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1.
Biofabrication ; 15(4)2023 09 22.
Artículo en Inglés | MEDLINE | ID: mdl-37703870

RESUMEN

Gaucher disease (GD), the most prevalent lysosomal disorder, is caused byGBA1gene mutations, leading to deficiency of glucocerebrosidase, and accumulation of glycosphingolipids in cells of the mononuclear phagocyte system. While skeletal diseases are the leading cause of morbidity and reduced quality of life in GD, the pathophysiology of bone involvement is not yet fully understood, partly due to lack of relevant human model systems. In this work, we present the first 3D human model of GD using aspiration-assisted freeform bioprinting, which enables a platform tool with a potential for decoding the cellular basis of the developmental bone abnormalities in GD. In this regard, human bone marrow-derived mesenchymal stem cells (obtained commercially) and peripheral blood mononuclear cells derived from a cohort of GD patients, at different severities, were co-cultured to form spheroids and differentiated into osteoblast and osteoclast lineages, respectively. Co-differentiated spheroids were then 3D bioprinted into rectangular tissue patches as a bone tissue model for GD. The results revealed positive alkaline phosphatase (ALP) and tartrate-resistant ALP activities, with multi-nucleated cells demonstrating the efficacy of the model, corroborating with gene expression studies. There were no significant changes in differentiation to osteogenic cells but pronounced morphological deformities in spheroid formation, more evident in the 'severe' cohort, were observed. Overall, the presented GD model has the potential to be adapted to personalized medicine not only for understanding the GD pathophysiology but also for personalized drug screening and development.


Asunto(s)
Enfermedad de Gaucher , Humanos , Leucocitos Mononucleares , Calidad de Vida , Huesos , Diferenciación Celular
2.
JIMD Rep ; 59(1): 60-68, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33977031

RESUMEN

Population studies point to regional and ethnicity-specific differences in genetic predisposition for some lysosomal storage disorders (LSDs). The aim of the study was to determine the prevalence of the three treatable forms of lysosomal storage disorders (Gaucher disease [GD], Pompe disease [PD], and Fabry disease [FD]) in a cohort of mostly urban-dwelling individuals of African ancestry, a previously unknown genetic landscape for LSDs. Large-scale selective multistep biochemical and genetic screening was performed in patients seeking healthcare for various health concerns. Fluorimetric enzyme assays for GD, PD, and FD were performed on dried blood spots. Targeted gene sequencing was performed on samples that showed significantly lower enzyme activities (<10% of control mean) after two tiers of enzymatic screening. A total of 5287 unique samples representing a cross section of patients who visited Howard University Hospital and College of Medicine from 2015 to 2017 were included in the study. Study samples were obtained from a population where ~90% reported as African-American, ~5% Hispanic, and <5% Caucasian or other. Regarding GD, three subjects had either homozygous or heterozygous mutations in the GBA gene. As to PD, eight subjects were either homozygous or compound heterozygous for GAA mutations, including three novel mutations: (a) c.472 A > G; p.T158A, (b) c.503G > T; p.R168L, (c) c.1985del. Regarding FD, two subjects had pathogenic GLA mutations, and four had single nucleotide polymorphisms in the 5'UTR, previously implicated in modulating gene expression. The findings highlight a higher incidence of abnormal enzyme levels and pathogenic mutations in the target population reflecting ancestry-based specific genotype and phenotype variations.

3.
Int J Mol Sci ; 21(19)2020 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-33003611

RESUMEN

Fabry disease (FD) is an X-linked lysosomal disorder caused by mutations in GLA gene resulting in lack of or faulty α-galactosidase A (α-GalA) enzyme. Enzyme replacement therapy (ERT) with recombinant human α-GalA enzyme (agalsidase) is the standard treatment option for FD. Infusion-related reactions (IRRs), with symptoms ranging from rigors, to fever, pain, vomiting, angioedema and diarrhea, are often seen due to immune response against the exogenous enzyme. To elucidate the mechanisms causing the IRRs in FD, eight patients who developed IRRs were investigated. All, except one, tested negative for agalsidase-specific IgE and had normal tryptase levels. Circulating dendritic cells were drastically reduced during IRRs, suggesting possible sequestration to the sites of inflammation. An increase in NK cells and a decrease in T cells were also observed. Cytokines IL-4, IL-8 and TNF-α showed a significant increase, indicating nonspecific degranulation of mast cells. All IRRs were managed successfully using a combination of standard premedications and mast cell stabilizers without any interruption of therapy. Taken together, the results indicate crosstalk between immune cells resulting in IgE-independent mast-cell-specific allergic inflammation. Mast cell stabilizers could be used to control IRRs and for safe reintroduction of agalsidase in patients previously treated with ERT.


Asunto(s)
Enfermedad de Fabry/tratamiento farmacológico , Enfermedad de Fabry/inmunología , Inflamación/inmunología , Isoenzimas/inmunología , Proteínas Recombinantes/inmunología , alfa-Galactosidasa/inmunología , Inmunidad Adaptativa/efectos de los fármacos , Inmunidad Adaptativa/inmunología , Adolescente , Adulto , Niño , Terapia de Reemplazo Enzimático/efectos adversos , Enfermedad de Fabry/genética , Enfermedad de Fabry/patología , Humanos , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/inmunología , Inmunoglobulina E/inmunología , Inflamación/patología , Reacción en el Punto de Inyección/genética , Reacción en el Punto de Inyección/inmunología , Isoenzimas/administración & dosificación , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Persona de Mediana Edad , Proteínas Recombinantes/administración & dosificación , Adulto Joven , alfa-Galactosidasa/administración & dosificación
4.
Life (Basel) ; 10(9)2020 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-32932790

RESUMEN

There is limited data on pregnancy outcomes in Pompe Disease (PD) resulting from deficiency of the lysosomal enzyme acid alpha-glucosidase. Late-onset PD is characterized by progressive proximal muscle weakness and decline of respiratory function secondary to the involvement of the respiratory muscles. In a cohort of twenty-five females, the effects of both PD on the course of pregnancy and the effects of pregnancy on PD were investigated. Reproductive history, course of pregnancy, use of Enzyme replacement therapy (ERT), PD symptoms, and outcomes of each pregnancy were obtained through a questionnaire. Among 20 subjects that reported one or more pregnancies, one subject conceived while on ERT and continued therapy through two normal pregnancies with worsening of weakness during pregnancy and improvement postpartum. While fertility was not affected, pregnancy may worsen symptoms, or cause initial symptoms to arise. Complications with pregnancy or birth were not higher, except for an increase in the rate of stillbirths (3.8% compared to the national average of 0.2-0.7%). Given small sample size and possible bias of respondents being only women who have been pregnant, further data may be needed to better analyze the effects of pregnancy on PD, and the effects of ERT on pregnancy outcomes.

5.
Biomolecules ; 10(4)2020 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-32244296

RESUMEN

Gaucher disease (GD) is caused by mutations in the GBA gene, leading to deficient activity of the lysosomal enzyme glucocerebrosidase. Among all the symptoms across various organ systems, bone disease is a major concern as it causes high morbidity and reduces quality of life. Enzyme replacement therapy (ERT) is the most accepted treatment; however, there are still unmet needs. As an alternative, substrate reduction therapy (SRT) was developed using glucosylceramide synthase inhibitors. In the current study, the effects of ERT vs. SRT were compared, particularly the immunological and bone remodeling aspects. GD subjects were divided into three cohorts based on their treatment at initial visit: ERT, SRT, and untreated (UT). Immunophenotyping showed no significant immune cell alterations between the cohorts. Expression of RANK/RANKL/Osteoprotegerin pathway components on immune cells and the secreted markers of bone turnover were analyzed. In the ERT cohort, no significant changes were observed in RANK, RANKL or serum biomarkers. RANKL on T lymphocytes, Osteopontin and MIP-1ß decreased with SRT treatment indicating probable reduction in osteoclast activity. Other secreted factors, Osteocalcin and RANKL/Osteoprotegerin did not change with the treatment status. Insights from the study highlight personalized differences between subjects and possible use of RANK pathway components as markers for bone disease progression.


Asunto(s)
Huesos/patología , Terapia de Reemplazo Enzimático , Enfermedad de Gaucher/inmunología , Enfermedad de Gaucher/terapia , Adolescente , Adulto , Huesos/efectos de los fármacos , Niño , Femenino , Enfermedad de Gaucher/patología , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven
6.
Am J Transl Res ; 11(3): 1683-1696, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30972193

RESUMEN

Fabry disease (FD) is a rare X-linked genetic disorder caused by mutations in the GLA gene encoding the lysosomal enzyme, α-galactosidase A (α-gal A). The mutations lead to lack of or faulty enzyme causing accumulation of globotriaosylceramide (Gb3) and related glycosphingolipids including globotriaosylsphingosine (lyso-Gb3). Treatment options for FD include enzyme replacement therapy. There are two different recombinant α-gal A enzymes, where agalsidase beta has been approved by FDA for use in the USA while both agalsidase beta and agalsidase alfa are being prescribed in many other countries. Several FD patients in the USA were switched to agalsidase alfa for a certain period of time due to supply shortage of agalsidase beta but were switched back to agalsidase beta upon availability. Due to the fact that some glycolipids may serve as antigens, various immune abnormalities have been associated with several lysosomal storage disorders (LSDs). In the present clinical study we evaluated alterations in peripheral immune cell subsets in patients with FD (n=27) compared to healthy control group (n=27). Patients with FD showed persistent T cell associated abnormalities, including skewed T helper to cytotoxic T cell ratio and elevated fraction of memory T cells and expression of activation markers on T cell subsets. Further, the study elucidated the effect of switching from agalsidase alfa to agalsidase beta on immune system as well as other clinical markers. While there was relative decrease in plasma lyso-Gb3 as well as urine lyso-Gb3 over time, their levels remained above the reference values. The immune abnormalities did not correlate with gender, age or lyso-Gb3 levels, indicating that these persistent changes were inherent to FD irrespective of the extent of substrate accumulation.

7.
Blood Cells Mol Dis ; 68: 81-85, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-27839980

RESUMEN

Gaucher disease (GD) is caused by mutations in the GBA gene encoding lysosomal enzyme, ß-glucocerebrosidase (GCase). GCase deficiency results in accumulation of its substrates in cells of macrophage lineage, affecting multiple organ systems. Enzyme replacement therapy (ERT) with recombinant human GCase is the standard of care to treat GD. In GD, it is well established that there are immune alterations, clinically presenting as lymphadenopathy, gammopathies, and predisposition to hematological cancers. We examined the effect of ERT on immune dysregulations in treatment-naïve GD patients longitudinally after the initiation of ERT. Immunophenotyping was performed in peripheral blood samples obtained before and after ERT. T and B lymphocyte subsets, NK, NKT and dendritic cells were evaluated. In all treatment naïve patients at baseline, transitional B cells, characterized by CD21low expression were markedly elevated. After establishment of stable-dose therapy, CD21low cells were significantly reduced and subsequent increase in CD21Hi B lymphocytes indicated improved B cell maturation. Class-switching and memory B cell defects which were noted prior to treatment were found to be normalized. An increase in dendritic cells also resulted after the treatment. Our data shows that GD affects across various immune cell types and ERT or its effects directly improve affected immunological parameters.


Asunto(s)
Linfocitos B/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Terapia de Reemplazo Enzimático , Enfermedad de Gaucher/tratamiento farmacológico , Glucosilceramidasa/uso terapéutico , Adulto , Linfocitos B/inmunología , Linfocitos B/patología , Células Dendríticas/inmunología , Células Dendríticas/patología , Terapia de Reemplazo Enzimático/métodos , Femenino , Enfermedad de Gaucher/inmunología , Enfermedad de Gaucher/patología , Humanos , Inmunofenotipificación , Masculino
8.
Blood Cells Mol Dis ; 68: 100-105, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-27839983

RESUMEN

Deficiency of the lysosomal enzyme, ß-glucocerebrosidase, and accumulation of its substrate in cells of the reticuloendothelial system affects multiple organ systems in patients with Gaucher disease (GD). Lipid laden macrophages turn into Gaucher cells (GC) which are the pathological characteristic of GD. GC focally accumulate in the liver, spleen and at extraosseous sites to form benign lesions called Gaucheromas. Gaucheromas pose diagnostic and therapeutic challenges. We studied the pathophysiology of extraosseous Gaucheroma formation in a cohort of patients with GD. Among 63 patients followed at a single center, 3 patients with genotypes L444P/L444P and N370S/N370S, were diagnosed with extraosseous Gaucheromas. Flow cytometry revealed a higher expression of CD16+/CCR4+ non-classical monocytes in blood of GD patients who have developed Gaucheromas. A biopsy showed infiltration of GC, which reactivity against CD163, CD68 and VEGF. The cell proliferative marker Ki67 and CCL2, a factor anti-tumor activity, were negative. Our study indicates that extraosseous Gaucheromas are comprised of cellular elements with characteristics of tumor-associated macrophages, the major players in cancer related inflammation. The occurrence of non-classical CD16+/CCR4+ monocytes reflect the underlying cause for the accumulation of the macrophages capable of migrating to distant sites outside the reticuloendotheial system, and giving rise to tumor-like Gaucheromas.


Asunto(s)
Carcinogénesis/patología , Enfermedad de Gaucher/complicaciones , Enfermedad de Gaucher/patología , Macrófagos/patología , Adulto , Anciano , Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Carcinogénesis/genética , Estudios de Cohortes , Femenino , Enfermedad de Gaucher/genética , Genotipo , Humanos , Macrófagos/metabolismo , Masculino , Receptores CCR4/análisis , Receptores de Superficie Celular/análisis , Factor A de Crecimiento Endotelial Vascular/análisis , Adulto Joven
9.
PLoS One ; 11(12): e0168135, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27942037

RESUMEN

Gaucher disease (GD) patients often present with abnormalities in immune response that may be the result of alterations in cellular and/or humoral immunity. However, how the treatment and clinical features of patients impact the perturbation of their immunological status remains unclear. To address this, we assessed the immune profile of 26 GD patients who were part of an enzyme replacement therapy (ERT) study. Patients were evaluated clinically for onset of GD symptoms, duration of therapy and validated outcome measures for ERT. According to DS3 disease severity scoring system criteria, they were assigned to have mild, moderate or severe GD. Flow cytometry based immunophenotyping was performed to analyze subsets of T, B, NK, NKT and dendritic cells. GD patients showed multiple types of immune abnormalities associated to T and B lymphocytes with respect to their subpopulations as well as memory and activation markers. Skewing of CD4 and CD8 T cell numbers resulting in lower CD4/CD8 ratio and an increase in overall T cell activation were observed. A decrease in the overall B cells and an increase in NK and NKT cells were noted in the GD patients compared to controls. These immune alterations do not correlate with GD clinical type or level of biomarkers. However, subjects with persistent immune alterations, especially in B cells and DCs correlate with longer delay in initiation of ERT (ΔTX). Thus, while ERT may reverse some of these immune abnormalities, the immune cell alterations become persistent if therapy is further delayed. These findings have important implications in understanding the immune disruptions before and after treatment of GD patients.


Asunto(s)
Terapia de Reemplazo Enzimático , Enfermedad de Gaucher/inmunología , Enfermedad de Gaucher/terapia , Tiempo de Tratamiento , Adolescente , Adulto , Anciano , Niño , Femenino , Citometría de Flujo , Enfermedad de Gaucher/fisiopatología , Humanos , Activación de Linfocitos , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Resultado del Tratamiento , Adulto Joven
10.
Blood Cells Mol Dis ; 59: 8-15, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27282561

RESUMEN

Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the gene encoding acid-ß-glucosidase, resulting in functional disruptions in degradation of glycosphingolipids and lysosomal accumulation of the substrates. The most frequent clinical presentations of GD are thrombocytopenia, splenomegaly and bone pain. Prior to advent of enzyme replacement therapy, splenectomy was performed for complications of hypersplenism such as severe thrombocytopenia and transfusion dependency. Though there is evidence about worsening bone disease after splenectomy, there is no systematic study to assess its effects on the immune system in GD patients. In order to investigate the long-term immunological effects of splenectomy, we used flow cytometry to compare the immunophenotypes of GD patients who had undergone splenectomy (SGD) to those with intact spleen. The results show that SGD patients have significantly fewer CD27(+)/IgM(+) B-cells but more CD4(+)/CD45RO(+) and CD8(+)/CD45RO(+) T-cells. The most surprising finding was an almost complete absence of circulating dendritic cells in SGD patients. In addition, splenectomized subjects had comorbidities, the most common being monoclonal gammopathy of undetermined significance (MGUS). Taken together, these results highlight the persistence of multiple immune alterations and comorbidities coexisting in higher frequency in the SGD group and they are not affected by GD specific therapy.


Asunto(s)
Comorbilidad , Enfermedad de Gaucher/cirugía , Sistema Inmunológico , Esplenectomía/efectos adversos , Adulto , Anciano , Linfocitos B/inmunología , Estudios de Casos y Controles , Células Dendríticas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Gammopatía Monoclonal de Relevancia Indeterminada/etiología , Linfocitos T/inmunología
11.
PLoS One ; 10(3): e0113483, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25789989

RESUMEN

Eosinophilic disorders of the gastrointestinal tract are an emerging subset of immune pathologies within the spectrum of allergic inflammation. Eosinophilic Esophagitis (EoE), once considered a rare disease, is increasing in incidence, with a rate of over 1 in 10,000 in the US, for unknown reasons. The clinical management of EoE is challenging, thus there is an urgent need for understanding the etiology and pathophysiology of this eosinophilic disease to develop better therapeutic approaches. In this open label, single arm, unblinded study, we evaluated the effects of an anti-IgE treatment, omalizumab, on local inflammation in the esophagus and clinical correlates in patients with EoE. Omalizumab was administered for 12 weeks to 15 subjects with long standing EoE. There were no serious side effects from the treatment. Esophageal tissue inflammation was assessed both before and after therapy. After 3 months on omalizumab, although tissue Immunoglobulin E (IgE) levels were significantly reduced in all but two of the subjects, we found that full remission of EoE, which is defined as histologic and clinical improvement only in 33% of the patients. The decrease in tryptase-positive cells and eosinophils correlated significantly with the clinical outcome as measured by improvement in endoscopy and symptom scores, respectively. Omalizumab-induced remission of EoE was limited to subjects with low peripheral blood absolute eosinophil counts. These findings demonstrate that in a subset of EoE patients, IgE plays a role in the pathophysiology of the disease and that anti-IgE therapy with omalizumab may result in disease remission. Since this study is open label there is the potential for bias, hence the need for a larger double blind placebo controlled study. The data presented in this pilot study provides a foundation for proper patient selection to maximize clinical efficacy.


Asunto(s)
Antialérgicos/administración & dosificación , Esofagitis Eosinofílica/tratamiento farmacológico , Omalizumab/administración & dosificación , Adolescente , Adulto , Anciano , Niño , Esofagitis Eosinofílica/sangre , Esofagitis Eosinofílica/patología , Eosinófilos/metabolismo , Eosinófilos/patología , Femenino , Humanos , Inmunoglobulina E/sangre , Recuento de Leucocitos , Masculino
13.
Tissue Eng Part A ; 16(4): 1363-8, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19912046

RESUMEN

The efficient culture of stem cells from epithelial tissues such as skin and corneas is important for both experimental studies and clinical applications of tissue engineering. We now demonstrate that treatment of human-skin-derived keratinocytes with a Rho-associated protein kinase inhibitor Y-27632 for the initial 6 days of primary culture can increase the number of keratinocytes that possess stem cell properties to form colonies during in vitro culture of freshly isolated cells and subsequent passage (50-fold). Further, we show that Y-27632 treatment can increase the total number of prostate epithelial cells derived from human prostate specimens. Therefore, the use of Y-27632 during primary cultures offers a simple and effective way to prepare a large number of epithelial stem cells from various human epithelial tissues.


Asunto(s)
Amidas/farmacología , Inhibidores Enzimáticos/farmacología , Piridinas/farmacología , Células Madre/citología , Células Madre/efectos de los fármacos , Quinasas Asociadas a rho/antagonistas & inhibidores , Células 3T3 , Animales , Separación Celular , Células Cultivadas , Técnicas de Cocultivo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Prepucio/citología , Humanos , Recién Nacido , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/enzimología , Masculino , Ratones , Próstata/citología , Células Madre/enzimología , Ingeniería de Tejidos
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