Posttraumatic Stress Disorder in Our Migrant Youth.

There is an ongoing diagnostic and treatment challenge for migrant youth with posttraumatic stress disorder (PTSD) that many clinicians face. Current studies have helped clinicians to develop a better understanding of the migrant youth's journey including potentially traumatic and adverse events they encounter. This includes determining if premigration, migration, and postmigration stressors have had an impact on the individual. This has also helped clinicians, educators, and legal advocates to use a collaborative approach to address the migrant youth's needs for managing the severity of PTSD symptoms.

Practical and Statistical Considerations for the Long Term Follow-Up of Gene Therapy Trial Participants.

Study sponsors and market authorization holders are required by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) to enroll patients administered a gene therapy product, whether in a trial setting or post-licensure, in a long term follow-up safety study to continue the safety assessments of their product. These follow-up studies range between 5 and 15 years after dosing. This unprecedented duration of engagement with patients and caregivers raises logistical challenges that will require innovation and collaboration across sponsors and regulators. In this paper we delineate some of the key considerations for designing long term follow-up protocols in the gene therapy setting, with an eye toward platform and master protocol approaches, and offer guidance for innovative operational and statistical methods that can help assess the safety profile and durability of response for these novel therapeutics.

Do antibullying laws in the United States have an impact?

Bullying is an omnipresent problem with high prevalence in children and adolescents. There are many antibullying state laws and practices in place however its real level impact is ambiguous at best. We studied the prevalence of bullying in the United States, examine the practices, laws, and interventions in place to prevent bullying and its real-life impact. Various studies have reported that the ground-level change brought about by these policies and programs varies and is not quite significant. The effectiveness of existing policies and legislations remains unclear and measurable objective change or impact is not yet observed. Therefore, a rethink of the strategies is needed. The focus must be on community-level programs and interventions with inclusive approaches involving all the stakeholders are critical for addressing this menace.

Many hops, many stops: care-seeking "loops" for diabetes and hypertension in three urban informal settlements in the Mumbai Metropolitan Region.

Background: The burden of Non-Communicable Diseases (NCDs) in urban informal settlements across Lower and Middle Income Countries is increasing. In recognition, there has been interest in fine-tuning policies on NCDs to meet the unique needs of people living in these settlements. To inform such policy efforts, we studied the care-seeking journeys of people living in urban informal settlements for two NCDs-diabetes and hypertension. The study was done in the Mumbai Metropolitan Region, India. Methods: This qualitative study was based on interviews with patients having diabetes and hypertension, supplemented by interactions with the general community, private doctors, and public sector staff. We conducted a total of 47 interviews and 6 Focus Group Discussions. We synthesized data thematically and used the qualitative software NVivo Version 10.3 to aid the process. In this paper, we report on themes that we, as a team, interpreted as striking and policy-relevant features of peoples' journeys. Results: People recounted having long and convoluted care-seeking journeys for the two NCDs we studied. There were several delays in diagnosis and treatment initiation. Most people's first point of contact for medical care were local physicians with a non-allopathic degree, who were not always able to diagnose the two NCDs. People reported seeking care from a multitude of healthcare providers (public and private), and repeatedly switched providers. Their stories often comprised multiple points of diagnosis, re-diagnosis, treatment initiation, and treatment adjustments. Advice from neighbors, friends, and family played an essential role in shaping the care-seeking process. Trade-offs between saving costs and obtaining relief from symptoms were made constantly. Conclusion: Our paper attempts to bring the voices of people to the forefront of policies on NCDs. People's convoluted journeys with numerous switches between providers indicate the need for trusted "first-contact" points for NCD care. Integrating care across providers-public and private-in urban informal settlements-can go a long way in streamlining the NCD care-seeking process and making care more affordable for people. Educating the community on NCD prevention, screening, and treatment adherence; and establishing local support mechanisms (such as patient groups) may also help optimize people's care-seeking pathways.

Examining the Long-Term Sequelae of SARS-CoV2 Infection in Patients Seen in an Outpatient Psychiatric Department.

Background: The acute phase of Coronavirus disease-19 (COVID-19) is well known. However, there is now an increasing number of patients suffering from the post-acute sequelae of COVID-19 (PASC Post COVID-19 condition occurs in individuals with a history of probable or confirmed SARS CoV-2 infection, usually 3 months from the onset of COVID-19 with symptoms and that last for at least 2 months and cannot be explained by an alternative diagnosis), including neuropsychiatric symptoms. The purpose of this report is to describe the sociodemographic, diagnostic and treatment characteristics of patients evaluated in an outpatient psychiatric setting for PASC. Methods: A retrospective review of 30 individuals with documented COVID-19 illness treated at a university hospital-based Post-COVID-19 Recovery Program were referred to an outpatient psychiatric department for consultation and treatment from December 2020 to July 2021. All individuals complained of neuropsychiatric symptoms including anxiety, depression, fatigue and cognitive problems. Data on sociodemographic characteristics, psychiatric diagnosis, prominent psychological themes and treatment prescribed were described and, where applicable, analyzed with SPSS software. Results: The study population consisted of patients between 25 and 82 years old, with a predominance of women between 46 and 60 years. Approximately half of the patient population had a primary diagnosis of major depressive disorder, often combined with prominent anxiety. Over two-thirds of the patient population reported a combination of depression, fatigue and cognitive complaints, predominantly memory and slowed processing speed. Prominent stressors and psychological themes included social and occupational decline, isolation, lack of empathy and understanding from family, friends and employers, and apprehension about future ability to return to their baseline level of function. Treatments recommended included individual and group psychotherapy, medication and cognitive rehabilitation. Modafinil and antidepressants, often in combination, were the most commonly used medications, intended to target the pervasive fatigue, depressive, and anxiety these individuals were facing. Conclusion: Clinical experience from this patient population underscored the significant medical, emotional, neurocognitive and functional sequelae of PASC. Management of these individuals requires a collaborative approach with the availability of psychotherapeutic interventions, pharmacologic treatment, neurocognitive assessment and remediation to address these symptoms.

Community interventions to prevent violence against women and girls in informal settlements in Mumbai: the SNEHA-TARA pragmatic cluster randomised controlled trial.

BACKGROUND: In a cluster randomised controlled trial in Mumbai slums, we will test the effects on the prevalence of violence against women and girls of community mobilisation through groups and individual volunteers. One in three women in India has survived physical or sexual violence, making it a major public health burden. Reviews recommend community mobilisation to address violence, but trial evidence is limited. METHODS: Guided by a theory of change, we will compare 24 areas receiving support services, community group, and volunteer activities with 24 areas receiving support services only. These community mobilisation activities will be evaluated through a follow-up survey after 3 years. Primary outcomes will be prevalence in the preceding year of physical or sexual domestic violence, and prevalence of emotional or economic domestic violence, control, or neglect against women 15-49 years old. Secondary outcomes will describe disclosure of violence to support services, community tolerance of violence against women and girls, prevalence of non-partner sexual violence, and mental health and wellbeing. Intermediate theory-based outcomes will include bystander intervention, identification of and support for survivors of violence, changes described in programme participants, and changes in communities. DISCUSSION: Systematic reviews of interventions to prevent violence against women and girls suggest that community mobilisation is a promising population-based intervention. Already implemented in other areas, our intervention has been developed over 16 years of programmatic experience and 2 years of formative research. Backed by public engagement and advocacy, our vision is of a replicable community-led intervention to address the public health burden of violence against women and girls. TRIAL REGISTRATION: Controlled Trials Registry of India, CTRI/2018/02/012047. Registered on 21 February 2018. ISRCTN, ISRCTN84502355. Registered on 22 February 2018.

Community-Based Management of Acute Malnutrition to Reduce Wasting in Urban Informal Settlements of Mumbai, India: A Mixed-Methods Evaluation.

BACKGROUND: We evaluated an adaptation of a large-scale community-based management of acute malnutrition program run by an NGO with government partnerships, in informal settlements of Mumbai, India. The program aimed to reduce the prevalence of wasting among children under age 3 and covered a population of approximately 300,000. METHODS: This study used a mixed-methods approach including a quasi-experimental design to compare prevalence estimates of wasting in intervention areas with neighboring informal settlements. Cross-sectional data were collected from March through November 2014 for the baseline and October through December 2015 for the endline. Endline data were analyzed using mixed-effects logistic regression models, adjusting for child, maternal, and household characteristics. In addition, we conducted in-depth interviews with 37 stakeholders (13 staff and 24 mothers) who reported on salient features that contributed to successful implementation of the program. RESULTS: We interviewed 2,578 caregivers at baseline and 3,455 at endline in intervention areas. In comparison areas, we interviewed 2,082 caregivers at baseline and 2,122 at endline. At endline, the prevalence of wasting decreased by 28% (18% to 13%) in intervention areas and by 5% (16.9% to 16%) in comparison areas. Analysis of the endline data indicated that children in intervention areas were significantly less likely to be malnourished (adjusted odds ratio, 0.81; confidence interval, 0.67 to 0.99). Stakeholders identified 4 main features as contributing to the success of the program: (1) tailoring and reinforcement of information provided to caregivers in informal settings, (2) constant field presence of staff, (3) holistic case management of issues beyond immediate malnourishment, and (4) persistence of field staff in persuading reluctant families. Staff capabilities were enhanced through training, stringent monitoring mechanisms, and support from senior staff in tackling difficult cases. CONCLUSION: NGO-government partnerships can revitalize existing community-based programs in urban India. Critical to success are processes that include reinforced knowledge-building of caregivers, a high level of field support and encouragement to the community, and constant monitoring and follow-up of cases by all staff levels.

Autoantibodies to thrombopoietin and the thrombopoietin receptor in patients with immune thrombocytopenia.

Autoantibodies to thrombopoietin (TPO, also termed THPO) or the TPO receptor (cMpl, also termed MPL) could play a pathological role in immune thrombocytopenia (ITP). In this study, we tested for autoantibodies against TPO, cMpl, or the TPO/cMpl complex in ITP and other thrombocytopenic disorders. Using an inhibition step with excess TPO in fluid-phase to improve binding specificity, the prevalence of anti-TPO autoantibodies was: active ITP: 9/32 (28%); remission ITP: 0/14 (0%); non-immune thrombocytopenias: 1/10 (10%); and healthy controls: 1/11 (9%). Similarly, using an inhibition step with excess cMpl, the prevalence of specific anti-cMpl autoantibodies was: active ITP: 7/32 (22%); remission ITP: 1/14 (7%); non-immune thrombocytopenias: 3/10 (30%); and healthy controls: 0/11 (0%). Two active ITP patients had autoantibodies against the TPO/cMpl complex, but not against TPO or cMpl alone. Anti-TPO or anti-cMpl autoantibodies were found in 44% of ITP patients, and in 40% of patients with other thrombocytopenic disorders. These autoantibodies did not correlate with ITP disease severity or number of ITP treatments received; however, in this cohort, 3 patients failed to respond to TPO receptor agonist medications, and of those, 2 had anti-TPO autoantibodies. This suggests that anti-TPO and anti-cMpl autoantibodies are associated with thrombocytopenia, and may be clinically relevant in a subset of ITP patients.

cAMP-Coupled riboflavin trafficking in placental trophoblasts: a dynamic and ordered process.

Riboflavin (RF, vitamin B(2)), an essential micronutrient central to cellular metabolism through formation of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors, is internalized, at least in part, via a proposed receptor-mediated endocytic (RME) process. The purpose of this study was to delineate the cellular RF distribution using human placental trophoblasts and evaluate the regulatory role of cAMP in this process. Subcellular fractionation and three-dimensional confocal microscopy analyses were carried out to define the RF accumulation profile. Biochemical assays evaluating the cAMP dependence of this pathway were also performed. This study records an intracellular RF distribution pattern that shows dynamic accumulation of the ligand predominantly in the endosomal and lysosomal compartments and to a lesser extent in the Golgi and mitochondria. In contrast, transferrin (TF) colocalizes rapidly within endosomes with minimal accumulation in the other organelles. The temporal and spatial distribution of RF and TF colocalized with unique markers of the endocytic machinery provides added morphological evidence in support of the RME process with ultimate translocation to the mitochondrial domain. Colocalized staining with the Golgi also suggests a possible recycling or exocytic mechanism for this ligand. Furthermore, this study demonstrates cAMP regulation of the putative ligand-bound RF receptor and its association into endocytic vesicles. Delineating the dynamics of the process governing cellular RF homeostasis presents an untapped resource that can be further exploited in improving our current understanding of nutritional biology and fetal growth and development, and perhaps in targeting the endogenous system for developing novel therapeutic approaches.

Cytoskeletal scaffolds regulate riboflavin endocytosis and recycling in placental trophoblasts.

Microfilaments and microtubules (MT) play a vital role in cellular endocytic processes. The present study evaluates the role of these cytoskeletal elements in the apical internalization and postendocytic fate of riboflavin (RF) in placental trophoblasts (BeWo cells). Biochemical modification of the actin and microtubule network by (1) okadaic acid (OA), which disrupts MT-based vesicular trafficking; (2) cytochalasin D and latrunculin B, which promote actin depolymerization; and (3) 2,3-butanedione monoxime (BDM), which inhibits myosin-actin interaction, was confirmed by immunofluorescence microscopy using actin- and tubulin-specific antibodies. Furthermore, involvement of the molecular motors dynein and kinesin was assessed in the presence of (1) sodium orthovanadate, which inhibits dynein-ATPase activity and (2) adenosine 5'-(beta,gamma-imido)triphosphate tetralithium salt hydrate, a non-hydrolyzable ATP analog, which results in defective kinesin-driven processes. RF internalization consequent to cytoskeletal alterations was compared with that of a clathrin-dependent endocytic marker ([125I]-transferrin [TF]), a caveolae-mediated endocytic substrate ([3H]-folic acid [FA]), and a fluid-phase endocytic marker ([125I]-horse radish peroxidase [HRP]). Apical recycling and bidirectional transport of RF and TF was measured following cytoskeletal alterations. Results indicate that uptake of RF, TF, FA and HRP are markedly reduced (approximately 30-65%) in the presence OA and BDM, suggesting differential sensitivities to modification of kinesin-driven microtubules. However, actin depolymerization negatively affected HRP endocytosis alone, while RF, FA and TF internalization remained unchanged. Disturbances in protein phosphorylation cascades also influenced apical recycling while net ligand transport across monolayers remained unaffected. In conclusion, apical RF trafficking in placental cells is tightly regulated by microtubules and supported by accessory actin involvement.

Recognition, co-internalization, and recycling of an avian riboflavin carrier protein in human placental trophoblasts.

Absorption of riboflavin (RF) across membrane barriers is essential to cellular oxidation reduction processes. Riboflavin carrier protein (RCP), a 37-kDa secretory protein, is proposed to play an important role in RF absorption, although information on the mammalian ortholog remains unclear. This study alludes to the existence of a mammalian RF carrier protein and further characterizes its carrier role and fate using avian RCP in human placental trophoblast (BeWo), another mammalian cell line, monkey kidney (COS-1), and the avian control, chicken hepatic (LMH/2A) cells. The presence of RCP and its involvement in RF internalization was analyzed by immunofluorescence and immunobinding assays using chicken RCP (cRCP) antibodies. In the presence of anti-cRCP, cellular RF uptake is significantly decreased (5% of control) in BeWo cells. Kinetic analyses of intracellular accumulation of (125)I-cRCP revealed a J(max) and K(m) of 28.56 +/- 2.70 pmol/mg protein/min and 142.43 +/- 82.16 nM, respectively, in BeWo cells and 75.14 +/- 7.6 pmol/mg protein/min and 104.37 +/- 23.96 nM in the species-specific control, LMH/2A cells. Subcellular fractionation studies revealed colocalization of both radiolabeled RF and cRCP within endosomal and lysosomal fractions, further elucidating RCP's role in trafficking RF through the cell. Following intracellular release of RF from the carrier complex, the protein is either subject to lysosomal breakdown or is conserved via recycling mechanisms for continued RF sequestration and uptake. In summary, mammalian placental trophoblasts exhibit specific carrier protein dependence that sequesters and essentially mediates RF internalization via the proposed receptor-mediated endocytic pathway.

In vitro and pharmacophore-based discovery of novel hPEPT1 inhibitors.

PURPOSE: The human proton-coupled small peptide carrier (hPEPT1) is a low-affinity, high-capacity transporter with broad substrate specificity. We have taken an iterative in vitro and in silico approach to the discovery of molecules with hPEPT1 affinity. METHODS: A pharmacophore-based approach was taken to identifying hPEPT1 inhibitors. The well-characterized and relatively high affinity ligands Gly-Sar, bestatin, and enalapril were used to generate a common features (HIPHOP) pharmacophore. This consisted of two hydrophobic features, a hydrogen bond donor, acceptor, and a negative ionizable feature. RESULTS: The pharmacophore was used to search the Comprehensive Medicinal Chemistry (CMC) database of more than 8000 drug-like molecules and retrieved 145 virtual hits mapping to the pharmacophore features. The highest scoring compounds within this set were selected and tested in a stably transfected CHO-hPepT1 cell model. The antidiabetic repaglinide and HMG CoA reductase inhibitor fluvastatin were found to inhibit hPEPT1 with sub-millimolar potency (IC(50) 178 +/- 1.0 and 337 +/- 4 microM, respectively). The pharmacophore was also able to identify known hPEPT1 substrates and inhibitors in further database mining of more than 500 commonly prescribed drugs. CONCLUSIONS: This study demonstrates the potential of combining computational and in vitro approaches to determine the affinity of compounds for hPEPT1 and, in turn, provides insights into key molecular interactions with this transporter.

Functional characterization of the peptide transporter PEPT2 in primary cultures of human upper airway epithelium.

This study characterizes the expression and function of the peptide transporter hPepT2 (SLC15A2) in differentiated primary cultures of human upper airway lung epithelia obtained from six human donors. Genotype analysis of a SNP in exon 15 of hPepT2 genotypes in six donors revealed an expected distribution of the two main variants present at similar frequency (two AA homozygotes, two BB homozygotes, and two AB heterozygotes). Real-time PCR analysis of the hPepT2 mRNA message revealed no significant differences among genotypes. hPEPT2 was expressed on the apical membrane in all donor specimens, demonstrated by cell surface biotinylation and Western analysis (104 kD). We then compared transepithelial transport of the prototypical substrate (3)H-glycylsarcosine in all donor cultures in the absence and presence of known inhibitors of hPEPT2 to ascertain the phenotype of functionally expressed hPepT2 in the upper airway epithelium. An array of inhibitors included dipeptides, beta-lactam antibiotics, bestatin, and ACE inhibitors. hPEPT2 exhibited saturable Michaelis-Menten-type kinetic parameters for GlySar, corroborating previously reported values for K(T) and J(max). Donor-to-donor variation of transport for different substrates did not correlate with hPepT2 haplotypes in this sample cohort. These findings demonstrate functional hPEPT2 transporter expression in primary cultures of human lung epithelial cells. hPEPT2-mediated transport could serve as a strategy for noninvasive systemic delivery of peptides and peptidomimetics drugs.

High glucose concentration in isotonic media alters caco-2 cell permeability.

Caco-2 cell permeability was evaluated in isotonic media containing high (25 mM) or physiological (5.5 mM) glucose concentrations. Transepithelial electrical resistance (TEER) and membrane fluidity were measured to assess glucose-induced alterations in physical barrier properties. In parallel, distribution of the actin filament (F-actin) and zonula occludens-1 (ZO-1) proteins was assessed by confocal microscopy. Transepithelial fluxes of mannitol, hydrocortisone, digoxin, and glycyl sarcosine (Gly-Sar) that permeate the intestinal mucosa by various pathways were measured to quantify the effect of glucose-induced changes on Caco-2 cell permeability. High glucose decreased maximum TEER of cell monolayers by 47%, whereas membrane fluidity at the hydrophobic core and lipid/polar head interphase was significantly increased. F-actin distribution in high glucose cells appeared more diffuse while ZO-1 was unchanged. Mannitol and hydrocortisone fluxes across Caco-2 cells cultured in high glucose increased by 65% and 24%, respectively. In addition, high glucose decreased the maximum transport capacity (Vmax) of PepT-1. P-glycoprotein activity, however, was unchanged. In conclusion, high extracellular glucose concentration in isotonic media significantly alters physical barrier properties of Caco-2 cell monolayers, which predominantly affects transepithelial transport of solutes permeating the cell barrier by paracellular and transcellular passive diffusion and facilitated transport mediated by the proton-dependent oligopeptide transporter (PepT-1).

Extracellular glucose concentration alters functional activity of the intestinal oligopeptide transporter (PepT-1) in Caco-2 cells.

The objective of this study was to determine the effect of different cell culture media glucose concentrations on the functional activity of PepT-1 in Caco-2 cells. Uptake kinetics of Gly-Sar into Caco-2 cells that were maintained in iso-osmotic media containing 25 or 5.5 mM glucose were determined in the presence and absence of amino acid-selective chemical modifiers and dithiothreitol. Inhibition of Gly-Sar uptake into Caco-2 cells was measured in the presence of dipeptides and xenobiotics exhibiting various binding affinities for the PepT-1. The effect of extracellular glucose on PepT-1 gene expression was assessed using comparative RT-PCR. Long-term exposure of Caco-2 cells to 25 mM glucose reduced maximum transport capacity for Gly-Sar uptake without altering PepT-1 gene expression. In contrast, binding affinity of Gly-Sar and other dipeptides or xenobiotics was not significantly changed. Chemical modification of Lys and Tyr residues decreased V(max), while Cys modification increased the maximum transport capacity of the carrier. Preincubation of Caco-2 cells with dithiothreitol restored PepT-1 activity in cells maintained at 25 mM glucose. In conclusion, cell culture media containing 25 mM glucose decreases maximum transport capacity of PepT-1 in Caco-2 cells without affecting substrate recognition, at least in part, mediated via an oxidative pathway.