and immune cells for the generation of adaptive T cell responses. The cGAS-STING axis activation by therapeutic agents that induce DNA damage, such as certain chemotherapies, continues to be reported, highlighting the importance of the interplay of this signaling pathway and the DNA damage response in cancer immunity/immunotherapy. We have developed a multi-targeted mannosylated cationic liposomal immunomodulatory system (DS) which contains low doses of the chemotherapeutic cytarabine (Ara-C). In this work, we show that entrapment of non-cytotoxic doses of Ara-C within the DS improves its ability to induce DNA double strand breaks in human ovarian and colorectal cancer cell lines, as well as in various immune cells. Importantly, for the first time we demonstrate that the DNA damage induced by Ara-C/DS translates into cGAS-STING axis activation. We further demonstrate that Ara-C/DS-mediated DNA damage leads to upregulation of surface expression of immune ligands on cancer cells, coinciding with priming of cytotoxic lymphocytes as assessed using an ex vivo model of peripheral blood mononuclear cells from colorectal cancer patients, as well as an in vitro NK cell model. Overall, the results highlight a broad immunotherapeutic potential for Ara-C/DS by enhancing tumor-directed inflammatory responses.

Neuroprotective Effects of Transferrin in Experimental Glaucoma Models.

Iron is essential for retinal metabolism, but an excess of ferrous iron causes oxidative stress. In glaucomatous eyes, retinal ganglion cell (RGC) death has been associated with dysregulation of iron homeostasis. Transferrin (TF) is an endogenous iron transporter that controls ocular iron levels. Intraocular administration of TF is neuroprotective in various models of retinal degeneration, preventing iron overload and reducing iron-induced oxidative stress. Herein, we assessed the protective effects of TF on RGC survival, using ex vivo rat retinal explants exposed to iron, NMDA-induced excitotoxicity, or CoCl2-induced hypoxia, and an in vivo rat model of ocular hypertension (OHT). TF significantly preserved RGCs against FeSO4-induced toxicity, NMDA-induced excitotoxicity, and CoCl2-induced hypoxia. TF protected RGCs from apoptosis, ferroptosis, and necrosis. In OHT rats, TF reduced RGC loss by about 70% compared to vehicle-treated animals and preserved about 47% of the axons. Finally, increased iron staining was shown in the retina of a glaucoma patient's eye as compared to non-glaucomatous eyes. These results indicate that TF can interfere with different cell-death mechanisms involved in glaucoma pathogenesis and demonstrate the ability of TF to protect RGCs exposed to elevated IOP. Altogether, these results suggest that TF is a promising treatment against glaucoma neuropathy.

Markers of systemic inflammation are positively associated with influenza vaccine antibody responses with a possible role for ILT2(+)CD57(+) NK-cells.

BACKGROUND: With increasing age, overall health declines while systemic levels of inflammatory mediators tend to increase. Although the underlying mechanisms are poorly understood, there is a wealth of data suggesting that this so-called "inflammaging" contributes to the risk of adverse outcomes in older adults. We sought to determine whether markers of systemic inflammation were associated with antibody responses to the seasonal influenza vaccine. RESULTS: Over four seasons, hemagglutination inhibition antibody titres and ex vivo bulk peripheral blood mononuclear cell (PBMC) responses to live influenza viruses assessed via interferon (IFN)-γ/interleukin (IL)-10 production, were measured pre- and 4-weeks post-vaccination in young adults (n = 79) and older adults randomized to standard- or high-dose inactivated vaccine (n = 612). Circulating tumour necrosis factor (TNF), interleukin (IL)-6 and C-reactive protein (CRP) were also measured pre-vaccination. Post-vaccination antibody titres were significantly associated with systemic inflammatory levels; specifically, IL-6 was positively associated with A/H3N2 titres in young adults (Cohen's d = 0.36), and in older high-dose, but not standard-dose recipients, all systemic inflammatory mediators were positively associated with A/H1N1, A/H3N2 and B titres (d = 0.10-0.45). We further show that the frequency of ILT2(+)CD57(+) CD56-Dim natural killer (NK)-cells was positively associated with both plasma IL-6 and post-vaccination A/H3N2 titres in a follow-up cohort of older high-dose recipients (n = 63). Pathway analysis suggested that ILT2(+)CD57(+) Dim NK-cells mediated 40% of the association between IL-6 and A/H3N2 titres, which may be related to underlying participant frailty. CONCLUSIONS: In summary, our data suggest a complex relationship amongst influenza vaccine responses, systemic inflammation and NK-cell phenotype in older adults, which depends heavily on age, vaccine dose and possibly overall health status. While our results suggest that "inflammaging" may increase vaccine immunogenicity in older adults, it is yet to be determined whether this enhancement contributes to improved protection against influenza disease.

Comparative Analysis of Urso- and Tauroursodeoxycholic Acid Neuroprotective Effects on Retinal Degeneration Models.

Ursodeoxycholic (UDCA) and tauroursodeoxycholic (TUDCA) acids have shown neuroprotective properties in neurodegenerative diseases, but differential effects of the two bile acids have been poorly explored. The aim of this study was to evaluate the neuroprotective effects of UDCA versus TUDCA in a neuroretinal degeneration model and to compare transcriptionally regulated pathways. The WERI-Rb-1 human cone-like cell line and retinal explants were exposed to albumin and TUDCA or UDCA. Viability, cell death, and microglial activation were quantified. Transcriptionally regulated pathways were analyzed after RNA sequencing using the edgeR bioconductor package. Pre-treatment of cone-like cells with UDCA or TUDCA significantly protected cells from albumin toxicity. On retinal explants, either bile acid reduced apoptosis, necroptosis, and microglia activation at 6 h. TUDCA induced the regulation of 463 genes, whilst 31 genes were regulated by UDCA. Only nineteen common genes were regulated by both bile acids, mainly involved in iron control, cell death, oxidative stress, and cell metabolism. As compared to UDCA, TUDCA up-regulated genes involved in endoplasmic reticulum stress pathways and down-regulated genes involved in axonal and neuronal development. Either bile acid protected against albumin-induced cell loss. However, TUDCA regulated substantially more neuroprotective genes than UDCA.

Understanding the Victimization-Delinquency Overlap Among Latino Adolescents: The Role of Cultural and Mental Health Factors.

The aim of this analysis was to evaluate whether cultural factors and mental health variables distinguish Latino youth who are delinquent-victims, primarily victims, or primarily delinquent. This study used data from the Dating Violence among Latino Adolescents (DAVILA) Study, which surveyed 1,525 Latino youth and queried participants about past year victimization, delinquency, psychological distress, and cultural factors. Using multinomial logistic regression, we evaluated whether these variables differentiated youth who were delinquent-victims, primarily victims, primarily delinquent, or neither delinquent nor victims. Results suggest that delinquent-victim Latino youth are differentiated from other groups primarily by the degree of familial support and anger/hostility. Other cultural and mental health variables do not appear to differentiate the groups, suggesting a greater degree of similarity among them based on the variables used in the analysis. The results indicate that Latino youth that are victimized and engage in delinquent behavior are primarily differentiated by the degree of anger/hostility they experience. Familial support, as has been seen with non-Latino groups, appears to present a significant protective quality and likely can serve as a prevention strategy, particularly for delinquent-victim youth. Study limitations include challenges with retrospective self-report and sampling using RDD methodologies.

NK- and T-cell granzyme B and K expression correlates with age, CMV infection and influenza vaccine-induced antibody titres in older adults.

Granzymes are a family of serine-proteases that act as critical mediators in the cytolytic and immunomodulatory activities of immune cells such as CD8+ T-cells and natural killer (NK) cells. Previous work indicates that both granzyme B (GZB) and K (GZK) are increased with age in CD8+ T-cells, and in the case of GZB, contribute to dysfunctional immune processes observed in older adults. Here, we sought to determine how GZB and GZK expression in NK-cells, and CD4+, CD8+, and gamma-delta T-cells, quantified in terms of positive cell frequency and mean fluorescence intensity (MFI), differed with age, age-related health-traits and the antibody response to high-dose influenza vaccine. We found that the frequency and MFI of GZB-expressing NK-cells, and CD8+ and Vδ1+ T-cells, and GZK-expressing CD8+ T-cells was significantly higher in older (66-97 years old; n = 75) vs. younger (24-37 years old; n = 10) adults by up to 5-fold. There were no significant associations of GZB/GZK expression with sex, frailty or plasma levels of TNF or IL-6 in older adults, but those who were seropositive for cytomegalovirus (CMV) exhibited significantly higher frequencies of GZB+ NK-cells, and CD4+, CD8+ and Vδ1+ T-cells, and GZK+ CD8+ T-cells (Cohen's d = .5-1.5). Pre-vaccination frequencies of GZB+ NK-cells were positively correlated with vaccine antibody responses against A/H3N2 (d = .17), while the frequencies of GZK+ NK and CD8+ T-cells were inversely associated with A/H1N1 (d = -0.18 to -0.20). Interestingly, GZK+ NK-cell frequency was inversely correlated with pre-vaccination A/H1N1 antibody titres, as well as those measured over the previous 4 years, further supporting a role for this subset in influencing vaccine antibody-responses. These findings further our understanding of how granzyme expression in different lymphoid cell-types may change with age, while suggesting that they influence vaccine responsiveness in older adults.

The impact of coaching on feigned psychiatric and medical symptoms: A meta-analysis using the MMPI-2.

Coaching individuals on test-taking strategies presents legal and ethical concerns and threatens the validity of psychological test score interpretations, which could lead to inaccuracies in clinical settings and injustices within the legal system. This meta-analysis examined the impact of coaching on the detection of symptom exaggeration or feigning on the MMPI-2. A total of 99 feigning studies (N = 19,536) comparing validity subscale scores between genuine and nongenuine (coached or non-coached) responders were analyzed. Potential moderating effects of control group, type of symptoms, publication status, financial incentive, and non-content validity screening were also examined regarding their impact on the effectiveness of coaching. Findings suggested that detection-based coaching (i.e., coaching regarding the presence of validity scales and detection avoidance strategies within the MMPI-2) improved individuals' ability to elude detection by the MMPI-2 validity scales. Nonetheless, the MMPI-2 validity scales still generated moderate to very large effect sizes in detecting symptom exaggeration or feigning even in the context of coaching (range g = .89 to 1.95). The findings provide reassurance for detection efforts, indicating that while the effectiveness of the MMPI-2 is somewhat diminished, it remains useful in detecting non-genuine responders even in the context of coaching. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Long-Term Oral Treatment with Non-Hypoglycemic Dose of Glibenclamide Reduces Diabetic Retinopathy Damage in the Goto-KakizakiRat Model.

Diabetic retinopathy (DR) remains a major cause of vision loss, due to macular edema, retinal ischemia and death of retinal neurons. We previously demonstrated that acute administration of glibenclamide into the vitreous, or given orally at a non-hypoglycemic dose, protected the structure and the function of the retina in three animal models that each mimic aspects of diabetic retinopathy in humans. In this pilot study, we investigated whether one year of chronic oral glibenclamide, in a non-hypoglycemic regimen (Amglidia®, 0.4 mg/kg, Ammtek/Nordic Pharma, 5 d/week), could alleviate the retinopathy that develops in the Goto-Kakizaki (GK) rat. In vivo, retinal function was assessed by electroretinography (ERG), retinal thickness by optical coherence tomography (OCT) and retinal perfusion by fluorescein and indocyanin green angiographies. The integrity of the retinal pigment epithelium (RPE) that constitutes the outer retinal barrier was evaluated by quantitative analysis of the RPE morphology on flat-mounted fundus ex vivo. Oral glibenclamide did not significantly reduce the Hb1Ac levels but still improved retinal function, as witnessed by the reduction in scotopic implicit times, limited diabetes-induced neuroretinal thickening and the extension of ischemic areas, and it improved the capillary coverage. These results indicate that low doses of oral glibenclamide could still be beneficial for the prevention of type 2 diabetic retinopathy. Whether the retinas ofpatients treated specifically with glibenclamideare less at risk of developing diabetic complications remains to be demonstrated.

Novel immunomodulatory properties of low dose cytarabine entrapped in a mannosylated cationic liposome.

Cancer treatment remains unsatisfactory with high rates of recurrence and metastasis. Immunomodulatory agents capable of promoting cellular antitumor immunity while inhibiting the local immunosuppressive tumor microenvironment could greatly improve cancer treatment. We have developed a multi-targeted mannosylated cationic liposome delivery system containing muramyl dipeptide (DS) and low doses of the chemotherapeutic agent cytarabine (Ara-C). Immunomodulation of primary immune cells and immortalized cancer cell lines by Ara-C/DS was assessed by measuring cytokine levels and surface marker expression. As a proof of concept, the generation of targeted cellular immunity was investigated in the context of responses to viral antigens. This report is the first demonstrating that Ara-C combined with DS can modulate immune responses and revert immunosuppression as evidenced by increased IFN-γ and IL-12p40 without changes in IL-10 in peripheral blood mononuclear cells, and increased CD80 and decreased CD163 on immunosuppressive macrophages. Furthermore, Ara-C/DS increased MHC class I expression on cancer cells while increasing the production of antigen-specific IFN-γ+ CD8+ T cells in viral peptide-challenged lymphocytes from both humans and vaccinated mice. Taken together, these results are the first to document immunomodulatory properties of Ara-C linked with recognition of antigens and potentially the generation of antitumor immune memory.

Oral Ursodeoxycholic Acid Crosses the Blood Retinal Barrier in Patients with Retinal Detachment and Protects Against Retinal Degeneration in an Ex Vivo Model.

Rhegmatogenous retinal detachment (RD) is a threatening visual condition and a human disease model for retinal degenerations. Despite successful reattachment surgery, vision does not fully recover, due to subretinal fluid accumulation and subsequent photoreceptor cell death, through mechanisms that recapitulate those of retinal degenerative diseases. Hydrophilic bile acids are neuroprotective in animal models, but whether they can be used orally for retinal diseases is unknown. Ursodeoxycholic acid (UDCA) being approved for clinical use (e.g., in cholestasis), we have evaluated the ocular bioavailability of oral UDCA, administered to patients before RD surgery. The level of UDCA in ocular media correlated with the extent of blood retinal barrier disruption, evaluated by the extent of detachment and the albumin concentration in subretinal fluid. UDCA, at levels measured in ocular media, protected photoreceptors from apoptosis and necrosis in rat retinal explants, an ex vivo model of RD. The subretinal fluid from UDCA-treated patients, collected during surgery, significantly protected rat retinal explants from cell death, when compared to subretinal fluid from control patients. Pan-transcriptomic analysis of the retina showed that UDCA upregulated anti-apoptotic, anti-oxidant, and anti-inflammatory genes. Oral UDCA is a potential neuroprotective adjuvant therapy in RD and other retinal degenerative diseases and should be further evaluated in a clinical trial.

MDSCs, ageing and inflammageing.

The challenge of distinguishing between changes attributable to ageing and those attributable to pathology is even greater for the immune system than for many other organs, and this is especially true for myeloid-derived suppressor cells (MDSCs). Hematopoiesis is different in older adults with a bias towards myelopoiesis, and older adults also manifest "inflammageing" exacerbated by disease and contributing to MDSC induction. Hence, at least in humans, one can only investigate MDSCs in the context of ageing and disease states, and not in the context of ageing processes per se. This contribution provides a brief overview of the literature on MDSCs and ageing in humans.

How Clinicians Incorporate Suicide Risk Factors Into Suicide Risk Assessment.

Background: The literature has focused on explicit communications of suicidal ideation, although such communications are infrequent and less overt symptoms are comparable indicators of suicide risk. Aims: Our aim was to understand how clinicians consider inexplicit suicide risk factors in assessments of risk. Method: A national sample of 75 psychiatric crisis clinicians were provided with three vignettes, which varied in nine variables related to suicide risk. Clinicians rated imminent suicide risk and identified an appropriate level of care for each hypothetical patient. Results: Prior suicide attempt, intent to die, presence of a suicide plan, desire to die, hopelessness, burdensomeness, and passive suicidal ideation (defined as a combination of hopelessness, burdensomeness, desire to die, and anhedonia) significantly impacted risk perception while depression and anhedonia did not. Level of care was significantly associated with passive suicidal ideation, suicide plan, desire to die, and hopelessness. Limitations: Limitations of the study include its small sample size, experienced clinicians, and reliance on hypothetical patients. The study design did not allow for all variables to be compared against one another. Conclusion: Clinicians considered less overt risk factors for suicide when assessing risk. Future research should examine whether this knowledge is applied in real-life scenarios and if less experienced clinicians equally consider these risk factors.