Cellular and Molecular Probing of Intact Human Organs.

Optical tissue transparency permits scalable cellular and molecular investigation of complex tissues in 3D. Adult human organs are particularly challenging to render transparent because of the accumulation of dense and sturdy molecules in decades-aged tissues. To overcome these challenges, we developed SHANEL, a method based on a new tissue permeabilization approach to clear and label stiff human organs. We used SHANEL to render the intact adult human brain and kidney transparent and perform 3D histology with antibodies and dyes in centimeters-depth. Thereby, we revealed structural details of the intact human eye, human thyroid, human kidney, and transgenic pig pancreas at the cellular resolution. Furthermore, we developed a deep learning pipeline to analyze millions of cells in cleared human brain tissues within hours with standard lab computers. Overall, SHANEL is a robust and unbiased technology to chart the cellular and molecular architecture of large intact mammalian organs.

Th17 cell plasticity towards a T-bet-dependent Th1 phenotype is required for bacterial control in Staphylococcus aureus infection.

Staphylococcus aureus is frequently detected in patients with sepsis and thus represents a major health burden worldwide. CD4+ T helper cells are involved in the immune response to S. aureus by supporting antibody production and phagocytosis. In particular, Th1 and Th17 cells secreting IFN-γ and IL-17A, are involved in the control of systemic S. aureus infections in humans and mice. To investigate the role of T cells in severe S. aureus infections, we established a mouse sepsis model in which the kidney was identified to be the organ with the highest bacterial load and abundance of Th17 cells. In this model, IL-17A but not IFN-γ was required for bacterial control. Using Il17aCre × R26YFP mice we could show that Th17 fate cells produce Th17 and Th1 cytokines, indicating a high degree of Th17 cell plasticity. Single cell RNA-sequencing of renal Th17 fate cells uncovered their heterogeneity and identified a cluster with a Th1 expression profile within the Th17 cell population, which was absent in mice with T-bet/Tbx21-deficiency in Th17 cells (Il17aCre x R26eYFP x Tbx21-flox). Blocking Th17 to Th1 transdifferentiation in Th17 fate cells in these mice resulted in increased S. aureus tissue loads. In summary, we highlight the impact of Th17 cells in controlling systemic S. aureus infections and show that T-bet expression by Th17 cells is required for bacterial clearance. While targeting the Th17 cell immune response is an important therapeutic option in autoimmunity, silencing Th17 cells might have detrimental effects in bacterial infections.

The CCR6/CCL20 axis expands RORγt+ Tregs to protect from glomerulonephritis.

Previous studies have identified a unique Treg population, which expresses the Th17 characteristic transcription factor RORγt. These RORγt+ Tregs possess enhanced immunosuppressive capacity, which endows them with great therapeutic potential. However, as a caveat, they are also capable of secreting pro-inflammatory IL-17A. Since the sum function of RORγt+ Tregs in glomerulonephritis (GN) remains unknown, we studied the effects of their absence. Purified CD4+ T cell populations, containing or lacking RORγt+ Tregs, were transferred into immunocompromised RAG1 knockout mice and the nephrotoxic nephritis model of GN was induced. Absence of RORγt+ Tregs significantly aggravated kidney injury, demonstrating overall kidney-protective properties. Analyses of immune responses showed that RORγt+ Tregs were broadly immunosuppressive with no preference for a particular type of T cell response. Further characterization revealed a distinct functional and transcriptional profile, including enhanced production of IL-10. Expression of the chemokine receptor CCR6 marked a particularly potent subset, whose absence significantly worsened GN. As an underlying mechanism, we found that chemokine CCL20 acting through receptor CCR6 signaling mediated expansion and activation of RORγt+ Tregs. Finally, we also detected an increase of CCR6+ Tregs in kidney biopsies, as well as enhanced secretion of chemokine CCL20 in 21 patients with anti-neutrophil cytoplasmic antibody associated GN compared to that of 31 healthy living donors, indicating clinical relevance. Thus, our data characterize RORγt+ Tregs as anti-inflammatory mediators of GN and identify them as promising target for Treg directed therapies.

Community-based accompaniment for adolescents transitioning to adult HIV care in urban Peru: a pilot study.

We piloted a community-based intervention to improve outcomes among adolescents living with HIV who were transitioning to adult-oriented care in Lima, Peru. We assessed feasibility and potential effectiveness, including within-person changes in self-reported adherence, psychosocial metrics (NIH Toolbox), and transition readiness ("Am I on TRAC" questionnaire, "Got Transition" checklist). From October 2019 to January 2020, we enrolled 30 adolescents (15-21 years). The nine-month intervention consisted of logistical, adherence and social support delivered by entry-level health workers and group sessions to improve health-related knowledge and skills and social support. In transition readiness, we observed within-person improvements relative to baseline. We also observed strong evidence of improvements in adherence, social support, self-efficacy, and stress, which were generally sustained three months post-intervention. All participants remained in treatment after 12 months. The intervention was feasible and potentially effective for bridging the transition to adult HIV care. A large-scale evaluation, including biological endpoints, is warranted.

RESUMEN: Piloteamos una intervención comunitaria para mejorar los resultados de adolescentes viviendo con el VIH que se encontraban en transición a la atención de VIH orientada a adultos en Lima, Perú. Evaluamos la viabilidad y la eficacia potencial, incluidos los cambios personales en la adherencia auto-reportada, criterios psicosociales (NIH Toolbox), y preparación para la transición (cuestionario "Estoy en el TRAC", lista de verificación "Got Transition"). Desde octubre de 2019 hasta enero de 2020, enrolamos a 30 adolescentes (15 a 21 años). La intervención de nueve meses consistió en apoyo logístico, de adherencia y social brindado por técnicos de enfermería y sesiones grupales para mejorar tanto el conocimiento y las habilidades relacionados con la salud, como el apoyo social. En cuanto a la preparación para la transición, observamos mejoras comparada con el basal. También observamos una fuerte evidencia de mejoras en la adherencia, apoyo social, autoeficacia y estrés, que se mantuvieron, generalmente, tres meses después del fin de la intervención. Todos los participantes seguían en tratamiento después de 12 meses. La intervención fue factible y potencialmente efectiva para tender un puente en la transición a la atención del VIH para adultos. Esto amerita una evaluación a mayor escala y que incluya criterios clínicos.

Parietal epithelial cell dysfunction in crescentic glomerulonephritis.

Crescentic glomerulonephritis represents a group of kidney diseases characterized by rapid loss of kidney function and the formation of glomerular crescents. While the role of the immune system has been extensively studied in relation to the development of crescents, recent findings show that parietal epithelial cells play a key role in the pathophysiology of crescent formation, even in the absence of immune modulation. This review highlights our current understanding of parietal epithelial cell biology and the reported physiological and pathological roles that these cells play in glomerular lesion formation, especially in the context of crescentic glomerulonephritis.

Referral Process for Surgical Management of Tuberculosis in Lima: A Qualitative Study.

BACKGROUND: Lung resection surgery can be a complementary therapy for managing tuberculosis (TB) complications, but access is lacking in high-burden areas. The referral process for surgical evaluation is not well described. This study aimed to elucidate the TB surgery referral process in Peru. METHODS: A qualitative study was conducted using focus groups and interviews of health care providers from the Peruvian National TB Program. A semi-structured interview guide was developed with local partners. Focus groups and individual interviews were recorded and transcribed. Thematic analysis was used to reconstruct the referral process and identify barriers as well as areas for improvement. RESULTS: A total of 12 sessions were recorded (7 interviews and 5 focus groups; 36 participants total). The main themes identified were: (1) Surgical referral workflow, (2) Unstandardized selection criteria for surgery, (3) Limited inter-institutional communication, and (4) Material barriers to surgical management. CONCLUSION: Health care providers involved in the referral process of surgical management of tuberculosis in Lima reported a hierarchical referral workflow. Interinstitutional communication may be a critical interventional point to improve a patient's quality of care during the referral process.

Feasibility and Acceptability of an Adolescent-Friendly Rap Video to Improve Health Literacy Among HIV-Positive Youth in Urban Peru.

Clinical outcomes among adolescents living with HIV (ALHIV) might be improved by interventions aimed at addressing limited health literacy. We developed a Spanish-language rap video on HIV concepts and examined its acceptability and feasibility as a learning tool among ALHIV in Lima, Peru. Twenty-eight ALHIV receiving care at an urban pediatric hospital and ten stakeholders engaged in the care of adolescents watched the video. Adolescents completed a pre- and post-video questionnaire. We conducted focus groups with ALHIV and in-depth interviews with stakeholders and analyzed transcripts to identify themes. ALHIV described concepts of CD4 cell count and viral load as they were portrayed. Participants reported the video was relatable, accessible, and provided hope that ALHIV could lead healthy lives and advocated for future videos to address topics such as transmission and sexual health. Questionnaires indicated some improvement in viral load knowledge. An HIV health literacy music video intervention was feasible to implement and accepted by ALHIV and their healthcare providers. Communicating HIV knowledge via music videos may be promising; further study is needed to optimize implementation.

RESUMEN: Los resultados clínicos entre los adolescentes que viven con el VIH (AVVIH) podrían mejorarse mediante intervenciones dirigidas a abordar la limitada alfabetización sanitaria. Desarrollamos un video de rap en español sobre los conceptos del VIH y examinamos su aceptabilidad y viabilidad como herramienta de aprendizaje entre los AVVIH en Lima, Perú. Veintiocho AVVIH que reciben atención en un hospital pediátrico urbano y diez interesados involucrados en la atención de adolescentes vieron el video. Los adolescentes completaron un cuestionario previo y posterior al video. Realizamos grupos focales con AVVIH y entrevistas a profundidad a los interesados y analizamos las transcripciones para identificar los temas. Los AVVIH describieron conceptos de recuento de células CD4 y carga viral tal como se retrataron. Los participantes informaron que el video era identificable, accesible y brindaba la esperanza de que los AVVIH pudiera llevar una vida saludable y abogaron por videos futuros para abordar temas como la transmisión y la salud sexual. Los cuestionarios indicaron cierta mejora en el conocimiento de la carga viral. Una intervención de video musical para educación en salud sobre el VIH fue factible de implementar y fue aceptada por los AVVIH y sus proveedores de atención médica. La comunicación de conocimientos sobre el VIH a través de videos musicales puede ser prometedora; se necesitan más estudios para optimizar la implementación.

Quality Improvement to Address Surgical Burden of Disease at a Large Tertiary Public Hospital in Peru.

BACKGROUND: In resource-limited settings, there is a unique opportunity for using process improvement strategies to address the lack of access to surgical care. By implementing organizational changes in the surgical admission process, we aimed to decrease wait times, increase surgical volume, and improve patient satisfaction for elective general surgery procedures at a public tertiary hospital in Lima, Peru. METHODS: During the first phase of the intervention, Plan-Do-Study-Act (PDSA) cycles were performed to ensure the surgery waitlist included up-to-date clinical information. In the second phase, Lean Six Sigma methodology was used to adapt the admission and scheduling process for elective general surgery patients. After six months, outcomes were compared to baseline data using Wilcoxon rank-sum test. RESULTS: At the conclusion of phase one, 87.0% (488/561) of patients on the new waitlist had all relevant clinical data documented, improved from 13.3% (2/15) for the pre-existing list. Time from admission to discharge for all surgeries improved from 5 to 4 days (p<0.05) after the intervention. Median wait times from admission to operation for elective surgeries were unchanged at 4 days (p=0.076) pre- and post-intervention. There was a trend toward increased weekly elective surgical volume from a median of 9 to 13 cases (p=0.24) and increased patient satisfaction rates for elective surgery from 80.5 to 83.8% (p=0.62), although these were not statistically significant. CONCLUSION: The process for scheduling and admitting elective surgical patients became more efficient after our intervention. Time from admission to discharge for all surgical patients improved significantly. Other measured outcomes improved, though not with statistical significance. Main challenges included gaining buy-in from all participants and disruptions in surgical services from bed shortages.

Understanding health-related behavior among adolescents living with HIV in Lima, Peru.

BACKGROUND: The global HIV burden among adolescents ages 10-19 is growing. This population concurrently confronts the multifaceted challenges of adolescence and living with HIV. With the goal of informing future interventions tailored to this group, we assessed sexual activity, HIV diagnosis disclosure, combination antiretroviral therapy (cART) adherence, and drug use among adolescents living with HIV (ALHIV) in Lima, Peru. METHODS: Adolescents at risk or with a history of suboptimal cART adherence completed a self-administered, health behaviors survey and participated in support group sessions, which were audio recorded and used as a qualitative data source. Additionally, we conducted in-depth interviews with caregivers and care providers of ALHIV. Thematic content analysis was performed on the group transcripts and in-depth interviews and integrated with data from the survey to describe adolescents' health related behaviors. RESULTS: We enrolled 34 ALHIV, of which 32 (14 male, 18 female, median age 14.5 years) completed the health behavior survey. Nine (28%) adolescents reported prior sexual intercourse, a minority of whom (44%) reported using a condom. cART adherence was highest in the 10-12 age group with 89% reporting ≤2 missed doses in the last month, compared to 36% in adolescents 13 years or older. Over 80% of adolescents had never disclosed their HIV status to a friend or romantic partner. Adolescents, caregivers, and health service providers described sexual health misinformation and difficulty having conversations about sexual health and HIV. CONCLUSIONS: In this group of ALHIV, adherence to cART declined with age and condom use among sexually active adolescents was low. Multifactorial interventions addressing sexual health, gaps in HIV-related knowledge, and management of disclosure and romantic relationships are urgently needed for this population.

Community-Based Accompaniment with Supervised Antiretrovirals for HIV-Positive Adults in Peru: A Cluster-Randomized Trial.

We conducted a cluster-randomized trial to estimate effects of directly observed combination antiretroviral therapy (DOT-cART) on retention with viral suppression among HIV-positive adults in Peru. We randomly allocated facilities to receive the 12-month intervention plus the standard of care, including adherence support provided through accompaniment. In the intervention arm, health workers supervised doses, twice daily, and accompanied patients to appointments. Among 356 patients, intention-to-treat analyses showed no statistically significant benefit of DOT, relative to no-DOT, at 12 or 24 months (adjusted probability of primary outcome: 0.81 vs. 0.73 and 0.76 vs. 0.68, respectively). A statistically significant benefit of DOT was found in per-protocol and as-treated analyses at 12 months (0.83 for DOT vs. 0.73 for no DOT, p value: 0.02 per-protocol, 0.01 as-treated), but not 24 months. Rates of retention with viral suppression were high in both arms. Among adults receiving robust adherence support, the added effect of time-limited DOT, if any, is small-to-moderate.

Validation of a Three-Dimensional Method for Counting and Sizing Podocytes in Whole Glomeruli.

Podocyte depletion is sufficient for the development of numerous glomerular diseases and can be absolute (loss of podocytes) or relative (reduced number of podocytes per volume of glomerulus). Commonly used methods to quantify podocyte depletion introduce bias, whereas gold standard stereologic methodologies are time consuming and impractical. We developed a novel approach for assessing podocyte depletion in whole glomeruli that combines immunofluorescence, optical clearing, confocal microscopy, and three-dimensional analysis. We validated this method in a transgenic mouse model of selective podocyte depletion, in which we determined dose-dependent alterations in several quantitative indices of podocyte depletion. This new approach provides a quantitative tool for the comprehensive and time-efficient analysis of podocyte depletion in whole glomeruli.

Copy-number variation associated with congenital anomalies of the kidney and urinary tract.

BACKGROUND: The most common cause of end-stage renal disease in children can be attributed to congenital anomalies of the kidney and urinary tract (CAKUT). Despite this high incidence of disease, the genetic mutations responsible for the majority of CAKUT cases remain unknown. METHODS: To identify novel genomic regions associated with CAKUT, we screened 178 children presenting with the entire spectrum of structural anomalies associated with CAKUT for submicroscopic chromosomal imbalances (deletions or duplications) using single-nucleotide polymorphism (SNP) microarrays. RESULTS: Copy-number variation (CNV) was detected in 10.1 % (18/178) of the patients; in 6.2 % of the total cohort, novel duplications or deletions of unknown significance were identified, and the remaining 3.9 % harboured CNV of known pathogenicity. CNVs were inherited in 90 % (9/10) of the families tested. In this cohort, patients diagnosed with multicystic dysplastic kidney (30 %) and posterior urethral valves (24 %) had a higher incidence of CNV. CONCLUSIONS: The genes contained in the altered genomic regions represent novel candidates for CAKUT. This study has demonstrated that a significant proportion of patients with CAKUT harbour submicroscopic chromosomal imbalances, warranting screening in clinics for CNV.

Retention and Viral Suppression Among Adolescents Newly Initiating Antiretroviral Therapy in Adult HIV Care in Lima, Peru: A Retrospective Cohort Study.

PURPOSE: Published data on outcomes among adolescents newly initiating antiretroviral treatment in the Latin American context are sparse. We estimated the frequency of sustained retention with viral load suppression (i.e., successful transition) and identified predictors of successful transition into adult care among youth (aged 14-21 years) with recently acquired HIV in Lima, Peru. METHODS: A retrospective cohort study was conducted among 184 adolescents and young adults who initiated antiretroviral therapy in an adult public sector HIV clinic between June 2014 and June 2019. Sustained retention (no loss-to-follow-up or death) with viral suppression was calculated for the first 12 and 24 months following treatment initiation. We conducted regression analyses to assess factors associated with successful transition to adult HIV care, including gender, age, occupation, nationality, pregnancy, same-sex sexual behavior, presence of treatment supporter, number of living parents, and social risk factors that may adversely influence health (e.g., lack of social support, economic deprivation). RESULTS: Patients were predominantly male (n = 167, 90.8%). Median age was 19 years (interquartile range: 18-21). Frequency of sustained retention with viral load suppression was 42.4% (78/184) and 35.3% (30/85) at 12 and 24 months following treatment initiation. In multivariable analyses, working and/or studying was inversely associated with successful transition into adult care at 12 months; number of known living parents (relative risk: 2.20; 95% confidence interval: 1.12, 4.34) and absence of social risk factors (relative risk: 1.68; 95% confidence interval: 0.91, 3.11) were positively associated with successful transition at 24 months. DISCUSSION: Sustained retention in HIV care was uncommon. Parental support and interventions targeting social risk factors may contribute to successful transition into adult HIV care in this group.

Apoptotic cell identity induces distinct functional responses to IL-4 in efferocytic macrophages.

Macrophages are functionally heterogeneous cells essential for apoptotic cell clearance. Apoptotic cells are defined by homogeneous characteristics, ignoring their original cell lineage identity. We found that in an interleukin-4 (IL-4)-enriched environment, the sensing of apoptotic neutrophils by macrophages triggered their tissue remodeling signature. Engulfment of apoptotic hepatocytes promoted a tolerogenic phenotype, whereas phagocytosis of T cells had little effect on IL-4-induced gene expression. In a mouse model of parasite-induced pathology, the transfer of macrophages conditioned with IL-4 and apoptotic neutrophils promoted parasitic egg clearance. Knockout of phagocytic receptors required for the uptake of apoptotic neutrophils and partially T cells, but not hepatocytes, exacerbated helminth infection. These findings suggest that the identity of apoptotic cells may contribute to the development of distinct IL-4-driven immune programs in macrophages.

The Dynamics of Intimate Partner Violence and Its Impact on HIV Care: A Cross-Sectional Study of People of Mixed Gender and Sexual Preference in Lima, Peru.

Intimate partner violence (IPV) is associated with a higher risk of contracting HIV and developing worse HIV outcomes. This cross-sectional, mixed methods study presents data on IPV using the Conflicts Tactics Scale (CTS2-S) among 180 persons with HIV in Lima, Peru, as well as qualitative interviews with 7 of them and 18 of their community caregivers. This study used data collected for a randomized controlled trial (RCT), CASAommunity Based Accompaniment with Supervised Antiretrovirals (CASA) Community-based Accompaniment with Supervised Antiretrovirals (CASA). Physical or sexual IPV was self-reported in 82 (45.6%) of participants reporting having been in a relationship in the last year and 59,8% of those were involved in bidirectional violence. Coping subscales, social support, and stigma were associated with IPV. Intimate partner violence negatively impacted patient adherence to medication and care, particularly during times of severe conflict. In conclusion, profound psychosocial vulnerability-including low social support, substance use as coping, and HIV stigma-contextualize IPV among people with HIV. Bidirectional violence often evolved over time as victims negotiated inter-personal strategies for survival, including retaliation. Interventions should focus on a deeper understanding IPV and facilitating of coping mechanisms to help people with HIV stay in care.

Adolescent, caregiver and provider perspectives on tuberculosis treatment adherence: a qualitative study from Lima, Peru.

OBJECTIVES: To understand the perspectives of adolescents (10-19 years old), their caregivers and healthcare providers regarding factors that impact adherence to tuberculosis (TB) treatment among adolescents. DESIGN: We conducted in-depth interviews using semistructured interview guides based on the World Health Organization (WHO)'s Five Dimensions of Adherence framework, which conceptualises adherence as being related to the health system, socioeconomic factors, patient, treatment and condition. We applied framework thematic analysis. SETTING: Between August 2018 and May 2019, at 32 public health centres operated by the Ministry of Health in Lima, Peru. PARTICIPANTS: We interviewed 34 adolescents who completed or were lost to follow-up from treatment for drug-susceptible pulmonary TB disease in the preceding 12 months; their primary caregiver during treatment; and 15 nurses or nurse technicians who had ≥6 months' experience supervising TB treatment. RESULTS: Participants reported numerous treatment barriers, the most common of which were the inconvenience of health facility-based directly observed therapy (DOT), long treatment duration, adverse treatment events and symptom resolution. The support of adult caregivers was critical for helping adolescents overcome these barriers and carry out the behavioural skills (eg, coping with the large pill burden, managing adverse treatment events and incorporating treatment into daily routines) needed to adhere to treatment. CONCLUSION: Our findings support a three-pronged approach to improve TB treatment adherence among adolescents: (1) reduce barriers to adherence (eg, home-based or community-based DOT in lieu of facility-based DOT, reducing pill burden and treatment duration when appropriate), (2) teach adolescents the behavioural skills required for treatment adherence and (3) strengthen caregivers' ability to support adolescents.

Multicytokine-producing CD4+ T cells characterize the livers of patients with NASH.

A role of CD4+ T cells during the progression from nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH) has been suggested, but which polarization state of these cells characterizes this progression and the development of fibrosis remain unclear. In addition, a gut-liver axis has been suggested to play a role in NASH, but the role of CD4+ T cells in this axis has just begun to be investigated. Combining single-cell RNA sequencing and multiple-parameter flow cytometry, we provide the first cell atlas to our knowledge focused on liver-infiltrating CD4+ T cells in patients with NAFLD and NASH, showing that NASH is characterized by a population of multicytokine-producing CD4+ T cells. Among these cells, only those with a Th17 polarization state were enriched in patients with advanced fibrosis. In parallel, we observed that Bacteroides appeared to be enriched in the intestine of NASH patients and to correlate with the frequency of multicytokine-producing CD4+ T cells. In short, we deliver a CD4+ T cell atlas of NAFLD and NASH, providing the rationale to target CD4+ T cells with a Th17 polarization state to block fibrosis development. Finally, our data offer an early indication to test whether multicytokine-producing CD4+ T cells are part of the gut-liver axis characterizing NASH.

Accumulation of α-synuclein mediates podocyte injury in Fabry nephropathy.

Current therapies for Fabry disease are based on reversing intracellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosomal dysfunction. However, their effect in the reversal of end-organ damage, like kidney injury and chronic kidney disease, remains unclear. In this study, ultrastructural analysis of serial human kidney biopsies showed that long-term use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/Cas9-mediated α-galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified α-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.