Evaluation of resazurin phenoxazine dye as a highly sensitive cell viability potency assay for natural killer cell-derived extracellular vesicle-based cancer biotherapeutics.

Natural killer cell-derived extracellular vesicles (NK-EVs) are candidate biotherapeutics against various cancers. However, standardised potency assays are necessary for a reliable assessment of NK-EVs' cytotoxicity. This study aims to thoroughly evaluate a highly sensitive resazurin phenoxazine-based cell viability potency assay (measurement of the cellular redox metabolism) for quantifying the cytotoxicity of NK-EVs against leukaemia K562 cells (suspension model) and breast cancer MDA-MB-231 cells (adherent model) in vitro. The assay was evaluated based on common analytical parameters setforth by regulatory guidelines, including specificity, selectivity,accuracy, precision, linearity, range and stability. Our results revealed that this resazurin-based cell viability potency assay reliably and reproducibly measured a dose-response of NK-EVs' cytotoxic activity against both cancer models. The assay showed precision with 5% and 20% variation for intra-run and inter-run variability. The assay signal showed specificity and selectivity of NK-EVs against cancer target cells, as evidenced by the diminished viability of cancer cells following a 5-hour treatment with NK-EVs, without any detectable interference or background. The linearity analysis of target cancer cells revealed strong linearity for densities of 5000 K562 and 1000 MDA-MB-231 cells per test with a consistent range. Importantly, NK-EVs' dose-response for cytotoxicity showed a strong correlation (|ρ| ∼ 0.8) with the levels of known cytotoxic factors associated with the NK-EVs' corona (FasL, GNLY, GzmB, PFN and IFN-γ), thereby validating the accuracy of the assay. The assay also distinguished cytotoxicity changes in degraded NK-EVs, indicating the ability of the assay to detect the potential loss of sample integrity. Compared to other commonly reported bioassays (i.e., flow cytometry, cell counting, lactate dehydrogenase release assay, DNA-binding reporter assay and confluence assay), our results support this highly sensitive resazurin-based viability potency assay as a high-throughput and quantitative method for assessing NK-EVs' cytotoxicity against both suspension and adherent cancer models for evaluating NK-EVs' biotherapeutics.

A summary of the U.S. Marine Recruit Assessment Program (RAP) procedures and survey from 2003 to 2021

The Recruit Assessment Program (RAP) is a cross-sectional, baseline survey of U.S. Marine recruits administered at Marine Corps Recruit Depot, San Diego. This report presents RAP study procedures and survey content that was administered to 229,015 participants between 2003 and 2021. Self-reported data were collected on recruit demographics, physical and mental health, adverse life experiences, lifestyle and risky behaviors, and substance use. In 2013, the survey was updated to remove questions with other linkable and reliable sources and those with low completion rates and low relevance to Marine health research; the removal of these items allowed for the addition of instrument measures for major depression, post-traumatic stress disorder, anger, and resilience with no significant change to overall survey length. Average completion rates are approximately 95%. Multiple studies have shown the utility of RAP data collected thus far as a robust data repository of pre-service health and behavioral measures.

Culturally competent social robots target inclusion in Africa.

Embedding culturally sensitive body, hand, and facial gestures in social robots will make them more acceptable in Africa.

Pre-service factors associated with sexual misconduct among male U.S. Marines.

PURPOSE: Sexual assault is a prevalent and persistent problem in the military, yet few studies have examined predictors of sexual offenses. The study aim was to determine pre-service factors associated with sexual offense conviction among U.S. Marines. METHODS: This retrospective cohort study analyzed data from male active duty U.S. Marines (2003-2018). Pre-service factors were assessed using survey data from the Recruit Assessment Program, obtained prior to recruit training at the Marine Corps Recruit Depot, San Diego, California. These survey data were linked with sexual offense conviction data obtained from the Naval Criminal Investigative Service Consolidated Law Enforcement Operations Center. RESULTS: Of the 146,307 participants, the majority were 18-19 years old (66.7%) and non-Hispanic, White (62.1%) with a high school education or less (76.8%); 107 received convictions for a sexual offense. In unadjusted analyses, race and ethnicity, parental education, type of primary caregiver, parental death, family economic status, childhood emotional trauma, childhood physical abuse, childhood sexual abuse, and unprotected sex were associated with a sexual offense conviction. In the final multivariable model, race and ethnicity (American Indian/Alaskan Native, odds ratio [OR]: 5.28, 95% confidence interval [CI]: 1.86-14.98; Hispanic, OR: 1.83, 95% CI: 1.06-3.18; multiracial/other, OR: 3.28, 95% CI: 1.56-6.89), education (≤ high school, OR: 2.65; 95% CI: 1.21-5.80), parental death (OR: 2.27; 95% CI: 1.16-4.45), unprotected sex (OR: 1.78; 95% CI: 1.03-3.05), and school suspension/expulsion (OR: 1.64; 95% CI: 1.02-2.65) were significant predictors of a subsequent sexual offense conviction. CONCLUSIONS: Results underscore the importance of understanding factors associated with sexual offense and highlight the large discrepancy between self-reported estimates of sexual assault and sexual offense convictions. Findings may inform the development of effective strategies to reduce sexual misconduct, such as technology-facilitated programs that provide private, targeted education; supportive assistance; and prevention materials to individuals who may have elevated sexual misconduct risk.

Assessing cognition in people with severe mental disorders in low- and middle-income countries: a systematic review of assessment measures.

BACKGROUND: Cognitive difficulties are common in people with severe mental disorders (SMDs) and various measures of cognition are of proven validity. However, there is a lack of systematic evidence regarding the psychometric properties of these measures in low- and middle-income countries (LMICs). OBJECTIVE: To systematically review the psychometric properties of cognitive measures validated in people with SMDs in LMICs. METHODS: We conducted a systematic review of the literature by searching from four electronic databases. Two authors independently screened studies for their eligibility. Measurement properties of measures in all included studies were extracted. All eligible measures were assessed against criteria set for clinical and research recommendations. Results are summarized narratively and measures were grouped by measurement type and population. RESULTS: We identified 23 unique measures from 28 studies. None of these was from low-income settings. Seventeen of the measures were performance-based. The majority (n = 16/23) of the measures were validated in people with schizophrenia. The most commonly reported measurement properties were: known group, convergent, and divergent validity (n = 25/28). For most psychometric property, studies of methodological qualities were found to be doubtful. Among measures evaluated in people with schizophrenia, Brief Assessment of Cognition in Schizophrenia, Cognitive Assessment Interview, MATRICS Consensus Cognitive Battery, and CogState Schizophrenia Battery were with the highest scores for clinical and research recommendation. CONCLUSIONS: Studies included in our review provide only limited quality evidence and future studies should consider adapting and validating measures using stronger designs and methods. Nonetheless, validated assessments of cognition could help in the management and allocating therapy in people with SMDs in LMICs.

Anti-CD52 blocks EAE independent of PD-1 signals and promotes repopulation dominated by double-negative T cells and newly generated T and B cells.

Lymphocyte depletion using anti-CD52 antibody effectively reduces relapses of multiple sclerosis (MS). To begin to understand what mechanisms might control this outcome, we examined the effect of a murine-CD52-specific mAb on the depletion and repopulation of immune cells in mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. We tested whether the tolerance-promoting receptor programmed cell death protein-1 (PD-1) is required for disease remission post anti-CD52, and found that PD-1-deficient mice with a more severe EAE were nevertheless effectively treated with anti-CD52. Anti-CD52 increased the proportions of newly generated T cells and double-negative (DN) T cells while reducing newly generated B cells; the latter effect being associated with a higher expression of CD52 by these cells. In the longer term, anti-CD52 caused substantial increases in the proportion of newly generated lymphocytes and DN T cells in mice with EAE. Thus, the rapid repopulation of lymphocytes from central lymphoid organs post anti-CD52 may limit further disease. Furthermore, these data identify DN T cells, a subset with immunoregulatory potential, as a significant hyperrepopulating subset following CD52-mediated depletion.

Alcohol use disorder among prisoners in Debre Berhan prison, Ethiopia: a cross-sectional study.

BACKGROUND: Several studies reported that history of alcohol use among prisoners is higher than the prevalence in the general population. Criminality is found to be associated with alcohol use disorder (AUD) in previous studies. In Ethiopia, there is limited information on the prevalence and associated factors of AUD among prisoners. Therefore, this study aimed to assess the prevalence and associated factors of AUD among prisoners of Debre Berhan Prison. METHODS: A cross-sectional survey was conducted to assess history of AUD among prisoners at Debre Berhan Prison, before imprisonment. We selected 347 prisoners with a systematic sampling technique and interviewed using Alcohol Use Disorder Identification Test (AUDIT) to screen for AUD in May 2017. Data entry was done using Epi-Data version 3.1 software, and bivariate and multivariate analyses were done using Stata version 13 software. Crude and adjusted odds ratios, with 95% confidence intervals and p-values are reported. RESULTS: About six out of ten prisoners (59.1%) had AUD before imprisonment. Factors associated with increased odds of AUD were perception that the current offence is related to using substances (AOR = 4.2; 95% CI = 2.3, 7.8), and family history of substance use (AOR = 8.7; 95% CI = 1.7, 44.9). Being married had lower odds of AUD compared to the unmarried (AOR = 0.5; 95% CI = 0.2, 0.9). CONCLUSION: We found that the prevalence of AUD 1 year before imprisonment in this population is high. AUD is found to be associated with a family history of substance use and perception that the current offence is related to using a substance. We recommend community-based study with different kind of study designs to see the relationship between AUD and crime for planning interventions.

Prevalence and associated factors of burnout among Debre Berhan University medical students: a cross-sectional study.

BACKGROUND: Burnout, a measure of professional distress, is more common among medical professionals. About half of medical students have this problem. However, little is known about the burnout status of medical students in Ethiopia. Therefore, the aim of this study was to assess the prevalence and associated factors of burnout among medical students of Debre Berhan University (DBU). METHODS: A cross-sectional study was conducted on randomly selected 151 medical students of DBU. Burnout was assessed using the Maslach Burnout Inventory-Human Services Survey (MBI-HSS). Participants were reported as having burnout if they scored ≥27 on Emotional Exhaustion (EE), ≥13 on Depersonalization (DP) sub-scales, and ≤ 31 on Personal Accomplishment (PA) sub-scale of the MBI-HSS. EpiData version 3.1 was used for data entry while SPSS version 20 and STATA version 13 for windows were used for data analysis. Both univariable and multivariable binary logistic regression analyses were conducted. The degree of association between variables was assessed using odds ratio (OR) with 95% confidence interval (CI) at two-tailed p-value of < 0.05. RESULT: Of 144 medical students took part, 34.0% had symptoms of burnout. Regarding domains of burnout, 61.8% scored high on EE, 47.9% scored high on DP and 59.7% scored low on PA. Dissatisfaction with practice lecturer (AOR = 3.8, 95% CI (1.3, 11.6)), moderate social support (AOR = 0.2, 95% CI (0.1, 0.8)), and satisfaction with their education (AOR = 0.1 95% CI (0.0, 0.7)) were associated with burnout. CONCLUSION: More than one-third of medical students at DBU had burnout. Individual and organizational level interventions targeting students who had poor social support, dissatisfied by their lecturer at the hospitals and their education are recommended.

Factors Affecting Psychological Distress among People Living with HIV/AIDS at Selected Hospitals of North Shewa Zone, Amhara Region, Ethiopia.

BACKGROUND: The new advances for the treatment of HIV infection using Highly Active Antiretroviral Therapy (HAART) have dramatically improved disease prognosis. However, they are living longer with a chronic condition that increases the risk for psychiatric and psychosocial problems. Various studies have linked HIV/AIDS with a number of psychological problems, depression being the most common. Moreover, studies have found that chronically ill people are at increased risk of psychological problems. Thus, this study aimed at assessing the level of psychological distress and its associated factors among people living with HIV/AIDS in selected Hospitals of North Sowa Zone of Amhara region, Ethiopia, 2017. METHOD: Institution based cross-sectional study design with systematic random sampling method was used. Data was collected by structured interviewer-based Amharic version questionnaire. A total of 422 people living with HIV/AIDS were involved in the study from 1 to 30 May 2017. Data analysis was done with the help of a computer program (SPSS version 16.0). Binary logistic regression analysis was used for bivariate and multivariate analysis. The strength of the association was presented by odds ratio with a 95% confidence interval. RESULT: The prevalence of psychological distress was 7.8% (95% CI: 5.25%, 10.39%). Being female (AOR = 3.02; 95% CI: 1.16, 7.82), illiterates (AOR = 3.91; 95% CI: 1.31, 6.45), participants who currently use alcohol (AOR = 2.70; 95% CI: 1.23, 5.88), respondents whose CD4 count is less than 500 cells/µl (AOR = 2.28; 95% CI: 1.02, 5.11), and participants who are considered stigmatized (AOR = 2.41; 95% CI: 1.11, 5.22) were positively associated with psychological distress. CONCLUSION: The prevalence of psychological distress was low as compared to other studies conducted in Ethiopia. This may affect the quality of life of people living with HIV/AIDS and their families. Being female, illiteracy, alcohol use, and having lower CD4 count and perceived stigma increased the odds of psychological distress. Thus, concerned stakeholders should collaborate on the integration of HIV/AIDs treatment and mental health services.

Immunization service availability and readiness in primary health care in pastoral and semi-pastoral CGPP Ethiopia implementation districts

Background: The availability of immunization services and the readiness of skilled health workers in health institutions to deliver potent vaccines to end users when required to do so are important inputs that contribute to the reduction of child morbidity and mortality from vaccine-preventable diseases(VPDs). Objective: Assess immunization service availability and readiness in primary health care units (PHCUs) in pastoral and semi-pastoral regions of CGPP Ethiopia implementation districts. Methods: A facility-based cross-sectional survey was employed on 14­23 August 2016 in all health centers (HCs) and three randomly selected health posts (HPs) in each HC catchment area in 85 CGPP implementation districts. An observation checklist was filled in by trained data collectors for all study PHCUs. Results: Immunization service availability and service delivery, based on 19 tracer items ,were assessed in 860 PHCUs in both pastoral and semi-pastoral areas. In total, 92%of the PHCUs reported providing an immunization service. However, only 18.1% of the PHCUs were observed and 32.4% reported providing immunization on the day data were collected. Overall,immunization service readiness was 56.6%: 85% of the HCs and 46.6% of the HPs were ready for immunization service over the study period. The proportion of PHCUs found to have functional refrigerators was 65%. Conclusions and recommendations: Great variability observed in terms of service readiness among HCs and HPs in this study. All PHCUs should be equipped with functional refrigerators that are regularly maintained; all immunization antigens and schedule immunization services should be available at the PHCUs daily to avoid missed opportunities; cold chain managers/immunization service providers should be given supervisory support to ensure that they record refrigerator temperatures

Prior to Peripheral Tolerance, Newly Generated CD4 T Cells Maintain Dangerous Autoimmune Potential: Fas- and Perforin-Independent Autoimmunity Controlled by Programmed Death-1.

Lymphopenia can result from various factors, including viral infections, clinical interventions, or as a normal property of the fetal/neonatal period. T cells in a lymphopenic environment undergo lymphopenia-induced proliferation (LIP) to fill the available "niche" as defined by peptide-MHC and homeostatic cytokine resources. We recently reported systemic autoimmunity following reconstitution of the lymphoid compartment of Rag1-/- mice with PD-1-/- hematopoietic stem cells or by transfer of thymocytes, but not splenocytes, suggesting that programmed death-1 (PD-1) plays a crucial role in controlling recent thymic emigrants (RTE) and preventing autoimmunity upon their LIP. However, it is unclear whether RTE residing within the periphery of a lymphoreplete host maintain enhanced autoimmune generating potential or if this property only manifests if RTE experience a lymphopenic periphery immediately after export from the thymus. Furthermore, it is unclear which of a variety of T cell effector mechanisms generate pathology when control of RTE by PD-1 is lacking. Herein, we determined that PD-1 is upregulated on CD4 T cells undergoing the natural LIP characteristic of the neonatal period. Newly generated T cells lacking PD-1 maintained an enhanced autoimmune potential even after residence in a lymphoreplete periphery, emphasizing the importance of PD-1 in the establishment of peripheral tolerance. Neither Fas nor perforin-dependent killing mechanisms were required for autoimmunity, while host MHC-II expression was critical, suggesting that LIP-driven autoimmunity in the absence of PD-1 may primarily result from a CD4 T cell-mediated systemic cytokinemia, a feature potentially shared by other autoimmune or inflammatory syndromes associated with immune reconstitution and LIP.

Insomnia and Its Temporal Association with Academic Performance among University Students: A Cross-Sectional Study.

INTRODUCTION: Studies show that 9.4% to 38.2% of university students are suffering from insomnia. However, research data in developing countries is limited. Thus, the aim of the study was to assess insomnia and its temporal association with academic performance. METHODS AND MATERIALS: Institution based cross-sectional study was conducted with 388 students at Debre Berhan University. Data were collected at the nine colleges. Logistic and linear regression analysis was performed for modeling insomnia and academic performance with a p value threshold of 0.05, respectively. Data were entered using EPI-data version 3.1 and analyzed using SPSS version 20. RESULTS: The prevalence of insomnia was 61.6%. Field of study (p value = 0.01), worshiping frequency (p value = 0.048), marital status (p value = 0.03), and common mental disorder (p value < 0.001) were identified associated factors of insomnia. There was no significant association between insomnia and academic performance (p value = 0.53, ß = -0.04). Insomnia explained 1.2% (r2 = 0.012) of the difference in academic performance between students. CONCLUSIONS: Nearly 3 out of 5 students had insomnia. We recommended that universities would endorse sleep quality and mental health illness screening programs for students.

Mental and Reproductive Health Correlates of Academic Performance among Debre Berhan University Female Students, Ethiopia: The Case of Premenstrual Dysphoric Disorder.

BACKGROUND: Globally 3 to 8% of reproductive age women are suffering from premenstrual dysphoric disorder (PMDD). Several mental and reproductive health-related factors cause low academic achievement during university education. However, limited data exist in Ethiopia. The aim of the study was to investigate mental and reproductive health correlates of academic performance. METHODS: Institution based cross-sectional study was conducted with 667 Debre Berhan University female students from April to June 2015. Academic performance was the outcome variable. Mental and reproductive health characteristics were explanatory variables. Two-way analysis of variance (ANOVA) test of association was applied to examine group difference in academic performance. RESULT: Among 529 students who participated, 49.3% reported mild premenstrual syndrome (PMS), 36.9% reported moderate/severe PMS, and 13.8% fulfilled PMDD diagnostic criteria. The ANOVA test of association revealed that there was no significant difference in academic performance between students with different level of PMS experience (F-statistic = 0.08, p value = 0.93). Nevertheless, there was a significant difference in academic performance between students with different length of menses (F-statistic = 5.15, p value = 0.006). CONCLUSION: There was no significant association between PMS experience and academic performance, but on the other hand, the length of menses significantly associated with academic performance.

Common mental disorder and its association with academic performance among Debre Berhan University students, Ethiopia.

BACKGROUND: Common mental disorder (CMD) is prevalent in industrialized and non-industrialized countries. The prevalence of CMD among university students was 28.8-44.7% and attributed to several risk factors, such as schooling. The aim of this study was to assess the prevalence and risk factors of CMD. In addition, the association between CMD and academic performance was tested. METHODS: Institution based cross-sectional study was conducted with 422 students at Debre Berhan university from March to April 2015. CMD was the primary outcome variable whereas academic performance was the secondary outcome variable. Kessler psychological distress (K10) scale was used to assess CMD. Bivariate and multiple logistic regression analysis were performed for modeling the primary outcome variable; independent samples T test and linear regression analysis were carried out for modeling the secondary outcome variable. The strength of association was interpreted using odds ratio and regression coefficient (ß) and decision on statistical significance was made at a p value of 0.05. Data were entered using EPI-data version 3.1 software and analyzed using the Statistical Package for the Social Sciences (SPSS) version 20.01 software. RESULTS: The prevalence of CMD was 63.1%. Field of study (p = 0.008, OR = 0.2, 95% CI 0.04-0.61), worshiping (p = 0.04, OR = 1.8, 95% CI 1.02-3.35), insomnia (p < 0.001, OR = 3.8, 95% CI 2.21-6.57), alcohol drinking (p = 0.006, OR = 2.7, 95% CI 1.33-5.66), and headache (p = 0.02, OR = 2.1, 95% CI 1.10-3.86) were identified risk factors for CMD. The mean cumulative grade point average of students with CMD was lower by 0.02 compared to those without CMD, but not statistically significant (p = 0.70, ß = -0.02, 95% CI -0.15 to 0.10). CMD explained only 0.8% (r2 = 0.008) of the difference in academic performance between students. CONCLUSIONS: At least three out of five students fulfilled CMD diagnostic criteria. The statistically significant risk factors were field of study, worshiping, insomnia, alcohol drinking, and headache. Moreover, there was no statistically significant association between CMD and academic performance. Undertaking integrated evidence-based intervention focusing on students with poor sleep quality, poor physical health, and who drink alcohol is essential if the present finding confirmed by a longitudinal study.

Rab32 connects ER stress to mitochondrial defects in multiple sclerosis.

BACKGROUND: Endoplasmic reticulum (ER) stress is a hallmark of neurodegenerative diseases such as multiple sclerosis (MS). However, this physiological mechanism has multiple manifestations that range from impaired clearance of unfolded proteins to altered mitochondrial dynamics and apoptosis. While connections between the triggering of the unfolded protein response (UPR) and downstream mitochondrial dysfunction are poorly understood, the membranous contacts between the ER and mitochondria, called the mitochondria-associated membrane (MAM), could provide a functional link between these two mechanisms. Therefore, we investigated whether the guanosine triphosphatase (GTPase) Rab32, a known regulator of the MAM, mitochondrial dynamics, and apoptosis, could be associated with ER stress as well as mitochondrial dysfunction. METHODS: We assessed Rab32 expression in MS patient and experimental autoimmune encephalomyelitis (EAE) tissue, via observation of mitochondria in primary neurons and via monitoring of survival of neuronal cells upon increased Rab32 expression. RESULTS: We found that the induction of Rab32 and other MAM proteins correlates with ER stress proteins in MS brain, as well as in EAE, and occurs in multiple central nervous system (CNS) cell types. We identify Rab32, known to increase in response to acute brain inflammation, as a novel unfolded protein response (UPR) target. High Rab32 expression shortens neurite length, alters mitochondria morphology, and accelerates apoptosis/necroptosis of human primary neurons and cell lines. CONCLUSIONS: ER stress is strongly associated with Rab32 upregulation in the progression of MS, leading to mitochondrial dysfunction and neuronal death.

Sociodemographic, clinical, and psychosocial factors associated with depression among type 2 diabetic outpatients in Black Lion General Specialized Hospital, Addis Ababa, Ethiopia: a cross-sectional study.

BACKGROUND: Depression is a common comorbidity among patients with type 2 diabetes. There are several reports supporting a bidirectional association between depression and type 2 diabetes. However, there is limited data from non-western countries. Therefore, the aim of this study was to assess the sociodemographic, clinical, and psychosocial factors associated with co-morbid depression among type 2 diabetic outpatients presenting to Black Lion General Specialized Hospital, Addis Ababa, Ethiopia. METHODS: This institution based cross-sectional study design was conducted on a random sample of 276 type 2 diabetic outpatients. Type 2 diabetes patients were evaluated for depression by administering a validated nine-item Patient Health Questionnaire (PHQ-9). Risk factors for depression among type 2 diabetes patients were identified using multiple logistic regression analysis. RESULT: In total, 264 study participants were interviewed with a response rate of 95.6%. The prevalence of depression was 44.7%. In the multivariate analysis, the statistically significant risk factors for depression were monthly family income ≤ 650 (p-value = 0.056; OR = 2.0; 95% CI = 1.01, 4.2), presence of ≥3 diabetic complications (p-value = 0.03; OR = 3.3; 95% CI = 1.1, 10.0), diabetic nephropathy (p-value = 0.01; OR = 2.9; 95% CI = 1.2, 6.7), negative life events (p-value = 0.01; OR = 2.4; 95% CI = 1.2, 4.5), and poor social support (p-value = 0.001; OR = 2.7; 95% CI = 1.5, 5.0). CONCLUSION: This study demonstrated that depression is a common co-morbid health problem with a prevalence rate of 44.7%. The presence of diabetic complications, low monthly family income, diabetic nephropathy, negative life event, and poor social support were the statistically significant risk factors associated with depression. We presume that the burden of mental health especially depression is high in the population with type 2 diabetes mellitus co-morbidity. Therefore, specific attention is needed to diagnose early and treat promptly.

Granzyme B-inhibitor serpina3n induces neuroprotection in vitro and in vivo.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune inflammatory and neurodegenerative disease of the central nervous system (CNS). It is widely accepted that inflammatory cells play major roles in the pathogenesis of MS, possibly through the use of serine protease granzyme B (GrB) secreted from the granules of cytotoxic T cells. We have previously identified GrB as a mediator of axonal injury and neuronal death. In this study, our goal was to evaluate the effect of GrB inhibition in the human system in vitro, and in vivo in EAE using the newly isolated GrB-inhibitor serpina3n. METHODS: We used a well-established in vitro model of neuroinflammation characterized by a co-culture system between human fetal neurons and lymphocytes. In vivo, we induced EAE in 10- to 12-week-old female C57/BL6 mice and treated them intravenously with serpina3n. RESULTS: In the in vitro co-culture system, pre-treatment of lymphocytes with serpina3n prevented neuronal killing and cleavage of the cytoskeletal protein alpha-tubulin, a known substrate for GrB. Moreover, in EAE, 50 µg serpina3n substantially reduced the severity of the disease. This dose was administered intravenously twice at days 7 and 20 post EAE induction. serpina3n treatment reduced axonal and neuronal injury compared to the vehicle-treated control group and maintained the integrity of myelin. Interestingly, serpina3n treatment did not seem to reduce the infiltration of immune cells (CD4(+) and CD8(+) T cells) into the CNS. CONCLUSION: Our data suggest further studies on serpina3n as a potentially novel therapeutic strategy for the treatment of inflammatory-mediated neurodegenerative diseases such as MS.

Reprogramming of HUVECs into induced pluripotent stem cells (HiPSCs), generation and characterization of HiPSC-derived neurons and astrocytes.

Neurodegenerative diseases are characterized by chronic and progressive structural or functional loss of neurons. Limitations related to the animal models of these human diseases have impeded the development of effective drugs. This emphasizes the need to establish disease models using human-derived cells. The discovery of induced pluripotent stem cell (iPSC) technology has provided novel opportunities in disease modeling, drug development, screening, and the potential for "patient-matched" cellular therapies in neurodegenerative diseases. In this study, with the objective of establishing reliable tools to study neurodegenerative diseases, we reprogrammed human umbilical vein endothelial cells (HUVECs) into iPSCs (HiPSCs). Using a novel and direct approach, HiPSCs were differentiated into cells of central nervous system (CNS) lineage, including neuronal, astrocyte and glial cells, with high efficiency. HiPSCs expressed embryonic genes such as nanog, sox2 and Oct-3/4, and formed embryoid bodies that expressed markers of the 3 germ layers. Expression of endothelial-specific genes was not detected in HiPSCs at RNA or protein levels. HiPSC-derived neurons possess similar morphology but significantly longer neurites compared to primary human fetal neurons. These stem cell-derived neurons are susceptible to inflammatory cell-mediated neuronal injury. HiPSC-derived neurons express various amino acids that are important for normal function in the CNS. They have functional receptors for a variety of neurotransmitters such as glutamate and acetylcholine. HiPSC-derived astrocytes respond to ATP and acetylcholine by elevating cytosolic Ca2+ concentrations. In summary, this study presents a novel technique to generate differentiated and functional HiPSC-derived neurons and astrocytes. These cells are appropriate tools for studying the development of the nervous system, the pathophysiology of various neurodegenerative diseases and the development of potential drugs for their treatments.

Characterization of the NT2-derived neuronal and astrocytic cell lines as alternative in vitro models for primary human neurons and astrocytes.

Primary human fetal neurons and astrocytes (HFNs and HFAs, respectively) provide relevant cell types with which to study in vitro the mechanisms involved in various human neurological diseases, such as multiple sclerosis, Parkinson's disease, and Alzheimer's disease. However, the limited availability of human fetal cells poses a significant problem for the study of these diseases when a human cell model system is required. Thus, generating a readily available alternative cell source with the essential features of human neurons and astrocytes is necessary. The human teratoma-derived NTera2/D1 (NT2) cell line is a promising tool from which both neuronal and glial cells can be generated. Nevertheless, a direct comparison of NT2 neurons and primary HFNs in terms of their morphology physiological and chemical properties is still missing. This study directly compares NT2-derived neurons and primary HFNs using immunocytochemistry, confocal calcium imaging, high-performance liquid chromatography, and high-content analysis techniques. We investigated the morphological similarities and differences, levels of relevant amino acids, and internal calcium fluctuations in response to certain neurotransmitters/stimuli. We also compared NT2-derived astrocytes and HFAs. In most of the parameters tested, both neuronal and astrocytic cell types exhibited similarities to primary human fetal neurons and astrocytes. NT2-derived neurons and astrocytes are reliable in vitro tools and a renewable cell source that can serve as a valid alternative to HFNs/HFAs for mechanistic studies of neurological diseases.

IL-25 prevents T cell-mediated neurotoxicity by decreasing LFA-1 expression.

Autoimmune diseases such as multiple sclerosis (MS) are thought to develop due to a dysregulation in the normal T(H)1-T(H)17/T(H)2 immune system balance, where pro-inflammatory responses with a T(H)1/T(H)17 prevalence develop. Some therapeutic treatments in MS promote a shift toward a TH2-prevalent environment and this has been shown to be protective. However, not all patients respond to current immunomodulatory treatments in MS so that new immunomodulatory drugs that can promote a shift of the immune system into an anti-inflammatory T(H)2 status are needed. IL-25 is a cytokine of the IL-17 family with powerful anti-inflammatory properties. This study demonstrates that IL-25 exerts neuroprotective functions by reducing T cell-mediated killing of human fetal neurons. The mechanism of action of this IL-25-mediated neuroprotective effect appears to be linked to reduction in the expression of the adhesion molecule LFA-1, which is relevant in stabilizing the immune synapse during cytotoxicity.