An Experimental Protocol for the Boyden Chamber Invasion Assay With Absorbance Readout.

The phenomenon of cell invasion is an essential step in angiogenesis, embryonic development, immune responses, and cancer metastasis. In the course of cancer progression, the ability of neoplastic cells to degrade the basement membrane and penetrate neighboring tissue (or blood vessels and lymph nodes) is an early event of the metastatic cascade. The Boyden chamber assay is one of the most prevalent methods implemented to measure the pro- or anti-invasive effects of drugs, investigate signaling pathways that modulate cell invasion, and characterize the role of extracellular matrix proteins in metastasis. However, the traditional protocol of the Boyden chamber assay has some technical challenges and limitations. One such challenge is that the endpoint of the assay involves photographing and counting stained cells (in multiple fields) on porous filters. This process is very arduous, requires multiple observers, and is very time-consuming. Our improved protocol for the Boyden chamber assay involves lysis of the dye-stained cells and reading the absorbance using an ELISA reader to mitigate this challenge. We believe that our improved Boyden chamber methodology offers a standardized, high-throughput format to evaluate the efficacy of various drugs and test compounds in influencing cellular invasion in normal and diseased states. We believe that our protocol will be useful for researchers working in the fields of immunology, vascular biology, drug discovery, cancer biology, and developmental biology. Key features • Measurement of tumor invasion using human cancer cells. • Ability to measure the pro-invasive/anti-invasive activity of small molecules and biological modifiers. • Measurement of chemotaxis, chemokines, trafficking of immune cells, and proteolytic activity of matrix metalloproteinases, lysosomal hydrolysates, collagenases, and plasminogen activators in physiological and pathological conditions. • Investigation of the role of extracellular matrix proteins in the crosstalk between endothelial, epithelial, muscle, or neuronal cells and their adjacent stroma.

Treatment patterns in patients with newly diagnosed COPD in the USA.

BACKGROUND: Prompt and effective management with maintenance therapy (single or dual bronchodilator therapy) is recommended after the initial diagnosis of chronic obstructive pulmonary disease (COPD) to maintain lung function and prevent exacerbations. Contrary to guideline-based recommendations, most patients are not prescribed maintenance treatment at initial diagnosis. The current study assessed the pharmacologic treatment patterns and outcomes of newly diagnosed patients with COPD in the USA. METHODS: This retrospective, noninterventional study used de-identified data from the Inovalon Insights' database (Commercial, Medicaid Managed Care, and Medicare Advantage-insured individuals) between January 1, 2015, and December 31, 2021. The "patient journey" from initial diagnosis was followed over a 4-year period. The primary outcome measure was the number of moderate or severe exacerbations. Secondary outcome measures included the cumulative incidence of exacerbations, mean cumulative count of moderate and severe exacerbations, rates of moderate and severe exacerbations in patients who remained untreated after diagnosis in 12-month time periods for 4 years, sociodemographic and clinical characteristics, and pharmacologic treatment patterns. RESULTS: The cohort consisted of 238,158 newly diagnosed patients with COPD (female [52.9%]; mean age 63.8 years). The majority of patients with COPD had Medicaid as their primary insurance (46.2%). Overall, during the 4-year follow-up period, 32.9% of the patients had at least one moderate or severe exacerbation, and 25.8% and 13.8% experienced moderate and severe exacerbations, respectively. At diagnosis, 86.2% of the patients were untreated and most remained untreated by the end of the follow-up (63.8%). Most patients (62.0%) received long-acting beta-agonist (LABA)/inhaled corticosteroids (ICS) as their initial treatment at diagnosis, and LABA/ICS continued to be the most common initial treatment during the 4-year period (64.0% at year 1; 58.0% at year 4). CONCLUSIONS: Most patients with COPD were not treated at initial diagnosis and remained untreated during follow-up. Our data highlight a lack of adherence to recommendations for clinical practice.

An Improved Protocol for the Matrigel Duplex Assay: A Method to Measure Retinal Angiogenesis.

Neovascular diseases of the retina, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD), are proliferative retinopathies involving the growth of new blood vessels on the retina, which in turn causes impairment and potential loss of vision. A drawback of conventional angiogenesis assays is that they are not representative of the angiogenic processes in the retina. In the retina, the new blood vessels grow (from pre-existing blood vessels) and migrate into a non-perfused region of the eye including the inner limiting membrane of the retina and the vitreous, both of which contribute to vision loss. The Matrigel Duplex Assay (MDA) measures the migration of angiogenic capillaries from a primary Matrigel layer to a secondary Matrigel layer, which resembles the pathological angiogenesis in AMD and DR. The methodology of MDA is comprised of two steps. In the first step, the human retinal microvascular endothelial cells (HRMECs) are mixed with phenol red-containing Matrigel (in a 1:1 ratio) and seeded in the center of an 8-well chamber slide. After 24 h, a second layer of phenol red-free Matrigel is overlaid over the first layer. Over the course of the next 24 h, the HRMECs invade from the primary Matrigel layer to the secondary layer. Subsequently, the angiogenic sprouts are visualized by brightfield phase contrast microscopy and quantified by ImageJ software. The present manuscript measures the angiogenesis-inhibitory activity of the Src kinase inhibitor PP2 in primary HRMECs using the MDA. The MDA may be used for multiple applications like screening anti-angiogenic drugs, measuring the pro-angiogenic activity of growth factors, and elucidating signaling pathways underlying retinal angiogenesis in normal and disease states.

The burden of anxiety among a nationally representative US adult population.

BACKGROUND: Anxiety disorders are highly prevalent and are associated with a significant humanistic and economic burden. This study evaluates the impact of anxiety symptoms on direct and indirect costs and quality of life in individuals with self-reported and unrecognized anxiety symptoms. METHODS: The 2019 US National Health and Wellness Survey database was analyzed to compare individuals with anxiety symptoms to individuals without symptoms, stratified by responses to a yes/no question about experiencing anxiety symptoms, and further stratified by severity of symptoms based on GAD-7 scores. Individuals who responded 'yes' were characterized as having self-reported anxiety symptoms, and those who responded 'no' were screened for unrecognized anxiety symptoms. RESULTS: Overall, 44.0 % of the population experienced anxiety symptoms, of which 32.5 % self-reported experiencing anxiety, while an additional 11.5 % had mild to severe symptoms but did not self-identify as having anxiety. Both groups experienced significantly worse quality of life, and higher direct and indirect costs than a control group who had no anxiety symptoms (GAD-7 ≤ 4). Individuals with more severe anxiety symptoms experienced worse outcomes. LIMITATIONS: The data were cross-sectional, so causality could not be determined. Outcomes were based on self-report, and are therefore subject to reporting and recall bias. Prevalence and severity were assessed using the GAD-7, and not clinically validated. CONCLUSION: A substantial proportion of the population experiences anxiety symptoms without recognizing it. Anxiety symptoms had a significant impact on quality of life, direct costs, and indirect costs, representing a considerable burden that increased with severity of illness.

The economic and humanistic burden of bipolar disorder in adults in the United States.

BACKGROUND: Bipolar disorder is associated with functional impairment and diminished health-related quality of life (HRQoL). The purpose of this study was to estimate the annual per patient direct healthcare costs, indirect costs, and HRQoL of patients with bipolar disorder by depressive symptom severity and overall compared to the general population in the US. METHODS: This cross-sectional study used self-reported data from the 2020 US National Health and Wellness Survey. Adult respondents who reported bipolar disorder symptoms in the past 12 months and/or a diagnosis of bipolar disorder were identified (bipolar disorder cohort) and were further classified by depressive symptom severity based on Patient Health Questionnaire (PHQ-9) scores (none/mild = 0-9, moderate = 10-14, severe = 15-27). Annualized direct healthcare costs and indirect costs were calculated from 6-month healthcare resource utilization and work productivity, respectively. A general population cohort was constructed using 2:1 propensity score matching. Multivariate regression models of all-cause hospitalizations in the past 6 months, annualized direct healthcare costs, annualized indirect costs, and HRQoL (eg, EuroQol 5-Dimension Health Questionnaire (EQ-5D)) controlled for confounders (demographic and clinical characteristics). RESULTS: Of 3583 adults meeting pre-specified criteria for bipolar disorder, 1401 (39.1%) reported none/mild, 889 (24.8%) moderate, and 1293 (36.1%) severe depressive symptom severity. Additionally, 3285 (91.7%) were matched to 6570 adults in the general population. Compared to the general population, adjusted mean hospitalizations (0.53 vs. 0.30), annualized per patient direct healthcare costs ($20,846 vs. $11,391), and indirect costs ($14,795 vs. $9274) were significantly greater for the bipolar disorder cohort (all p < 0.001); adjusted HRQoL (EQ-5D: 0.69 vs. 0.79) was significantly worse (p < 0.001). By depressive symptom severity, adjusted mean hospitalizations (none/mild = 0.30, moderate = 0.50, severe = 0.46), direct healthcare costs ($14,389, $22,302, $21,341), and indirect costs ($10,799, $17,109, $18,470) were significantly greater for moderate and severe compared to none/mild depressive symptom severity (all p < 0.01); adjusted HRQoL (EQ-5D: 0.77, 0.67, 0.59) was significantly worse (p < 0.001). CONCLUSIONS: Among respondents with bipolar disorder, those with moderate to severe depression had greater direct healthcare costs and indirect costs as well as worse HRQoL than those with mild or no depressive symptoms. Treatment targeting reduction in depressive symptoms may reduce the economic and humanistic burden of bipolar disorder.

The Associations between Insomnia Severity and Health Outcomes in the United States.

Little is known about the associations between insomnia severity, insomnia symptoms, and key health outcomes. Using 2020 United States National Health and Wellness Survey (NHWS) data, we conducted a retrospective, cross-sectional analysis to determine the associations between insomnia severity and a number of health outcomes germane to patients (health-related quality of life (HRQoL), employers and government (workplace productivity), and healthcare payers (healthcare resource utilization (HCRU)). The Insomnia Severity Index (ISI) questionnaire was used to evaluate overall insomnia severity. HRQoL was assessed using the physical and mental component summary scores of the Short Form-36v2 (SF-36v2) questionnaire, and health utility status was measured using the Short Form-6D (SF-6D) and EuroQoL-5D (EQ-5D) questionnaires. Workplace productivity was measured using the Work Productivity and Activity Impairment (WPAI) questionnaire. After adjusting for confounders, greater insomnia severity was significantly associated with worsened quality of life, decreased productivity, and increased HCRU in an apparent linear fashion. These findings have important implications for future research, including the need for specific assessment of insomnia symptoms and their impact on key health outcomes.

Anticancer Activity of Region B Capsaicin Analogs.

The heterocyclic vanilloid compound capsaicin is responsible for the spicy and pungent flavor of chili peppers. Several convergent studies have shown that capsaicin suppresses the growth of multiple human cancers. Apart from capsaicin, natural and synthetic capsaicin-like compounds display growth suppressive activity in human cancers. The pharmacophore of capsaicin is comprised of three regions, namely region A (the aromatic ring), region B (the amide bond), and region C (the side chain). The present manuscript describes the isolation and synthesis of capsaicin analogs which have structural modifications in region B of the molecule. Furthermore, the pharmacokinetic properties, anticancer activity of region B capsaicin analogs, as well as the signaling pathways (underlying the growth-inhibitory effects of region B capsaicin analogs) have also been described. The discovery of novel, second-generation region B capsaicin analogs may foster the hope of innovative nutrition-based combination therapies in human cancers.

Mapping the Insomnia Severity Index Instrument to EQ-5D Health State Utilities: A United Kingdom Perspective.

OBJECTIVE: This study aimed to map the Insomnia Severity Index (ISI) to the EQ-5D-3L utility values from a UK perspective. METHODS: Source data were derived from the 2020 National Health and Wellness Survey (NHWS) for France, Germany, Italy, Spain, the UK and the US. Ordinary least squares regression, generalised linear model (GLM), censored least absolute deviation, and adjusted limited dependent variable mixture model (ALDVMM) were employed to explore the relationship between ISI total summary score and EQ-5D utility while accounting for adjustment covariates derived from the NHWS. Fitting performance was assessed using standard metrics, including mean-squared error (MSE) and coefficient of determination (R2). RESULTS: A total of 17,955 respondent observations were included, with a mean ISI score of 12.12 ± 5.32 and a mean EQ-5D-3L utility (UK tariff) of 0.71 ± 0.23. GLM gamma-log and ALDVMM were the two best performing models. The ALDVMM had better fitting performance (R2 = 0.320, MSE 0.0347) than the GLM gamma-log (R2 = 0.303, MSE 0.0353); in train-test split-sample validation, ALDVMM also slightly outperformed the GLM gamma-log model, with an MSE of 0.0351 versus 0.0355. Based on fitting performance, ALDVMM and GLM gamma-log were the preferred models. CONCLUSIONS: In the absence of preference-based measures, this study provides an updated mapping algorithm for estimating EQ-5D-3L utilities from the ISI summary total score. This new mapping not only draws its strengths from the use of a large international dataset but also the incorporation of adjustment variables (including sociodemographic and general health characteristics) to reduce the effects of confounders.