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1.
J Exp Med ; 221(6)2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38634869

RESUMO

We previously reported two siblings with inherited PD-1 deficiency who died from autoimmune pneumonitis at 3 and 11 years of age after developing other autoimmune manifestations, including type 1 diabetes (T1D). We report here two siblings, aged 10 and 11 years, with neonatal-onset T1D (diagnosed at the ages of 1 day and 7 wk), who are homozygous for a splice-site variant of CD274 (encoding PD-L1). This variant results in the exclusive expression of an alternative, loss-of-function PD-L1 protein isoform in overexpression experiments and in the patients' primary leukocytes. Surprisingly, cytometric immunophenotyping and single-cell RNA sequencing analysis on blood leukocytes showed largely normal development and transcriptional profiles across lymphoid and myeloid subsets in the PD-L1-deficient siblings, contrasting with the extensive dysregulation of both lymphoid and myeloid leukocyte compartments in PD-1 deficiency. Our findings suggest that PD-1 and PD-L1 are essential for preventing early-onset T1D but that, unlike PD-1 deficiency, PD-L1 deficiency does not lead to fatal autoimmunity with extensive leukocytic dysregulation.


Assuntos
Antígeno B7-H1 , Diabetes Mellitus Tipo 1 , Criança , Pré-Escolar , Humanos , Recém-Nascido , Autoimunidade , Antígeno B7-H1/deficiência , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Homozigoto , Receptor de Morte Celular Programada 1/deficiência , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia
2.
Cytometry A ; 101(4): 351-360, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34967113

RESUMO

Mislabeling samples or data with the wrong participant information can affect study integrity and lead investigators to draw inaccurate conclusions. Quality control to prevent these types of errors is commonly embedded into the analysis of genomic datasets, but a similar identification strategy is not standard for cytometric data. Here, we present a method for detecting sample identification errors in cytometric data using expression of human leukocyte antigen (HLA) class I alleles. We measured HLA-A*02 and HLA-B*07 expression in three longitudinal samples from 41 participants using a 33-marker CyTOF panel designed to identify major immune cell types. 3/123 samples (2.4%) showed HLA allele expression that did not match their longitudinal pairs. Furthermore, these same three samples' cytometric signature did not match qPCR HLA class I allele data, suggesting that they were accurately identified as mismatches. We conclude that this technique is useful for detecting sample-labeling errors in cytometric analyses of longitudinal data. This technique could also be used in conjunction with another method, like GWAS or PCR, to detect errors in cross-sectional data. We suggest widespread adoption of this or similar techniques will improve the quality of clinical studies that utilize cytometry.


Assuntos
Estudos Transversais , Alelos , Humanos , Reação em Cadeia da Polimerase em Tempo Real
3.
Int J Gynaecol Obstet ; 157(3): 604-612, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34358336

RESUMO

OBJECTIVE: To investigate whether angiogenic biomarker concentrations differ between women who deliver small-for-gestational-age (SGA) infants (<10th centile birth weight for gestational age) compared with controls, because identifying SGA risk early could improve outcomes. METHODS: This case-control study compared serum concentrations of angiogenic biomarkers before 24 weeks of pregnancy from 62 women who delivered SGA infants (cases) and 62 control women from an urban Zambian cohort. Odds of delivering an SGA infant were calculated using conditional logistic regression. RESULTS: Placental growth factor (PlGF), soluble fms-like tyrosine kinase (sFLT-1) and soluble endoglin (sEng) in controls were 37.74 pg/mL (interquartile range [IQR] 23.12-63.15), 2525.18 pg/mL (IQR 1502.21-4265.54) and 2408.18 pg/mL (IQR 1854.87-3017.94), respectively. SGA cases had higher PlGF (40.50 pg/mL, IQR 22.81-67.94) and sFLT-1 (2613.06 pg/mL, IQR 1720.58-3722.50), and lower sEng (2038.06 pg/mL, IQR 1445.25-3372.26). Participants with sEng concentration below and concomitant sFLT-1 concentration above their respective thresholds (n = 40) had five-fold higher odds of SGA (adjusted odds ratio 4.77, 95% confidence interval 1.61-14.1; P = 0.005). CONCLUSION: Biomarker concentrations were similar between cases and controls. Participants with concomitant low sEng and high sFLT-1 had the highest odds of SGA, suggesting that a combination of biomarkers may better for predicting SGA than single biomarkers.


Assuntos
Recém-Nascido Pequeno para a Idade Gestacional , Pré-Eclâmpsia , Biomarcadores , Estudos de Casos e Controles , Endoglina , Feminino , Humanos , Lactente , Recém-Nascido , Fator de Crescimento Placentário , Gravidez , Zâmbia
4.
Lancet HIV ; 8(10): e605-e613, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34509197

RESUMO

BACKGROUND: Women with HIV face an increased risk of preterm birth. 17 alpha-hydroxyprogesterone caproate (17P) has been shown in some trials to reduce early delivery among women with a history of spontaneous preterm birth. We investigated whether 17P would reduce this risk among women with HIV. METHODS: We did a randomised, double-blind, placebo-controlled trial in pregnant women with HIV at the University Teaching Hospital and Kamwala District Health Centre in Lusaka, Zambia. Eligible patients were women aged 18 years or older with confirmed HIV-1 infection, viable intrauterine singleton pregnancy at less than 24 weeks of gestation, and were receiving or intending to commence antiretroviral therapy during pregnancy. Exclusion criteria were major uterine or fetal anomaly; planned or in situ cervical cerclage; evidence of threatened miscarriage, preterm labour, or ruptured membranes at screening; medical contraindication to 17P; previous participation in the trial; or history of spontaneous preterm birth. Eligible participants provided written informed consent and were randomly assigned (1:1) to receive 250 mg intramuscular 17P or placebo once per week, starting between 16 and 24 weeks of gestation until delivery, stillbirth, or reaching term (37 weeks). Participants and study staff were masked to assignment, except for pharmacy staff who did random assignment and prepared injections but did not interact with participants. The primary outcome was a composite of delivery before 37 weeks or stillbirth at any gestational age. Patients attended weekly visits for study drug injections and antenatal care. We estimated the absolute and relative difference in risk of the primary outcome and safety events between treatment groups by intention to treat. This trial is registered with ClinicalTrials.gov, NCT03297216, and is complete. FINDINGS: Between Feb 7, 2018 and Jan 13, 2020, we assessed 1042 women for inclusion into the study. 242 women were excluded after additional assessments, and 800 eligible patients were enrolled and randomly assigned to receive intramuscular 17P (n=399) or placebo (n=401). Baseline characteristics were similar between groups. Adherence to study drug injections was 98% in both groups, no patients were lost to follow-up, and the final post-partum visit was on Aug 6, 2020. 36 (9%) of 399 participants assigned to 17P had preterm birth or stillbirth, compared with 36 (9%) of 401 patients assigned to placebo (risk difference 0·1, 95% CI -3·9 to 4·0; relative risk 1·0, 95% CI 0·6 to 1·6; p=0·98). Intervention-related adverse events were reported by 140 (18%) of 800 participants and occurred in similar proportions in both randomisation groups. No serious adverse events were reported. INTERPRETATION: Although 17P seems to be safe and acceptable to participants, available data do not support the use of the drug to prevent preterm birth among women whose risk derives solely from HIV infection. The low risk of preterm birth in both randomisation groups warrants further investigation. FUNDING: US National Institutes of Health and the Bill and Melinda Gates Foundation.


Assuntos
Infecções por HIV , Nascimento Prematuro , Caproato de 17 alfa-Hidroxiprogesterona/uso terapêutico , Adolescente , Método Duplo-Cego , Feminino , Idade Gestacional , Infecções por HIV/tratamento farmacológico , Infecções por HIV/prevenção & controle , Humanos , Recém-Nascido , Gravidez , Nascimento Prematuro/tratamento farmacológico , Nascimento Prematuro/epidemiologia , Nascimento Prematuro/prevenção & controle , Zâmbia
5.
BMC Pregnancy Childbirth ; 21(1): 534, 2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34320947

RESUMO

BACKGROUND: Maternal HIV increases the risk of adverse birth outcomes including preterm birth, fetal growth restriction, and stillbirth, but the biological mechanism(s) underlying this increased risk are not well understood. We hypothesized that maternal HIV may lead to adverse birth outcomes through an imbalance in angiogenic factors involved in the vascular endothelial growth factor (VEGF) signaling pathway. METHODS: In a case-control study nested within an ongoing cohort in Zambia, our primary outcomes were serum concentrations of VEGF-A, soluble endoglin (sEng), placental growth factor (PlGF), and soluble fms-like tyrosine kinase-1 (sFLT-1). These were measured in 57 women with HIV (cases) and 57 women without HIV (controls) before 16 gestational weeks. We used the Wilcoxon rank-sum and linear regression controlling for maternal body mass index (BMI) and parity to assess the difference in biomarker concentrations between cases and controls. We also used logistic regression to test for associations between biomarker concentration and adverse pregnancy outcomes (preeclampsia, preterm birth, small for gestational age, stillbirth, and a composite of preterm birth or stillbirth). RESULTS: Compared to controls, women with HIV had significantly lower median concentrations of PlGF (7.6 vs 10.2 pg/mL, p = 0.02) and sFLT-1 (1647.9 vs 2055.6 pg/mL, p = 0.04), but these findings were not confirmed in adjusted analysis. PlGF concentration was lower among women who delivered preterm compared to those who delivered at term (6.7 vs 9.6 pg/mL, p = 0.03) and among those who experienced the composite adverse birth outcome (6.2 vs 9.8 pg/mL, p = 0.02). Median sFLT-1 concentration was lower among participants with the composite outcome (1621.0 vs 1945.9 pg/mL, p = 0.04), but the association was not significant in adjusted analysis. sEng was not associated with either adverse birth outcomes or HIV. VEGF-A was undetectable by Luminex in all specimens. CONCLUSIONS: We present preliminary findings that HIV is associated with a shift in the VEGF signaling pathway in early pregnancy, although adjusted analyses were inconclusive. We confirm an association between angiogenic biomarkers and adverse birth outcomes in our population. Larger studies are needed to further elucidate the role of HIV on placental angiogenesis and adverse birth outcomes.


Assuntos
Endoglina/sangue , Infecções por HIV/sangue , Fator de Crescimento Placentário/sangue , Complicações Infecciosas na Gravidez/sangue , Resultado da Gravidez/epidemiologia , Fator A de Crescimento do Endotélio Vascular/sangue , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/sangue , Adulto , Indutores da Angiogênese , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Humanos , Placenta/irrigação sanguínea , Gravidez , Nascimento Prematuro/epidemiologia , Zâmbia/epidemiologia
6.
AIChE J ; 65(12)2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32921797

RESUMO

Many cell behaviors are significantly affected by cell culture geometry, though it remains unclear which geometry from two- to three-dimensional (2D to 3D) culture is appropriate for probing a specific cell function and mimicking native microenvironments. Toward addressing this, we established a 2.5D culture geometry, enabling initial cell spreading while reducing polarization to bridge between 2D and 3D geometries, and examined the responses of wound healing cells, human pulmonary fibroblasts, within it. To achieve this, we used engineered biomimetic hydrogels formed by photopolymerization, creating robust layered hydrogels with spread fibroblasts at the interface. We found that fibroblast responses were similar between 2D and 2.5D culture and different from 3D culture, with some underlying differences in mechanotransduction. These studies established the 2.5D cell culture geometry in conjunction with biomimetic synthetic matrices as a useful tool for investigations of fibroblast activation with relevance to the study of other cell functions and types.

7.
ACS Biomater Sci Eng ; 4(9): 3304-3316, 2018 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-32494587

RESUMO

Synthetic hydrogels with well-defined mechanical properties have become invaluable tools for probing cell response to extracellular cues including matrix stiffness and integrin binding. These synthetic matrices are often decorated with either proteins or integrin-binding peptides to promote cell adhesion and to direct or probe cell behavior. For example, both collagen I-functionalized polyacrylamide and peptide-functionalized poly(ethylene glycol) hydrogels have been instrumental in elucidating the role of the elasticity or 'stiffness' of the matrix in promoting fibroblast activation in wound healing and fibrosis. However, the two methods of promoting integrin binding are not often directly compared in the same system, partly owing to differences in material designs, despite the potential differences in the way cells interact with whole proteins and protein mimetic peptides. We hypothesized that such a comparison could provide insight into the ways integrin binding affects fibroblast activation within commonly utilized in vitro cell culture models, and more broadly, to inform the design of materials to modulate fibroblast activation in studies of wound healing and disease. To enable this comparison, we developed a method to conjugate whole proteins to step-growth poly(ethylene glycol) (PEG) hydrogels and investigated fibroblast response to protein-peptide pairs: fibronectin and PHSRN(G)10RGDS or collagen I and (POG)3POGFOGER(POG)4, which are important in matrix remodeling and relevant to fibroblast activation. With this approach, we observed that human pulmonary fibroblasts adopted a similar morphology on fibronectin and PHSRN(G)10RGDS, although with a slight increase in the percentage of alpha smooth muscle actin (αSMA) expressing cells on PHSRN(G)10RGDS. Interestingly, we observed that fibroblasts formed activated clusters on the collagen mimic (POG)3POGFOGER(POG)4 while exhibiting less activation on collagen I. This cell activation and clustering is reminiscent of fibroblast foci that are observed in lung fibrosis, suggesting the relevance of these well-defined polymer-peptide hydrogels for investigating fibrosis and decoupling biochemical and biophysical cues.

8.
ACS Macro Lett ; 7(9): 1105-1110, 2018 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-32832198

RESUMO

Synthetic hydrogels have been widely adopted as well-defined matrices for three-dimensional (3D) cell culture, with increasing interest in systems that enable the co-culture of multiple cell types for probing both cell-matrix and cell-cell interactions in studies of tissue regeneration and disease. We hypothesized that the unique dynamic covalent chemistry of self-healing hydrogels could be harnessed for not only the encapsulation and culture of human cells but also the subsequent construction of layered hydrogels for 3D co-cultures. To test this, we formed hydrogels using boronic acid-functionalized polymers and demonstrated their self-healing in the presence of physiologically-relevant cell culture media. Two model human cell lines, MDA-MB-231 breast cancer cells and CCL151 pulmonary fibroblasts, were encapsulated within these dynamic materials, and good viability was observed over time. Finally, self-healing of cut hydrogel 'blocks' laden with these different cell types was used to create layered hydrogels for the generation of a dynamic co-culture system. This work demonstrates the utility of self-healing materials for multi-dimensional cultures and establishes approaches broadly useful for a variety of biological applications.

9.
Biomater Sci ; 2(5): 634-650, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25379176

RESUMO

Wound healing results from complex signaling between cells and their environment in response to injury. Fibroblasts residing within the extracellular matrix (ECM) of various connective tissues are critical for matrix synthesis and repair. Upon injury or chronic insult, these cells activate into wound-healing cells, called myofibroblasts, and repair the damaged tissue through enzyme and protein secretion. However, misregulation and persistence of myofibroblasts can lead to uncontrolled accumulation of matrix proteins, tissue stiffening, and ultimately disease. Extracellular cues are important regulators of fibroblast activation and have been implicated in their persistence. Hydrogel-based culture models have emerged as useful tools to examine fibroblast response to ECM cues presented during these complex processes. In this Mini-Review, we will provide an overview of these model systems, which are built upon naturally-derived or synthetic materials, and mimic relevant biophysical and biochemical properties of the native ECM with different levels of control. Additionally, we will discuss the application of these hydrogel-based systems for the examination of fibroblast function and fate, including adhesion, migration, and activation, as well as approaches for mimicking both static and temporal aspects of extracellular environments. Specifically, we will highlight hydrogels that have been used to investigate the effects of matrix rigidity, protein binding, and cytokine signaling on fibroblast activation. Last, we will describe future directions for the design of hydrogels to develop improved synthetic models that mimic the complex extracellular environment.

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