A perfusable, vascularized kidney organoid-on-chip model.

The ability to controllably perfuse kidney organoids would better recapitulate the native tissue microenvironment for applications ranging from drug testing to therapeutic use. Here, we report a perfusable, vascularized kidney organoid on chip model composed of two individually addressable channels embedded in an extracellular matrix (ECM). The channels are respectively seeded with kidney organoids and human umbilical vein endothelial cells that form a confluent endothelium (macrovessel). During perfusion, endogenous endothelial cells present within the kidney organoids migrate through the ECM towards the macrovessel, where they form lumen-on-lumen anastomoses that are supported by stromal-like cells. Once micro-macrovessel integration is achieved, we introduced fluorescently labeled dextran of varying molecular weight and red blood cells into the macrovessel, which are transported through the microvascular network to the glomerular epithelia within the kidney organoids. Our approach for achieving controlled organoid perfusion opens new avenues for generating other perfused human tissues.

Immune-infiltrated kidney organoid-on-chip model for assessing T cell bispecific antibodies.

T cell bispecific antibodies (TCBs) are the focus of intense development for cancer immunotherapy. Recently, peptide-MHC (major histocompatibility complex)-targeted TCBs have emerged as a new class of biotherapeutics with improved specificity. These TCBs simultaneously bind to target peptides presented by the polymorphic, species-specific MHC encoded by the human leukocyte antigen (HLA) allele present on target cells and to the CD3 coreceptor expressed by human T lymphocytes. Unfortunately, traditional models for assessing their effects on human tissues often lack predictive capability, particularly for "on-target, off-tumor" interactions. Here, we report an immune-infiltrated, kidney organoid-on-chip model in which peripheral blood mononuclear cells (PBMCs) along with nontargeting (control) or targeting TCB-based tool compounds are circulated under flow. The target consists of the RMF peptide derived from the intracellular tumor antigen Wilms' tumor 1 (WT1) presented on HLA-A2 via a bivalent T cell receptor-like binding domain. Using our model, we measured TCB-mediated CD8+ T cell activation and killing of RMF-HLA-A2-presenting cells in the presence of PBMCs and multiple tool compounds. DP47, a non-pMHC-targeting TCB that only binds to CD3 (negative control), does not promote T cell activation and killing. Conversely, the nonspecific ESK1-like TCB (positive control) promotes CD8+ T cell expansion accompanied by dose-dependent T cell-mediated killing of multiple cell types, while WT1-TCB* recognizing the RMF-HLA-A2 complex with high specificity, leads solely to selective killing of WT1-expressing cells within kidney organoids under flow. Our 3D kidney organoid model offers a platform for preclinical testing of cancer immunotherapies and investigating tissue-immune system interactions.

Programming Cellular Alignment in Engineered Cardiac Tissue via Bioprinting Anisotropic Organ Building Blocks.

The ability to replicate the 3D myocardial architecture found in human hearts is a grand challenge. Here, the fabrication of aligned cardiac tissues via bioprinting anisotropic organ building blocks (aOBBs) composed of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) is reported. A bioink composed of contractile cardiac aOBBs is first generated and aligned cardiac tissue sheets with linear, spiral, and chevron features are printed. Next, aligned cardiac macrofilaments are printed, whose contractile force and conduction velocity increase over time and exceed the performance of spheroid-based cardiac tissues. Finally, the ability to spatially control the magnitude and direction of contractile force by printing cardiac sheets with different aOBB alignment is highlighted. This research opens new avenues to generating functional cardiac tissue with high cell density and complex cellular alignment.

Health Education about Rheumatic Heart Disease: A Community-Based Cluster Randomized Trial: Rheumatic Heart Disease Educational Strategies.

Introduction: The burden of rheumatic heart disease (RHD) is still high in Brazil. Lack of population awareness about the disease limits the efficacy of prevention programs. We aimed to evaluate the effectiveness of education on RHD in schools, comparing the conventional expository teaching method with tablet-based worked examples. Method: A prospective, cluster randomized trial was conducted over eight months in six randomly selected low-income Brazilian public schools. Each class was considered a cluster (total: 90), being randomized 1:1 to receive one of the educational methods. Pre-test evaluated students' prior knowledge on RHD. Post-tests, 10 days, and three months later, evaluated retention of knowledge. Results: At total 1,301 students (52% female) completed the study, being 63% from high school. Baseline knowledge about RHD was universally low (average score expository classes [G1] 33.9% vs. worked examples [G2] 32.5%, p = 0.23). A significant but similar improvement was observed in both groups in the immediate post-test (pre- vs. post: p < 0.001): G1 57.5% vs. G2 56.7%, p = 0.69. In the late post-test, a significant 20% worsening was observed in both groups and the final scores were again similar: G1 45.0% vs. G2 45.9%, p = 0.87. Highschool students had higher scores (p < 0.001), and girls had better overall performances than boys (p < 0.001). Conclusion: The novel technology of tablet-based worked examples had similar results compared with expository classes for RHD education in schools. Both educational processes resulted in modest gains in knowledge, with low retention. More studies are needed to determine if increased knowledge leads to behavioral changes that could reduce RHD burden. Highlights: In a cluster-randomized trial, two different educational strategies about Rheumatic Heart Disease (RHD) for children - standard expository classes and worked examples based on interactive modules in tablet computers - were compared in public schools of underserved Brazilian neighborhoods.Baseline knowledge was low, and the novel tablet-based technology had similar results compared with traditional teaching for RHD education in schools, with no differences in acquisition and mid-term retention of knowledge.Both educational interventions resulted in similar 71% improvement in the immediate post-test, although with suboptimal retention, with over 20% worsening in three months in both groups.Our data suggests that the optimal strategy for RHD education is yet to be determined, and future studies should be warranted to determine if increased knowledge leads to behavioral changes that could reduce disease burden in endemic areas.

Validation of a simplified score for predicting latent rheumatic heart disease progression using a prospective cohort of Brazilian schoolchildren.

OBJECTIVES: Echocardiographic (echo) screening is an important tool to estimate rheumatic heart disease (RHD) prevalence, but the natural history of screen-detected RHD remains unclear. The PROVAR+ (Programa de RastreamentO da VAlvopatia Reumática) study, which uses non-experts, telemedicine and portable echo, pioneered RHD screening in Brazil. We aimed to assess the mid-term evolution of Brazilian schoolchildren (5-18 years) with echocardiography-detected subclinical RHD and to assess the performance of a simplified score consisting of five components of the World Heart Federation criteria, as a predictor of unfavourable echo outcomes. SETTING: Public schools of underserved areas and private schools in Minas Gerais, southeast Brazil. PARTICIPANTS: A total of 197 patients (170 borderline and 27 definite RHD) with follow-up of 29±9 months were included. Median age was 14 (12-16) years, and 130 (66%) were woman. Only four patients in the definite group were regularly receiving penicillin. PRIMARY AND SECONDARY OUTCOME MEASURES: Unfavourable outcome was based on the 2-year follow-up echo, defined as worsening diagnostic category, remaining with mild definite RHD or development/worsening of valve regurgitation/stenosis. RESULTS: Among patients with borderline RHD, 29 (17.1%) progressed to definite, 49 (28.8%) remained stable, 86 (50.6%) regressed to normal and 6 (3.5%) were reclassified as other heart diseases. Among those with definite RHD, 13 (48.1%) remained in the category, while 5 (18.5%) regressed to borderline, 5 (18.5%) regressed to normal and 4 (14.8%) were reclassified as other heart diseases. The simplified echo score was a significant predictor of RHD unfavourable outcome (HR 1.197, 95% CI 1.098 to 1.305, p<0.001). CONCLUSION: The simple risk score provided an accurate prediction of RHD status at 2-year follow-up, showing a good performance in Brazilian schoolchildren, with a potential value for risk stratification and monitoring of echocardiography-detected RHD.

Atrial fibrillation detection with a portable device during cardiovascular screening in primary care.

INTRODUCTION: A novel handheld dual-electrode stick is a portable atrial fibrillation (AF) screening device (AFSD). We evaluated AFSD performance in primary care patients referred for echocardiogram (echo). METHODS: The AFSD has a light indication of irregular rhythm and single-lead ECG recording. Patients were instructed to hold the device for 1 min, and AF indication was recorded. A 12-lead ECG was performed for all AFSD-positive patients and 250 patients with negative AFSD screen. Echos were performed based on a clinical risk score: all high-risk patients and a sampling of low-risk patients underwent complete echo. Intermediate risk patients first had a screening echocardiogram, with a follow-up complete study if abnormality was suspected. RESULTS: In 5 days, 1518 patients underwent clinical evaluation and cardiovascular risk stratification: mean age 58±16 years, 66% women. The AFSD was positive in 6.4%: 12.6% high risk, 6.1% intermediate risk and 2.2% low risk. Older age was a risk factor (9.3% vs 4.8% in those more than and less than 65 years, p=0.001). AFSD positive was independently associated with heart disease in echo (OR=3.9, 95% CI 2.1 to 7.2, p<0.001). Compared with 12-lead ECG, the AFSD had sensitivity of 90.2% (95% CI 77.0% to 97.3%) and specificity of 84.0% (95% CI 79.3% to 88.0%) for AF detection. CONCLUSION: AFSD demonstrated high sensitivity for AF detection in primary care patients referred for echo. AF prevalence was substantial and independently associated with structural or functional heart disease, suggesting that AFSD screening could be a useful primary care tool to stratify risk and prioritise echo.

Altered In Vivo Lipid Fluxes and Cell Dynamics in Subcutaneous Adipose Tissues Are Associated With the Unfavorable Pattern of Fat Distribution in Obese Adolescent Girls.

Patterns of abdominal fat distribution (for example, a high vs. low visceral adipose tissue [VAT]/[VAT + subcutaneous adipose tissue (SAT)] ratio), independent of obesity, during adolescence carry a high risk for insulin resistance and type 2 diabetes. Longitudinal follow-up of a cohort of obese adolescents has recently revealed that a high ratio (high VAT/[VAT + SAT]) is a major determinant of fatty liver and metabolic impairment over time, with these effects being more pronounced in girls than in boys. To unravel the underlying metabolic alterations associated with the unfavorable VAT/(VAT + SAT) phenotype, we used the 2H2O labeling method to measure the turnover of adipose lipids and cells in the subcutaneous abdominal and gluteal/femoral adipose tissue (SAT) of weight-stable obese adolescent girls with a similar level of obesity but discordant VAT/(VAT + SAT) ratios. Girls with the unfavorable (high VAT/[VAT + SAT]) phenotype exhibited higher in vivo rates of triglyceride (TG) turnover (representing both lipolysis and synthesis at steady state), without significant differences in de novo lipogenesis in both abdominal and gluteal depots, compared with obese girls with the favorable phenotype. Moreover, mature adipocytes had higher turnover, with no difference in stromal vascular cell proliferation in both depots in the metabolically unfavorable phenotype. The higher TG turnover rates were significantly correlated with higher intrahepatic fat stores. These findings are contrary to the hypothesis that impaired capacity to deposit TGs or proliferation of new mature adipocytes are potential mechanisms for ectopic fat distribution in this setting. In summary, these results suggest that increased turnover of TGs (lipolysis) and of mature adipocytes in both abdominal and gluteal SAT may contribute to metabolic impairment and the development of fatty liver, even at this very early stage of disease.

Relation of the degree of obesity in childhood to adipose tissue insulin resistance.

AIMS: In this study, we investigated whether adipose tissue insulin resistance (IR) is affected by the degree of obesity during the fasting and post-prandial state, independent of glucose tolerance among obese children and adolescents. We also tested whether systemic subclinical inflammation is associated with adipose tissue IR. METHODS: Subjects were recruited to the Yale Pathophysiology of Type 2 Diabetes in Youth Study (NCT01967849). An oral glucose-tolerance test was performed to establish glucose-tolerance status and blood samples were drawn for measurement of free fatty acids (FFAs), to calculate the area under the curve (AUC) of FFA. Adipose tissue insulin resistance was calculated as the product of insulin and FFA concentrations. RESULTS: In total, 671 children and adolescents (58.6% females) were included with a mean age of 13.3(2.7) years and BMI Z score of 2.45(0.31). The degree of obesity emerged as an independent predictor of both fasting and post-prandial adipose IR, p < 0.0001. Higher degree of obesity was associated with greater AUC FFA (lower suppression) compared to lower degree of obesity, p = 0.01. Furthermore, higher levels of IL-6 were positively associated with post-prandial adipose tissue IR, p = 0.02. CONCLUSIONS: The degree of obesity in childhood and adolescence is strongly associated with adipose tissue IR independent of glucose tolerance. This is reflected not only in calculated indices of adipose IR but also in lower suppression of FFAs during the OGTT regardless of glucose tolerance or fasting adipose tissue IR. Furthermore, markers of subclinical inflammation such as IL-6 are associated with adipose tissue IR, independent of other factors.

A low visceral fat proportion, independent of total body fat mass, protects obese adolescent girls against fatty liver and glucose dysregulation: a longitudinal study.

BACKGROUND: The relative proportion of visceral fat (VAT) to subcutaneous fat (SAT) has been described as a major determinant of insulin resistance (IR). Our study sought to evaluate the effect of body fat distribution on glucose metabolism and intrahepatic fat content over time in a multiethnic cohort of obese adolescents. SUBJECTS/METHODS: We examined markers of glucose metabolism by oral glucose tolerance test, and body fat distribution by abdominal MRI at baseline and after 19.2 ± 11.4 months in a cohort of 151 obese adolescents (88 girls, 63 boys; mean age 13.3 ± 3.4 years; mean BMI z-score 2.15 ± 0.70). Hepatic fat content was assessed by fast-gradient MRI in a subset of 93 subjects. We used the median value of VAT/(VAT + SAT) ratio within each gender at baseline to stratify our sample into high and low ratio groups (median value 0.0972 in girls and 0.118 in boys). RESULTS: Female subjects tended to remain in their VAT/(VAT + SAT) category over time (change over follow-up P = 0.14 among girls, and P = 0.04 among boys). Baseline VAT/(VAT + SAT) strongly predicted the hepatic fat content, fasting insulin, 2-h glucose, and whole-body insulin sensitivity index at follow-up among girls, but not in boys. CONCLUSIONS: The VAT/(VAT + SAT) ratio is a major determinant of impaired glucose metabolism and hepatic fat accumulation over time, and its effects are more pronounced in girls than in boys.

Effects of Fish Oil on HIV-Related Inflammation and Markers of Immunosenescence: A Randomized Clinical Trial.

OBJECTIVE: To explore the safety and efficacy of fish oil to modulate parameters of inflammation and immunosenescence in HIV-infected older adults. DESIGN: This study uses a randomized, controlled, double-blind clinical trial. SETTING: The study was conducted in an outpatient HIV/AIDS clinic in a large urban Midwestern city in the United States. SUBJECTS: A total of 37 clinically stable HIV-infected adults between the ages of 40 and 70 years of age participated. INTERVENTIONS: Fish oil 1.6 g/day was administered for 12 weeks or placebo. OUTCOME MEASURES: Inflammatory cytokine production, surface markers of immunosenescence, and adverse events were measured. RESULTS: After 12 weeks of supplementation, there were no significant differences between the treatment and control groups on any measures of inflammation or immunosenescence in both CD4+ and CD8+ T lymphocytes. More participants in the treatment group reported adverse gastrointestinal events compared with the control group. CONCLUSIONS: A 12-week supplementation regimen of 1.6 g/day of fish oil did not favorably modulate parameters of inflammation or immune senescence in HIV-infected adults. Future studies should test agents that directly target mechanisms that underlie HIV-related inflammation to determine whether reducing inflammation can reverse immunosenescence.

Metabolic Features of Nonalcoholic Fatty Liver (NAFL) in Obese Adolescents: Findings From a Multiethnic Cohort.

We conducted a prospective study in a large, multiethnic cohort of obese adolescents to characterize clinical and genetic features associated with pediatric nonalcoholic fatty liver (NAFL), the most common cause of chronic liver disease in youth. A total of 503 obese adolescents were enrolled, including 191 (38.0%) whites, 134 (26.6%) blacks, and 178 (35.4%) Hispanics. Participants underwent abdominal magnetic resonance imaging (MRI) to quantify hepatic fat fraction (HFF), an oral glucose tolerance test (OGTT) to assess glucose tolerance and insulin sensitivity, and the genotyping of three single-nucleotide polymorphisms (SNPs) associated with nonalcoholic fatty liver disease (NAFLD) (patatin-like phospholipase domain-containing protein 3 [PNPLA3] rs738409, glucokinase regulatory protein [GCKR] rs1260326, and transmembrane 6 superfamily member 2 [TM6SF2] rs58542926). Assessments were repeated in 133 subjects after a 2-year follow-up. Prevalence of nonalcoholic fatty liver (NAFL) was 41.6% (209 patients) and ranged widely among ethnicities, being 42.9% in whites, 15.7% in blacks, and 59.6% in Hispanics (P < 0.0001). Among adolescents with NAFL, blacks showed the highest prevalence of altered glucose homeostasis (66%; P = 0.0003). Risk factors for NAFL incidence were white or Hispanic ethnicity (P = 0.021), high fasting C-peptide levels (P = 0.0006), and weight gain (P = 0.0006), whereas baseline HFF (P = 0.004) and weight loss (P = 0.032) predicted resolution of NAFL at follow-up. Adding either gene variant to these variables improved significantly the model predictive performance. CONCLUSION: Black obese adolescents are relatively protected from liver steatosis, but are more susceptible to the deleterious effects of NAFL on glucose metabolism. The combination of ethnicity/race with markers of insulin resistance and genetic factors might help identify obese youth at risk for developing NAFL.

Methamphetamine decreases CD4 T cell frequency and alters pro-inflammatory cytokine production in a model of drug abuse.

The reason co-morbid methamphetamine use and HIV infection lead to more rapid progression to AIDS is unclear. We used a model of methamphetamine self-administration to measure the effect of methamphetamine on the systemic immune system to better understand the co-morbidity of methamphetamine and HIV. Catheters were implanted into the jugular veins of male, Sprague Dawley rats so they could self-administer methamphetamine (n=18) or be given saline (control; n=16) for 14 days. One day after the last operant session, blood and spleens were collected. We measured serum levels of pro-inflammatory cytokines, intracellular IFN-γ and TNF-α, and frequencies of CD4(+), CD8(+), CD200(+) and CD11b/c(+) lymphocytes in the spleen. Rats that self-administered methamphetamine had a lower frequency of CD4(+) T cells, but more of these cells produced IFN-γ. Methamphetamine did not alter the frequency of TNF-α-producing CD4(+) T cells. Methamphetamine using rats had a higher frequency of CD8(+) T cells, but fewer of them produced TNF-α. CD11b/c and CD200 expression were unchanged. Serum cytokine levels of IFN-γ, TNF-α and IL-6 in methamphetamine rats were unchanged. Methamphetamine lifetime dose inversely correlated with serum TNF-α levels. Our data suggest that methamphetamine abuse may exacerbate HIV disease progression by activating CD4 T cells, making them more susceptible to HIV infection, and contributing to their premature demise. Methamphetamine may also increase susceptibility to HIV infection, explaining why men who have sex with men (MSM) and frequently use methamphetamine are at the highest risk of HIV infection.

Development of IgG Mediated Antibody Dependent Cell-mediated Cytotoxicity (ADCC) in the Serum and Genital Mucosa of HIV Seroconverters.

BACKGROUND: We measured antibody-dependent cell mediated cytotoxicity (ADCC) activity in serum and genital fluids of heterosexually exposed women during HIV seroconversion. METHODS: Plasma and cervico-vaginal lavage (CVL) fluid from 11 seroconverters (SC) were analyzed biannually from one year pre- to 6 year post-seroconversion using a 51Cr-release assay to measure HIV-1 gp120 specific ADCC. RESULTS: No SC had significant HIV specific CVL ADCC activity before seroconversion or until 1.5 yr after seroconversion. One individual had a %Specific Release (SR) of 25.4 at 2 years, 26.7 at 3 years and 21.0 at 4 years after seroconversion in CVL. Another sample had 4.7% SR at 2 years, 5.3 at 3 years, 10.9 at 4 years, and 8.4 at 5 years after seroconversion in CVL. A third had no activity until 17% SR 5 years after seroconversion in CVL. A fourth showed activity of 36.5% SR at 6.5 years after seroconversion. Seven women had no ADCC activity in their CVL. Paired serum samples showed HIV specific ADCC activity prior to the appearance of CVL ADCC activity. CONCLUSIONS: HIV specific ADCC activity in CVL rose 2 years after seroconversion; ADCC was present in the serum prior to this time. These data suggest that genital tract ADCC activity is not present until well after acute infection.

Effects of cryopreservation on effector cells for antibody dependent cell-mediated cytotoxicity (ADCC) and natural killer (NK) cell activity in (51)Cr-release and CD107a assays.

Freshly isolated PBMC are broadly used as effector cells in functional assays that evaluate antibody-dependent cell mediated cytotoxicity (ADCC) and NK activity; however, they introduce natural-individual donor-to-donor variability. Cryopreserved PBMC provide a more consistent source of effectors than fresh cells in cytotoxicity assays. Our objective was to determine the effects of cryopreservation of effector PBMC on cell frequency, and on the magnitude and specificity of ADCC and NK activity. Fresh, frozen/overnight rested and frozen/not rested PBMC were used as effector cells in (51)Cr-release and CD107a degranulation assays. Frozen/overnight rested PBMC had higher ADCC and NK activity in both assays when compared to fresh PBMC; however, when using frozen/not rested PBMC, ADCC and NK activities were significantly lower than fresh PBMC. Background CD107a degranulation in the absence of target cell stimulation was greater in PBMC that were frozen/not rested when compared to fresh PBMC or PBMC that were frozen overnight and rested. The percentages of CD16(+)CD56(dim) NK cells and CD14(+) monocytes were lower in PBMC that were frozen and rested overnight than in fresh PBMC. CD16 expression on CD56(dim) NK cells was similar for all PBMC treatments. PBMC that were frozen and rested overnight were comparable to fresh PBMC effectors. PBMC that were frozen and used immediately when evaluating ADCC or NK activity using either a (51)Cr-release assay or a CD107a degranulation assay had the lowest activity. Clinical studies of antibodies that mediate ADCC would benefit from using effector cells that have been frozen, thawed and rested overnight prior to assay.

Comparison of antibodies that mediate HIV type 1 gp120 antibody-dependent cell-mediated cytotoxicity in asymptomatic HIV type 1-positive men and women.

Recent studies suggest that HIV-specific antibody-dependent cell-mediated cytotoxicity (ADCC) antibodies contribute to protective immunity against HIV. An important characteristic of future HIV vaccines will, therefore, be the ability to stimulate production of these antibodies in both men and women. Early studies suggest that men may have a better ADCC antibody response against HIV than women. Our objective was to determine whether men and women differ with respect to their ADCC response to HIV-1 gp120. HIV-positive, asymptomatic untreated men and women were matched for race, age, CD4(+) T cell number, HIV-1 viral load, and treatment and HIV-1 gp120 ADCC antibody titers were compared. A standard (51)Cr-release assay was used to determine HIV-1 gp120 ADCC antibody titers in HIV-1-seropositive individuals from the Multicenter AIDS Cohort Study (MACS; n=32) and the Women's Interagency HIV Study (WIHS; n=32). Both sexes had high ADCC titers against HIV-1 gp120: 34.4% (n=11) and 40.6% (n=13) of men and women, respectively, had titers of 10,000; 62.5% (n=20) and 56.3% (n=18) had titers of 100,000. Groups did not differ in percent specific release (% SR), lytic units (LU), correlations of titer to viral load, or titer to CD4(+) T cells in men or women. Both groups also had similar cross-clade ADCC antibody responses (p>0.5 for % SR and LU). Comparable groups of asymptomatic HIV-1-infected men and women had comparable HIV-1 gp120 ADCC antibodies. Both sexes had significant cross-clade reactivity. Differences between men and women may become evident as disease progresses; this should be evaluated at later stages of HIV-1 infection.